WO2006105602A1

WO2006105602A1 - Animal models and cells with a modified gene encoding transthyretin-related protein and applications thereof

Info

Publication number: WO2006105602A1
Application number: PCT/AU2006/000456
Authority: WO
Inventors: William S. Stevenson; Andrew Roberts; Douglas J. Hilton; Warren S. Alexander; Andrienne A. Hilton
Original assignee: The Walter And Eliza Hall Institute Of Medical Research
Priority date: 2005-04-06
Filing date: 2006-04-06
Publication date: 2006-10-12

Abstract

The presenting invention discloses, inter alia, modified cells or non-human animals comprising them having a modified TRP gene, genetic constructs derived therefrom and methods for screening for agents useful in the treatment of cancer, particularly liver cancer, and thrombocytoses.

Description

Animal models and cells with a modified gene encoding transthyretin-related protein and applications thereof

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to compositions comprising agents that modulate cellular activity and in particular agents that modulate cancer (tumor) development and the development of haemopoietic lineages such as platelet production. The present invention also provides animal models, cellular models and agents, drug targets, and methods for screening for and testing agents useful in the modulation of cellular activity and treatment and prevention of cancer.

DESCRIPTION OF THE PRIOR ART Bibliographic details of references in the subject specification are also listed at the end of the specification.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Advances in our knowledge relating to the functions and synthesis of biopolymers such a nucleic acids, proteins and polysaccharides are facilitating the development of new therapeutic and diagnostic agents. Of particular interest are those agents, which regulate cellular activities such as proliferation and differentiation.

Cancer is one widespread example of a disease or condition which is associated with uncontrolled cellular proliferation. The most widely applied treatment for primary and metastatic cancer is a combination of surgery, radiotherapy and chemotherapy. Some cancers have a viral aetiology, for example, hepatitis B and C are causal agents in liver cancer. Chemotherapy affects rapidly dividing cells and a frequent side effect of chemotherapy is thrombocytopenia (low platelet numbers) due to destruction of cells including megakaryocytes and their progenitors in the bone marrow. Platelets are required for blood clotting and haemostasis. Thrombocytopenia may also occur as an inherited disease, as a result of autoimmune disease or viral infection.

The steady state platelet count in humans is predominantly genetically determined (Buckley M. F. et al, Thromb. Haemost. ,§3:480-484, 2000). However, the genes that are important in platelet production, release, circulation and clearance which collectively determine inter-individual variation in platelet counts are largely unknown.

Thrombopoietin (TPO) is the principal growth factor that regulates steady state platelet production via the stimulation of megakaryocyte and megakaryocyte progenitor proliferation and differentiation through the cellular receptor c-Mpl (Bartley T. D. et al, Cell 77:1117-1124, 1994; de Sauvage F. J. et al., Nature 3(59:533-538, 1994; Kaushansky K. et al., Nature 3(59:568-571 1994; Lok S. et al., Nature 369:565-568, 1994). The primary role of this hemopoietic cytokine is well illustrated in the Tpo and c-Mpl knock-out mice (Tpo^";' and MpI^''^" respectively) that are phenotypically identical and display profound thrombocytopenia (de Sauvage F. et al, J. Exp. Med. 183:651-656, 1996; Gurney A. L. et al., Science 2(55:1445- 1447, 1994). Thrombopoietin is produced by a number of organs but the most important physiological sources are the liver and kidneys. TPO transcription appears to be constant, and the level of the cytokine in the body is thought to be regulated by the rate of receptor-mediated uptake and degradation by c-Mpl-expressing platelets and megakaryocytes (Stoffel R. et al., Blood §7:567-573, 1996; Fielder P. J. et al, Blood 57:2154-2161, 1996). In thrombocytopenic states, the decrease in c-Mpl-mediated TPO uptake by platelets results in an increased concentration of TPO available to the bone marrow to drive accelerated (or emergency) thrombopoiesis. However, there is also some evidence that other physiological mechanisms may be important in TPO regulation. For example, thrombopoietin has been demonstrated to be normal or elevated in reactive thrombocytosis in some studies (Cerutti A. et al, Br. J. Haematol. 99:281-284, 1997) and there is evidence that TPO production may be differentially regulated in other sites such as the bone marrow stroma (Sungaran R. et al, Blood 59:101-107, 1997).

The need remains for agents which may be used therapeutically or as a prophylactic to regulate cellular activity or diagnostically to monitor cellular status. Animal models are useful for investigating the precise nature of molecular interactions which determine cellular proliferation or cellular differentiation, and for testing the efficacy of drugs or drug targets. SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. Genes and other genetic material (eg mRNA, constructs etc) are represented in italics and their proteinaceous products are represented in non-italicised form. Thus, TRP polypeptide is the product of the TRP gene. The term "TRP" or "TRP" is used to encompass all homologs, including orthologs and paralogs and variants in any species including, unless otherwise stated, TRP-PLT2. In some embodiments, the invention includes a human TRP homolog. Accordingly, homologous animal including avian and fish TRP (and TRP-PLT2 forms and their products are encompassed in the terms TRP and TRP. Mammalian TRP polypeptide is preferred.

The transthyretin (TTR) and transthyretin related protein (TRP) families have been reviewed, for example, by Eneqvist T. et al, in Eur. J. Biochem. 270:518-532, 2003. The present invention describes a further form of TRP and variants thereof not previously recognised, which comprises sequence encoded by an additional 5' exon. The TTR family and TRP family members share approximately 35 % amino acid sequence identity and both families are characterized by a set of conserved amino acid residues as shown in Figure 10. TRP family members share at least 30% to 95% sequence identity and are generally distinguished from TTRs by the presence of a C-terminal YRGS motif. TTR family members are homotetrameric transport proteins which bind to and transport thyroxine and retinol- binding protein in the plasma. TTR is associated in man with amyloidosis which is a group of conditions characterized by amlyoid deposits in one or more tissues or organs of the body. The structures of TRPs from various mammalian, amphibian, fish, plant, bacteria, parasitic, fungal and mycobacterial species have been described (see Figure 10) The function and ligands of mammalian TRPs have not previously been elucidated and the present finding that a mammalian TRP/TRP is a tumor suppressor notably in the liver and modulates TPO-mediated cellular differentiation pathways provides new therapeutic and diagnostic applications inter alia for TRP and TRP, and variants, mimetics, analogues, binding partners, receptors, ligands, agonists and antagonists thereof.

