WO2008052016A2 - Récepteur associé à la sortiline sorl1 présentant un lien fonctionnel et génétique avec la maladie d'alzheimer - Google Patents

Récepteur associé à la sortiline sorl1 présentant un lien fonctionnel et génétique avec la maladie d'alzheimer Download PDF

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WO2008052016A2
WO2008052016A2 PCT/US2007/082308 US2007082308W WO2008052016A2 WO 2008052016 A2 WO2008052016 A2 WO 2008052016A2 US 2007082308 W US2007082308 W US 2007082308W WO 2008052016 A2 WO2008052016 A2 WO 2008052016A2
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snp
linkage disequilibrium
sorl1
haplotypes
patient
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WO2008052016A3 (fr
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Richard Mayeux
Ekaterina Rogaeva
Peter St. George-Hyslop
Lindsay Farrer
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Columbia University
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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
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates methods for determining if a patient is at risk of developing Alzheimer's Disease by analyzing DNA from the patient and determining whether there are any nucleotide variants in the region of the gene for SORL1 at SNP sites 4 or 8-12 and 19-26.
  • amyloid beta peptide a neurotoxic proteolytic derivative of the amyloid precursor protein (APP) is a central event in the pathogenesis of Alzheimer's Disease (AD) 1 .
  • AD Alzheimer's Disease
  • APP amyloid precursor protein
  • PSl presenilin 1
  • PS2 presenilin 2
  • APOE apolipoprotein E
  • AD Alzheimer's Disease
  • AD Alzheimer's disease
  • FIG. 1 A Top panel: Domain diagram of the SORL1 protein.
  • VPS10 vacuolar protein sorting 10-like domain
  • YWTD low density lipoprotein receptor YWTD domain
  • EGF EGF-like domain
  • LDLa low density lipoprotein receptor class A domain
  • FN3 fibronectin type 3 domain
  • TM transmembrane domain.
  • Bottom panel Genomic map of SORL1 gene showing the location of SNPs genotyped in this study. Orange bars represent the 5'UTR and 3'UTR, red bar represents intragenic regions, and vertical bars represent each of the 48 exons. SNPs 1, 28 and 29 are located in extragenic intervals.
  • B Diagram of APP processing pathways.
  • APP holoprotein is synthesized in the endoplasmic reticulum (ER) and Golgi. Proteolytic cleavage through the A ⁇ peptide domain by ADAM17 and other ⁇ -secretase enzymes generates N-terminal soluble APPs ⁇ and membrane-bound APP-CTF ⁇ fragments. Sequential cleavage by BACE1 ( ⁇ -secretase) generates N-terminal APPs ⁇ and membrane bound APP-CTF ⁇ fragments. The latter undergoes presenilin-dependent ⁇ -secretase cleavage to generate A ⁇ and amyloid intracellular domain (AICD). SORL1 binds both APP holoprotein (see FIG.
  • VPS35 acts as a sorting receptor for APP holoprotein. Absence of SORL1 switches the APP holoprotein away from the retromer recycling pathway., The APP holoprotein is instead directed into the ⁇ -secretase cleavage pathway, increasing APPs ⁇ production (FIG 2c) and then into the ⁇ -secretase cleavage pathway to generate A ⁇ (see FIG. 2b).
  • retromer complex proteins such as VPS26 (FIG. 2d) or VPS35, or Golgi-localized gamma-ear-containing ARF-binding (GGA) adaptor proteins
  • FIG. 2 A Small quantities of endogenous APP holoprotein but not APP C- terminal fragments (APP-CTFs, generated by ⁇ - or ⁇ -secretase) can be co- immunoprecipitated with endogenous SORL1 (Top panel). Conversely small quantities of endogenous SORL1 can be co-precipitated with endogenous APP holoprotein (Bottom panel).
  • B SORL1 does not interact with BACE1 ( ⁇ -secretase). Co-immunoprecipitations with antibodies to over-expressed BACE1-V5 fail to capture SORL1 (Bottom panel). Conversely, SORL1 -directed antibodies do not co-immunoprecipitate BACE1 (Top panel) even though BACE1 also traffics through the endosome to Golgi pathway.
  • FIG. 3 A Over-expression of SORL1 reduces A ⁇ 40 (and A ⁇ 42 not shown) secretion (p ⁇ 0.05).
  • Upper panel Representative data of Western blot for SORL1 and APP in HEK293 cells stably expressing APP Swe , and transiently transfected with empty vector
  • C anti-SORL1 siRNA treatment results in significant increases in APPs ⁇ secreted into the media, but no significant change in APPs ⁇ levels.
  • Left panel Western blots of conditioned media from cells treated with nonsense siRNA oligo-nucleotides (Controls #1 and #2) or with anti-SORL1 siRNA oligonucleotides investigated with the 2H3 antibody to
  • FIG. 4 Linkage disequilibrium(LD) structure of SORL1. Relative locations of single nucleotide polymorphisms (SNPs) included in each dataset are shown on two parallel stickdiagrams, with LD maps for the TGEN dataset located above and the north European familial AD dataset in the Primary Study below the gene structure. The measure of LD(D0) among all possible pairs of SNPs is shown graphically according to the shade of red where white represents very low D0 and dark red represents very highD0. High D0 estimates associated with a large confidence interval (most likely owing to one of the alleles being rare) are denoted by blue squares. TGEN: Translational Genomics Research Institute.
  • a method for determining if a patient is at risk of developing Alzheimer's Disease by a. obtaining a DNA sample from the patient, b. determining if the DNA sample shows a high risk nucleotide variant in an SNP that is a member of the group comprising SNPs rs560573, rs985421, rs593769, rs12364988, rs668387 [SNPs 8-12], rs4935775, 17 rs12285364, rs2298813 [SNPs 16-18], rs11600231, rs2276346, rs10502262, SORL1-T833T, rs556349, rs7131432, rs11218340 and rs10892756.
  • SORL1 sortilin-related receptor low density lipoprotein receptor class A
  • high risk nucleotide variants include the high risk nucleotide variant is an allele variant that is a member selected from the group that includes C at SNP, G at SNP rs985421, C at SNP rs593769, G at SNP rs1l600231, T at SNP rs556349, C at SNP rs668387, T at SNP rs668387, G at SNP rs2276346, and G at SNP s10892756.
