WO2006022636A1 - Méthodes d’identification du risque d’apparition de diabètes de type ii et traitements associés - Google Patents

Méthodes d’identification du risque d’apparition de diabètes de type ii et traitements associés Download PDF

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WO2006022636A1
WO2006022636A1 PCT/US2004/023933 US2004023933W WO2006022636A1 WO 2006022636 A1 WO2006022636 A1 WO 2006022636A1 US 2004023933 W US2004023933 W US 2004023933W WO 2006022636 A1 WO2006022636 A1 WO 2006022636A1
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diabetes
type
polymorphic
nucleic acid
subject
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PCT/US2004/023933
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Maria L. Langdown
Matthew Roberts Nelson
Rikard Henry Reneland
Stefan M. Kammerer
Andreas Braun
Carolyn R. Hoyal-Wrightson
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Sequenom, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to genetic methods for identifying predisposition to type II diabetes, also known as non-insulin dependent diabetes, and treatments that specifically target the disease.
  • Type I diabetes insulin-dependent diabetes
  • pancreatic ⁇ -cells pancreatic ⁇ -cells with subsequent insulin deficiency.
  • Type II diabetes non-insulin dependent diabetes
  • Type II diabetes represents 90-95% of the affected population, more than 100 million people worldwide. Approximately 17 million Americans suffer from type II diabetes, although 6 million don't even know they have the disease. The prevalence of the disease has jumped 33% in the last decade and is expected to rise further as the baby boomer generation gets older and more overweight. The global figure of people with diabetes is set to rise to an estimated 150 to 220 million in 2010, and 300 million in 2025. The widespread problem of diabetes has crept up on an unsuspecting health care community and has already imposed a huge burden on health-care systems (Zimmet et al (2001) Nature 414: 782-787).
  • the onsebof type II diabetes can be insidious, or even clinically unapparent, making diagnosis difficult. Even when the disease is properly diagnosed, many of those treated do not have adequate control over their diabetes, resulting in elevated sugar levels in the bloodstream that slowly destroys the kidneys, eyes, blood vessels and nerves. This late damage is an important factor contributing to mortality in diabetics.
  • Type II diabetes is associated with peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion (DeFronzo, R. A. (1988) Diabetes 37:667- 687), although the primary pathogenic lesion on type II diabetes remains elusive. Many have suggested that primary insulin resistance of the peripheral tissues is the initial event. Genetic epidemiological studies have supported this view. Similarly, insulin secretion abnormalities have been argued as the primary defect in type II diabetes. It is likely that both phenomena are important in the development of type II diabetes, and genetic defects predisposing to both are likely to be important contributors to the disease process (Rimoin, D.L., et al. (1996) Emery and Rimoin's Principles and Practice of Medical Genetics 3rd Ed. 1: 1401-1402).
  • polymorphic variations in human genomic DNA are associated with the occurrence of type II diabetes, also known as non-insulin dependent diabetes.
  • polymorphic variants in loci containing SLC22A1, TTN, LOCI 12609 (also known as C6orfll7), TRPSl, PRAME, ZNF221, FOXA2, JAMS and GLLS regions in human genomic DNA have been associated with risk of type II diabetes.
  • identifying a subject at risk of type II diabetes and/or a risk of type II diabetes in a subject which comprise detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in or around loci described herein in a human nucleic acid sample.
  • two or more polymorphic variations are detected and in some embodiments, 3 or more, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more polymorphic variants are detected.
  • nucleic acids that include one or more polymorphic variations associated with occurrence of type II diabetes, as well as polypeptides encoded by these nucleic acids.
  • methods for identifying candidate therapeutic molecules for treating type II diabetes and other insulin-related disorders as well as methods for treating type II diabetes in a subject by identifying a subject at risk of type II diabetes and treating the subject with a suitable prophylactic, treatment or therapeutic molecule.
  • compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleic acid, with a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence, or a nucleic acid that hybridizes to such a nucleotide sequence under stringent conditions.
  • the RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid is designed from a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence that includes one or more type II diabetes associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence.
  • nucleic acids bound to a solid surface in which one or more nucleic acid molecules of the array have a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing.
  • compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAM3 or GLB polypeptide, with an antibody that specifically binds to the polypeptide.
  • the antibody specifically binds to an epitope in the polypeptide that includes a non-synonymous amino acid modification associated with type II diabetes (e.g., results in an amino acid substitution in the encoded polypeptide associated with type II diabetes).
  • the antibody specifically binds to an epitope comprising a glycine corresponding to position 557 in a ZNF221 polypeptide (e.g., a polypeptide having an amino acid sequence in SEQ ID NO: 27).
  • Figures 1 A-IC show proximal SNPs in a SLC22A1 region in genomic DNA for females, males and combined results, respectively.
  • Figures 2A-2C show proximal SNPs in a TTN region in genomic DNA for females, males and combined results, respectively.
  • Figures 3A-3C show proximal SNPs in a LOCI 12609 region in genomic DNA for females, males and combined results, respectively.
  • Figures 4A-4C show proximal SNPs in a TRPSl region in genomic DNA for females, males and combined results, respectively.
  • Figures 5A-5C show proximal SNPs in a PRAME region in genomic DNA for females, males and combined results, respectively.
  • Figures 6A-6C show proximal SNPs in a ZNF221 region in genomic DNA for females, males and combined results, respectively.
  • Figures 7A-7C show proximal SNPs in a FOXA2 region in genomic DNA for females, males and combined results, respectively.
  • Figures 8A-8C show proximal SNPs in a JAMS region in genomic DNA for females, males and combined results, respectively.
  • Figures 9A-9C show proximal SNPs in a GLB region in genomic DNA for females, males and combined results, respectively.
  • each SNP in the chromosome is shown on the x-axis and the y-axis provides the negative logarithm of the p-value comparing the estimated allele frequency in the cases to that of the control group. Also shown in the figures are exons and introns of the genes in approximate chromosomal positions.
  • Figures 1 OA-I OG show results of an odds-ratio meta analysis for SLC22A1, LOCI 12609, PRAME, ZNF221, FOXA2, JAM3 and GLB regions, respectively.
  • polymorphic variants described in a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB locus in human genomic DNA are associated with occurrence of type II diabetes in subjects.
  • detecting genetic determinants in and around this locus associated with an increased risk of type II diabetes occurrence can lead to early identification of a risk of type II diabetes and early application of preventative and treatment measures.
  • Associating the polymorphic variants with type II diabetes also has provided new targets for diagnosing type II diabetes, and methods for screening molecules useful in diabetes treatments and diabetes preventatives.
  • SLC22A1 encodes solute carrier family 22, member 1, which is a polyspecific organic cation transporters in the liver, kidney, intestine, and other organs are critical for elimination of many endogenous small organic cations as well as a wide array of drugs from the systemic circulation, maintaining body fluid homeostasis, and acting as a defense system against toxic agents.
  • This gene is one of three similar cation transporter genes located in a cluster on chromosome 6.
  • the encoded protein contains twelve putative transmembrane domains and is a plasma integral membrane protein.
  • TTN encodes a 3-megadalton protein composed of 244 copies of immunoglobulin and fibronectin type III domains.
  • TITIN is stringlike in structure and in vivo spans from the M to Z lines, important for structural integrity of the myofibril and passive tension.
  • Titin phosphorylates Titin-cap (a 19 kDa protein) a protein that associates with the N-terminus of titin.
  • TRPSl trichorhinophalangeal syndrome I
  • FOXA2 encodes a polypeptide that is a member of a forkhead class of DNA-binding proteins referred to as forkhead box A2.
  • FOXA2 consists of two transcript variants, which differ in the 5' UTR but encode the same protein.
  • FOXA2 serves as a transcription factor involved in the regulation of pancreatic development. Its expression is known to be regulated by HNF6 in human hepatoma cells.
  • the JAM3 gene encodes a protein that is a member of the junctional adhesion molecule protein family and acts as a receptor for another member of this family.
  • the protein encoded by this immunoglobulin superfamily gene member is localized in the tight junctions between high endothelial cells. Unlike other proteins in this family, this protein is unable to adhere to leukocyte cell lines and only forms weak homotypic interactions. Tight junctions represent one mode of cell- to-cell adhesion in epithelial or endothelial cell sheets, forming continuous seals around cells and serving as a physical barrier to prevent solutes and water from passing freely through the paracellular space.
  • JAM3 gene contains 9 exons and spans more than 88 kb.
  • the deduced 310- arnino acid protein is more than 30% identical to JAM2 and JAMl . It possesses a signal sequence; 2 Ig-like folds, one a V type and the other a C2 type, containing 6 cysteines; 2 potential N- glycosylation sites; and a 46-amino acid intracellular tail with a C-terminal binding motif for PDZ domains and a phosphorylation site.
  • Northern blot analysis revealed wide expression of an approximately 3.3-kb transcript, with highest levels in placenta, brain, and kidney. Expression was also detected in cultured endothelial cells.
  • the GLB gene encodes a protein which belongs to the C2H2-type zinc finger proteins subclass of the GIi family. They are characterized as a transcription factor that binds to DNA through zinc finger motifs. These motifs have conserved H-C links. GIi family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling. The protein encoded by this gene localizes in the cytoplasm and activates patched Drosophila homolog (PTCH) gene expression. It is also thought to play a role during embryogenesis. The encoded protein is associated with several phenotypes- Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial Polydactyly type IV, postaxial Polydactyly types Al and B.
  • Type II diabetes refers to non-insulin-dependent diabetes.
  • Type II diabetes refers to an insulin-related disorder in which there is a relative disparity between endogenous insulin production and insulin requirements, leading to elevated hepatic glucose production, elevated blood glucose levels, inappropriate insulin secretion, and peripheral insulin resistance.
  • Type II diabetes has been regarded as a relatively distinct disease entity, but type II diabetes is often a manifestation of a much broader underlying disorder (Zimmet et al (2001) Nature 414: 782-787), which may include metabolic syndrome (syndrome X), diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes, autoimmune diabetes), hyperinsulinemia, hyperglycemia, impaired glucose tolerance (IGT), hypoglycemia, B-cell failure, insulin resistance, dyslipidemias, atheroma, insulinoma, hypertension, hypercoagulability, microalbuminuria, and obesity and obesity-related disorders such as visceral obesity, central fat, obesity-related type II diabetes, obesity-related atherosclerosis, heart disease, obesity-related insulin resistance, obesity- related hypertension, microangiopathic lesions resulting from obesity-related type II diabetes, ocular lesions caused by microangiopathy in obese individuals with obesity-related type II diabetes, and renal lesions caused by microangiopathy in obese individuals with obesity-related type II
  • type II diabetes Some of the more common adult onset diabetes symptoms include fatigue, excessive thirst, frequent urination, blurred vision, a high rate of infections, wounds that heal slowly, mood changes and sexual problems. Despite these known symptoms, the onset of type II diabetes is often not discovered by health care professionals until the disease is well developed. Once identified, type II diabetes can be recognized in a patient by measuring fasting plasma glucose levels and/or casual plasma glucose levels, measuring fasting plasma insulin levels and/or casual plasma insulin levels, or administering oral glucose tolerance tests or hyperinsulinemic euglycemic clamp tests.
  • individuals having type II diabetes can be selected for genetic studies. Also, individuals having no history of metabolic disorders, particularly type II diabetes, often are selected for genetic studies as controls. The individuals selected for each pool of case and controls, were chosen following strict selection criteria in order to make the pools as homogenous as possible. Selection criteria for the study described herein included patient age, ethnicity, BMI, GAD (Glutamic Acid Decarboxylase) antibody concentration, and HbAIc (glycosylated hemoglobin AIc) concentration.
  • GAD antibody is present in association ⁇ with islet cell destruction, and therefore can be utilized to differentiate insulin dependent diabetes (type I diabetes) from non-insulin dependent diabetes (type II diabetes). HbAIc levels will reveal the average blood glucose over a period of 2-3 months or more specifically, over the life span of a red blood cell, by recording the number of glucose molecules attached to hemoglobin.
  • polymorphic site refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals.
  • a polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example.
  • a polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region.
  • a polymorphic site is often one nucleotide in length, which is referred to herein as a "single nucleotide polymorphism" or a "SNP.”
  • each nucleotide sequence is referred to as a "polymorphic variant" or "nucleic acid variant.”
  • polymorphic variants represented in a minority of samples from a population is sometimes referred to as a “minor allele” and the polymorphic variant that is more prevalently represented is sometimes referred to as a "major allele.”
  • minor allele the polymorphic variant represented in a minority of samples from a population
  • major allele the polymorphic variant that is more prevalently represented
  • Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as
  • allelotyped and/or genotyped refers to a process for determining the allele frequency for a polymorphic variant in pooled DNA samples from cases and controls. By pooling DNA from each group, an allele frequency for each SNP in each group is calculated. These allele frequencies are then compared to one another.
  • genotyped refers to a process for determining a genotype of one or more individuals, where a “genotype” is a representation of one or more polymorphic variants in a population.
  • a genotype or polymorphic variant may be expressed in terms of a "haplotype," which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population.
  • haplotype refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population.
  • two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation.
  • Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position.
  • the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.
  • phenotype refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like.
  • An example of a phenotype is occurrence of type II diabetes.
  • a polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10% or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population.
  • a polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid.
  • a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5 ' untranslated region (UTR), a 3 ' UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide.
  • DNA e.g., genomic DNA (gDNA) and complementary DNA (cDNA)
  • RNA e.g., mRNA, tRNA, and rRNA
  • Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs456598, rs3798168, rs662138, rs644992, rs637841, rs622342, rs650284 and rs2297374.
  • a guanine at position 24609, a thymine at position 45495, a cytosine at position 50164, a guanine at position 51691, an adenine at position 53067, an adenine at position 58554, an adenine at position 60223, and a thymine at position 61673 were associated with risk of type II diabetes.
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs2195070, rs967507, rs2366577, rs3829747, rs2857265, rs2303539, rs3731750, rs3731749, rs747122, rs2366751, rs3813247, rs3731745, rs3813246, rs3813245, rs744426, rs2042996, rsl560221, rs2288569, rs2288568, rsl001238, rs2288566, rs2288563, rs2243452, rs2246932, rs2562844, rsl 366677, rs2742355, rs2562846, rs2163008, rs2562848, rs2562849, rs2742353, rs
  • Polymorphic variants at the following positions were associated with an increased risk of type ⁇ diabetes: rs2480187, rs6926656, rs2497120, rs2480195, rs6914226, rs6922368, rs2480196, rs2497136, rs2497131, rs2480197, rs3219, rs2497126, rs2480201, rs2476913, rs9449794, rs7741955, and rs2497150.