In some embodiments, the present invention pertains to the identification of a role for

TRP or TRP in modulating TPO activity. Specifically, a mutation in TRP leads to up regulation of TPO-dependent pathways, particularly in the liver or other tissues where

TPO/Mpl function to regulate cellular activity. In other embodiments, the present invention identifies and pertains to TRP as a tumor suppressor gene, notably in the liver.

In one aspect, genetically modified cells or non-human organisms comprising such cells are also provided by the present invention. In one embodiment, the cells comprise genetically modified TRP or produce modified TRP. Such cells and animals are useful in in vivo or in vitro cellular model systems to identify and isolate, inter alia, modulators of TRP or TRP. Such cells are also useful in cell therapy, including transplantation.

Genetically modified non-human organisms may be provided in the form of embryos for transplantation. Embryos are preferably maintained in a frozen state and may optionally be sold with instructions for use. Targeting constructs and genetically modified cells are also preferably maintained in a frozen state and may optionally be sold with instructions for use. All such cells are referred to herein as an in vivo or in vitro cellular model system.

In accordance with the present invention, regulation of the level or activity of TRP and TRP will be important in modulating cellular activities such as the development of cancer or the development of haematopoietic lineages, such as platelet development. In some embodiments, therefore, the present invention provides a method for modulating cellular activity in a cell, tissue or subject comprising administering an agent which modulates the level or activity of TRP or TRP. In some embodiments, cellular activity is cancer development while in other embodiments, cellular activity is megakaryocyte differentiation or megakaryocyte progenitor proliferation or development and platelet production. In some embodiments, modulation is up regulation of the level or activity of TRP or TRP. In certain embodiments, down regulation of the level or activity of TRP or TRP will be undertaken.

In another embodiment, the present invention provides compositions comprising agents, which modulate the level or activity of TRP or TRP. Such agents are useful in modulating cellular activity, such as cell proliferation and differentiation. In an illustrative embodiment, down regulation of the level or activity of TRP causes megakaryocyte and megakaryocyte progenitor differentiation and platelet production. Conversely, agents which up regulate the level of TRP or TRP are proposed for lowering platelet levels or production in a subject, as required.

In some embodiments, agents which modulate the level or activity of TRP or TRP comprise TRP or TRP or variants, derivatives, mimetics and analogs thereof. Thus, in some embodiments the present invention contemplates administering TRP polypeptide or an agent from which TRP polypeptide is producible. In other embodiments, the agents are ligands, receptors, regulatory molecules and other binding partners, agonists or antagonists and variants, derivatives, mimetics and analogs thereof. Such agents are identified inter alia through screening assays which are routinely performed by the skilled artisan using all or part of TRP or TRP. In some embodiments, the agents are used in the manufacture of medicaments for the treatment or prevention of cancer. In other embodiments, medicaments are suitable for the regulation of haematopoiesis. The agents may be used in conjunction with other cancer treatments to enhance their efficacy or reduce side-effects.

In other embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding all or a part of a TRP-PLT2 (also referred to as the long form of TRP) polypeptide having an amino acid sequence substantially as set out in SEQ ID NO: 4 (Figure 9) or a sequence of amino acids having at least 60% sequence identity thereto. In some embodiments, the sequence of amino acids has at least 60% similarity to about 20 to 30 contiguous amino acids at the N-terminal end of the polypeptide. In another embodiment, the invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set out in SEQ ID NO: 3 or its complement or which has about 60% sequence identity to all or a part thereof or which hybridises thereto under conditions of low or medium stringency. In another embodiment, the nucleotide sequence has at least about 60% sequence identity in the 5' end portion having about 60 to 100 contiguous nucleotides and/or hybridises to this 5' portion under conditions of medium or high stringency.

Methods of risk assessment for cancer are contemplated comprising screening for mutations in TRP or TRP. Any form of cancer is contemplated, although in one embodiment cancer in tissues such as the liver are particularly contemplated. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a representation of data showing that plt2/plt2 animals display thrombocytosis and hepatomegaly. (A) Mice homozygous for the plt2 mutation display thrombocytosis. (B) Liver weight expressed as a proportion of total weight is increased in mice homozygous for the plt2 mutation. (C) The F2 generation produced by intercrossing plt2/plt2 and MpI^'1' animals produced a wide range of platelet counts that was bimodal in distribution. The platelet counts of wild-type mice (WT), mice with the c-Mpl knock-out allele (MpI) and mice inferred to be homozygous for the plt2 mutation based on liver size are included for comparison. (D) The platelet phenotype of tht plt2/plt2 MpI^'1' compound mutant was identical to MpI^'1" mice (E) Gating on the megakaryocyte population in the bone marrow (rectangular gate) allowed the distribution of ploidy to be displayed in a frequency histogram for wild-type (P) and plt2/plt2 (■) mice. Megakaryocyte numbers in the bone marrow were quantitated with beads (elliptical gate).

Figure 2 is a representation of data showing that the pH2 Mutation acts extrinsically on the hemopoietic system. (A) 8 week post-transplantation platelet counts from 9 wild-type and 9 plt2/plt2 recipient mice after they received bone marrow from either a plt2/plt2 (M) or wild- type donor (L^" S). (B) The high purity of the megakaryocyte suspension after purification is illustrated in this photomicrograph of a cytocentrifuge preparation stained with acetylcholinesterase and then counterstained with hematoxylin. The cells were visualized with an Axioplan2 microscope (Zeiss, Jena Germany) and the image captured with an Axiocam camera with Axiovision3.1 software (Zeiss) under 2Ox original magnification. (C) Southern Blot of amplified megakaryocyte DNA probed with a marker for the Y chromosome (Sry) and Pdgfr from an autosome. A female (F) and male (M) bone marrow recipient demonstrate the presence of both Sry and Pdgfr alleles. A control female mouse that did not receive a marrow transplant (J) has no evidence of the Sry allele and a semi-quantitative control of 1 part male megakaryocyte suspension mixed with 9 parts female suspension (m/f) demonstrates the presence of a signal at the Siγ allele.

Figure 3 is a representation of data showing that thrombopoietin is present in excess in plt2/plt2 mice. (A) Serum Thrombopoietin ELISA in wild-type C57BL/6 mice (n=15), plt2/plt2 mice (n=18) and wild-type C57BL/6 mice with rebound thrombocytosis during recovery from 5FU cytotoxicity (i3;n=6). (B) Tpo transcript expression as measured by quantitative real-time PCR of reverse-transcribed RNA extracted from the liver of 4 wild-type mice and 4 plt2/plt2 animals. The relative Tpo expression is presented as fold change in gene expression normalised to two separate housekeeping genes (Hmbs and Polr2a) relative to one of the control liver samples. (C) Quantitative real-time PCR of Tpo transcript in a panel of tissues normalised to Polr2a and relative to a control kidney sample for 3 wild-type mice (w) and 3 plt2/plt2 mice (p). (D) Thrombopoietin content of liver lysates was measured by ELISA for 8 wild-type mice and 8 plt2/plt2 mice.