  • high risk nucleotide variants include a haplotype that is a member selected from the group that includes CGC at SNP rs560573, rs985421, and rs593769, respectively, or at a site in linkage disequilibrium with it; CGC at SNP rs985421, rs593769, and rs12364988, respectively, or at a site in linkage disequilibrium with it, GCC at SNP rs985421, rs593769, and rs12364988, respectively, or at a site in linkage disequilibrium with it, ATA at SNP CTT at SNP rs560573, rs985421, and rs593769, respectively, or at a site in linkage disequilibrium with it, CTT SORL1-T833T, rs556349, rs7131432, respectively, or at a site in linkage disequilibrium
  • the low risk nucleotide variants include a haplotype variant that is a member selected from the group that includes GCG at SNP rs560573, rs985421, and rs593769, respectively; or in linkage disequilibrium with it, GCG at SNP rs985421, rs593769, and rs12364988, respectively, or in linkage disequilibrium with it, CGG at SNP rs985421, rs593769, and rs12364988, respectively, or in linkage disequilibrium with it, TAT at SNP CTT at SNP rs560573, rs985421, and rs593769, respectively, or in linkage disequilibrium with it, GAA SORL1-T833T, rs556349, rs7131432, respectively, or in linkage disequilibrium with it, AAG at SNP rs556349,
  • Another aspect is directed to a method for confirming a diagnosis of Alzheimer's Disease in a patient having a diagnosis of probable or possible Alzheimer's Disease by: a. obtaining a DNA sample from the patient, b. determining if the DNA sample shows a high risk nucleotide variant in an SNP that is a member of the group comprising SNPs rs560573, rs985421, rs593769, rs12364988, rs668387 [8-12], rs4935775, 17 rs12285364, rs2298813 [16-18], rs11600231, rs2276346, rs10502262, SORL1-T833T, rs556349, rs7131432, rs11218340 and rs10892756.
  • the method further includes: d. obtaining a cell sample from the patient, e. determining the level of SORL1 expression in the patient cell sample, f. comparing the level of expression of SORL1 in the patient cell sample to the level of expression of SORL1 in a cell sample taken from a normal patient, and g. determining that the patient has Alzheimer's Disease if the level of SORL1 expression in patient sample is significantly lower than the level in the cell sample from the normal patient.
  • SORL1 sortilin-related receptor low density lipoprotein receptor class A
  • Another aspect of the invention is directed to a method for determining if a patient is at risk of developing AD, by a. obtaining a cell sample from the patient, b. determining the level of SORL1 expression in the patient cell sample, c. comparing the level of expression of SORL1 in the patient cell sample to the level of expression of SORL1 in a cell sample taken from a normal patient, and d. determining that the patient is at risk for Alzheimer's Disease if the level of SORL1 expression in patient sample is significantly lower than the level in the cell sample from the normal patient.
  • Another aspect of the invention is directed to a method for treating Alzheimer's Disease in a patient, by administering SORL1 (preferably human recombinant SORL1) in a therapeutically effective amount that reduces the level of amyloid beta in the serum, plasma or cerebrospinal fluid (csf) of the patient.
  • SORL1 preferably human recombinant SORL1
  • the SORL1 is formulated in a pharmaceutical composition that crosses the blood brain barrier and is recombinant human SORL1.
  • Another aspect of the invention is directed to a pharmaceutical composition for treating or preventing Alzheimer's Disease that includes human recombinant SORL1 in a formulation that crosses the blood brain barrier.
  • haplotype is meant a is a set of closely linked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination). Some haplotypes may be in linkage disequilibrium, haplotype can be identified by patterns of SNPs. Maps of SNPs (Haplotype maps) can be used to identify complex genetic variations of inherited diseases. A haplotype is a contraction of the phrase "haploid genotype”.
  • linkage disequilibrium is meant the case where the observed frequencies of haplotypes in a population does not agree with haplotype frequencies predicted by multiplying together the frequency of individual genetic markers in each haplotype.
  • geneotype is meant the specific allelic composition of a gene.
  • Significantly lower means that the difference is statistically significant.
  • protein or “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • biological activity is meant the functional activity of SORL1 in a standardized quantity of tissue or cells.
  • Peptide variants means polypeptides that may contain one or more substitutions, additions, deletions and/or insertions such that the therapeutic, antigenic and/or immunogenic properties of the peptides encoded by the variants are not substantially diminished, relative to the corresponding peptide.
  • modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183, 1983).
  • the antigenicity or immunogenicity of a peptide variant is not substantially diminished.
  • Variants also include what are sometimes referred to as "fragments.” Fragments also include peptides that may contain one or more amino acid substitutions, additions, deletions and/or insertions, such that the biologic activity, therapeutic, antigenic and/or immunogenic properties of the peptide variants are not substantially diminished, relative to the corresponding peptide.
  • SORL1 When SORL1 is discussed in the context of expression, activity or secretion, or therapeutic use the term includes biologically active fragments and variants thereof, and recombinant or synthesized proteins. The preferred therapeutic form of SORL1 is human recombinant SORL1.
  • the term "therapeutically effective" or “effective amount” is intended to mean an amount of a compound sufficient to substantially improve some symptom associated with a disease or a medical condition.
  • a therapeutic amount of SORL1 includes an amount that decreases amyloid beta and APPsBeta production.
  • a therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe, or recovery is accelerated in an individual.
  • nucleotide variants within two distinct regions of the sortilin-related receptor low density lipoprotein receptor class A gene are associated with both familial and sporadic Alzheimer's disease.
  • SORL1 gene the sortilin-related receptor low density lipoprotein receptor class A gene
  • Our results show that certain genetic variants in the genomic interval containing SNPs 4 or 8-12, and 16-26 (defined by the dbSNP rs numbers listed in Table 1C) were associated with Alzheimer's Disease, particularly familial Alzheimer's Disease.