  • Li the TRPSl locus polymorphic variants at positions selected from the group consisting of rs2625681, rs2625680, rs2625678, rs2625676, rs2737202, rsl905376, rs2178946, rs800919, rs2142327, rs2884365, rs800918, rs800917, rs800916, rs2625683, rs2625682, rsl483583, rs3808432, rsl483584, rsl483585, rs800912, rs3779878, rs3808433, rs3808434, rs3808435, rs3808436, rs3808437, rs3824201, rs3808438, rs3808439, rs3808440, rsl351744, rsl483589, rs
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rsl905376, rs2178946, rsl483583, rs3779878, rs3808435, rs3808437, rs3808438, rs3808440, rsl483589, rs3802219, rs2884366, rs3779879, rs3808447, rs3808448, rs3808450, rs2223103, rsl 160333, rslO28274, rsl963677, rs3779881, rs2358021, rs2049864, rs2049866, rs3808456, rs3808457,,rs719422, rs2178949, rs2272619, rs3808461, rs2737203, rs2737205, rs2721929, r
  • polymorphic variants at positions selected from the group consisting of rsl 1109, rs361737, AA at position 522, rs2236729, rs2236730, rs2051486, rs2051487, rs2051488, rs965764, rs2006783, rs2032416, rs362139, AB at position 19890, rs361901, rs361676, rs362184, rs361797, rs361907, rs362004, rs362176, rs361570, rs361595, rs361778, rs361709, rs361558, rs361762, rs361737, rs362003, rs362011, rs361721, rs361959, rs362230, rs361970, rs362012,
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rsl 1109, rs361709, rs362012, rs362167, rs361976, rs361864, rs362107, rs361940, rs361828, rs361829, rs2156888, rs2156889, rs3827305, rs7104, rsl3604, rslO24372, rs2283799, rs2877079, rs2266991, rs3827306, rs460608, rs457967, rs2236732 and rs715516.
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs374652, rs445865, rs2356537, rs2356538, rs2284243, rs442543, rs397276, rs365745, rs366111, rs2191565, rs381888, rs415913, rs451388, rs430107 and rs2191566.
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs2404167, rs2021681, rsl203868, rs945982, rsl203871, rsl203873, rsl203876, rsl203877, rsl203879, rsl203880, rsl203881, rsl203883, rsl203884, rsl203886, rs2471, rsl203889, rs2277763, rsl203892, rs932560, rsl203894, rsl203896, rsl203898, rsl203899, rsl203905, rsl203907, rsl 1482531, rsl055080, rs2277764, rsl337918, rsl209523, rsl337919, r
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs470780, rs2510345, rsl784500, rs470372, rs470474, rs470536, rs470522, rs470639, rs470565, rs470953, rs639165, rs626717, rs658544, rs470882, rs470425, rs610684, rs625740, rs624504, rs470547, rs470500, rs470713, rs470570, rs470936, rs470600, rs470809, rs595584, rs610382, rs597320, and rs2014708.
  • Polymorphic variants at the following positions were associated with an increased risk of type II diabetes: rs699493, rs846265, rs7785287, rs846263, rs2282920, rs2330284, rs917229, rs2237422, rs2237420, rsl527499, rs6959829, rs2237415, rs6959294, rs7793034, rsl405750, rs4724092, rs4724093, rs3801168, rslO24552, rs846291, rs846290, rs846315, rs846312, rs846311, rs846310, rs846309, rs846308, rs846306, rs2108165, rs3801174, rs3801177, rs3839733, rs3801183, r
  • methods for identifying a polymorphic variation associated with type II diabetes that is proximal to an incident polymorphic variation associated with type II diabetes which comprises identifying a polymorphic variant proximal to the incident polymorphic variant associated with type II diabetes, where the incident polymorphic variant is in a SLC22A1, TIN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence.
  • the nucleotide sequence often comprises a polynucleotide sequence selected from the group consisting of (a) a polynucleotide sequence of SEQ ID NO: 1-9; (b) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 1-9; and (c) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-9 or a polynucleotide sequence 90% or more identical to the polynucleotide sequence of SEQ ID NO: 1-9.
  • the presence or absence of an association of the proximal polymorphic variant with type II diabetes is determined using a known association method, such as a method described in the Examples hereafter.
  • the incident polymorphic variant is a polymorphic variant associated with type II diabetes described herein.
  • the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database, hi other embodiments, the polymorphic variant identified is not publicly disclosed and is discovered using a known method, including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic samples.
  • a known method including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic samples.
  • the proximal polymorphic variant often is identified in a region surrounding the incident polymorphic variant, hi certain embodiments, this surrounding region is about 50 kb flanking the first polymorphic variant (e.g. about 50 kb 5' of the first polymorphic variant and about 50 kb 3' of the first polymorphic variant), and the region sometimes is composed of shorter flanking sequences, such as flanking sequences of about 40 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 7 kb, about 5 kb, or about 2 kb 5' and 3' of the incident polymorphic variant, hi other embodiments, the region is composed of longer flanking sequences, such as flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 90 kb, about 95 kb, or about 100 kb
  • polymorphic variants associated with type II diabetes are identified iteratively. For example, a first proximal polymorphic variant is associated with type II diabetes using the methods described above and then another polymorphic variant proximal to the first proximal polymorphic variant is identified (e.g., publicly disclosed or discovered) and the presence or absence of an association of one or more other polymorphic variants proximal to the first proximal polymorphic variant with type II diabetes is determined.
  • the methods described herein are useful for identifying or discovering additional polymorphic variants that may be used to further characterize a gene, region or loci associated with a condition, a disease (e.g., type II diabetes), or a disorder.
  • allelotyping or genotyping data from the additional polymorphic variants may be used to identify a functional mutation or a region of linkage disequilibrium.
  • polymorphic variants identified or discovered within a region comprising the first polymorphic variant associated with type II diabetes are genotyped using the genetic methods and sample selection techniques described herein, and it can be determined whether those polymorphic variants are in linkage disequilibrium with the first polymorphic variant.
  • the size of the region in linkage disequilibrium with the first polymorphic variant also can be assessed using these genotyping methods.
  • methods for determining whether a polymorphic variant is in linkage disequilibrium with a first polymorphic variant associated with type II diabetes can be used in prognosis/diagnosis methods described herein.
  • a nucleic acid variant may be represented on one or both strands in a double-stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous)).
  • nucleic acid includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, siRNA and tRNA) and analogs of DNA or RNA, for example, by use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded and it is often double-stranded.
  • isolated or purified nucleic acid refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid.
  • isolated includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated.
  • An "isolated” nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the term "gene” refers to a nucleotide sequence that encodes a polypeptide.
  • the nucleic acid often comprises a part of or all of a nucleotide sequence in SEQ ID NO: 1-20, or a substantially identical sequence thereof.
  • a nucleotide sequence sometimes is a 5' and/or 3' sequence flanking a polymorphic variant described above that is 5-1000 nucleotides in length, or in some embodiments 5-500, 5-100, 5-75, 5-50, 5-45, 5-40, 5-35, 5-30, 5-25 or 5-20 nucleotides in length.
  • the nucleic acid comprises one or more of the following nucleotides: a guanine at position 522 of SEQ ID NO:5 or a thymine at position 19890 of SEQ ID NO: 5.
  • Other embodiments are directed to methods of identifying a polymorphic variation at one or more positions in a nucleic acid (e.g., genotyping at one or more positions in the nucleic acid), such as at a position corresponding to SNP AA or SNP AB in the PRAME gene.
  • nucleic acid fragments are also included herein. These fragments often are a nucleotide sequence identical to a nucleotide sequence of SEQ ID NO: 1-20, a nucleotide sequence substantially identical to a nucleotide sequence of SEQ ID NO: 1-20, or a nucleotide sequence that is complementary to the foregoing.
  • the nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in a nucleotide sequence of SEQ ID NO: 1-9, and may encode a domain or part of a domain of a polypeptide. Sometimes, the fragment will comprises one or more of the polymorphic variations described herein as being associated with type II diabetes.
  • the nucleic acid fragment is often 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 10000, 15000, or 20000 base pairs in length.
  • a nucleic acid fragment that is complementary to a nucleotide sequence identical or substantially identical to a nucleotide sequence in SEQ ID-NO: 1-20 and hybridizes to such a nucleotide sequence under stringent conditions is often referred to as a "probe.”
  • Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymorphic site as described hereafter.
  • JAM3 nucleic acid fragments sometimes encode an immunoglobulin domain cell adhesion molecule (cam) subfamily region from positions 283 to 534 and 577 to 858 of the mRNA sequence (positions 87 to 170 and 185 to 278, respectively, of the polypeptide sequence) or neural cell adhesion molecule Ll region from positions 340 to 843 of the mRNA sequence (positions 106 to 273 of the polypeptide sequence), for example.
  • cam immunoglobulin domain cell adhesion molecule
  • GLI3 nucleic acid fragments sometimes encode a C2H2-type Zn- finger protein region from positions 1111 to 1935 of the mRNA sequence (positions 353 to 627 of the polypeptide sequence) or a zinc finger region from positions 1492 to 1950 of the mRNA sequence (positions 480 to 632 of the polypeptide sequence), for example.
  • An example of a nucleic acid fragment is an oligonucleotide.
  • oligonucleotide refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides.
  • oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism. Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.
  • Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABF M 3900 High Throughput DNA Synthesizer and the EXPEDITETM 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, CA). Analogs and derivatives are exemplified in U.S. Pat. Nos.
  • Oligonucleotides may also be linked to a second moiety.
  • the second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage Ml 3 universal tail sequence), and others.
  • the second moiety may be a non-nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide.
  • labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like.
  • the second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.
  • Nucleic acid coding sequences may be used for diagnostic purposes for detection and control of polypeptide expression.
  • oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide.
  • Antisense techniques and RNA interference techniques are known in the art and are described herein.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • hammerhead motif ribozyme molecules may be engineered that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAM3 or GLB nucleotide sequences.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including type II diabetes, resulting from aberrant expression of a target gene described herein.
  • the nucleic acid sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or Northern blot analysis, in situ hybridization assays).
  • the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide.
  • production of functionally impaired polypeptide is the cause of various disease states, such as type II diabetes.
  • In situ hybridizations using polypeptide as a probe may be employed to predict problems related to type II diabetes.
  • administration of human active polypeptide, recombinantly produced as described herein may be used to treat disease states related to functionally impaired polypeptide.
  • gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.
  • nucleic acid vectors often expression vectors, which contain a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLIS nucleotide sequence or a substantially identical sequence thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.
  • a vector can include a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLI3 nucleotide sequence in a form suitable for expression of an encoded target polypeptide or target nucleic acid in a host cell.
  • a "target polypeptide” is a polypeptide encoded by & SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLIS nucleotide sequence or a substantially identical nucleotide sequence thereof.
  • the recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. Expression vectors can be introduced into host cells to produce target polypeptides, including fusion polypeptides.
  • Recombinant expression vectors can be designed for expression of target polypeptides in prokaryotic or eukaryotic cells.
  • target polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.
  • GST glutathione S-transferase
  • fusion polypeptides can be used in screening assays and to generate antibodies specific for target polypeptides.
  • fusion polypeptide expressed in a retroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed ⁇ e.g., six (6) weeks).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type (e.g.', tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific promoters include an albumin promoter (liver-specific; Pinkert et al, Genes Dev. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol.
  • promoters of T cell receptors (Winoto & Baltimore, EMBO J. 8: 729-733 (1989)) promoters of immunoglobulins (Banerji et al, Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-948 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci.
  • pancreas-specific promoters Eslund et al, Science 230: 912-916 (1985)
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters (Kessel & Grass, Science 249: 31A-119 (1990)) and the ⁇ -fetopolypeptide promoter (Campes & Tilghman, Genes Dev. 3: 537-546 (1989)).
  • a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleic acid may also be cloned.into an expression vector in an antisense orientation.
  • Regulatory sequences e.g., viral promoters and/or enhancers
  • operatively linked to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleic acid cloned in the antisense orientation can be chosen for directing constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types.
  • Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus.
  • Antisense genes see, e.g., Weintraub et ah, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) (1986).
  • host cells that include a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLI3 nucleotide sequence within a recombinant expression vector or a fragment of such a nucleotide sequence which facilitate homologous recombination into a specific site of the host cell genome.
  • host cell and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a target polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vectors can be introduced into host cells via conventional transformation or transfection techniques.
  • a host cell provided herein can be used to produce (i.e., express) a target polypeptide or a substantially identical polypeptide thereof. Accordingly, further provided are methods for producing a target polypeptide using host cells described herein. In one embodiment, the method includes culturing host cells into which a recombinant expression vector encoding a target polypeptide has been introduced in a suitable medium such that a target polypeptide is produced. In another embodiment, the method further includes isolating a target polypeptide from the medium or the host cell.
  • cells or purified preparations of cells which include a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB transgene, or which otherwise misexpress target polypeptide.
  • Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells.
  • the cell or cells include a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB transgene (e.g., a heterologous form of a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAM3 or GLL3 gene, such as a human gene expressed in non-human cells).
  • the transgene can be misexpressed, e.g., overexpressed or underexpressed.
  • the cell or cells include a gene which misexpress an endogenous target polypeptide (e.g., expression of a gene is disrupted, also known as a knockout).
  • a gene which misexpress an endogenous target polypeptide e.g., expression of a gene is disrupted, also known as a knockout.
  • Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed alleles or for use in drug screening.
  • human cells e.g., a hematopoietic stem cells transformed with a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid.
  • cells or a purified preparation thereof e.g. , human cells
  • an endogenous SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid is under the control of a regulatory sequence that does not normally control the expression of the endogenous gene corresponding to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAM3 or GLB nucleotide sequence.
  • an endogenous gene within a cell can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the corresponding endogenous gene.
  • a heterologous DNA regulatory element e.g., a gene which is "transcriptionally silent,” not normally expressed, or expressed only at very low levels
  • an endogenous corresponding gene may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell.
  • Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, published on May 16, 1991.
  • Non-human transgenic animals that express a heterologous target polypeptide (e.g., expressed from a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid or substantially identical sequence thereof) can be generated. Such animals are useful for studying the function and/or activity of a target polypeptide and for identifying and/or evaluating modulators of the activity of SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acids and encoded polypeptides.
  • a heterologous target polypeptide e.g., expressed from a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acids and encoded polypeptides.
  • a "transgenic animal” is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird ⁇ e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophil ⁇ mel ⁇ nog ⁇ stef), in which one or more of the cells of the animal includes a transgene.