Figure 4 is a representation of data showing that the plt2 locus lies between D7Wehi28 and D7Mit46 on mouse chromosome 7. (A) Genetic linkage for thrombocytosis was observed at D7Mitl89 after a genome wide scan was performed on 89 N2 backcross mice with 162 SSLP markers. (B) The presence of a homozygous C57BL/6 allele at D7Mit46 separates the thrombocytosis phenotype from the range of platelet counts displayed by the 282 N2 backcross mice. (C) SSLP markers telomeric to D7Mit71 were homozygous C57BL/6 (■) in the 50 backcross mice with the highest platelet counts (>2217xlO⁹/L) and heterozygous for the C57BL/6 and the Balb/c allele (LJ) in the 50 mice with the lowest platelet counts (<1272xlO⁹/L). 15 intercross mice with high (>2194xlO⁹/L) or low platelet counts (<1296xlO⁹/L) were observed to have a recombination event between D7Wehi28 and D7Mit46. Progeny testing was performed on 2 mice that defined the telomeric end and one mouse that defined the centromeric end of the interval in the low platelet group (*).

Figure 5 is a graphical representation of data showing that homozygous plt2/plt2 mice develop liver tumors (hepatomas) at increased frequency after natural aging or after radiation, compared to wild-type mice (n=9 in each group).

Figure 6 is a graphical representation of nucleotide sequencing data showing the A to G mutation in the genetic region encoding TRP and causing a tyrosine to cysteine modification in plt2 animals. For sequencing, total cellular RNA was isolated from liver samples from a wild-type and plt2/plt2 animal after they were snap-frozen into liquid nitrogen and homogenised in TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was then purified using the RNeasy kit (Qiagen GmbH, Germany) according to the manufacturer's protocol. First strand cDNA synthesis was performed using Superscript II Reverse Transcriptase (Invitrogen). The cDNA was the used in a 35 cycle PCR reaction using a PFU polymerase (Promega) and primers specific for the predicted short and long versions of the AK00470 (TRP) gene (short 5'- acggactggctgatcactct-3', 5'- caaagcccatgatttgtgtg-3' and long 5'- tgcacagaccagagcttcag-3', 5'- caggcagatagatggctttctt-3'). lμL of the PCR product was then used in a second PCR reaction with nested primers (short 5'- agctggcgcgccaggctaccgagagcagtccc-3', 5'- agctacgcgtactcccccggtaggtggtg-3' and long 5'- agctggcgcgccagagttccaggaccgccccg-3', 5'- agctacgcgtactcccccggtaggtggtg-3') and the product of this second reaction sequenced in a reaction using Big Dye Terminator and analysed on an Applied Biosystems automatic sequencer. Figure 7 is a graphical representation of data showing allelic discrimination between plt2 homozygous, heterozygous and wild type genotypes. Genomic DNA was prepared from a tail biopsy taken from experimental mice at approximately 3 weeks of age. DNA was amplified using primers specific for exon 2 of AK004470 (77??) (5'- GGCACCTATAAGCTGTTCTTCGA-3' and 5'-ACCCTGACACTCACCTCTACATAG-S'). The PCR product was then identified as mutant or wild type by measuring specific fluorescence associated with the mutant or wild-type fluorescent-tagged oligonucleotide probe (wild-type: VIC- CAGAGCGCTACTGGAAA, plt2 mutant: FAM- AGCGCTGCTGGAAA). The PCR reaction and alleleic discrimination detection were performed on an ABI Prism 7900HT Sequence Detection System. Figure 8 is a diagrammatic representation of the exon structure of the gene affected by the plt2 mutation.

Figure 9 is a representation of data showing the nucleotide and predicted amino acid sequence of TRP family members identified herein, (a) the nucleotide sequence of short wild- type form of mouse TRP as set forth in SEQ ID NO:1. (b) the nucleotide sequence of the long wild-type form of mouse TRP as set forth in SEQ ID NO.3. (c) the nucleotide sequence (cDNA) NCBI Accession No. AK00447 as set forth in SEQ ID NO: 5. (d) amino acid sequence of short wild-type mouse TRP protein as set forth in SEQ ID NO: 2. (e) amino acid sequence of long wild-type mouse TRP protein as set forth in SEQ ID NO: 4.

Figure 10 is a multiple sequence alignment TRP and TTR family members extracted from Figure 1 of Eneqvist T. et al, 2003 {supra) incorporated herein in its entirety. Amino acid sequences of TTR-related proteins from 47 species aligned and compared with TTR sequences from 20 species (reviewed by Eneqvist T. et al., Amyloid: Int. J. Exp CHn. Invest. 5:149-168, 2001). Similarity was defined as amino acid substitutions within one of the following groups: FYW, IVLM, RK, DE, GA, TS, and NQ. Positions that are more than 80% identical are red, and those more than 80% similar are pink. Residues displaying an identity of 80% or higher within the TRP family are shown in dark green, while those more than 80% similar are light green. Similarly, positions displaying above 80% identity and 80% similarity in the TTR family are shown in dark and light blue, respectively. Confirmed or predicted signal peptides are indicated with yellow background colouring. Numbering and secondary structure elements are based on human TTR and are shown as green arrows β-strands) and a red box α-helix). Residues lining the hormone-binding channel in TTR are marked with blue stars. The N-terminal sequences of TRPs (residues preceding 10 according to human TTR numbering) were not aligned, whereas these residues in TTR were aligned manually.

Figure 11 is diagrammatic representation of data as a phylogenetic tree of TRP and TTR members extracted from Figure 9 of Eneqvist, T. et al., 2003 {supra) incorporated herein in its entirety by reference. The tree was based on the multiple sequence alignment comprising 49 TRP sequences and 20 TTR sequences presented in Figure 10. TRP sequences from species where it is unclear if a functional TRP gene exists and those with predicted signal peptides are marked with (?) and (SP), respectively. The TTR family branch represented by vertebrates is also indicated.

Figure 12 is a graphical representation of data showing that plt2/plt2 mice have an aberrant liver gene expression profile. Liver gene expression from four plt2/plt2 mice were compared to four wildtype mice. The Y axis scale is logarithmic fold change in gene expression between plt2/plt2 replicates and wild-type replicates. The X axis is logarithmic change in signal intensity. The three statistically most differentially expressed genes between plt2/plt2 and wild-type mice are listed (Sult2a2, Igβp2, Scd2).