  • SNPs 4 or 8-12 are clustered near the 5' end of the gene and SNPs 16-26 are clustered near the 3' end.
  • nucleotide variants at these SNP regions are further associated with a decreased expression of the SORL1 gene in patients having AD, which in turn causes an increase in amyloid beta and APPsBeta levels, as is described below.
  • SNPs For convenience we have consecutively numbered the SNPs we analyzed in the three studies described below.
  • associated with is meant here that a particular SORL1 allele, genotype or haplotype is disproportionately represented in individuals with Alzheimer's disease compared with control subjects or in individuals having a higher/larger outcome for a measureable trait compared with individuals having a lower/smaller outcome for that trait.
  • Tables IA (allele variants) and lB(haplotype variants) list high and low risk factors at specific SNPs in the SORL1 gene.
  • the haplotypes listed in Table 1B also include haplotypes that are in linkage disequilibrium with them. Where there is a high risk for a given haplotype (e.g. CGC at SNPs 8-10), the reverse is also true: there is a low risk for the opposite haplotype (e.g. GCG at SNPs 8-10).
  • Certain embodiments of the invention are directed to a method for determining if a patient is at risk of developing Alzheimer's Disease, by a. obtaining a DNA sample from the patient, b. determining if the DNA sample shows a nucleotide variant in an SNP that is a member of the group that includes SNPs rs661057 (SNP 4), rs560573, rs985421, rs593769, rs12364988, rs668387 [8-12], rs4935775, 17 rs12285364, rs2298813 [16-18], rs11600231, rs2276346, rs10502262, SORL1-T833T, rs556349, rs7131432, rs11218340 and rs10892756.
  • SNP 4 SNP 4
  • rs560573 rs985421
  • rs593769 rs12364988, r
  • [19-26] of the sortilin-related receptor low density lipoprotein receptor class A (SORL1) gene and c. if a high risk nucleotide variant is detected concluding that the patient is at high risk of developing Alzheimer's Disease, or if a low risk variant is detected concluding that the patient is at low risk of developing Alzheimer's Disease.
  • Patients at risk of developing AD can also be identified by testing to see if the patient's SORL1 levels are below normal, and concluding that the patient is at risk of developing AD if the SORL1 levels are below normal.
  • Patient samples suitable for obtaining a DNA sample include any cell or tissue, preferably fibroblasts, lymphoblasts, or epidermal cells. If a patient has a high risk genetic variant in the SORL1 gene or low SORL1 levels or both, his or her risk of developing AD is heightened. Should there be a familial incidence of AD, such a patient should be carefully monitored for early intervention.
  • another embodiment of the invention is directed to a method for confirming a diagnosis of Alzheimer's Disease in a patient having a diagnosis of probable or possible Alzheimer's Disease by determining if a DNA sample from the patient has a nucleotide variant in an SNPs 4, 8-12 or 16-26, and if a high risk nucleotide variant (Table 1Aor B) is detected, concluding that the patient has Alzheimer's Disease, or if a low risk nucleotide variant is detected concluding that the patient does not have Alzheimer's Disease, but perhaps another form of dementia.
  • a positive diagnosis of AD can also be confirmed by further testing to see if the patient's SORL1 levels are below normal, and concluding that the patient has AD if the patient has a diagnosis of probable AD and the SORL1 levels are below normal.
  • both testing for genetic variants and below normal levels of SORL1 can be done to confirm a diagnosis of probable AD. These tests can also be used to identify individuals having a mild cognitive disorder who will progress to AD.
  • a therapeutically effective amount is an amount that decreases amyloid beta in plasma, serum or csf.
  • a ⁇ neurotoxic amyloid ⁇ -peptide
  • AD Alzheimer's Disease
  • VPS35 (16q12); VPS26 (10q21); sortilin SORT1 (1p21-p13); sortilin-related VPS10 domain containing receptors 1-3 SORCS1 (10q23-q25), SORCS3 (10q23-q25), SORCS2 (4p16); and sortilin-related receptor, low density lipoprotein receptor class A repeats-containing; SORL1 (11q23-q24)].
  • SNPs single nucleotide polymorphisms
  • This replication cohort contained (i) northern European individuals from a case-control study (178 individuals with sporadic Alzheimer disease and 242 controls of self-identified Caucasian European ancestry) 20 , (ii) MIRAGE Caucasian sibships (276 Caucasian sibships from the MIRAGE Study) 23 ' 24 , (iii) MIRAGE African American sibships (238 African American sibships from the MIRAGE Study) 23 ' 24 and (iv) Israeli Arab affected individuals and controls (all 111 individuals with Alzheimer disease and 114 normal controls were from the Wadi Ara population study) 19 ' 25 .
  • Alzheimer disease was associated with the C, G and C alleles at SNPs 8, 9 and 10, respectively, in the Caribbean Hispanic FAD (P 1 ⁇ 4 0.013, 0.017 and 0.021, respectively), Israeli Arab case- control (P 1 ⁇ 4 0.002, 0.007 and 0.005, respectively) and north European case-control data sets (P 1 ⁇ 4 0.021, 0.040 and 0.067, respectively; Table 2 and Supplementary Table 4).
  • Alzheimer disease was associated with the G and T alleles at SNPs 19 and 23, respectively, in the north European FAD (P 1 ⁇ 4 0.031 and 0.0031, respectively) and north European case-control data sets (P 1 ⁇ 4 0.00082 and 0.00073, respectively; Table 2 and Supplementary Table 4).
  • Post hoc statistical adjustment for APOE genotype, age and gender did not alter the conclusions that (i) there were allelic associations between Alzheimer's disease and two clusters of SNPs in distinct regions of SORL1 in different data sets and (ii) that these associations replicated in multiple independent data sets.
  • Alzheimer disease was associated with the C, G and C alleles at SNPs 8, 9 and 10, respectively, in the Caribbean Hispanic FAD (P 1 ⁇ 4 0.013, 0.017 and 0.021, respectively), Israeli Arab case-control (P 1 ⁇ 4 0.002, 0.007 and 0.005, respectively) and north European case-control data sets (P 1 ⁇ 4 0.021, 0.040 and 0.067, respectively; Table 2 and Supplementary Table 4).