  • a mammal e.g., a non-human primate such as chimpanzee, baboon, or macaque
  • an ungulate such as an equine, bovine, or caprine
  • a rodent such as a rat, a mouse, or
  • a transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal.
  • a transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout).
  • a transgenic animal can be one in which an endogenous nucleic acid homologous to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.
  • a cell of the animal e.g., an embryonic cell of the animal
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene.
  • One or more tissue-specific regulatory sequences can be operably linked to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence to direct expression of an encoded polypeptide to particular cells.
  • a transgenic founder animal can be identified based upon the presence of a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence in its genome and/or expression of encoded mRNA in tissues or cells of the animals.
  • transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a SLC22A1, TTN, LOCI 12609, TRPSl, FRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence can further be bred to other transgenic animals carrying other transgenes.
  • Target polypeptides can be expressed in transgenic animals or plants by introducing, for example, a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleic acid into the genome of an animal that encodes the target polypeptide.
  • the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal.
  • tissue specific promoter e.g., a milk or egg specific promoter
  • a population of cells from a transgenic animal e.g., a milk or egg specific promoter
  • isolated target polypeptides which are encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence (e.g., SEQ ID NO: 1-20) or a substantially identical nucleotide sequence thereof, such as the polypeptides having amino acid sequences in SEQ ID NO: 21-30.
  • polypeptide as used herein includes proteins and peptides.
  • An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language "substantially free” means preparation of a target polypeptide having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-target polypeptide (also referred to herein as a "contaminating protein"), or of chemical precursors or non-target chemicals.
  • the target polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation.
  • Isolated or purified 1 target polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight.
  • the polypeptide fragment may be a domain or part of a domain of a target polypeptide.
  • a fragment sometimes is a FOXA2 polypeptide domain encoded by a nucleotide sequence referenced in SEQ ID NO: 17 or 18.
  • the polypeptide fragment may have increased, decreased or unexpected biological activity.
  • the polypeptide fragment is often 50 or fewer, 100 or fewer, or 200 or fewer amino acids in length, and is sometimes 300, 400, 500, 600, 700, or 900 or fewer amino acids in length.
  • Substantially identical target polypeptides may depart from the amino acid sequences of target polypeptides in different manners. For example, conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of target polypeptides.
  • a "conservative amino acid substitution” is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known.
  • amino acids with basic side chains ⁇ e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • non-essential amino acids may be replaced.
  • a "non-essential" amino acid is one that can be altered without abolishing or substantially altering the biological function of a target polypeptide, whereas altering an "essential” amino acid abolishes or substantially alters the biological function of a target polypeptide.
  • Amino acids that are conserved among target polypeptides are typically essential amino acids.
  • target polypeptides may exist as chimeric or fusion polypeptides.
  • a target "chimeric polypeptide” or target “fusion polypeptide” includes a target polypeptide linked to a non-target polypeptide.
  • a "non-target polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the target polypeptide, which includes, for example, a polypeptide that is different from the target polypeptide and derived from the same or a different organism.
  • the target polypeptide in the fusion polypeptide can correspond to an entire or nearly entire target polypeptide or a fragment thereof.
  • the non-target polypeptide can be fused to the N-terminus or C-terminus of the target polypeptide.
  • Fusion polypeptides can include a moiety having high affinity for a ligand.
  • the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the target polypeptide is fused at the N- or C-terminus to a string of histidine residues.
  • Such fusion polypeptides can facilitate purification of recombinant target polypeptide.
  • Fusion polypeptides are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a nucleotide sequence in SEQ ID NO: 1-20, or a substantially identical nucleotide sequence thereof, can be cloned into an expression vector such that the fusion moiety is linked in- frame to the target polypeptide.
  • the fusion polypeptide can be a target polypeptide containing a heterologous signal sequence at its N-terminus.
  • expression, secretion, cellular internalization, and cellular localization of a target polypeptide can be increased through use of a heterologous signal sequence.
  • Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).
  • Target polypeptides can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these target polypeptides can be used to affect the bioavailability of a substrate of the target polypeptide and may effectively increase target polypeptide biological activity in a cell.
  • Target fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a target polypeptide; (ii) mis-regulation of the gene encoding the target polypeptide; and (iii) aberrant post-translational modification of a target polypeptide.
  • target polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify target polypeptide ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of a target polypeptide with a substrate.
  • polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al, (1984) Nature July 12 -18;310(5973):105-ll).
  • a relative short fragment can be synthesized by use of a peptide synthesizer.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3- amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b- alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be
  • Polypeptides and polypeptide fragments sometimes are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like.
  • Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.
  • the polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.
  • chemically modified derivatives of polypeptides that can provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see e.g., U.S. Pat. No: 4,179,337.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • polymers should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.
  • attachment methods available to those skilled in the art (e.g., EP 0 401 384 (coupling PEG to G-CSF) and Malik et al. (1992) Exp Hematol. September;20(8): 1028-35 (pegylation of GM-CSF using tresyl chloride)).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • the attachment sometimes is at an amino group, such as attachment at the N-terminus or lysine group.
  • Proteins can be chemically modified at the N-terminus.
  • polyethylene glycol as an illustration of such a composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N- terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • nucleotide sequences and polypeptide sequences that are substantially identical to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence and the target polypeptide sequences encoded by those nucleotide sequences, respectively, are included herein.
  • the term "substantially identical” as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively.
  • nucleotide sequences or polypeptide sequences that are 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3% or 4% variability) identical to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence or the encoded target polypeptide amino acid sequences.
  • One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.
  • sequence identity is often performed as follows. Sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, or 100% of the length of the reference sequence.
  • the nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences.
  • the nucleotides or amino acids are deemed to be identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences.
  • Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. MoI. Biol.
  • nucleic acids Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions.
  • stringent conditions refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. , 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used.
  • stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50 0 C.
  • Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55°C.
  • a further example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 0 C.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • An example of a substantially identical nucleotide sequence to a nucleotide sequence in SEQ ID NO: 1-20 is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the nucleotide sequence in SEQ ED NO: 1-20.
  • Another example is a nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes more than 75% or more, 80% or more, or 85% or more identical to, and often more than 90% or more and 95% or more identical to a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO: 1-20.
  • SEQ ID NO: 1-20 typically refers to one or more sequences in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20. Many of the embodiments described herein are applicable to (a) a nucleotide sequence of SEQ DO NO: 1-20; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-20; (d) a fragment of a nucleotide sequence of (a), (b), or (c); and/or a nucleotide sequence complementary to the nucleo
  • nucleotide sequences examples include nucleotide sequences from subjects that differ by naturally occurring genetic variance, which sometimes is referred to as background genetic variance ⁇ e.g., nucleotide sequences differing by natural genetic variance sometimes are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one another).
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25(17): 3389- 3402 (1997).
  • default parameters of the respective programs e.g., XBLAST and NBLAST
  • a nucleic acid that is substantially identical to a nucleotide sequence in SEQ ID NO: 1- 20 may include polymorphic sites at positions equivalent to those described herein when the sequences are aligned.
  • SNPs in a sequence substantially identical to a sequence in SEQ ID NO: 1-20 can be identified at nucleotide positions that match with or correspond to (i.e., align) nucleotides at SNP positions in each nucleotide sequence in SEQ ID NO: 1-20.
  • insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.
  • Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring.
  • Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
  • Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50% or more, about 55% or more, often about 70-75% or more or about 80-85% or more, and sometimes about 90-95% or more identical to the amino acid sequences of target polypeptides or a fragment thereof. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID NO: 1-20 or a fragment of this sequence.
  • nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID NO: 1-20 can further be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID NO: 1-20.
  • substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a target polypeptide in a particular expression system.
  • the nucleic acid can be one in which one OF more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae), human (e.g., 293 cells), insect or rodent (e.g., hamster)- cells.
  • nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-20; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence
  • polymorphic variants at the positions described herein are detected for determining a risk of type II diabetes, and polymorphic variants at positions in linkage disequilibrium with these positions are detected for determining a risk of type II diabetes.
  • SEQ ID NO: 1-20 refers to individual sequences in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20.
  • Results from prognostic tests may be combined with other test results to diagnose type II diabetes related disorders, including metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia.
  • prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to type II diabetes, the patient sample is analyzed, and the results of the analysis may be utilized to diagnose the type II diabetes related condition (e.g., metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia).
  • type II diabetes diagnostic methods can be developed from studies used to generate prognostic methods in which populations are stratified into subpopulations having different progressions of a type II diabetes related disorder or condition.
  • prognostic results may be gathered, a patient's risk factors for developing type II diabetes (e.g., age, weight, race, dief ⁇ analyzed, and a patient sample may be ordered based on a determined predisposition to type II diabetes.
  • a patient's risk factors for developing type II diabetes e.g., age, weight, race, dief ⁇ analyzed
  • a patient sample may be ordered based on a determined predisposition to type II diabetes.
  • Risk of type II diabetes sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor.
  • the risk sometimes is expressed as a relative risk with respect to a population average risk of type II diabetes, and sometimes is expressed as a relative risk with respect to the lowest risk group.
  • Such relative risk assessments often are based upon penetrance values determined by statistical methods and are particularly useful to clinicians and insurance companies for assessing risk of type II diabetes (e.g., a clinician can target appropriate detection, prevention and therapeutic regimens to a patient after determining the patient's risk of type II diabetes, and an insurance company can fine tune actuarial tables based upon population genotype assessments of type II diabetes risk).
  • Risk of type II diabetes sometimes is expressed as an odds ratio, which is the odds of a particular person having a genotype has or will develop type II diabetes with respect to another genotype group (e.g., the most disease protective genotype or population average).
  • the risk often is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested.
  • two or more polymorphic variations are detected in a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB locus.
  • nucleic acid sample typically is isolated from a biological sample obtained from a subject.
  • nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue.
  • the nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2.
  • the term "subject” refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys ⁇ , reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms.
  • the nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.
  • the presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S.
  • mismatch sequence dete ⁇ nination methods e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958
  • microarray sequence determination methods e.g., restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625. and 6,013,499), PCR- based assays (e.g., TAQMAN ® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.
  • RFLP restriction fragment length polymorphism
  • SSCP single strand conformation polymorphism detection
  • PCR- based assays e.g., TAQMAN ® PCR System (Applied Biosystems)
  • nucleotide sequencing methods may be used.
  • Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation.
  • PCR polymerase chain reaction
  • One oligonucleotide primer is complementary to a region 3' of the polymorphism and the other is complementary to a region 5' of the polymorphism.
  • a PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example.
  • PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP ® Systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLIS nucleotide sequence using knowledge available in the art.
  • extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation.
  • adjacent refers to the 3' end of the extension oligonucleotide being often 1 nucleotide from the 5' end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid.
  • extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present.
  • Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039.
  • Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144, and a method often utilized is described herein in Example 2.
  • a microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample.
  • a microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos.
  • the microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions.
  • the oligonucleotides may also be linked to the solid support directly or by a spacer molecule.
  • a microarray may comprise one or more oligonucleotides complementary to a polymorphic site set forth herein.
  • a kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample.
  • a kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of SEQ ID NO: 1-20 or a substantially identical sequence thereof, where the fragment includes a polymorphic site.
  • the kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664.
  • the kit often comprises an elongation oligonucleotide that hybridizes to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLIS nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site.
  • the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide.
  • chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like.
  • the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides.
  • Kits optionally include buffers, vials, microtiter plates, and instructions for use.
  • An individual identified as being at risk of type II diabetes may be heterozygous or homozygous with respect to the allele associated with a higher risk of type II diabetes.
  • a subject homozygous for an allele associated with an increased risk of type II diabetes is at a comparatively high risk of type II diabetes
  • a subject heterozygous for an allele associated with an increased risk of type II diabetes is at a comparatively intermediate risk of type II diabetes
  • a subject homozygous for an allele associated with a decreased risk of type II diabetes is at a comparatively low risk of type II diabetes.
  • a genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.
  • the antibody specifically binds to an epitope comprising a glycine at position 557 in a ZNF221 polypeptide.
  • Pharmacogenomics is a discipline that involves, tailoring a treatment for a subject according to the subject's genotype as a particular treatment regimen may exert a differential effect depending upon the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects).
  • a particular treatment regimen can exert a differential effect depending upon the subject's genotype.
  • a candidate therapeutic exhibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction)
  • such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele.
  • a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but ia comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele
  • the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.
  • the methods described herein are applicable to pharmacogenomic methods for preventing, alleviating or treating type II diabetes conditions such as metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia.
  • type II diabetes conditions such as metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia.
  • a nucleic acid sample from an individual may be subjected to a prognostic test described herein.
  • information for preventing or treating type II diabetes and/or one or more type II diabetes treatment regimens then may be prescribed to that subject.
  • a treatment or preventative regimen is specifically prescribed and/or administered to individuals, who will most benefit from it based upon their risk of developing type II diabetes assessed by the methods described herein.
  • a treatment or preventative regimen is specifically prescribed and/or administered to individuals, who will most benefit from it based upon their risk of developing type II diabetes assessed by the methods described herein.
  • certain embodiments are directed to a method for reducing type II diabetes in a subject, which comprises: detecting the presence or absence of a polymorphic variant associated with type II diabetes in a nucleotide sequence in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-20; (b)- a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-20; and (d) a fragment of a polynucle
  • Certain preventative treatments often are prescribed to subjects having a predisposition to type II diabetes and where the subject is diagnosed with type II diabetes or is diagnosed as having symptoms indicative of early stage type II diabetes, ⁇ e.g., impaired glucose tolerance, or IGT).
  • IGT impaired glucose tolerance
  • the treatment sometimes is preventative (e.g., is prescribed or administered to reduce the probability that a type II diabetes associated condition arises or progresses);, sometimes is therapeutic, and sometimes delays, alleviates or halts the progression of a type II diabetes associated condition. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of a type II diabetes associated disorder is prescribed and/or administered.
  • the treatment sometimes includes changes in diet, increased exercise, and the administration of therapeutics such as sulphonylureas (and related insulin secretagogues), which increase insulin release from pancreatic islets; metformin (GlucophageTM), which acts to reduce hepatic glucose production; peroxisome proliferator-activated receptor-gamma (PPAR) agonists (thiozolidinediones such as Avandia® and Actos®), which enhance insulin action; alpha- glucosidase inhibitors (e.g., Precose®, Voglibose®, and Miglitol®), which interfere with gut glucose absorption; and insulin itself, which suppresses glucose production and augments glucose utilization (Moller Nature 414, 821-827 (2001)).