Figure 13 is a diagrammatic representation of the nucleotide and amino acid sequence of long and short forms of TRP cloned from mouse liver that confirmed the presence of a mutation at Y98C. (A) Nucleotide sequence demonstrating the open reading frame from the short transcript cloned from wild-type mouse liver. In plt2/plt2 mice, the short transcript is identical except at nucleotide 224, where an A to G point mutation has occurred (underlined). (B) Nucleotide sequence demonstrating the open reading frame from the long transcript cloned from wild-type mouse liver. In plt2/plt2 mice, the long transcript is identical except at nucleotide 293, where an A to G point mutation has occurred (underlined). (C) Translation of the transcript presented in (A) below aligned to the transcript presented in (B) above. Both predicted proteins are identical, except the clone of TRP short is lacking the amino acid residues translated from the first exon of TRP long. The amino acid that is mutated mplt2/pU2 mice (Y98C) is underlined.

Figure 14 is a photographical representation of data showing TRP transcript are expressed in a range of tissues. (A) Reverse transcriptase PCR was performed on cDNA from a panel of tissues from wild-type and plt2/plt2 mice. Primers specific for the 5' UTR of TRP short and TRP long were selected to allow differentiation of short and long transcripts to be determined. At relatively low PCR cycle number, short and long TRP cDNA was present in samples derived from liver from both wild-type and plt2/plt2 mice. At higher cycle number, TRP cDNA was apparent from a range of organs. The presence of amplified cDNA was determined by staining with ethidium bromide after the PCR product had been electrophoresed on an agarose gel. The presence of PCR product after amplification with hprt -specific primers indicates the presence of cDNA in each tissue sample. No template controls were repeatedly negative in this experiment. (B) Protein lysates derived from 3T3 cells transfected with FLAG tagged constructs of TRP short and long were electrophoresed in parallel with liver lysates from seven different wild-type mice. A polyclonal antisera raised against peptide sequences from TRP exon 1 and 2 (αTRP) recognised a single band of approximately 15 kDa in all wildtype liver lysates studied. It was not possible to determine if this single endogenous band represented the protein product from TRP short or long, as both constructs were of different size to endogenous protein because of the addition of a FLAG tag. Figure 15 is a photographical representation of data showing TRP is markedly reduced in plt2/plt2 hepatocytes. (A) Western blot performed on protein lysates derived from a panel of tissues from wild-type and plt2/plt2 mice. A polyclonal antisera raised against TRP (αTRP) recognises a single 15 kDa band in wild-type liver lysate that was absent in plt2/plt2 liver. Adequate protein loading was determined by staining protein lysates for heat shock protein 70 (αHSP70). (B) Isolated hepatocytes from wild-type and plt2/plt2 mice were stained with isotype control antibody, αTRP antisera and sera from the same rabbit prior to immunisation. Cell nuclei were stained with DAPI (blue) and the rabbit protein detected with Alexa-Fluor (green). Images were captured with a Leica TCS4 SP2 spectral confocal scanner.

Figure 16 is a diagrammatical representation of an alignment of amino acid sequence showing protein homology and hydrophobicity comparison between TRP and transthyretin (TTR). Predicted protein homology of the translated murine TRP long transcript with predicted genes from worms (Caenorhabditis elegans), flies {Drosophila melanogaster), plants (Arabidopsis thaliand) and bacteria {Escherichia coli). TRP is related to transthyretin and the transthyretin protein for humans and mice is included for comparison. The tyrosine that is mutated by the plt2 mutation is marked (*). TRP sequence from lower organisms was identified by a BLAST search (www.ncbi.nlm.nih.gov) with transcript from murine TRP. Nucleotide sequence was then used to align predicted proteins with Clustal W software (www.ebi.ac.uk) and the amino acid output has been colour-coded based on protein hydrophobicity using ASAD software (bioinf.wehi.edu.au/software/ASAD/).

Figure 17 is a diagrammatical representation of the predicted protein structure of wild- type and modified TRP. Predicted protein structure of murine wild-type and mutant TRP with the Y98C substitution caused by the plt2 mutation. Tyrosine-98 (blue) is predicted to be positioned in the highly structured helix (arrow). The structure of murine TRP was modelled on the crystal structure of transthyretin from Spams aurata (Folli et al., FEBS Lett., 555:279- 284, 2003). This was performed using Swiss model software (Guex et al, Electrophoresis, 75:2714-2723, 1997) and then images were generated using the pymol program (http://www.pymol.org).

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides a description of the SEQ ID NOs provided herein.

Table 2 provides an amino acid sub-classification.

Table 3 provides exemplary amino acid substitutions. Table 4 provides a list of non-natural amino acids contemplated in the present invention.

Table 5 (including supplemental Table 5) provides the hematological profile of plt2/plt2 mice.

Table 6 provides a list of the SSLP markers used for the genome wide scan for linkage described in Example 7.

Table 7 tabulates the details of mice informative for further fine mapping of plt2 mutation described in Example 10.

Table 8 tabulates the details of primers and the method used (SSLP or SNP) for further fine mapping described in Example 10. Table 9 provides the details of individual genes in the genetic interval between

CeleraSNP12 and Celera SNP17 based on the prediction by UCSC Genome Browser (May 2004 genome assembly). Exon and exon-intron boundaries were sequenced in two animals that were homozygous for C57BL/6 markers across the region of interest, one intercross animal that was Balb/C across the region of interest and one control C57BL/6 mouse as described in Example 11.

Table 10 provides nucleotide sequences of primers used in PCR and sequencing reactions for each of the genes sequences as described in Example 11.

Table 11 provides twenty most differentially expressed genes between plt2/plt2 and wild-type forms. Table 12 provides the gene ontology groups for the most differentially expressed genes between plt2/plt2 and wild-type forms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated, in part, upon the identification and analysis of a pedigree of mice, called plt2, which displays recessive thrombocytosis associated with increased thrombopoietin production. Furthermore, homozygous plt2/plt2 mice develop tumors at increased frequency after natural aging or after radiation, compared to wild-type mice, identifying the encoding region as comprising a tumor suppressor. Using markers polymorphic between different inbred mouse strains the locus bearing this mutation was in accordance with the present invention mapped to an 8.6 Mb region of the telomeric end of chromosome 7. By exploiting intercrosses of the mutagenised pedigree with MpI knock-out mice on the same genetic background, a number of different steady state platelet count phenotypes were generated which provide insight to the regulation of the TPO-dependent pathway of platelet production. Further mapping analysis identified the plt2 mutation as a point mutation in a gene whose nucleotide sequence appears in the University College Santa Cruz (UCSC) 2004 mouse assembly and as NCBI Accession No. AK00447. The mutation causes a tyrosine to cysteine substitution in a conserved region of a Transthyretin related protein (TRP) and a previously unknown form thereof (herein referred to as "TRP-PLT2" or the "long form") which comprises an amino acid sequence encoded by an additional 5' exon not present in the NCBI gene database entry for AK00447.