  • Alzheimer disease was associated with the G and T alleles at SNPs 19 and 23, respectively, in the north European FAD (P 1 ⁇ 4 0.031 and 0.0031, respectively) and north European case-control data sets (P 1 ⁇ 4 0.00082 and 0.00073, respectively; Table 2 and Supplementary Table 4).
  • Post hoc statistical adjustment for APOE genotype, age and gender did not alter the conclusions that (i) there were allelic associations between Alzheimer disease and two clusters of SNPs in distinct regions of SORL1 in different data sets and (ii) that these associations replicated in multiple independent data sets.
  • the CGC haplotype at SNPs 8, 9 and 10 was associated with Alzheimer disease in the Caribbean Hispanic FAD (global P 1 ⁇ 4 0.0098, haplotype P 1 ⁇ 4 0.0053, haplotype frequency estimated by FBAT 1 ⁇ 4 0.638 versus 0.583 in unrelated controls), the Israeli Arab case-control (global P 1 ⁇ 4 0.023, haplotype P 1 ⁇ 4 0.0085, frequency 1 ⁇ 4 0.661 in cases versus 0.539 in controls) and the north European case- control data set (haplotype P 1 ⁇ 4 0.045, frequency 1 ⁇ 4 0.638 in cases versus 0.566 in controls; Table 3 and Supplementary Table 5).
  • the TAT haplotype at SNPs 8, 9 and 10 was associated with decreased risk of Alzheimer's disease in these data sets (Hispanic FAD: haplotype P 1 ⁇ 4 0.0086; haplotype frequency estimated by FBAT 1 ⁇ 4 0.317 versus 0.394 in unrelated controls; Israeli Arab case-control: frequency 1 ⁇ 4 0.301 in affected individuals versus 0.434 in controls, and haplotype P 1 ⁇ 4 0.0037; north European Caucasian case control: frequency 1 ⁇ 4 0.351 in affected individuals versus 0.417 in controls, and haplotype P 1 ⁇ 4 0.068).
  • Hispanic FAD haplotype P 1 ⁇ 4 0.0086
  • haplotype frequency estimated by FBAT 1 ⁇ 4 0.317 versus 0.394 in unrelated controls Israeli Arab case-control: frequency 1 ⁇ 4 0.301 in affected individuals versus 0.434 in controls, and haplotype P 1 ⁇ 4 0.0037
  • north European Caucasian case control frequency 1 ⁇ 4 0.351 in affected individuals versus 0.417 in controls
  • both the Mayo data set and the overall Caucasian case-control data set also detected association with SNP 4 (P 1 ⁇ 4 0.009 and P 1 ⁇ 4 0.002, respectively), a result not evident in the individual data sets.
  • Risk is relative to the comparison group. In a case-control study or in families you are comparing the odds that an individual with a certain genotype is a case (affected with AD) compared to the odds that he is a control (unaffected). The ratio of the odds in large studies is roughly equivalent to risk ratio. Risk is then defined by the presence or absence of the risk factor - in this situation a particular genotype or allele or a particular haplotype.
  • Tables IA (allele variants) and Table lB(haplotype variants) list high and low risk factors at specific SNPs in the SORL1 gene.
  • the haplotypes listed in Table IB also include haplotypes that are in linkage disequilibrium with them. Where there is a high risk for a given haplotype (e.g. CGC at SNPs 8-10), the reverse is also true: there is a low risk for the opposite haplotype (e.g. GCG at SNPs 8-10).
  • Certain embodiments are directed to a method for determining if a patient is at risk of developing Alzheimer's Disease, by a. obtaining a DNA sample from the patient, b.
  • SORL1 sortilin-related receptor low density lipoprotein receptor class A
  • a high risk nucleotide variant is detected concluding that the patient is at high risk of developing Alzheimer's Disease, or if a low risk variant is detected concluding that the patient is at low risk of developing Alzheimer's Disease.
  • Patients at risk of developing AD can also be identified by testing to see if the patient's SORL1 levels are below normal, and concluding that the patient is at risk of developing AD if the SORL1 levels are below normal.
  • Patient samples suitable for obtaining a DNA sample include any cell or tissue, preferably fibroblasts, lymphoblasts, or epidermal cells. If a patient has both a high risk genetic variant in the SORL1 gene and low SORL1 levels, his or her risk of developing AD is heightened.
  • another embodiment of the invention is directed to a method for confirming a diagnosis of Alzheimer's Disease in a patient having a diagnosis of probable or possible Alzheimer's Disease by determining if a DNA sample from the patient has a nucleotide variant in an SNPs 4, 8-12 or 16-26, and if a high risk nucleotide variant (Table 1 Aor B) is detected, concluding that the patient has Alzheimer's Disease, or if a low risk nucleotide variant is detected concluding that the patient does not have Alzheimer's Disease, but perhaps another form of dementia.
  • a positive diagnosis of AD can also be confirmed by further testing to see if the patient's SORL1 levels are below normal, and concluding that the patient has AD if the patient has a diagnosis of probably AD and the SORL1 levels are below normal.
  • both testing for genetic variants and below normal levels of SORL1 can be done to determine if a patient with a probable diagnosis of AD actually has this disease.
  • SORL1 did not bind to other type 1 membrane proteins (for example, BACEl (ref. 31) and FIG. 3) or to VPS26 (which links VPS35 to the other structural elements of the retromer (ref. 30 and data not shown).
  • siRNA suppression of SORL1 expression which we speculate mimics the effects of Alzheimer disease-associated variants in SORL1, accordingly resulted in deflection of APP holoprotein away from the retromer recycling pathway and into the late endosome-lysosome pathway.
  • siRNA suppression of SORL1 led to (i) overproduction of the soluble N-terminal ectodomain of APP (APPsb) generated by BACEl cleavage of APP holoprotein (149.45% ⁇ 9.66 of control (mean ⁇ s.e.m.), P o 0.0001, n 1 ⁇ 4 5 replications; FIG.
  • association with different SNPs in different populations does not indicate a spurious result.
  • the association of disease with a single allele in all data sets is not a universal observation for either complex or monogenic diseases 17 .