  • therapeutics such as sulphonylureas (and related insulin secretagogues), which increase insulin release from pancreatic islets; metformin (GlucophageTM), which acts to reduce hepatic glucose production;
  • type II diabetes preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing type II diabetes or those that have early stages of type II diabetes), provided herein is a method for preventing or reducing the risk of developing type II diabetes in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject with a predisposition to type II diabetes, whereby the presence of the polymorphic variation is indicative of a predisposition to type II diabetes in the subject; and (c) if such a predisposition is identified, providing the subject with information about methods or products to prevent or reduce type II diabetes or to delay the onset of type II diabetes.
  • Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition which comprises: (a) detecting the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with type II diabetes; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.
  • Pharmacogenomics methods also may be used to analyze and predict a response to a type II diabetes treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a type II diabetes treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.
  • the response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen. In an embodiment, a response to metformin is predicted based upon the genotype of a nucleic acid sample from a subject.
  • detecting the presence of a cytosine at a position corresponding to position 50104 in SEQ ID NO: 1 in a nucleic acid sample is predictive of the subject not being responsive to metformin, and the drug typically is not prescribed to a subject having such a genotype.
  • the tests described herein also are applicable to clinical drug trials.
  • One or more polymorphic variants indicative of response to an agent for treating type II diabetes or to side effects to an agent for treating type II diabetes may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
  • another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug.
  • the polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a nucleotide sequence of SEQ ID NO: 1-20; (ii) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20; (iii) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-20; and (iv) a fragment of a polynucleotide sequence of (i), (ii), or (iii) comprising of a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-20; and (iv) a fragment of a poly
  • step (c) optionally comprises- administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
  • a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with type II diabetes; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above.
  • composition comprising a cell from a subject having type II diabetes or at risk of type II diabetes and one or more molecules specifically directed and targeted to a nucleic acid comprising a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence or amino acid sequence.
  • Such directed molecules include, but are not limited to, a compound that binds to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence or amino acid sequence referenced herein; a nucleic acid that hybridizes to a SLC22A 1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid under conditions of high stringency; a RNAi or siRNA molecule having a strand complementary to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence; an antisense nucleic acid complementary to an RNA encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence
  • the diabetes directed molecule interacts with a nucleic acid or polypeptide variant associated with diabetes, such as variants referenced herein.
  • the diabetes directed molecule interacts with a polypeptide involved in a signal pathway of a polypeptide encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, FRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence, or a nucleic acid comprising such a nucleotide sequence.
  • compositions sometimea include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response.
  • Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or ⁇ - interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in-water emulsion (e.g., an emulsion stabilized by Tween SO and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation
  • compositions are useful for generating an immune response against a diabetes directed molecule (e.g., an HLA- binding subsequence within a polypeptide encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence).
  • a diabetes directed molecule e.g., an HLA- binding subsequence within a polypeptide encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence.
  • a peptide having an amino acid subsequence of a polypeptide encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleotide sequence is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response.
  • the peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide.
  • the cell may be in a group of cells cultured in vitro or in a tissue maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human).
  • a composition comprises a component from a cell such as a nucleic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example.
  • a nucleic acid molecule e.g., genomic DNA
  • the aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in diabetes therapeutics described hereafter. Certain diabetes directed molecules are described in greater detail below.
  • Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al, J. Med. Chem.37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound” library methods; and synthetic library methods using affinity chromatography selection.
  • Biolibrary and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)).
  • Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al, J. Med. Chem.
  • a compound sometimes alters expression and sometimes alters activity of a polypeptide target and may be a small molecule.
  • Small molecules include, but are not limited to, peptides, peptidomimetics ⁇ e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds ⁇ i.e., including heteroorganic and organometallic compounds) having, a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • an "antisense” nucleic acid refers to a nucleotide sequence complementary to a "sense" nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mKNA sequence.
  • the antisense nucleic acid can be complementary to an entire coding strand ⁇ e.g., SEQ ID NO: 8-16 ⁇ , or to a portion thereof or a substantially identical sequence thereof.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence ⁇ e.g., 5' and 3' untranslated regions in SEQ ID NO: 1-9).
  • An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence ⁇ e.g., SEQ ID NO: 1-20), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • the antisense nucleic acids which include the ribozymes described hereafter, can be designed to target a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAM3 or GLB nucleotide sequence, often a variant associated with diabetes, or a substantially identical sequence thereof.
  • minor alleles and major alleles can be targeted, and those associated with a higher risk of diabetes are often designed, tested, and administered to subjects.
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures.
  • an antisense nucleic acid ⁇ e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides, or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ⁇ i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • antisense nucleic acids When utilized as therapeutics, antisense nucleic acids typically are administered to a subject ⁇ e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular niRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation.
  • antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors, or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules, are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.
  • a strong promoter such as a pol II or pol III promoter
  • Antisense nucleic acid molecules sometimes, are *-anomeric nucleic acid molecules.
  • An *-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual *-units, the strands run parallel to each other (Gaultier et ah, Nucleic Acids. Res. 15: 6625-6641 (1987)).
  • Antisense nucleic acid molecules can also comprise a 2'-o-methylribonucleotide (Inoue et ah, Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et ah, FEBS Lett.
  • an antisense nucleic acid is a ribozyme.
  • a ribozyme having specificity for a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLIS nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No.
  • a derivative of a Tetrahymena L- 19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742).
  • target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261: 1411-1418 (1993)).
  • Diabetes directed molecules include in certain embodiments nucleic acids that can form triple helix structures with a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLIS nucleotide sequence or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide.
  • Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a nucleotide sequence referenced herein or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des.
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Diabetes directed molecules include RNAi and siRNA nucleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi.
  • dsRNA double-stranded RNA
  • RNAi RNA interference
  • Fire et al US Patent Number 6,506,559
  • Tuschl et al. PCT International Publication No. WO 01/75164
  • Bosher JM Labouesse, Nat Cell Biol 2000 Feb;2(2):E31-6.
  • RNA interference RNA interference
  • siRNA refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene.
  • siRNA refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the siRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence.
  • an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.
  • the targeted region When designing the siRNA molecules, the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al,. Methods 26:199-213 (2002). Initially, 5' or 3' UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(Nl 9)TT (N, an nucleotideX and regions with approximately 30% to 70% G/C-content (often about 50% G/C-content) often are selected.
  • the sequence of the sense siRNA sometimes corresponds to (N 19) TT or N21 (position 3 to 23 of the 23 -nt motif), respectively. In the latter case, the 3' end of the sense siRNA often is converted to TT.
  • the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
  • the antisense siRNA is synthesized as the complement to position 1 to 21 of the 23 -nt motif. Because position 1 of the 23- nt motif is not recognized sequence-specifically by the antisense siRNA, the 3 '-most nucleotide residue of the antisense siRNA can be chosen deliberately.
  • the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23 -nt motif) often is complementary to the targeted sequence.
  • TT often is utilized.
  • Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.
  • the sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof.
  • the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the siRNA sometimes is about 21 nucleotides in length.
  • siRNA molecules sometimes is of a different chemical composition as compared to native RNA that imparts increased stability in cells (e.g., decreased susceptibility to degradation), and sometimes includes one or more modifications in siSTABLE RNA described at the http address www.dharmacon.com.
  • Antisense, ribozyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules.
  • the nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et ah, Bioorganic & Medicinal Chemistry 4 (1): 5-23 (1996)).
  • peptide nucleic acid refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et ah, (1996) supra and Perry-O'Keefe et ah, Proc. Natl. Acad. Sci. 93: 14670-675 (1996).
  • PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as "artificial restriction enzymes" when used in combination with other enzymes, (e.g., Sl nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et ah, (1996) supra; Perry-O'Keefe supra).
  • oligonucleotides may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et ah, Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across cell membranes see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et ah, Proc. Natl. Acad. Sci
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al., Bio-Techniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm, Res. 5: 539-549 (1988) ).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • molecular beacon oligonucleotide primer and probe molecules having one or more regions complementary to a SLC22A1, TTN, LOCI 12609, TKPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence or a substantially identical sequence thereof, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample.
  • Molecular beacon nucleic acids are described, for example, in Lizardi et ah, U.S. Patent No. 5,854,033; Nazarenko et al, U.S. Patent No. 5,866,336, and Livak et al, U.S. Patent 5,876,930.
  • antibody refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • An antibody sometimes is a polyclonal, monoclonal, recombinant ⁇ e.g., a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody.
  • An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.
  • a full-length polypeptide or antigenic peptide fragment encoded by a nucleotide sequence referenced herein can be uaed as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like.
  • An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence referenced herein, or substantially identical sequence thereof, and encompasses an epitope.
  • Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.
  • Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity.
  • regions located on the surface of the polypeptide e.g., hydrophilic regions
  • an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production.
  • the antibody may bind an epitope on any domain or region on polypeptides described herein.
  • chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects.
  • Chimeric and humanized monoclonal antibodies comprising both human and non-human portions, can be made using standard recombinant DNA techniques.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al European Patent Application 173,494; Neuberger et al PCT International Publication No.
  • Completely human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995); and U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806.
  • companies such as Abgenix, Inc. (Fremont, CA) and Medarex, Inc. (Princeton, NJ ⁇ , can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Completely human antibodies that recognize a selected epitope also can be generated using a technique referred to as "guided selection.”
  • a selected non- human monoclonal antibody ⁇ e.g., a murine antibody
  • This technology is described for example by Jespers et al, Bio/Technology 12: 899-903 (1994).
  • An antibody can be a single chain antibody.
  • a single chain antibody (scFV) can be engineered (see, e.g., Colcher et al, Ann. N Y Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)).
  • Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.
  • Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor.
  • an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor (e.g., it has a mutagenized or deleted Fc receptor binding region).
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-ftuorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin ⁇ , bleomycin, mithramycin, and anthramycin (AMC)), and anti ⁇ mitotic agents (
  • Antibody conjugates can be used for modifying a given biological response.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,
  • An antibody e.g., monoclonal antibody
  • an antibody can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling).
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and
  • suitable radioactive material include 125 1, 131 1, 35 S or 3 H.
  • an antibody can be utilized as a test molecule for determining whether it can treat diabetes, and
  • An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.
  • a purified antigen or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.
  • the methods comprise contacting a test molecule with a target molecule in a system.
  • a "target molecule” as used herein refers to a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing.
  • the methods also comprise determining the presence or absence of an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a candidate type II diabetes therapeutic.
  • the interaction between the test molecule and the target molecule may be quantified.
  • Test molecules and candidate therapeutics include, but are not limited to, compounds, antisense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS or GLB nucleotide sequence, or a substantially identical sequence or fragment thereof, and immunotherapeutics. (e.g., antibodies and HLA-presented polypeptide fragments).
  • a test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system.
  • a “modulator” may agonize ⁇ i.e., up-regulates) or antagonize (i.e., down-regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing ⁇ e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing ⁇ e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis, of pro-polypeptides).
  • DNA replication and/or DNA processing e.g., DNA methylation or DNA repair
  • RNA transcription and/or RNA processing e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus
  • a modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of type II diabetes).
  • a candidate therapeutic increases glucose uptake in cells of a subject (e.g., in certain cells of the pancreas).
  • system refers to a cell free in vitro environment and a cell- based environment such as a collection of cells, a tissue, an organ, or an organism.
  • a system is "contacted” with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal.
  • interaction refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.
  • Test molecule/target molecule interactions can be detected and/or quantified using assays known in the art. For example, an interaction can be determined by labeling the test molecule and/or the target molecule, where the label is covalently or non-covaleiitly attached to the test molecule or target molecule.
  • the label is sometimes a radioactive molecule such as 125 1, 131 I, 35 S or ⁇ , which can be detected by direct counting of radioemission or by scintillation counting.
  • enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product.
  • a microphysiometer e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • cells typically include a SLC22A1, TTN, LOC112609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid, an encoded polypeptide, or substantially identical nucleic acid or polypeptide thereof, and are often of mammalian origin, although the cell can be of any origin.
  • Whole cells, cell homogenates, and cell fractions e.g., cell membrane fractions
  • soluble and/or membrane bound forms of the polypeptide may be utilized.
  • membrane-bound forms of the polypeptide it may be desirable to utilize a solubilizing agent.
  • solubilizing agents include non-ionic detergents such as n- octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-
  • An interaction between a test molecule and target molecule also can be detected by monitoring fluorescence energy transfer (FET) (see, e.g., Lakowicz et at, U.S. Patent No. 5,631,169; Stavrianopoulos et al. U.S. Patent No. 4,868,103).
  • FET fluorescence energy transfer
  • a fluorophore label on a first, "donor” molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the "donor" polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues.
  • Labels are chosen that emit different wavelengths of light, such that the "acceptor” molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal.
  • An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art ⁇ e.g., using a fluorimeter).
  • determining the presence or absence of an interaction between a test molecule and a target molecule can be effected by monitoring surface plasmon resonance ⁇ see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et al, Curr. Opin. Struct. Biol. 5: 699-705 (1995)).
  • surface plasmon resonance or “biomolecular interaction analysis (BIA)” can be utilized to detect biospecific interactions in real time, without labeling any of the interactants ⁇ e.g., BIAcore ⁇ .
  • the target molecule or test molecules are anchored to a solid phase, facilitating the detection of target molecule/test molecule complexes and separation of the complexes from free, uncomplexed molecules.
  • the target molecule or test molecule is immobilized to the solid support.
  • the target molecule is anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • test molecules may be desirable to immobilize a target molecule, an anti-target molecule antibody, and/or test molecules to facilitate separation of target molecule/test molecule complexes from uncomplexed forms, as well as to accommodate automation of the assay.
  • the attachment between a test molecule and/or target molecule and the solid support may be covalent or non-covalent ⁇ see, e.g., U.S. Patent No. 6,022,688 for non-covalent attachments).
  • the solid support may be one or more surfaces of the system, such as one or more surfaces in each well of a microtiter plate, a surface of a silicon wafer, a surface of a bead ⁇ see, e.g., Lam, Nature 354: 82-84 (1991))- that is optionally linked to another solid support, or a channel in a microfluidic device, for example.
  • Types of solid supports, linker molecules for covalent and non-covalent attachments to solid supports, and methods for immobilizing nucleic acids and other molecules to solid supports are well known ⁇ see, e.g., U.S. Patent Nos. 6,261,776; 5,900,481; 6,133,436; and 6,022,688; and WIPO publication WO 01/18234).
  • target molecule may be immobilized to surfaces via biotin and streptavidin.