Accordingly, in one aspect the present invention provides an isolated cell, or a non- human animal comprising such cells, wherein TRP or TRP is modified to effectively modulate its functional activity in the cell or animal compared to a non-modified animal of the same species.

Cells may be derived from human or non-human animal sources. The term "derived" does not necessarily mean that the cells are directly obtained from a particular source. Reference to a "cell" includes a system of cells such as a particular tissue or organ. In some embodiments, the modified cells are bacterial yeast or insect cells. Viral constructs comprising modified TRP are also contemplated including bacteriophage.

The term "modified" includes genetically modified but encompasses non-genetic or epigenetic modifications to affect TRP or TRP activity by, for example, the administration of an agent such as, without limitation, an organic or inorganic chemical agents, antibody, enzyme, peptide, genetic, oligosaccharide, lipid or proteinaceous molecule to effectively modulate the functional activity of TRP or TRP. Reference herein to "modulate" and "modulation" includes completely or partially inhibiting or reducing or down regulating all or part of TRP or TRP functional activity and enhancing or up regulating all or part its functional activity. Functional activity may be modulated by, for example, modulating TRP or TRP binding capabilities or TRP transcriptional or translational activity, or its half-life. With regard to TRP, its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell or membrane or its enzymatic capability. Thus, TRP level or activity may be modulated by modulating TRP expression, transcript stability, post translational modification, and the activity of regulatory molecules such as promoters, enhancers and such like. Reference to the "activity" or "functional activity" of TRP or TRP encompasses any relevant, measurable activity or characteristic of the molecule in proteinaceous or genetic form. Binding activity is a preferred activity, which may conveniently be assessed as described herein. Such assays may be conveniently adapted for high throughput monitoring using, for example, chromatographic methods such as HPLC or thin layer chromatography. TRP also binds to TPO-pathway members and binding assays are performed to determine whether this activity of TRP is modulated. Binding is conveniently assayed using antibodies to TRP or to other heterologous epitopes associated with the expressed polypeptide. Antibodies or antigen binding molecules specific to TRP are expressly contemplated. TRP is also required in some embodiments for platelet homeostasis. Accordingly, in vitro or in vivo assays may employ these outcomes as markers of TRP activity using, for example, the methods exemplified herein. For example, platelet levels or turnover may be measured using automated haemological analysis as described in Example 1.

The activity of TRP or TRP may be monitored using antibodies or other proteinaceous or genetic agents in a number of assays which are well known to those of skill in the art. Antibodies, for example, may be used to detect TRP by Western Blotting, histochemical or ELISA procedures. As discussed herein below, such agents may also distinguish between active and inactive forms of the TRP or between long and short forms of TRP or TRP. In accordance with the present invention, mutant forms of TRP or TRP are forms of TRP (found in a population of subjects) associated or linked with aberrant haematopoiesis, such as thrombocytopenia or thrombocytosis or a risk or presence of tumor development. Mutant forms of TRP may also be conveniently be detected using nucleic acid based assays well known in the art and as described herein. In some embodiments, low levels of active TRP may be produced as a result of mutations in TRP leading to altered expression levels, altered transcript stability or altered post-transcriptional or post-translational processing. Thus, TRP activity may be monitored indirectly by monitoring RNA production and/or stability, or the levels of regulatory molecules such as enhancers and repressors.

The term "genetically modified" refers to changes at the genome level and refers herein to a cell or animal that contains within its genome a specific gene which has been altered. Alternations may be single base changes such as a point mutation or may comprise deletion of the entire gene such as by homologous recombination. Genetic modifications includes alterations to regulatory regions, insertions of further copies of endogenous or heterologous genes, insertions or substitutions with heterologous genes or genetic regions etc. Alterations include, therefore, single of multiple nucleic acid insertions, deletions, substitutions or combinations thereof.

Cells and animals which carry a mutant TRP allele or where one or both alleles are mutated or deleted can be used as model systems to study the effects of TRP in megakarocytopoiesis and/or to test for substances which have potential as therapeutic agents when these function are impaired. Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant TRP alleles (including those carrying loxP flanking sequences), usually from a second animal of the same species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous TRP gene of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques. These animal models provide an extremely important testing vehicle for potential therapeutic products. The cells may be isolated from individuals with TRP mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the TRP allele, as described above. After a test substance is applied to the cells, the phenotype of the cell is determined. Any trait of the cells can be assessed.

Thus a genetically modified animal or cell includes animals or cells from a transgenic animal, a "knock in" or knock out" animal, conditional variants or other mutants or cells or animals susceptible to co-suppression, gene silencing or induction of RNAi.

Conveniently, targeting genetic constructs are initially used to generate the modified genetic sequences in the cell or organism. Targeting constructs generally but not exclusively modify a target sequence by homologous recombination. Alternatively, a modified genetic sequence may be introduced using artificial chromosomes. Targeting or other constructs are produced and introduced into target cells using methods well known in the art which are described in molecular biology laboratory manuals such as, for example, in Sambrook, Molecular Cloning: A Laboratory Manual, 3^rd Edition, CSHLP, CSH, NY, 2001; Ausubel (Ed) Current Protocols in Molecular Biology, 5^th Edition, John Wiley & Sons, Inc, NY, 2002. Targeting constructs may be introduced into cells by any method such as electroporation, viral mediated transfer or microinjection. Selection markers are generally employed to initially identify cells which have successfully incorporated the targeting construct.

Genetically modified organisms are generated using techniques well known in the art such as described in Hogan et ah, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbour Laboratory Press, CSH NY, 1986; Mansour et ah, Nature, 336:348-352, 1988; Pickert, Transgenic Animal Technology: A Laboratoiy Handbook, Academic Press, San Diago, CA, 1994. Stem cells including embryonic stem cells (ES cells) are introduced into the embryo of a recipient organism at the blastocyst stage of development. There they are capable of integration into the inner cell mass where they develop and contribute to the germ line of the recipient organism. ES cells are conveniently obtained from pre-implantation embryos maintained in vitro (Robertson et ah, Nature, 322:445-448, 1986). Once correct targeting has been verified, modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism. Alternatively, modified cells are allowed to aggregate with dissociated embryonic cells to form aggregation chimera. The chimeric organism is then implanted into a suitable female foster organism and the embryo allowed to develop to term. Chimeric progeny are bred to obtain offspring in which the genome of each cell contains the nucleotide sequences conferred by the targeting construct. Genetically modified organism may comprise a heterozygous modification or alternatively both alleles may be affected.