  • the occurrence of pathogenic mutations across multiple domains of disease genes allelic heterogeneity
  • the absence of these variants in some data sets are frequently observed in both monogenic and complex traits such as AD 34 ' 35 .
  • the absence of significant associations in two data sets does not negate the findings from the other data sets.
  • another embodiment of the invention is directed to the therapeutic administration of SORL1 to treat or prevent AD; preferably human recombinant SORL1 is administered in a form that increases its ability of this large molecule (Molecular Weight of SORL1 is 248441 Daltons, and the Length of SORL1 is: 2214 amino acids) to cross the blood brain barrier.
  • the effective amount of SORL1 can be determined by monitoring the amount of amyloid beta in serum, plasma or cerebrospinal fluid of an AD patient or a patient at risk of developing AD; SORL1 should be administered in an amount that decreases amyloid beta.
  • Sorll can be formulated and administered to treat or prevent AD by any means that produces contact of the active ingredient with the agent's site of action in the brain of a mammal.
  • SORL1 can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. It can be administered alone, but is generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice for facilitating passage of a therapeutic agent through the blood brain barrier.
  • Treatment of a subject having AD with a therapeutically effective amount of SORL1 can include a single treatment or, preferably, can include a series of treatments. The duration of administration and amount of drug can be influenced by the amelioration of the symptoms of the disease.
  • One embodiment is directed to a method for identifying a patient having mild cognitive impairment who will progress to AD, by determining if the patient DNA has a genetic variant in the 3' or 5' end of the gene for SORL1, particularly at the SNPs we have identified. If the patient has a high risk variant, he or she will likely progress to AD and should be treated with early intervention accordingly.
  • SNP12 which is located 12.2 kb from the SNP 8-10 cluster associated with AD in multiple datasets in the primary study, was significantly associated with AD in the African-Americans and Caribbean Hispanics.
  • SNP 20 is closely flanked by both SNP 19 (-93 b.p.), which was associated with AD in several datasets in our report and by SNP 21 (+5966 b.p.), which was associated with AD in the North European case control dataset in the primary study.
  • the TGEN database had 31 SORL1 SNPs, and eight of those overlapped the 29 SNPs in the primary study. These 31 SNPs are referred to by their sequential order on the physical map in TGEN database, such as T.1, T.2, T.n, T.31. Therefore, a total of 52 unique SNPs were analyzed in these two studies (Table 1). All SNPs are in Hardy- Weinberg equilibrium in control samples. The LD structures of SNPs in the 50 and 30 regions are similar in the north European family data in the primary study and TGEN data [Lee JH, et al., Arch Neurol 2007; 64:501-506.] (FIG. 4).
  • T.17, T.19, T.20, T.21, T.26, T.27 showed nominally significant association (0.01r Po0.05) with AD under at least one model (Table 10). These six SNPs span a region of approximately 35 kb including SNPs 21-25 near the 30 end of SORL1 which were strongly associated with AD in the primary study. TGEN SNPs T.17 and T.19 are located between SNPs 20 and 21, TGEN SNPs T.20 and T.21 are between SNPs 22 and 23, and TGEN SNPs T.26 and T.27 are between SNPs 24 and 25 (Table 10).
  • Table 1Aand Table IB summarize the nucleotide variants at SNPs 8-12 and 19-26 from all 3 studies with their associated risk f or developing AD.
  • Suitable routes of administration can include oral, intestinal, parenteral, transmucosal, transdermal, intramuscular, subcutaneous, transdermal, rectal, intramedullary, intrathecal, intravenous, intraventricular, intraatrial, intraaortal, intraarterial, or intraperitoneal administration.
  • compositions of the present invention can be administered to the subject by a medical device, such as, but not limited to, catheters, balloons, implantable devices, biodegradable implants, prostheses, grafts, sutures, patches, shunts, or stents.
  • a medical device such as, but not limited to, catheters, balloons, implantable devices, biodegradable implants, prostheses, grafts, sutures, patches, shunts, or stents.
  • the dosage administered will be a therapeutically effective amount of the compound sufficient to result in amelioration of symptoms of the AD and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the formulation of SORL1, its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
  • Treatment of a subject having AD with a therapeutically effective amount of SORL1 can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with enough SORL1 to cause plasma or csf levels of amyloid beta to go down. This can be monitored using methods known in the art.
  • the duration of administration and amount of drug can be influenced by the amelioration of the symptoms of the disease.
  • 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 ED50 (the dose therapeutically effective in 50% of the population). Since SORL1 is a naturally-occurring compound toxicity should be low. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds 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 pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, eth
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain preferably a water soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.
  • the compounds may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example as an emulsion in acceptable oil
  • ion exchange resins for example as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • standard pharmaceutical methods can be employed to control the duration of action.
  • controlled-release preparations can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate.
  • concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release.
  • the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.
  • Injectable A parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic with sodium chloride and sterilized.
  • Suspension An aqueous suspension is prepared for oral administration so that each 5 millimeters contain 100 milligrams of finely divided active ingredient, 200 milligrams of sodium carboxymethyl cellulose, 5 milligrams of sodium benzoate, 1.0 grams of sorbitol solution U. S. P. and 0.025 millimeters of vanillin.
  • the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in an animal body to achieve a particular effect (see, e.g., Rosenfeld et al, 1991, supra; Rosenfeld et al, 1991, Clin. Res., 39(2), 31 IA; Jaffe et al., supra; and Berkner, supra).
  • Rosenfeld et al, 1991, supra Rosenfeld et al, 1991, Clin. Res., 39(2), 31 IA
  • Jaffe et al., supra and Berkner, supra.
  • Local or systemic delivery can be accomplished by administration including application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral or intracranial introduction, and intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • a composition of the present invention can also be formulated as a sustained and/or timed release formulation.
  • sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference.
  • sustained release refers to release of a therapeutic or prophylactic amount of a drug or an active metabolite thereof over a period of time that is longer than a conventional formulation of the drug.
  • sustained release typically means release of the drug within the gastrointestinal tract lumen over a period of from about 2 to about 30 hours, more typically over a period of about 4 to about 24 hours.