  • biotinylated target polypeptide can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art ⁇ e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • a target polypeptide can be prepared as a fusion polypeptide.
  • glutathione-S-transferase/target polypeptide fusion can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the beads or microtiter plate wells are washed to remove any unbound components, or the matrix is immobilized in the case of beads, and complex formation is determined directly or indirectly as described above.
  • the complexes can be dissociated from the matrix, and the level of target molecule binding or activity is determined using standard techniques.
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that a significant percentage of complexes formed will remain immobilized to the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of manners. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface, e.g., by adding a labeled antibody specific for the immobilized component, where the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody.
  • an assay is performed utilizing antibodies that specifically bind target molecule or test molecule but do not interfere with binding of the target molecule to the test molecule.
  • Such antibodies can be derivitized to a solid support, and unbound target molecule may be immobilized by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Cell free assays also can be conducted in a liquid phase.
  • reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, e.g., Rivas, G., and Minton, Trends Biochem SciAug;18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion- exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology , J.
  • a cell or cell free mixture is contacted with a candidate compound and the expression of target mRNA or target polypeptide is evaluated relative to the level of expression of target mRNA or target polypeptide in the absence of the candidate compound.
  • the candidate compound is identified as an agonist of target mRNA or target polypeptide expression.
  • the candidate compound is. identified as an antagonist or inhibitor of target mRNA or target polypeptide expression.
  • the level of target mRNA or target polypeptide expression can be determined by methods described herein.
  • binding partners that interact with a target molecule are detected.
  • the target molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides in vivo, and these interacting molecules are referred to herein as "binding partners.”
  • Binding partners can agonize or antagonize target molecule biological activity.
  • test molecules that agonize or antagonize interactions between target molecules and binding partners can be useful as therapeutic molecules as they can up-regulate or down-regulated target molecule activity in vivo and thereby treat type II diabetes.
  • Binding partners of target molecules can be identified by methods, known in the art. For example, binding partners may be identified by lysing cells and analyzing cell lysates by electrophoretic techniques. Alternatively, a two-hybrid assay or three-hybrid assay can be utilized (see, e.g., U.S. Patent No. 5,283,317; Zervos et al., Cell 72:223-232 (1993); Madura et al, J. Biol. Chem.
  • a two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. The assay often utilizes two different DNA constructs.
  • a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid (sometimes referred to as the "bait") is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence from a library of DNA sequences that encodes a potential binding partner (sometimes referred to as the "prey") is fused to a gene that encodes an activation domain of the known transcription factor.
  • a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 or GLB nucleic acid can be fused to the activation domain. If the "bait" and the “prey” molecules interact in vivo, the DNA- binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to identify the potential binding partner.
  • a reporter gene e.g., LacZ
  • a reaction mixture containing the target molecule and the binding partner is prepared, under conditions and for a time sufficient to allow complex formation.
  • the reaction mixture often is provided in the presence or absence of the test molecule.
  • the test molecule can be included initially in the reaction mixture, or can be added at a time subsequent to the addition of the target molecule and its binding partner. Control reaction mixtures are incubated without the test molecule or with a placebo. Formation of any complexes between the target molecule and the binding partner then is detected.
  • Decreased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule antagonizes target molecule/binding partner complex formation.
  • increased formation of a complex in the reaction mixture containing test molecule as. compared to in a control reaction mixture indicates that the molecule agonizes target molecule/binding partner complex formation.
  • complex formation of target molecule/binding partner can be compared to complex formation of mutant target molecule/binding partner (e.g., amino acid modifications in a target polypeptide). Such a comparison can be important in those cases where it is desirable to identify test molecules that modulate interactions of mutant but not non-mutated target gene products.
  • the assays can be conducted in a heterogeneous or homogeneous format.
  • target molecule and/or the binding partner are immobilized to a solid phase, and complexes are detected on the solid phase at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase.
  • the order of addition of reactants can be varied to obtain different information about the molecules being tested.
  • test compounds that agonize target molecule/binding partner interactions can be identified by conducting the reaction in the presence of the test molecule in a competition format.
  • test molecules that agonize preformed complexes e.g., molecules with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
  • the target molecule or the binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly.
  • the anchored molecule can be immobilized by non-covalent or covalent attachments.
  • an immobilized antibody specific for the molecule to be anchored can be used to anchor the molecule to the solid surface.
  • the partner of the immobilized species is exposed to the coated surface with or without the test molecule. After the reaction is complete, unreacted components are removed (e.g., by washing) such that a significant portion of any complexes formed will remain immobilized on the solid surface.
  • the detection of label immobilized on the surface is indicative of complex.
  • an indirect label can be used to detect complexes anchored to the surface; e.g., by using a labeled antibody specific for the initially non- immobilized species.
  • test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of test molecule, where the reaction products are separated from unreacted components, and the complexes are detected (e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes)-.
  • test compounds that inhibit complex or that disrupt preformed complexes can be identified.
  • a homogeneous assay can be utilized. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared. One or both of the target molecule or binding partner is labeled, and the signal generated by the label(s) is quenched upon complex formation (e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). Addition of a test molecule that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target molecule/binding partner complexes can be identified.
  • Candidate therapeutics for treating type II diabetes are identified from a group of test molecules that interact with a target molecule.
  • Test molecules are normally ranked according to the degree with which they modulate (e.g., agonize or antagonize) a function associated with the target molecule (e.g., DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or biological function/activity), and then top ranking modulators are selected.
  • pharmacogenomic information described herein can determine the rank of a modulator. The top 10% of ranked test molecules often are selected for further testing as candidate therapeutics, and sometimes the top 15%, 20%, or 25% of ranked test molecules are selected for further testing as candidate therapeutics.
  • Candidate therapeutics typically are formulated for administration to a subject. Therapeutic Formulations
  • Formulations and pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier one or more target molecule modulators.
  • the modulator often is a test molecule identified as having an interaction with a target molecule by a screening method described above.
  • the modulator may be a compound, an antisense nucleic acid, a ribozyme, an antibody, or a binding partner.
  • formulations may comprise a target polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier
  • the term "pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition typically is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g. , intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Molecules which exhibit high therapeutic indices are preferred. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such molecules lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0.1 to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • the protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • polypeptide formulations featured herein is a method for treating type II diabetes in a subject, which comprises contacting one or more cells in the subject with a first polypeptide, where the subject comprises a second polypeptide having one or more polymorphic variations associated with cancer, and where the first polypeptide comprises fewer polymorphic variations associated with cancer than the second polypeptide.
  • the first and second polypeptides are encoded by a nucleic acid which comprises a nucleotide sequence in SEQ ID NO: 1-20; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence referenced in SEQ ID NO: 1-20; a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ED NO: 1-20 and a nucleotide sequence 90% or more identical to a nucleotide sequence in SEQ ID NO: 1-20.
  • the subject often is a human.
  • a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half- life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193 (1997).
  • Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, .alpha. -interferon, .beta.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration ⁇ see, e.g., U.S. Patent 5,328,470) or by stereotactic injection ⁇ see e.g., Chen et al, (1994) Proc. Natl Acad. ScL USA i?i:3054-3057).
  • Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.
  • a therapeutic formulation described above can be administered to a subject in need of a therapeutic for inducing a desired biological response.
  • Therapeutic formulations can be administered by any of the paths described herein. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein.
  • treatment is defined as the application or administration of a therapeutic formulation to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improye or affect type II diabetes, symptoms of type II diabetes or a predisposition towards type II diabetes.
  • a therapeutic formulation includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • Administration of a therapeutic formulation can occur prior to the manifestation of symptoms characteristic of type II diabetes, such that type II diabetes is prevented or delayed in its progression.
  • the appropriate therapeutic composition can be determined based on screening assays described herein.
  • embodiments include methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a polypeptide described herein, or a fragment thereof, or a therapeutic formulation described herein, wherein said biological response is selected from the group consisting of: (a) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (b) increasing cell or - tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; (c) inhibiting the progression from impaired glucose tolerance to insulin resistance; (d) increasing glucose uptake in skeletal muscle cells; (e) increasing glucose uptake in adipose cells; (f) increasing glucose uptake in neuronal cells; (g) increasing glucose uptake in red blood cells; (h) increasing glucose uptake in the brain; and (i) significantly reducing the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal
  • a pharmaceutical or physiologically acceptable composition can be utilized as an insulin sensitizer, or can be used in: a method to improve insulin sensitivity in some persons with type II diabetes in combination with insulin therapy; a method to improve insulin sensitivity in some persons with type II diabetes without insulin therapy; or a method of treating individuals with gestational diabetes.
  • Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.
  • the pharmaceutical or physiologically acceptable composition can be used in a method of treating individuals with impaired fasting glucose (IFG).
  • Impaired fasting glucose (IFG) is a condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes (i.e.
  • the pharmaceutical or physiologically acceptable composition can be used in a method of treating and preventing impaired glucose tolerance (IGT) in an individual.
  • IGT impaired glucose tolerance
  • the pharmaceutical or physiologically acceptable composition can be used in a method of treating a subject having polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population.
  • Insulin-sensitizing agents e.g., troglitazone
  • troglitazone have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100:1230), such as in insulin- resistant humans. Accordingly, provided are methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.
  • the pharmaceutical or physiologically acceptable composition can be used in a method of treating a subject having insulin resistance, where a subject having insulin resistance is treated to reduce or cure the insulin resistance.
  • a subject having insulin resistance is treated to reduce or cure the insulin resistance.
  • insulin resistance is also often associated with infections and cancer, preventing or reducing insulin resistance may prevent or reduce infections and cancer.
  • the pharmaceutical compositions and methods described herein are useful for: preventing the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin resistance; controlling blood glucose in some persons with type II diabetes in combination with insulin therapy; increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; inhibiting or preventing the progression from impaired glucose tolerance to insulin resistance; improving glucose control of type II diabetes patients alone, without an insulin secretagogue or an insulin sensitizing agent; and administering a complementary therapy to type II diabetes patients to improve their glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitizing (preferably oral form) agent.
  • an insulin secretagogue preferably oral form
  • an insulin sensitizing preferably oral form
  • the oral insulin secretagogue sometimes is l,l-dimethyl-2-(2- morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • the insulin sensitizing agent sometimes is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • FIG. 1 For embodiments, further embodiments include methods of administering a pharmaceutical or physiologically acceptable composition concomitantly or concurrently, with an insulin secretagogue or insulin sensitizing agent, for example, in the form of separate dosage units to be used simultaneously, separately or sequentially (e.g., before or after the secretagogue or before or after the sensitizing agent).
  • a pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitizing agent as a combined preparation for simultaneous, separate or sequential use for the improvement of glucose control in type II diabetes patients.
  • any test known in the art or a method described herein can be used to determine that a subject is insulin resistant, and an insulin resistant patient can then be treated according to the methods described herein to reduce or cure the insulin resistance.
  • the methods described herein also can be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.
  • modulators include, but are not limited to, small organic or inorganic molecules; antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab') 2 and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof); and peptides, phosphopeptides, or polypeptides.
  • antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level of target gene activity.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • Antisense, ribozyme and triple helix molecules are discussed above. It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype.
  • nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method.
  • the target gene encodes an extracellular polypeptide
  • nucleic acid molecules may be utilized in treating or preventing type II diabetes.
  • Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to ligands (see, e.g., Osborne, et al, Curr. Opin. Chem. Biol.1(1): 5-9 (1997); and Patel, D. J., Curr. Opin. Chem. Biol. Jim; 1(1): 32-46 (1997)).
  • gene therapy which can also be referred to as allele therapy.
  • a gene therapy method for treating type II diabetes in a subject which comprises contacting one or more cells in the subject or from the subject with a nucleic acid having a first nucleotide sequence.
  • Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with type II diabetes (e.g., the second nucleic acid has a nucleotide sequence in SEQ ID NO: 1-20).
  • the first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with type II diabetes than the second nucleotide sequence.
  • the first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof.
  • the subject is often a human. Allele therapy methods often are utilized in conjunction with a method of first determining whether a subject has genomic DNA that includes polymorphic variants associated with type II diabetes.
  • Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with type II diabetes (e.g., the second nucleic acid has a nucleotide sequence in SEQ ID NO: 1-20).
  • the first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with type II diabetes than the second nucleotide sequence.
  • the first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human.
  • antibodies can be generated that are both specific for target molecules and that reduce target molecule activity. Such antibodies may be administered in instances where antagonizing a target molecule function is appropriate for the treatment of type II diabetes.
  • the target molecule is intracellular and whole antibodies are used, internalizing antibodies may be preferred.
  • Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered.
  • Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see, e.g., Marasco et ah, Proc. Natl. Acad. ScL USA 90: 7889- 7893 (1993)).
  • Modulators can be administered to a patient at therapeutically effective doses to treat type ⁇ diabetes.
  • a therapeutically effective dose refers to an amount of the modulator sufficient to result in amelioration of symptoms of type II diabetes.
  • Toxicity and therapeutic efficacy of modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5O (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 ZED 50 .
  • Modulators that exhibit large therapeutic indices are preferred. While modulators that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such molecules to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.
  • Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be fo ⁇ nulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • Another example of effective dose determination for an individual is the ability to directly assay levels of "free" and "bound” compound in the serum of the test subject.
  • Such assays may utilize antibody mimics and/or "biosensors” that have been created through molecular imprinting techniques.
  • Molecules that modulate target molecule activity are used as a template, or "imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions.
  • Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes readily can be assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC 50 .
  • An example of such a "biosensor” is discussed in Kriz et at, Analytical Chemistry 67: 2142-2144 (1995).
  • SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS and GLlS polypeptides and polypeptide variants in vitro or in vivo.
  • SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, F0XA2, JAMS and GLlS nucleic acids or polypeptides and variants thereof are utilized for screening test molecules for those that interact with SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS and GLIS molecules.
  • Test molecules identified as interactors with SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS and GLIS molecules and variants are further screened in vivo to determine whether they treat breast cancer.
  • Example 1 Test molecules identified as interactors with SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAMS and GLIS molecules and variants are further screened in vivo to determine whether they treat breast cancer.
  • Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age-matched controls. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.
  • the solution was incubated at 37°C or room temperature if cell clumps were visible after mixing until the solution was homogeneous.
  • 2 ml of protein precipitation was added to the cell lysate.
  • the mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000 x g.
  • the supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100% isopropanol.
  • the samples were mixed by inverting the tubes gently until white threads of DNA were visible.