Another aspect of the present invention provides cells or animal comprising one, two or more genes or regions which are modified. For example, the genetically modified cells or animals may comprise a gene capable of functioning as a marker for detection of modified cells. Alternatively, the instant animals may be bred with other transgenic or mutant non- human animals to provide progeny some of which exhibit one or both traits or a modified trait/s. Chimeric animals are also contemplated. The terms "genetic material", "genetic forms", "nucleic acids", "nucleotide" and

"polynucleotide" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. α- anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

The present invention further contemplates recombinant nucleic acids including a recombinant construct comprising all or part of TRP. The recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosonal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic or synthetic origin which, by virtue of its origin or manipulation: (i) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (ii) is linked to a polynucleotide other than that to which it is linked in nature; or (iii) does not occur in nature. Where nucleic acids according to the invention include RNA, reference to the sequence shown should be construed as reference to the RNA equivalent with U substituted for T. Such constructs are useful to elevate TRP levels or to down-regulate TRP levels such as via antisense means or RNAi- mediated gene silencing. As will be well known to those of skill in the art, such constructs are also useful in generating animal models carrying a modified TRP allele. Genetically modified cells or non-human organisms may be provided in the form of cells or embryos for transplantation. Cells and embryos are preferably maintained in a frozen state and may optionally be distributed or sold with instructions for use.

In a further aspect, the present invention provides a genetically modified cell, or non- human animal comprising such cells, wherein a TRP gene is modified and the cell or animal produces a substantially enhanced level or activity of TRP, or substantially reduced level or activity of TRP compared to a non-modified animal of the same species, or is substantially incapable of producing TRP. The genetically modified cells and non-human animals may be a non-human primate, livestock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish, bird or other organism. Preferably the genetically modified non-human animal is a murine animal. In one aspect, the modified cell or non-human animal is genetically modified and produces a substantially reduced level of TRP, or is substantially incapable of producing TRP, or produces TRP having substantially reduced or no activity.

Preferably a TRP gene is modified. Modification may be in one or both alleles and may optionally be within a regulatory region of the gene. In another embodiment, the genetic modification resulting in a cell or animal capable of exhibiting a modified level or activity of TRP comprises genetic modification outside the TRP gene to cause expression of genetic or proteinaceous molecules which effectively modulate the activity of TRP or TRP.

In another aspect, the modified cell or non-human animal is genetically modified and substantially overproduces TRP having normal or altered activity relative to an unmodified cell or animal of the same species.

In yet another aspect, the invention provides a method of screening for or testing an agent capable of complementing a phenotype shown by a cell or non-human animal comprising a modified TRP or TRP and exhibiting a substantially modified level or activity of TRP. Preferably, the cell or animal is contacted with the agent and its effect on the phenotype of the cell or animal determined. In one aspect the method comprises screening for mutants which exhibit a complementing phenotype and then mapping and identifying the modifying gene. In another aspect, the method comprises screening for agents which enhance the level or activity of TRP in a normal or modified cell. In further embodiment, the subject invention provides a use of a cell or non-human animal comprising a modified TRP or TRP and exhibiting a substantially reduced level or activity of TRP in screening for or testing agents for use in the treatment or prophylaxis of haematological disorders such as thrombocytopenia and/or cancer.

The term "substantially" refers to a statistically significant change having a phenotypic or physiological effect. "Substantially enhanced level or activity" refers to significantly greater amounts having a phenotype or physiological effect. "Substantially reduced level or activity" refers to zero amounts to about 90% lower amounts compared to amounts detectable in a non- modified animal or cell. A substantially reduced level or activity of TRP or TRP is conveniently assessed in terms of a percent reduction relative to normal cells or animals or pre-treatment/pre- administration. A substantial reduction is one which results in detectable thrombocytosis in a subject or aberrant megakaryocytosis or cancer development. Preferably, the reduction is at least 20% compared to normal animals, more preferably about 25%, still more preferably at least about 30% reduction, more preferably at least about 40% reduction in TRP or TRP level or activity. The reduction may of course be complete loss of TRP activity in a cell or animal. A "modified" level or activity includes enhanced levels of TRP activity relative to pre- treatment levels and may equate to or exceed the level or activity of TRP or TRP detectable in healthy subjects or subjects unlikely to develop thrombocytopenia or cancer.

The present invention further provides a method for identifying agents useful in the treatment or prophylaxis of cancer or haematological disorders such as thrombocytopenia comprising screening compounds for their ability to modulate the functional activity of TRP or TRP. In a further aspect, the present invention provides a composition comprising an agent which down regulates the level or activity of TRP or TRP in a cell for use in modulating platelet production.

In another aspect, the present invention provides a composition comprising an agent which down regulates the level or activity of TRP in a subject for use in modulating platelet numbers in circulation.

The modulatory agents of the present invention may be chemical agents such as a synthetic or recombinant molecules, polypeptides, peptides or proteins, lipids, glycoproteins or other naturally or non-naturally occurring molecules or analogs thereof. Alternatively, genetic agents such as DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs )) ribozymes, aptamers, DNAzymes or other ribonuclease- type complexes may be employed.

Agents in accordance with this aspect of the invention may directly interact with TRP. Here, for example, antibodies or peptides, oligosaccharides, peptidomimetics or analogs and other such biomolecules may be conveniently employed. Alternatively, genetic mechanism are used to indirectly modulate the activity of TRP. Again, various strategies are well documented and include mechanisms for pre or post-transcriptional silencing. The expression of antisense molecules or co-suppression or RNAi or siRNA or DNA strategies are particularly contemplated.

Aptamers are also contemplated. RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9): 1628- 1650, 1999; Morris et al, Proc. Natl. Acad. ScI, USA, 95(6):2902-2907, 1998). Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in U.S. Pat. Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679.

An increasing number of DNA and RNA aptamers that recognize their non-nucleic acid targets have been developed by SELEX and have been characterized (Gold et al, Annu. Rev. Biochem., (54:763-797.1995; Bacher et al., Drug Discovery Today, 3(6):265-273, 1998).