  • Sustained release formulations achieve therapeutically effective concentrations of the drug in the systemic blood circulation over a prolonged period of time relative to that achieved by oral administration of a conventional formulation of the drug.
  • Dellayed release refers to release of the drug or an active metabolite thereof into the gastrointestinal lumen after a delay time period, typically a delay of about 1 to about 12 hours, relative to that achieved by oral administration of a conventional formulation of the drug.
  • compositions of the present invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microp articles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions of the invention.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.
  • SORL1 is a very large molecule
  • preferred embodiments are directed to pharmaceutical formulations that optimize delivery of the drug to the brain.
  • SORL1 is derivatized to enhance BBB penetration by formation of a reversible linkage with one or more suitable groups so as to yield "pro-drugs", i.e. chemical derivatives that, after having passed through the blood-brain barrier, are converted (back) to the original compound itself inside the patient's brain.
  • Pro-drugs i.e. chemical derivatives that, after having passed through the blood-brain barrier, are converted (back) to the original compound itself inside the patient's brain.
  • Liberation of the parent compound may be by chemical hydrolysis or enzymatic attack.
  • a derivative or pro-drug has an "enhanced blood-brain barrier permeability "according to the present invention or an “enhanced blood-brain barrier penetration” if, after administration of a pro-drug or derivative thereof to a living organism, a higher amount of the compound penetrates through the BBB, resulting in a higher level of effective agent in the brain, as compared to administration of the base compound without derivatization.
  • Known derivatives that facilitate penetration across the BBB include quaternary ammonium salts with a labile nitrogen-carbon bond at R5; mono- or diacyl derivatives (esters) of the hydroxyl groups of the base compounds (R1, R2); sugar derivatives, preferably glucuronides (R1, R2); derivatives coupled with nicotinic acid (R1, R2); and selected halogenides (R3).
  • Another derivative that increases BBB penetration is a lipophilic dihydropyridinium carrier. This Redox Chemical Delivery System (RCDS; Misra A. et al. 2003 J Pharm Pharmaceut Sci 6:252-273) is known to significantly enhance drug delivery through the BBB into the brain parenchyma.
  • the dihydropyridinium moiety is enzymatically oxidized to the corresponding ionic pyridinium salt. Subsequent cleavage of the original compound from the carrier leads to liberation of the original compound and to sustained levels of it in the brain tissue.
  • the derivatives obtained by chemical modification do not need to work as medicaments but rather may initially be prodrugs that, after penetration though the blood-brain barrier, are converted (e.g., by brain enzymes) to the parent compound or a metabolite thereof and work as such as a medicament.
  • the BBB has specific receptors that allow the transport from the blood to the brain of several macromolecules.
  • transporters include those that transport insulin, transferrin, insulin-like growth factors 1 and 2 (IGFl and IGF2), leptin, and lipoproteins.
  • IGFl and IGF2 insulin-like growth factors 1 and 2
  • lipoproteins include those that transport insulin, transferrin, insulin-like growth factors 1 and 2 (IGFl and IGF2), leptin, and lipoproteins.
  • One noninvasive approach for the delivery of drugs to the CNS is to attach the agent (SORL1 or a fragment, variant or derivative of it) of interest to a molecule that binds with receptors on the BBB. The molecule then serves as a vector for transporting the agent across the BBB.
  • MTH molecular Trojan horses
  • a MTH is an exogenous peptide or peptidomimetic moiety (e.g., a MAb to a transport receptor) capable of binding to an endogenous BBB receptor mediated transport system that traverses the BBB on the endogenous BBB receptor-mediated transport system.
  • AngioPep-1 SEQ ID NO.:67
  • aprotinin SEQ ID NO. 98
  • peptides having similar domains as aprotinine and Angiopep-1 and a modified form of Angiopep-1 are also used as potential carrier vectors. These derived peptides resemble aprotinine and Angiopep-1 but comprise different amino acid insertions and bear different charges. US application 20060189515.
  • compositions comprising micro particles having an average diameter ranging from 40 to 150 nm, consisting of one or more lipids, a drug and, optionally, a steric stabilizer have also been used successfully to transport large molecules across the blood-brain barrier.
  • Therapeutic SORL1 includes any biologically active fragment, epitope, modifications, derivatives or variants thereof.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of SORL1.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • Variants of SORL1 include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or other molecule that facilitates transport through the BBB . (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, or leader or secretary sequence, or a sequence facilitating purification.
  • additional amino acids such as an IgG Fc fusion region peptide, or leader or secretary sequence, or a sequence facilitating purification.
  • polypeptide variants are deemed to be within the scope of those skilled in the art from the teachings herein.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity; see, e.g. Pinckard, Clin. Exp. Immunol. 2 (1967), 331-340: Bobbins, Diabetes 36 (1987), 838-845; Cleland. Crit. Rev. Therapeutic Drug Carrier Systems 10 (1993), 307-377.
  • amino acid residue refers to an amino acid which is part of a polypeptide.
  • the amino acid residues described herein are preferably in the L" isomeric form. However, residues in the D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide.
  • amino acid residue is broadly defined to include the 20 amino acids commonly found in natural proteins, as well as modified and unusual amino acids, such as those referred to in 37 C.F.R.
  • substitutions are preferably made as follows: Original residue Conservative substitution Ala (A) GIy; Ser Arg (R) Lys Asn (N) GIn; His Cys (C) Ser GIn (Q) Asn GIu (E) Asp GIy (G) Ala; Pro His (H) Asn; GIn He (I) Leu; VaI Leu (L) Ile; VaI Lys (K) Arg; GIn; GIu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe VaI (V) He; Leu.
  • N-terminal amino group can be accomplished using a hydrophilic compound, such as hydroorotic acid or the like, or by reaction with a suitable isocyanate, such as methylisocyanate or isopropylisocyanate, to create a urea moiety at the N-terminus.
  • a hydrophilic compound such as hydroorotic acid or the like
  • a suitable isocyanate such as methylisocyanate or isopropylisocyanate
  • Other agents can also be N-terminally linked that will increase the duration of action of the SRIF analog as known in this art.