  • DNA was quantified by placing samples on a hematology mixer for at least 1 hour. DNA was serially diluted (typically 1:80, 1:160, 1:320, and 1:640 dilutions) so that it would be within the measurable range of standards. 125 ⁇ l of diluted DNA was transferred to a clear U- bottom microtitre plate, and 125 ⁇ l of IX TE buffer was transferred into each well using a multichannel pipette. The DNA and IX TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 ⁇ l of diluted DNA was added to wells A5-H12 of a black flat bottom microtitre plate.
  • DNA was serially diluted (typically 1:80, 1:160, 1:320, and 1:640 dilutions) so that it would be within the measurable range of standards.
  • 125 ⁇ l of diluted DNA was transferred to a clear U- bottom microtitre plate, and 125 ⁇ l of IX TE buffer was
  • the plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 nm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/ ⁇ l were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/ ⁇ l or less were re-measured for confirmation.
  • a Fluoroskan Ascent Machine microplate fluorometer produced by Labsystems
  • Samples were placed into one of four groups based on disease status.
  • the four groups were female case samples, female control samples, male case samples and male control samples.
  • a select set of samples from each group were utilized to generate pools, and one pool was created for each group.
  • Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA.
  • Inclusion or exclusion of samples for a pool was based upon the following criteria and detailed in the tables below: patient ethnicity, diagnosis with type II diabetes, GAD antibody concentration, HbAIc concentration, body mass (BMI), patient age, date of primary diagnosis, and age of individual as of primary diagnosis.
  • a whole-genome screen was performed to identify particular SNPs associated with occurrence of type II diabetes. As described in Example 1, two sets of samples were utilized: female individuals having type II diabetes (female cases) and samples from female individuals not having type II diabetes or any history of type II diabetes (female controls), and male individuals having type II diabetes (male cases) and samples from male individuals not having type II diabetes or any history of type II diabetes (male controls).
  • the initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another.
  • SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p-value was calculated to determine whether the case and control groups had statistically significantly differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level.
  • a whole-genome SNP screen began with an initial screen of approximately 25,000 SNPs over each set of disease and control samples using a pooling approach. The pools studied in the screen are described in Example 1.
  • the SNPs analyzed in this study were part of a set of 25,488 SNPs confirmed as being statistically polymorphic as each is characterized as having a minor allele frequency of greater than 10%.
  • the SNPs in the set reside in genes or in close proximity to genes, and many reside in gene exons. Specifically, SNPs in the set are located in exons, introns, and within 5,000 base-pairs upstream of a transcription start site of a gene.
  • SNPs were selected according to the following criteria: they are located in ESTs; they are located in Locuslink or Ensembl genes; and they are located in Genomatix promoter predictions. SNPs in the set were also selected on the basis of even spacing across the genome, as depicted in Table 3. An additional 3088 SNPs were included with these 25,488 SNPs and these additional SNPs had been chosen on the basis of gene location, with preference to non-synonymous coding SNPs located in disease candidate genes.
  • Table 4 includes information pertaining to the incident polymorphic variant associated with type II diabetes identified herein. Public information pertaining to the polymorphism and the genomic sequence that includes the polymorphism are indicated. The genomic sequences identified in Table 4 may be accessed at the http address www.ncbi.nih.gov/entrez/query.fcgi, for example, by using the publicly available SNP reference number (e.g., rs662138).
  • the "Contig Position” provided in Table 4 corresponds to a nucleotide position set forth in the contig sequence, and designates the polymorphic site corresponding to the SNP reference number.
  • the sequence containing the polymorphisms also may be referenced by the "Sequence Identification" set forth in Table 4.
  • the "Sequence Identification” corresponds to cDNA sequence that encodes associated target polypeptides (e.g., SLC22A1) of the invention.
  • the position of the SNP within the cDNA sequence is provided in the "Sequence Position" column of Table 4. Also, the allelic variation at the polymorphic site and the allelic variant identified as associated with type II diabetes is specified in Table 4. All nucleotide sequences referenced and accessed by the parameters set forth in Table 4 are incorporated herein by reference.
  • a MassARRAY® system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hMETM or homogeneous MassEXTENDTM (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTENDTM primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.
  • hMETM homogeneous, single-tube assay method
  • MassEXTENDTM primer which is complementary to the amplified target up to but not including the polymorphism
  • SpectroDESIGNERTM software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTENDTM primer was used to genotype the polymorphism.
  • Table 5 shows PCR primers and Table 6 shows extension primers used for analyzing polymorphisms.
  • the initial PCR amplification reaction was performed in a 5 ⁇ l total volume containing IX PCR buffer with 1.5 mM MgCl 2 (Qiagen), 200 ⁇ M each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest.
  • Qiagen PCR Primers
  • a primer extension reaction was initiated by adding a polymorphism-specific MassEXTENDTM primer cocktail to each sample.
  • Each MassEXTENDTM cocktail included a specific combination of dideoxynucleotides (ddNTPs ⁇ and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another.
  • ddNTPs dideoxynucleotides
  • dNTPs deoxynucleotides
  • the MassEXTENDTM reaction was performed in a total volume of 9 ⁇ l, with the addition of IX ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTENDTM primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP.
  • the deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94°C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72°C.
  • samples were desalted by adding 16 ⁇ l of water (total reaction volume was 25 ⁇ l), 3 mg of SpectroCLEANTM sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJETTM (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCFflP ® (Sequenom, Inc.)).
  • MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RTTM software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.
  • Genotyping results for the allelic variant set forth in Table 4 are shown for female pools in Table 7, for male pools in Table 8, and the combined results in Table 9.
  • F case and F control refer to female case and female control groups
  • M case and M control refer to male case and male control groups
  • AF refers to allele frequency.
  • SNP rs366111 is a coding non-synonymous SNP (G/A), which results in an amino acid change from arginine to glycine.
  • G/A coding non-synonymous SNP
  • the G allele is. associated with an increased risk of type II diabetes and codes for glycine; therefore, individuals with the glycine residue also have an increased risk of type II diabetes.
  • Odds ratio results are shown in Tables 7, 8 and 9. An odds ratio is an unbiased estimate of relative risk which can be obtained from most case-control studies.
  • Relative risk (RR) is an estimate of the likelihood of disease in the exposed group (susceptibility allele or genotype carriers) compared to the unexposed group (not carriers). It can be calculated by the following equation:
  • /A is the incidence of disease in the A carriers and /a is the incidence of disease in the non- carriers.
  • RR > 1 indicates the A allele increases disease susceptibility.
  • RR ⁇ 1 indicates the a allele increases disease susceptibility.
  • An odds ratio can be interpreted in the same way a relative risk is interpreted and can be directly estimated using the data from case-control studies, i.e., case and control allele frequencies.
  • the higher the odds ratio value the larger the effect that particular allele has on the development of type II diabetes. Possessing an allele associated with a relatively high odds ratio translates to having a higher risk of developing or having type II diabetes.
  • the single marker polymorphisms set forth in Table 4 were genotyped again in two replication cohorts to further validate their association with type II diabetes. Like the original study population described in Examples 1 and 2, the replication cohorts consisted of type II diabetics (cases.) and non-diabetics (controls). The case and control samples were selected and genotyped as described below.
  • Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age-matched controls. All of the samples were collected from individuals residing in Newfoundland, Canada. residents of Newfoundland represent a preferred population for genetic studies because of their relatively small founder population and resulting homogeneity.
  • Phenotypic trait information was gathered from individuals for each case and control sample, and genomic DNA was extracted from each of the blood samples for genetic analyses.
  • Samples were placed into one of four groups based on disease status. The four groups were female case samples, female control samples, male case samples, and male control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group.
  • Patients were included in the case pools if a) they were diagnosed with type II diabetes as documented in their medical record, b) they were treated with either insulin or oral hypoglycaemic agents, and c) they were of Caucasian ethnicity. Patients were excluded in the case pools if a) they were diabetic or had a history of diabetes, b) they suffered from diet controlled glucose intolerance, or c) they (or any their relatives) were diagnosed with MODY or gestational diabetes.
  • Phenotype information included, among others, patient ethnicity, country or origin of mother and father, diagnosis with type II diabetes (date of primary diagnosis, age of individual as of primary diagnosis), body weight, onset of obesity, retinopathy, glaucoma, cataracts, nephropathy, heart disease, hypertension, myocardial infarction, ulcers, required treatment (onset of insulin treatment, oral hypoglycemic agent), blood glucose levels, and MODY.
  • the polymorphisms described in Table 4 were genotyped again in a second replication cohort, consisting of individuals of Danish ancestry, to further validate their association with type II diabetes. Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age- matched controls.
  • Phenotypic trait information was gathered from individuals for each case and control sample, and genomic DNA was extracted from each of the blood samples for genetic analyses.
  • Samples were placed into one of four groups based on disease status. The four groups were female case samples, female control samples, male case samples, and male control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group.
  • Phenotype information included, among others, e.g. body mass index , waist/hip ratio, blood pressure, serum insulin, glucose, C-peptide, cholesterol, hdl, triglyceride, Hb A ic, urine, creatinine, free fatty acids (mmol/1), GAD antibodies.
  • the associated SNP from the initial scan was re-validated by genotyping the associated SNP across the replication cohorts described in Example 3. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and p-values were calculated to determine whether the case and control groups had statistically significant differences in allele frequencies for a particular SNP.
  • the replication genotyping results with a calculated p-value of less than 0.05 were considered particularly significant, which are set forth in bold text. See Tables 12-14 herein.
  • Genotyping of the replication cohort was performed using the same methods used for the original genotyping, as described herein.
  • a MassARRAY® system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion.
  • This genotyping platform was complemented by a homogeneous, single-tube assay method (hMETM or homogeneous MassEXTENDTM (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest.
  • a third primer (the MassEXTENDTM primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.
  • SpectroDESIGNERTM software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTENDTM primer which where used to genotype the polymorphism.
  • Other primer design software could be used or one of ordinary skill in the art could manually design primers based on his or her knowledge of the relevant factors and considerations in designing such primers.
  • Table 6 shows PCR primers and Table 7 shows extension probes used for analyzing (e.g., genotyping) polymorphisms in the replication cohorts.
  • the initial PCR amplification reaction was performed in a 5 ⁇ l total volume containing IX PCR buffer with 1.5 mM MgCl 2 (Qiagen), 200 ⁇ M each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest. [0255] Samples were incubated at 95°C for 15 minutes, followed by 45 cycles of 95 0 C for 20 seconds, 56 0 C for 30 seconds, and 72°C for 1 minute, finishing with a 3 minute final extension at 72°C.
  • shrimp alkaline phosphatase (0.3 units in a 2 ⁇ l volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 ⁇ l) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37°C, followed by 5 minutes at 85°C to denature the SAP.
  • SAP shrimp alkaline phosphatase
  • a primer extension reaction was initiated by adding a polymorphism-specific MassEXTENDTM primer cocktail to. each sample.
  • Each MassEXTENDTM cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another.
  • ddNTPs dideoxynucleotides
  • dNTPs deoxynucleotides
  • the MassEXTENDTM reaction was performed in a total volume of 9 ⁇ l, with the addition of IX ThermoSequenase buffer, ⁇ .576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTENDTM primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP.
  • the deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94°C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72°C.
  • samples were desalted by adding 16 ⁇ l of water (total reaction volume was 25 ⁇ l), 3 mg of SpectroCLEANTM sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJETTM (Sequenom, Inc.)) onto either 96-spot or 384-s.pot silicon chips containing a matrix that crystallized each sample (SpectroCHIP® (Sequenom, Inc.)).
  • MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RTTM software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.
  • TBN refers to the discovery cohort
  • NNL refers to the Newfoundland replication cohort
  • Steno refers to the Denmark replication cohort.
  • the boxes are centered over the odds ratio for each sample, with the size of the box correlated to the contribution of each sample to the combined meta analysis odds ratio.
  • the lines extending from each box are the 95% confidence interval values.
  • the diamond is centered over the combined meta analysis odds ratio with the ends of the diamond depicting the 95% confidence interval values.
  • the meta-analysis further illustrates the strong association each of the incident SNPs has with type II diabetes across multiple case and control samples.
  • the SNP rs662138 associated with type II diabetes lies within intron 7 of SLC22A1.
  • Ninety additional allelic variants proximal to rs662138 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 14.
  • the chromosome position provided in column four of Table 14 is based on Genome "Build 34" of NCBFs GenBank.
  • the "genome letter” corresponds to the particular allele that appears in NCBFs build 34 genomic sequence of the region (chromosome 6: positions 160423601-160523200), and the “deduced iupac” corresponds to the single letter IUPAC code for the SLC22A1 polymorphic variants as they appear in SEQ ID NO: 1. Also, the “genome letter” may differ from the alleles (A1/A2) provided in Table 14 (and in subsequent Tables that provide the same information) if the genome letter is on one strand and the alleles are on the complementary strand, thus having different strand orientations ⁇ i.e., reverse vs forward). TABLE 14
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures IA- 1C for females, males and combined, respectively. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 1A-1C can be determined by consulting Tables 17-19.
  • the left-most X on the left graph is at position 160423829.
  • the allele frequency associated with each symbol shown can be determined.
  • multiple lines have been added to the graph.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 20, 21 and 22 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • Metformin seems to ameliorate hyperglycemia by improving peripheral sensitivity to insulin, reducing gastrointestinal glucose absorption and hepatic glucose production (Caspary and Creutzfeldt, Diabetologia 7: 379-385, 1971; ⁇ un ⁇ al et aL, 2000 Diabetes 49: 2063-2069; Borst and Snellen, 2001 Life Sd 69: 1497- 1507), although the exact mechanism for its pharmacological action is not yet fully understood.
  • Table 23 shows that the major allele C has been selected for through evolution, maybe in times of lack of substantial food, as happened with the Pima Indians. The results show that having the major allele C individuals have a greater risk of developing diabetes. In addition, they are less likely to be treated with metformin. Accordingly, it is believed that patients were treated and did not respond. Also, it is believed that the polymorphism in the SLC22A1 gene results, in an alteration in the metformin uptake and or signaling cascade thereby preventing the substrate of SLC22A1 binding. Usually when metformin is administered to diabetics it results in a decrease in hepatic glucose production (HGP). This decrease in HGP may not be seen in the individual with the polymorphism in SLC22A1. Thus administering the drug to these individuals would result in no overall improvement in glucose homeostasis.
  • HGP hepatic glucose production
  • the SNP rs 1001238 is associated with type II diabetes in the examples above.
  • One hundred thirty-four additional allelic variants proximal to rs 1001238 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 24.