In some embodiments, as discussed above, agents which modulate the level or activity of TRP or TRP may be derived from TRP or TRP or be variants thereof. Alternatively, they may be identified in in vitro or in vivo screens. Natural products, combinatorial, synthetic/peptide/polypeptide or protein libraries or phage display technology are all available to screening for such agents. Natural products include those from coral, soil, plant, or the ocean or antarctic environments.

In each case the agent to be tested is contacted with a system comprising TRP or TRP. Then, the following may be assayed for: the presence of a complex between the agent and TRP or TRP, a change in the activity of the target, or a change in the level of activity of an indicator of the activity of the target. Competitive binding assays and other high throughput screening methods are well known in the art and are described for example in International Publication Nos. WO 84/03564 and WO 97/02048).

Antisense or other inhibitory or gene silencing polynucleotide sequences are useful agents in preventing or reducing the expression of TRP. Alternatively, morpholines may be used as described by Summerton and Weller (Antisense and Nucleic acid Drug Development:! 187-195, 1997). Antisense molecules may interfere with any function of nucleic acid molecule. The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of the TRP gene.

While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals. Double stranded DNA molecules are also usefully employed. In the context of the subject invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases. While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

High-throughput screening protocols are well used such as those described in Geysen (International Publication No. WO 84/03564). Briefly, large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods. A similar method involving peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, is described in U.S. Patent No. 4,631,211 and a related method is described in International Publication No. WO 92/00091. A significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, so as to facilitate identification of the amino acid sequence of each library member. These improved bead-based methods are described in International Publication No. WO 93/06121. Another chemical synthesis screening method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface wherein each unique peptide sequence is at a discrete, predefined location in the array. The identity of each library member is determined by its spatial location in the array. The locations in the array where binding interactions between a predetermined molecule and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location. These methods are described in U.S. Patent No. 5,143,854; International Publication Nos WO 90/15070 and WO 92/10092; Fodor et al, Science, 251:161, 1991. Of particular use are display systems, which enable a nucleic acid to be linked to the polypeptide it expresses. Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques. Such systems, in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, are useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen. The nucleotide sequences encoding the V_H and V_L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E. coli and the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or p VIII). Alternatively, antibody fragments are displayed externally on lambda phage capsids (phage bodies). An advantage of phage-based display systems is that selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.

Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (Kang et al, Proc. Natl. Acad. Sci. U.S.A., 88:4363, 1991; Clackson et al., Nature, 352:624, 1991; Lowman et al., Biochemistry, 30:10832, 1991; Burton et al., Proc. Natl. Acad. Sci U.S.A., SS/10134, 1991; Hoogenboom et al, 1991 Nucleic Acids Res., 19:4\33, 1991, incorporated herein by reference in their entirety). One particularly advantageous approach has been the use of scFv phage-libraries (Huston et al, Proc. Natl Acad. Sci U.S.A., <°5:5879-5883, 1988; Chaudhary et al, Proc. Natl. Acad. Sci. U.S.A., 57:1066-1070, 1990; Clackson et al, 1991, (supra)). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Further phage display approaches are also known, for example as described in International Publication Nos. WO 96/06213 and WO 92/01047 (Medical Research Council et al.) and International Publication No. WO 97/08320 (Morphosys) which are incorporated herein by reference. Other systems for generating libraries of polypeptides or nucleotides involve the use of cell-free enzymatic machinery for the in vitro synthesis of the library members. In one method, RNA molecules are selected by alternate rounds of selection against a target ligand and PCR amplification (Tuerk and Gold, Science, 249:505, 1990; Ellington and Szostak, Nature, 346:818, 1990). A similar technique may be used to identify DNA sequences which bind to carbohydrate, polysaccharide, proteoglycan, glucosaminoglycans and the like. Similarly, in vitro translation can be used to synthesize polypeptides as a method for generating large libraries. These methods which generally comprise stabilized polysome complexes, are described further in International Publication No. WO88/08453. Alternative display systems which are not phage- based, such as those disclosed in International Publication Nos. WO 95/22625 and WO 95/11922 (Affymax) use the polysomes to display polypeptides for selection. These and all the foregoing documents also are incorporated herein by reference. The genetic agents or compositions in accordance with this invention preferably comprise from about 8 to about 80 nucleobases or greater (i.e. from about 8 to about 80 or greater linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

As mentioned previously herein, the agents or compositions of the present invention may be TRP or parts thereof, or TRP or parts thereof or complementary forms or molecules derived or designed from TRP or TRP. Thus, the present invention provides a composition comprising TRP or TRP (ie the molecule in genetic or proteinaceous form) or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP. In some embodiments, the composition effectively modulates megakaryocytopoiesis and/or cancer development. The present invention provides a composition comprising TRP or TRP (ie the molecule in genetic or proteinaceous form) or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP for use in modulating megakaryocytopoiesis and/or cancer development. In another aspect, the present invention provides a composition comprising TRP or TRP or a functional variant, functionally equivalent derivative, mimetic, analog or homolog thereof which substantially enhances the activity of TRP or TRP in a subject for use in the treatment or prophylaxis of cancer. Particularly preferred compositions are pharmaceutical compositions comprising TRP or TRP or a functional part or functionally equivalent derivative thereof capable of enhancing TRP level or activity suitable for use in the treatment or prophylaxis of cancer.

Any subject who could benefit from the present methods or compositions is encompassed. The term "subject" includes, without limitation, humans and non-human primates, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds and any other organism. A subject, regardless of whether it is a human or non-human organism may be referred to as a patient, individual, subject, animal, host or recipient.

The term "composition" and terms such as "agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound or cellular composition which induces a desired pharmacological and/or physiological effect. The terms encompass pharmaceutically acceptable and pharmacologically active ingredients including but not limited to salts, esters, amides, pro-drugs, active metabolites, analogs and the like. The term includes genetic and proteinaceous or lipid molecules or analogs thereof as well as cellular compositions as previously mentioned. The instant compounds and compositions are for the manufacture of a medicament for the treatment and/or prevention of thrombocytopenia and/or cancer. In some embodiments, agents which modulate TRP polypeptide activity in a cell are useful reagents in vitro cell cultures or maintenance.

In relation to cellular compositions, the present invention extends to cellular compositions including genetically modified stem cells which are capable of regenerating tissues and/or organs, such as the liver, of an animal subject in situ or in vivo. Stem cells or stem cell-like cells are preferably multipotent or pluripotent. Cells may be directly derived from humans however, totipotent embryonic stem cells from human embryos are not encompassed. Other cellular compositions comprise vectors such as viral vectors for delivery of nucleic acid constructs as described later herein.