  • Reductive amination is the process by which ammonia is condensed with aldehydes or ketones to form imines which are subsequently reduced to amines.
  • reductive amination is a potentially useful method for conjugation to PEG.
  • Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities.
  • reductive amination is a potentially useful method for conjugation to PEG. Bentley et al, J Pharm ScL 1998 Nov;87( 11): 1446-9.
  • polypeptides are not always entirely linear.
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non- translational natural processes and by synthetic methods.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides. For instance, the amino-terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N- formy lmethionine .
  • the modifications can be a function of how the protein is made.
  • the modifications will be determined by the host cell posttranslational modification capacity and the modification signals in the polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells, and, for this reason, insect cell expression systems have been developed to efficiently express mammalian proteins having native patterns of glycosylation. Similar considerations apply to other modifications.
  • the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.
  • the north European case-control set is drawn from the same populations as the north European FAD data set 20 ' 22 .
  • the three Mayo data sets were drawn from Caucasian affected individuals and controls assessed in clinical series at the Rochester and Jacksonville Mayo Clinics or from Caucasian brains in which the presence or absence of Alzheimer disease was determined neuropathologically by autopsy ('AUT' in Supplementary Table 1).
  • Genotyping was performed using the GenomeLab SNP stream System®, and primer sets were as in Supplementary Table 3 (Beckman Coulter). We genotyped 100 DNA samples twice for every SNP marker (the concordance rate was499%). APOE was genotyped as described5. Genotyping of the Mayo samples was performed on an ABI 7900 instrument using TaqMan chemistry with primers and probes designed by Applied Biosystems. The entire ORF of the SORL1 gene was sequenced in 12 individuals with sporadic Alzheimer disease, 12 individuals with familial Alzheimer disease and two normal controls selected from the north European and Caribbean Hispanic data sets (Supplementary Tables 3 and 10).
  • spliced transcripts were sought by conventional RT-PCR in eight overlapping fragments using total RNA isolated from frontal cortex (16 normal controls and 17 individuals with sporadic Alzheimer disease from the Canadian Brain Tissue Bank and the New York Brain Bank; Supplementary Table 3).
  • SNP marker data were assessed for deviations from Hardy- Weinberg equilibrium (using Pedstats software) and for Mendelian inheritance errors (using Pedcheck software).
  • Single-point family-based association was assessed with FBAT vl.5.5 (ref. 36), using an additive genetic model with the null hypothesis of no linkage and no association. Allele frequencies were estimated by FBAT using the EM algorithm.
  • APOE e4 carrier status was included in the analyses using PBAT v2.6 (refs.
  • the w2 test (or the Fisher's exact test) was used to assess genotypic and allelic associations between Alzheimer disease. Multivariate logistic regression analysis was performed to adjust for APOE e4, sex and age-at-onset or age-at-examination.
  • Haplotype analyses were carried out with a sliding window of three contiguous SNPs using FBAT for family data and Haplo. stats v 1.1.1 for case-control data 16 ' 26 ' 42-44 . The analyses were repeated using sliding windows of two, four, five and six SNPs. Expression plasmids and cDNA constructs for human SORL1. The cDNA clones encoding APP K670N/M671L Swedish mutation (APPSwe) and BACE1 (V5-tagged at the C terminus) were as described previously 45 ' 46 .
  • RNA interference siRNA oligonucleotides were designed using the online siRNA Design Tool ® (Dharmacon Research). The siRNAs for SORL1 are in Supplementary Table 3. The siCONTROL Non-Targeting siRNAs #1 and #2 (Dharmacon Research) were used as a negative control.
  • PCR primer pairs targeting SORL1 exon 23 were as in Supplementary Table 3.
  • Total RNA (5 mg) was reverse transcribed using a random hexamer.
  • Real-time PCR was performed in a 384-well format using an ABI Prism 7900HT instrument and the Sybr Green detection method. Samples were analyzed in triplicate, and mean expression levels corresponding to SORL1 mRNA expression were normalized to b-actin mRNA levels.
  • Frozen brain tissue was obtained from 103 autopsy confirmed cases of AD, and from 17 elderly controls with a normal postmortem examination, and without a history of dementia or another neurological disorder. .
  • the average age of onset for the patients was 80.5 and 52.4% were women.
  • the mean age of the combined group of controls was 79.7 years and 48.4% were women.
  • the Institutional Review Boards of Columbia University Medical Center and the New York Psychiatric Institute approved recruitment, informed consent and study procedures.
  • Genotyping was performed using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (Sequenom). Detailed information on genotyping was previously described (1), and is available upon request.
  • the numbering system for the SNPs 1 to 29 reflects their relative order on the physical map of SORL1, and was the same system used in the primary study. We restricted the work to include only 12 of the previously genotyped SNPs to focus on 5' (SNPs 1, 2 and 7-10) and 3' (SNPs 13, 17 and 22-25) regions highlighted in the previous report.
  • SNP marker data were assessed for deviations from Hardy- Weinberg equilibrium using the HAPLOVIEW program ( Barrett JC, Fry B, Mailer J, Daly MJ.
  • Haploview analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-265.), and none deviated.
  • the ⁇ 2 test (or the Fisher's exact test) was used to for analysis of genotypic and allelic associations between AD and each of the SNP markers.
  • the HAPLOVIEW program was used to perform single point analysis, estimates of linkage disequilibrium (LD) structure and haplotype blocks. Haplotype analyses were performed with HAPLO. STATS vl.1.1 for case-control data using the same sliding window of three contiguous SNPs as described in the primary study.
  • the diagnosis of dementia was established at a consensus conference that included neurologists, neuropsychologists and psychiatrists and based on all available information gathered from the initial and follow-up assessments and medical records. The diagnosis was based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease Related Disorders Association (N1NCDS-AD RD A) criteria for probable AD. DSM-III and IV American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. Third Edition, revised ed: Washington, DC: American Psychiatric Association; 1987; McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM.