  • the chromosome position provided in column four of Table 24 is based on Genome "Build 34" of NCBPs GenBank.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 2A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 2A-C can be determined by consulting Tables 27-29. For example, the left-most X on the left graph is at position 179567944. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples.3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 30, 31 and 32 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • the SNP rs3219 associated with type II diabetes in the examples above falls in the untranslated region of the LOCI 12609 gene. Fifty-one additional allelic variants proximal to rs3219 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2. The polymorphic variants are set forth in Table 33. The chromosome position provided in column four of Table 33 is based on Genome "Build 34" of NCBPs GenBank.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 3A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 3A-C can be determined by consulting Tables 36-38. For example, the left-most X on the left graph is at position 84746396. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances, between SNPs.
  • Two other lines are drawn to expose linear trends, in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping, results are shown for female (F), male (M), and combined cases and controls in Table 39, 40 and 41 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • Some SNPs may be labeled "untyped" because of failed assays. TABLE 39: Female Replication Allelotyping Results
  • the SNP rsl 160333 associated with type II diabetes in the examples above falls within an intron of the trichorhinophalangeal syndrome I (TRPSl) gene.
  • TRPSl trichorhinophalangeal syndrome I
  • Sixty additional allelic variants proximal to rsl 160333 were identified and subsequently allelotyped in diabetes, case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 42.
  • the chromosome position provided in column four of Table 42 is based on Genome "Build 34" of NCBFs GenBank.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 4A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives, the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 4A-C can be determined by consulting Tables 45-47. For example, the left-most X on the left graph is at position 116488511. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 48, 49 and 50 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • the SNP rs 1024373 associated with type II diabetes in the examples above falls within an intron of the PRAME gene.
  • One hundred thirty-two additional allelic variants proximal to rslO24373 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 51.
  • the chromosome position provided in column four of Table 51 is based on Genome "Build 34" of NCBFs GenBank.
  • the amino acid changes provided in column 8 of Table 51 correspond to SNPs that code for non-synonymous amino acid changes, where changes with two * occur in the SUHW2 polypeptide (locus ID no. 140883), changes with one * occur in the SUHWl polypeptide (locus ID no. 129025), and the W7R change occurs in the PRAME polypeptide.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 5A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures.5 A-C can be determined by consulting Tables 54-56. For example, the left-most X on the left graph is at position 21166760. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 57, 58 and 59 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • SNP rs366111 associated with type II diabetes in the examples above falls within an exon of the ZNF221 gene.
  • SNP r&366111 is a coding non-synonymous SNP (G/A), which results in an amino acid change from arginine to glycine.
  • G/A coding non-synonymous SNP
  • the G allele is associated with an increased risk of type II diabetes and codes for glycine; therefore, individuals with the glycine residue also have an increased risk of type II diabetes.
  • One hundred forty-six additional allelic variants proximal to rs366111 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 60.
  • the chromosome position provided in column four of Table 60 is based on Genome "Build 34" of NCBI's GenBank.
  • the amino acid changes provided in column 8 of Table 60 correspond to SNPs that code for non-synonymous amino acid changes, where changes with one * occur in the ZNF221 polypeptide, changes with two * occur in the ZNFl 55 polypeptide (locus ID no. 7711), and changes with three * occur in the ZNF230 polypeptide (locus ID no. 7773).
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 6A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 6A-C can be determined by consulting Tables 63-65. For example, the left-most X on the left graph is at position 49113509. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 "8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 66, 61 and 68 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • the SNP rsl203877 associated with type II diabetes in the examples above falls near a member of the forkhead class of DNA-binding proteins called forkhead box A2 (FOXA2).
  • FOXA2 forkhead box A2
  • One hundred additional allelic variants proximal to rs 1203877 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 69.
  • the chromosome position provided in column four of Table 69 is based on Genome "Build 34" of NCBI's GenBank.
  • allelotyping results were considered particularly significant with a calculated p- value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 7A-C for females, males and combined, respectively. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 7A-C can be determined by consulting Tables 12-1 A. For example, the left-most X on the left graph is at position 22487606. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10 '8 were truncated at that value.
  • the proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 75, 76 and 77 respectively.
  • the allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency.
  • Tables 81-83 shows the case and control allele frequencies along with the p- values for all of the SNPs genotyped in female pools, male pools, and combined, respectively. P- values less than 0.05 are in bold.
  • the SNP rs470936 associated with type II diabetes in the examples above falls within an intron of the JAMS gene.
  • One hundred ten additional allelic variants proximal to rs470936 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2.
  • the polymorphic variants are set forth in Table 84.
  • the chromosome position provided in column four of Table 84 is based on Genome "Build 34" of NCBI's GenBank.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 8A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 8A-C can be determined by consulting Tables 87-89. For example, the left-most X on the left graph is at position 133498018. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two common significance levels, and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs. to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and T.J. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than ICF 8 were truncated at that value.
  • SNP rs846312 associated with type II diabetes in the examples above falls within • an intron of the GLI3 gene. Two hundred fifteen additional allelic variants proximal to rs846312 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2. The polymorphic variants are set forth in Table 90. SNP rs846266 codes for a non-synonymous amino acid change (Tl 83A). The chromosome position provided in column four of Table 90 is based on Genome "Build 34" of NCBFs GenBank.
  • allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold.
  • the allelotyping p-values were plotted in Figures 9A-C for females, males and combined, respectively.
  • the position of each SNP on the chromosome is presented on the x-axis.
  • the y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group.
  • the minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures 9A-C can be determined by consulting Tables 93-95. For example, the left-most X on the left graph is at position 41819727. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.
  • the broken horizontal lines are drawn at two. common significance levels, 0.05 and 0.01.
  • the vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs.
  • Two other lines are drawn to expose linear trends in the association of SNPs to the disease.
  • the light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W.S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and TJ. Hastie, Wadsworth & Brooks/Cole.).
  • the black line provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p- values that were less than 10 "8 were truncated at that value.
  • cDNA is. cloned into a pIVEX 2.3-MCS vector (Roche Biochem ⁇ using a directional cloning method.
  • a cDNA insert is prepared using PCR with forward and reverse primers having 5' restriction site tags, (in frame) and 5-6 additional nucleotides in addition to 3' gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length.
  • a Sal I restriction site is introduced by the forward primer and a Sma I restriction site is introduced by the reverse primer.
  • the ends of PCR products are cut with the corresponding restriction enzymes (i.e., Sal I and Sma I) and the products are gel-purified.
  • the pIVEX 2.3-MCS vector is linearized using the same restriction enzymes, and the fragment with the correct sized fragment is isolated by gel-purification. Purified PCR product is ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production.
  • E. coti lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer.
  • 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution.
  • 0.75 ml of Reaction Mix, 50 ⁇ l of Energy Mix, and 10 ⁇ g of the template DNA is added to the E. coli lysate.
  • reaction device (Roche Biochem) 1 ml of the Reaction Solution is loaded into the reaction compartment.
  • the reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 30 0 C for 24 hours with a stir bar speed of 150 rpm.
  • the pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the target polypeptide for the purpose of protein purification.
  • Target polypeptide is purified by contacting the contents of reaction device with resin modified with Ni 2+ ions.
  • Target polypeptide is eluted from the resin with a solution containing free Ni 2+ ions.
  • Nucleic acids are cloned into DNA plasmids having phage recombination cites and target polypeptides are expressed therefrom in a variety of host cells.
  • Alpha phage genomic DNA contains short sequences known as attP sites
  • E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new art sites, L and R, which flank the inserted prophage DNA. Phage excision from E.
  • coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis.
  • DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target DNA fragment into a backbone vector in a rapid in vitro reaction (GatewayTM Technology (Invitrogen, Inc.)).
  • a first step is to transfer the nucleic acid insert into a shuttle vector that contains attL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB.
  • a second method is to amplify the nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF.
  • PCR polymerase chain reaction
  • a third method is to utilize a topoisomerase-mediated process, in which the nucleic acid is amplified via PCR using gene-specific primers with the 5' upstream primer containing an additional CACC sequence ⁇ e.g.,
  • the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.
  • the nucleic acid can be cloned into an expression vector having attR sites.
  • vectors containing attR sites, for expression of target polypeptide as a native polypeptide, N-fusion polypeptide, and C-fusion polypeptides are commercially available (e.g. , pDEST (Invitrogen, Inc.)), and any vector can be converted into an expression vector for receiving a nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase).
  • the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37°C with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method.
  • expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N-terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the target polypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed a adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2.
  • test molecule refers to a molecule that is added to a system, where an agonist effect, antagonist effect, or lack of an effect of the molecule on SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3 function or a related physiological function in the system is assessed.
  • An example of a test molecule is a test compound, such as a test compound described in the section "Compositions Comprising Diabetes-Directed Molecules" above.
  • test molecule is a test peptide, which includes, for example, a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3- related test peptide such as a soluble, extracellular form of SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLB, a biologically active fragment of SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3, a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3, a SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3 binding partner or ligand, or a functional fragment of the foregoing.
  • a concentration range or amount of test molecule utilized in the assays and models is selected from a variety of available ranges and amounts.
  • a test molecule sometimes is introduced to an assay system in a concentration range between 1 nanomolar and 100 micromolar or a concentration range between 1 nanograms/mL and 100 micrograms/mL.
  • An effect of atest molecule on SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3 function or a related physiological function often is determined by comparing an effect in a system administered the test molecule against an effect in system not admininstered the test molecule. Described directly hereafter are examples of in vitro assays. Glucose Uptake Assay
  • GLUT4 an insulin- regulatable glucose transporter.
  • Insulin binding to insulin receptors on the cell surface results in autophosphorylation and activation of the intrinsic tyrosine kinase activity of the insulin receptor.
  • Phosphorylated tyrosine residues on the insulin receptor and its endogenous targets activate several intracellular signaling pathways that eventually lead to the translocation of GLUT4 from intracellular stores to the extracellular membrane.
  • Cells are plated in 6-welI dishes, and grown to confluency. Cells are then differentiated with DMEM plus 10% fetal calf serum (FCS) 5 10 ug/mL insulin, 390 ng/mL dexamethasone and 112 ug/mL isobutylmethylxanthine for 2 days. After 2 days of differentiation * media is changed to maintenance media DMEM plus. 10% FCS and 5 ug/mL insulin. Media is changed every 2 days thereafter. Cells are assayed for insulin-mediated glucose uptake 10 days after differentiation.
  • FCS fetal calf serum
  • recombinant rat SLC22A1, TTN 3 LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3/Fc chimeria is added to a concentration of 1.75 ug/mL, and anti-human IgG, Fc ⁇ fragment specific antibody to a final concentration of 17.5 ug/mL.
  • media is. replaced with 2 mL of preclustered SLC22A1, TTN 5 LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3, and incubated for 10, 40 and 90 min at 37 deg.
  • porcine insulin is added to a final concentration of 100 nM for 10 min at 37 deg.
  • 100 uL of PBS-2-DOG label is added to give a final concentration of 2 uCi.
  • Cells are immediately placed on ice, washed three times with ice cold PBS, and lysed with 0.7 mL of 0.2 N NaOH. Lysates are read in a Wallac 1450 Microbeta Liquid Scintillation and Luminescence Counter.
  • TGs triacylglycerol
  • a direct metabolic consequence of glucose transport intracellularly is its incorporation into the fatty acid and glycerol moieties of triacylglycerol (TG).
  • TGs are highly concentrated stores of metabolic energy, and are the major energy reservoir of cells.
  • the major site of accumulation of triacylglycerols is the cytoplasm of adipose cells.
  • Adipocytes are specialized for the synthesis, and storage of TG, and for their mobilization into fuel molecules that are transported to other tissues through the bloodstream. It is likely that changes in the transport of glucose intracellularly can affect cytoplasmic stores of triacylglycerols.
  • Cells are plated in 6-well dishes, and grown to confluency. When cells reached confluency, cells are differentiated with DMEM plus 10% fetal calf serum (FCS), 10 ug/mL insulin, 390 ng/mL dexamethasone and 112 ug/mL isobutylmethylxanthine for 2 days. After 2 days of differentiation, media is changed to maintenance media DMEM plus 10% FCS and 5 ug/mL insulin. On the day of the assay (day 9 post-differentiation), cells are washed once with PBS, and serum starved by adding 2 mL of DMEM plus 2 mg/niL BSA for 3 hours.
  • FCS fetal calf serum
  • recombinant rat SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3/Fc chimeric ligand is preclustered.
  • a solution of PBS plus 2 mg/mL BSA recombinant rat SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3/Fc chimeria is added to a concentration of 1.75 ug/mL, and anti-human IgG, Fc ⁇ fragment specific antibody to a final concentration of 17.5 ug/mL.
  • sample In a 96-well, flat bottom, transparent microtiter plate, 3 uL of sample are added to 300 uL of INFINITY Triglyceride Reagent. Samples are incubated at room temperature for 10 minutes. The assay is read at 500-550 nm.
  • Resistin is a secreted factor specifically expressed in white adipocyte. It was initially discovered in a screen for genes downregulated in adipocytes by PPAR gamma, and expression was found to be attenuated by insulin. Elevated levels of resistin have been measured in genetically obese, and high fat fed obese mice. It is therefore thought that resistin contributes to peripheral tissue insulin unresponsiveness, one of the pathological hallmarks of diabetes.
  • 3T3-L1 cells are differentiated for 3 days as previously described and maintained for three days prior to splitting. At day 5 post-differentiation, differentiated cells are plated in 10 cm dish at a cell density of 3X10 6 cells. Cells are then serum starved on day 7 after initiation of differentiation. On day 8, cells are treated with pre-clustered recombinant rat SLC22A1, TTN 5 LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLBfFc chimera as described above for 10 min and treated with 10 nM insulin for 2 hours.
  • Cells are harvested, mRNA extracted using magnetic DYNAL beads and reverse transcribed to cDNA using Superscript First-Strand Synthesis as described by the manufacturer. Appropriate primers are designed and used in 15 uL PCR reaction using 55 deg annealing temperature and 30 cycles of amplification.
  • C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, MD) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc) + 10% FCS. Two days later they become 80-90% confluent. At this time, the media is changed to DMEM+2% horse serum to allow differentiation. The media is changed daily. Abundant myotube formation occurs after 3-4 days of being in 2% horse serum, although the exact time course of C2C12 differentiation depends on how long they have been passaged and how they have been maintained, among other factors.
  • test molecules e.g., test peptides added in a range of 1 to 2.5 ⁇ g/mL
  • test molecules are added the day after seeding when the cells are still in DMEM with 10% FCS.
  • Two days after plating the cells one day after the test molecule was first added, at about 80-90% confluency, the media is changed to DMEM+2% horse serum plus the test molecule.