  • Genotyping was performed using the GenomeLab SNP stream System and primer sets were as described in Example 1. 100 DNA samples were genotyped twice for every SNP marker (concordance rate >99%). APOE was genotyped as previously described 5 . We numbered the SNPs 1 to 29 reflecting there relative order on the physical map of SORL1, and is the same nomenclature used in (Table 2).
  • Haplotype analyses were performed with HAPLO. STATS vl .1.1 for case-control data using the same sliding window of three contiguous SNPs as described in our previous publication. We designed this study to confirm the primary study. Under these circumstances, a nominal p-value of 0.05 is widely considered to be sufficient for confirmation. Ott J. Analysis of human genetic linkage. Baltimore, Johns Hopkins University Press; 1999. Consequently, nominal p-values are presented in Table 2 for single point analysis. However, to minimize the risk of a false positive finding from rare haplotypes, we computed empirical p-values by generating the null distribution based on 10,000 replicates of the haplotype analyses.
  • Marker genotype distributions in cases and controls were compared in several ways: (i) a genotypic test with two degrees of freedom, models assuming (ii) dominant and (iii) recessive inheritance, and (iv) the Cochran-Armitage trend test.
  • A1 Minor allele name (based on whole sample); F A: Frequency of this allele in cases; F U" Frequency of this allele in controls; A2" Major allele name. CHISQ" Chi- square test of allele; OR" estimated Odds ratio of Allele A1 ; Allele.P” p-value for allelic association; Geno.P” p-value for genotypic (2 df) test; DOM.P” p-value for genotypic (1 df) test with dominant model; RECP” p-value for genotypic (1 df) test with recessive model; TREND.P” Cochran-Armitage trend test.
  • A Genotype and allele frequencies (or raw genotype counts) of SORL1 single nucleotide polymorphisms (SNPs) for all nine datasets analyzed. Sibs - siblings.
  • F Linkage disequilibrium (LD) block structure around SORL1 estimated using the Haploview software.
  • the markers generally show weak evidence of inter-marker LD. However, there are several haplotype blocks.
  • Block 1 contains SNPs land 2 (1 kb); Block 2 contains SNPs 6 to 10 (20 kb); Block 3 contains SNPs 19 and 20 (0 kb); Block 4 contains SNPs 23 to 25 (7 kb); and Block 5 contains SNPs 28 and 29 (0 kb). Similar results were observed in all other datasets (A, B, C) and non-AD subjects used in the HapMap project.
  • siRNAs for SORL1 are:
  • siRNAs for VPS26 are:

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Abstract

La présente invention concerne des procédés permettant de déterminer si un patient présente un risque de développer la maladie d'Alzheimer en analysant l'ADN du patient et en déterminant si des variants nucléotidiques se situent dans la région du gène pour SORL1 au niveau des sites SNP 4 ou 8-12 et 19-26.
PCT/US2007/082308 2006-10-23 2007-10-23 Récepteur associé à la sortiline sorl1 présentant un lien fonctionnel et génétique avec la maladie d'alzheimer WO2008052016A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10308718B2 (en) 2015-04-07 2019-06-04 Alector Llc Anti-sortilin antibodies and methods of use thereof
US10849992B1 (en) 2015-04-07 2020-12-01 Alector Llc Methods of screening for sortilin binding antagonists
US11396546B2 (en) 2018-07-13 2022-07-26 Alector Llc Anti-Sortilin antibodies and methods of use thereof
WO2023275376A1 (fr) * 2021-07-02 2023-01-05 Aarhus Universitet Méthodes de traitement de la maladie d'alzheimer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050106573A1 (en) * 2003-11-13 2005-05-19 Council Of Scientific And Industrial Research Method of detection of predisposition to high altitude pulmonary edema (HAPE)
US20050177881A1 (en) * 2001-12-04 2005-08-11 Andreas Papassotiropoulos Methods of identifying genetic risk for and evaluating treatment of alzheimer's disease by determining single nucleotide polymorphisms
US20060134664A1 (en) * 2004-11-03 2006-06-22 Scherzer Clemens R Identification of dysregulated genes in patients with neurological diseases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050177881A1 (en) * 2001-12-04 2005-08-11 Andreas Papassotiropoulos Methods of identifying genetic risk for and evaluating treatment of alzheimer's disease by determining single nucleotide polymorphisms
US20050106573A1 (en) * 2003-11-13 2005-05-19 Council Of Scientific And Industrial Research Method of detection of predisposition to high altitude pulmonary edema (HAPE)
US20060134664A1 (en) * 2004-11-03 2006-06-22 Scherzer Clemens R Identification of dysregulated genes in patients with neurological diseases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 25 May 2006 Database accession no. (rs560573) *
DATABASE GENBANK [Online] 25 May 2006 Database accession no. (rs593769) *
DATABASE GENBANK [Online] 25 May 2006 Database accession no. (rs985421) *
ROGAEVA ET AL.: 'The Neuronal Sortilin-Related Receptor SORL1 is Genetically Associated with Alzheimer Disease' NATURE GENETICS vol. 39, no. 2, February 2007, pages 168 - 177 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10308718B2 (en) 2015-04-07 2019-06-04 Alector Llc Anti-sortilin antibodies and methods of use thereof
US10428150B2 (en) 2015-04-07 2019-10-01 Alector Llc Anti-sortilin antibodies and methods of use thereof
US10849992B1 (en) 2015-04-07 2020-12-01 Alector Llc Methods of screening for sortilin binding antagonists
US11186645B2 (en) 2015-04-07 2021-11-30 Alector Llc Isolated nucleic acids encoding anti-sortilin antibodies
US11208488B2 (en) 2015-04-07 2021-12-28 Alector Llc Methods of increasing progranulin levels using anti-Sortilin antibodies
US11339223B2 (en) 2015-04-07 2022-05-24 Alector Llc Methods of use of anti-Sortilin antibodies for treating a disease, disorder, or injury
US11396546B2 (en) 2018-07-13 2022-07-26 Alector Llc Anti-Sortilin antibodies and methods of use thereof
WO2023275376A1 (fr) * 2021-07-02 2023-01-05 Aarhus Universitet Méthodes de traitement de la maladie d'alzheimer

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