  • C2C12 cells are differentiated in the presence or absence of 2 ⁇ g/mL test molecules for 4 days. On day 4, oleate oxidation rates are determined by measuring conversion of l- 14 C-oleate (0.2 mM) to 14 CO 2 for 90 min. This experiment can be used to screen for active polypeptides and peptides as well as agonists and antagonists or activators and inhibitors of SLC22Al, TTN 1 LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLB polypeptides or binding partners.
  • test molecules on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, VA CRL-1772) and in a hepatocyte cell line (Hepal-6; ATCC, Manassas, VA CRL-1830). Cultured cells are maintained according to manufacturer's instructions.
  • the oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791). Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes becomes maximal.
  • DMEM low serum differentiation media
  • Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days. One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 ⁇ g/mL gentamycin) is added.
  • preincubation media MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 ⁇ g/mL gentamycin
  • test molecule e.g., 2.5 ⁇ g/mL of SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GL/3-related test peptide.
  • test molecule e.g., 2.5 ⁇ g/mL of SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GL/3-related test peptide.
  • 0.75 mL of the media is removed and assayed for 14 C-oxidation products as described below for the muscle FFA oxidation experiment.
  • Triglyceride and Protein Analysis, following Oleate Oxidation in Cultured Cells [0347] Following transfer of media for oleate oxidation assay, cells are placed on ice. To determine triglyceride and protein content, ceils are washed with 1 mL of Ix PBS to remove residual media. To each well 300 ⁇ L of cell dissociation solution (Sigma) is added and incubated at 37 0 C for 10 min. Plates are tapped to loosen cells, and 0.5 mL of Ix PBS is added. The cell suspension is transferred to an Eppendorf tube, each well is rinsed with an additional 0.5 mL of Ix PBS, and is transferred to the appropriate Eppendorf tube.
  • Samples are centrifuged at 1000 rpm for 10 minutes at room temperature. Each supernatant is discarded and 750 ⁇ L of Ix PBS/2% CHAPS is added to cell pellet. The cell suspension is vortexed and placed on ice for 1 hour. Samples are then centrifuged at 13000 rpm for 20 min at 4 0 C. Each supernatant is transferred to a new tube and frozen at -2O 0 C until analyzed. Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit.
  • the assay is performed in 48 well plate, 350 ⁇ L of sample volume is assayed, a control blank consists of 350 ⁇ L PBS/2% CHAPS, and a standard contains 10 ⁇ L standard provide in the kit with 690 ⁇ L PBS/2% CHAPS.
  • Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.
  • Protein analysis is carried out on 25 ⁇ L of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions. Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm. Stimulation of insulin secretion in HIT-Tl 5 cells
  • HIT-T 15 is an immortalized hamster insulin-producing cell line. It is known that stimulation of cAMP in HIT-T 15 cells causes an increase in insulin secretion when the glucose concentration in the culture media is changed from 3mM to 15 mM. Thus, test molecules also are tested for their ability to stimulate glucose-dependent insulin secretion (GSIS) in HIT-T15 cells. In this assay, 30,000 cells/well in a 12-well plate are incubated in culture media containing 3 mM glucose and no serum for 2 hours. The media is then changed, wells receive media containing either 3 mM or 15 mM glucose, and in both cases the media contains either vehicle (DMSO) or test molecule at a concentration of interest.
  • DMSO vehicle
  • Some wells receive media containing 1 micromolar forskolin as. a positive control. AU conditions are tested in triplicate. Cells are incubated for 30 minutes, and the amount of insulin secreted into the media is determined by ELISA, using a kit from either Peninsula Laboratories (Cat # ELIS-7536) or Crystal Chem Inc. (Cat # 90060).
  • IEQ islet equivalents
  • strainers Move strainers to next wells (Low 1) with 4 or 5 ml low glucose KRB. Incubate at 37° C for 30 minutes. Collect supernatants into low-binding polypropylene tubes pre- labelled for identification and keep cold.
  • Insulin determinations are performed as above, or by Linco Labs as a custom service, using a rat insulin RIA (Cat. # RI-13K).
  • mice Following is a representative rodent model for identifying thereapeutics for treating human diabetes. Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group). All mice are housed individually. The mice are maintained on a high fat diet throughout each experiment.
  • the high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal% 16, sucrose kcal% 26, and fat kcal% 58.
  • the fat is primarily composed of coconut oil, hydrogenated.
  • mice After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide test molecule, saline, and a control molecule (e.g., an irrelevant peptide) to the mice subcutaneously (s.c.) for 18 days.
  • a control molecule e.g., an irrelevant peptide
  • JAMS an d GZ/5-related test peptides are provided at doses of 100, 50, 25, and 2.5 ⁇ g/day and an irrelevant peptide is provided at 10 ⁇ g/day.
  • Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment. Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels. The amount of food consumed per day is also determined for each group.
  • mice are administered orally with dextrose at 5 g/kg dose.
  • Test molecule is delivered orally via a gavage needle (p.o. volume at 100 ml).
  • Control Ex-4 is delivered intraperitoneally.
  • Levels of blood glucose are determined at regular time points using Glucometer Elite XL (Bayer).
  • Reduction in blood glucose at each time point may be expressed as percentage of original glucose levels, averaged from the number of animals for each group. Results show the effect SLC22A1, TTN, LOCI 12609, TRPSl, PRAME, ZNF221, FOXA2, JAM3 and GLI3-related test peptides and test molecules for improving glucose homeostasis in diabetic animals.
  • test molecule is injected i.p. in 100 ⁇ L saline (e.g., 25 ⁇ g of test peptide).
  • saline e.g. 25 ⁇ g of test peptide
  • the same dose 25 ⁇ g/mL in lOO ⁇ L
  • Control animals are injected with saline (3xl00 ⁇ L). Untreated and treated animals are handled in an alternating mode.
  • Plasma samples are taken in hourly intervals, and are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 0 C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako). Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples. For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.
  • FFA free fatty acids
  • TG triglycerides
  • glucose is determined in duplicate using pooled samples. For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.
  • a test molecule i.p. in lOO ⁇ L saline (e.g., 25 ⁇ g of test peptide).
  • test molecule e.g. 50 ⁇ g of test peptide
  • control animals are injected with saline (e.g., 3xl00 ⁇ L). Untreated and treated animals are handled in an alternating mode.
  • Plasma samples are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 0 C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • FFA free fatty acids
  • TG triglycerides
  • glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • mice are injected with epinephrine.
  • epinephrine Two groups of mice are given epinephrine (5 ⁇ g) by intraperitoneal injection. A treated group is injected with a test molecule (e.g., 25 ⁇ g of test peptide) one hour before and again together with epinephrine, while control animals receive saline. Plasma is isolated and free fatty acids and glucose are measured as described above.
  • Muscles are rinsed for 30 min in incubation media with oxygenation. The muscles are then transferred to fresh media (1.5 mL) and incubated at 30 0 C in the presence of l ⁇ Ci/mL [1- 14 C] oleic acid (American Radiolabeled Chemicals). The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm x 11.5 cm) is suspended.
  • test molecules are added to the media (e.g., a final concentration of 2.5 ⁇ g/mL of test peptide) and maintained in the media throughout the procedure.
  • media e.g., a final concentration of 2.5 ⁇ g/mL of test peptide
  • mice are intravenously (tail vein) injected with 30 ⁇ L bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.
  • Intralipid is an intravenous fat emulsion used in nutritional therapy.
  • a treated group (treated with test molecule) is injected with a test molecule (e.g., 25 ⁇ g of a test peptide) at 30 and 60 minutes before Intralipid is given, while control animals receive saline. Plasma is isolated and FFAs are measured as described previously. The effect of a test molecule on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.
  • the db/db mice progressively develop insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled.
  • the state of the pancreas and its course vary according to the models. Since this is a model of type II diabetes mellitus, test molecules are tested for blood sugar and triglycerides lowering activities.
  • Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31 :1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp.
  • the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217- 220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA 88:7806, 1991).
  • Tubby tub/tub>mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).
  • leptin is reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al. Nature 401 : 73-76 (1999). Leptin is found to be less effective in a different lipodystrophic rodent model of lipoatrophic diabetes (Grajova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables).
  • STZ streptozotocin
  • the monosodium glutamate (MSG) model for chemically- induced obesity (Olney, Science 164:719, 1969; Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined.
  • a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.
  • Test molecules are tested for reducing hyperglycemia in any or all of the above rodent diabetes models or in humans with type II diabetes or other metabolic diseases described previously or models based on other mammals.
  • the test molecule sometimes is combined with another compatible pharmacologically active antidiabetic agent such as insulin, leptin (US provisional application No 60/155,506), or troglitazone, either alone or in combination.
  • test molecules are administered intraperitoneally, subcutaneously, intramuscularly or intravenously. Glucose and insulin levels of the mice are tested, food intake and liver weight monitored, and other factors, such as leptin, FFA, and TG levels, often are measured in these tests.
  • mice Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22° C and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch Basic Glucose Monitor System (Lifescan). Mice that have plasma glucose levels between 250 to 500 mg/dl are used.
  • Each treatment group consists, of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study, db/db mice are dosed by micro-osmotic pumps, inserted using isofturane anesthesia, to provide test molecules, saline, and an irrelevant peptide to the mice subcutaneously (s.c).
  • Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations.
  • Food is withdrawn from 0-4 h post dosing and reintroduced thereafter.
  • Individual body weights and mean food consumption are also measured after 24 h. Significant differences between groups (comparing test molecule treated to saline-treated) are evaluated using a Student t-test.
  • Tests of the efficacy of test molecules in humans are performed in accordance with a physician's recommendations and with established guidelines.
  • the parameters tested in mice are also tested in humans ⁇ e.g. food intake, weight, TG, TC, glucose, insulin, leptin, FFA). It is expected that the physiological factors are modified over the short term. Changes in weight gain sometimes require a longer period of time. In addition, diet often is carefully monitored.
  • Test molecules often are administered in daily doses (e.g., about 6 mg test peptide per 70 kg person or about 10 mg per day). Other doses are tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.
  • genomic nucleotide sequence for a SLC22A1 region.
  • the genomic nucleotide sequence is set forth in SEQ ID NO: 1.
  • the following nucleotide representations are used throughout: "A” or “a” is adenosine, adenine, or adenylic acid; “C” or “c” is cytidine, cytosine, or cytidylic acid; “G” or “g” is guanosine, guanine, or guanylic acid; “T” or “t” is thymidine, thymine, or thymidylic acid; and “I” or “i” is inosine, hypoxanthine, or inosinic acid.
  • SNPs are designated by the following convention: “R” represents A or G, “M” represents A or C; “W” represents A or T; “Y” represents C or T; “S” represents C or G; “K” represents G or T; “V” represents A, C or G; “H” represents A, C, or T; “D” represents A, G, or T; “B” represents. C, G, or T; and 'TST" represents A, G, C, or T.

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Abstract

La présente invention a pour objet des méthodes d'évaluation du risque d’apparition d’un diabète de type II chez un patient, ainsi que les réactifs et le matériel nécessaire à l'application desdites méthodes. La présente invention a également pour objet des méthodes d'identification de médicaments potentiellement utilisables dans un traitement des diabètes de type II, ainsi que des méthodes thérapeutiques et prophylactiques applicables aux diabètes de type II. Ces modes de l’invention sont basés sur une analyse des variations polymorphiques de séquences de nucléotides au sein du génome humain.
PCT/US2004/023933 2004-07-22 2004-07-22 Méthodes d’identification du risque d’apparition de diabètes de type ii et traitements associés WO2006022636A1 (fr)

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WO2008087209A1 (fr) * 2007-01-19 2008-07-24 Integragen Gène iglc de sensibilité au diabète humain
EP2021502A1 (fr) * 2006-05-09 2009-02-11 Oy Jurilab Ltd Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité
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US9886556B2 (en) 2015-08-20 2018-02-06 Aseko, Inc. Diabetes management therapy advisor
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US9897565B1 (en) 2012-09-11 2018-02-20 Aseko, Inc. System and method for optimizing insulin dosages for diabetic subjects
US11081226B2 (en) 2014-10-27 2021-08-03 Aseko, Inc. Method and controller for administering recommended insulin dosages to a patient

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DATABASE SNP [online] 27 July 2000 (2000-07-27), XP002994324, accession no. NCBI Database accession no. (RS662138) *
WANG D-S. ET AL: "Involvement of Organic Cation Transporter 1 in Hepatic Intestinal Distribution of Metformin", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 302, no. 2, 2002, pages 510 - 515, XP002994325 *

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EP2021502A1 (fr) * 2006-05-09 2009-02-11 Oy Jurilab Ltd Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité
EP2021502A4 (fr) * 2006-05-09 2010-08-25 Mas Metabolic Analytical Servi Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité
WO2008087209A1 (fr) * 2007-01-19 2008-07-24 Integragen Gène iglc de sensibilité au diabète humain
US10629294B2 (en) 2012-09-11 2020-04-21 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US9483619B2 (en) 2012-09-11 2016-11-01 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US11733196B2 (en) 2012-09-11 2023-08-22 Aseko, Inc. System and method for optimizing insulin dosages for diabetic subjects
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US9710611B2 (en) 2014-01-31 2017-07-18 Aseko, Inc. Insulin management
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US10811133B2 (en) 2014-01-31 2020-10-20 Aseko, Inc. System for administering insulin boluses to a patient
US9486580B2 (en) 2014-01-31 2016-11-08 Aseko, Inc. Insulin management
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US9233204B2 (en) 2014-01-31 2016-01-12 Aseko, Inc. Insulin management
US11158424B2 (en) 2014-01-31 2021-10-26 Aseko, Inc. Insulin management
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US11468987B2 (en) 2014-01-31 2022-10-11 Aseko, Inc. Insulin management
US11490837B2 (en) 2014-01-31 2022-11-08 Aseko, Inc. Insulin management
US10403397B2 (en) 2014-10-27 2019-09-03 Aseko, Inc. Subcutaneous outpatient management
US11678800B2 (en) 2014-10-27 2023-06-20 Aseko, Inc. Subcutaneous outpatient management
US11694785B2 (en) 2014-10-27 2023-07-04 Aseko, Inc. Method and dosing controller for subcutaneous outpatient management
US11081226B2 (en) 2014-10-27 2021-08-03 Aseko, Inc. Method and controller for administering recommended insulin dosages to a patient
US10128002B2 (en) 2014-10-27 2018-11-13 Aseko, Inc. Subcutaneous outpatient management
US9892234B2 (en) 2014-10-27 2018-02-13 Aseko, Inc. Subcutaneous outpatient management
US11574742B2 (en) 2015-08-20 2023-02-07 Aseko, Inc. Diabetes management therapy advisor
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