In relation to TRP, the terms functional form or variant, functionally equivalent derivative or homolog include molecules which hybridize to TRP or a complementary form thereof over all or part of the genetic molecule under conditions of low stringency at a defined temperature or range of conditions, or which have about 60% or greater sequence identity to the nucleotide sequence defining TRP.

In relation to TRP, the terms functional form or variant, functionally equivalent derivative or homolog include molecules which hybridize to TRP or a complementary form thereof over all or part of the genetic molecule under conditions of medium or high stringency at a defined temperature or range of conditions, or which have about 60% to 80% sequence identity to the nucleotide sequence defining TRP.

Illustrative TRP nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 1 (mouse TRP mRNA short form) and SEQ ID NO: 3 (mouse TRP mRNA long form) or their complements. For the avoidance of doubt however, it should be noted that the term "TRP" and "TRP gene" expressly encompass all forms of the gene including regulatory regions such as those required for expression of the coding sequence and genomic forms or specific fragments including probes and primers and constructs comprising same or parts thereof as well as cDNA or RNA and parts thereof. In relation to TRP, the terms "functional form" or "variant", "functionally equivalent derivatives" or "homologs" include polypeptides comprising a sequence of amino acids having about 60% sequence identity to the TRP polypeptide of SEQ ID NO: 2 or 4.

Functional or active forms or variants of TRP polypeptide are selected among variants which retain functional activity, for example, in regulating TPO levels, platelet levels or liver homeostasis. In other embodiments, functional forms retain functional domains such as a hormone binding domain, a retinol binding domain or structural domains such as are important in forming a barrel structure or a highly ordered helix structure as shown in Figure 17. In still further embodiments, functional forms retain the ability to modulate levels of proteins or their encoding genetic sequences such as those involved in lipid metabolism, protein metabolism, biotransformation, other metabolism, cell cycle control, acute phase response and blood co- aggulation and proteolysis and peptidolysis. In some embodiments in particular, functional forms of TRP polypeptide are capable of regulating sulphotransferases, insulin-like growth factor binding proteins, stearoyl-Coenzyme A desaturase, galactose binding lectin, cytochrome P450 and other molecules such as those set forth in Table 11 and Table 12. Exemplary TRP amino acid sequences include those comprising sequences set forth in

SEQ ID NO: 2 (mouse TRP) and SEQ ID NO: 4 (mouse TRP-PLT2). In one embodiment, the present invention provides an isolated TRP-PLT2 polypeptide comprising an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof. In a further embodiment, the present invention provides an isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a TRP-PLT2 polypeptide having an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof. In another embodiment, the amino acid sequence comprises about 60% or greter sequence identity to about 20 to 30 contiguous amino acid residues at the N- terminal region of the polypeptide. In yet another embodiment, a nucleic acid molecule comprising a sequence of nucleotides substantially as set out in SEQ ID NO: 3 or a functional variant thereof or their complementary forms.

In still another embodiment, the nucleic acid molecules have about 60% or greater sequence identity to SEQ ID NO: 3 or a complementary form thereof over at least a 5'-terminal portion comprising about 60 to 100 contiguous nucleotides.

Reference herein to a "low stringency" includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-3O⁰C to about 42⁰C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as "medium stringency", which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (Marmur et al, J. MoI. Biol., 5:109, 1962). However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner et al., Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20⁰C to 65⁰C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C. In some embodiments, the nucleic acid molecule encoding a TRP polypeptide comprise a sequence of nucleotides as set forth in SEQ ID NO: 3 or which hybridises thereto or to a complementary form thereof under medium or high stringency hybridisation conditions. Preferably the hybridisation region is about 12 to about 80 nucleobases or greater in length.

The terms "similarity" or "identity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity and amino acid sequence comparisons are made at the level of similarity.

Preferably, the percent similarity between a particular amino sequence and a reference sequence is about 30% or about 65% or about 70% or about 80% or about 85% or more preferably about 90% similarity or greater as about 95%, 96%, 97%, 98%, 99% or greater. Percent similarities between 30% and 100% are encompassed.

Preferably, the precent identity between a particular nucleotide sequence and a reference sequence is about 30%, or 65% or about 70% or about 80% or about 85% or more preferably about 90% similarity or greater as about 95%, 96%, 97%, 98%, 99% or greater. Percent identities between 60 and 100% are encompassed. A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al, Nucl. Acids Res, 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in Molecular Biology John Wiley & Sons Inc, 1994-1998, Chapter 15).

A percentage of sequence identity between nucleotide sequences, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity for amino acid sequences.

In some embodiments, the present invention contemplates the use of full-length TRP or biologically active portions of those polypeptides. Typically, biologically active TRP portions comprise one or more binding domain . A biologically active portion of a full-length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, or more amino acid residues in length. The TRP polypeptide of the present invention includes all biologically active or functionally naturally occurring forms of TRP as well as biologically active portions thereof and variants or derivatives of these.

The present invention also contemplates variant forms of the interacting molecules. "Variant" polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein (e.g., wound- treating activity). Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native TRP polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a TRP polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

A TRP polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a TRP polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 52:488-492, 1985), Kunkel et al, {Methods in Enzymol, 754:367-382, 1987), U.S. Pat. No. 4,873,192, Watson et al. ("Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (Natl. Biomed. Res. Found, 5:345-358,1978). See also Figure 10 showing an alignment and consensus sequences for TTR-related proteins. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of TRP polypeptides. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify TRP polypeptide variants (Arkin et al., Proc. Natl. Acad. Sci. USA, 59:7811-7815, 1992; Delgrave et al., Protein Engineering, (5:327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

Variant TRP polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to the parent TRP amino acid sequence. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan. As shown herein, loss of tyrosine from the α-helix of TRP polypeptide profoundly alters its ability to be active in vivo.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterises certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally- occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, 1978 {supra); and by Gonnet et al, Science, 25<5(5062):1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.

Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 2.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional TRP polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 3 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

Thus, a predicted non-essential amino acid residue in a TRP polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a TRP polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.

Accordingly, the present invention also contemplates variants of the naturally- occurring TRP polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference TRP polypeptide sequence as, for example, set forth in any one of SEQ ID NO: 2 or 4. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acids but which retain the properties of the reference TRP polypeptide are contemplated. TRP polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to TRP polynucleotide sequences, or the non-coding strand thereof.

In some embodiments, variant polypeptides differ from an TRP sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any one of SEQ ID NO: 2 or 4 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. ("Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution. The multiple sequence alignment for TTR and TTR related proteins from Eneqvist et al., 2003 (supra) represented in Figure 10 demonstrates conserved residues, including a conserved tyrosine in the α-helix, amoung others.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of an TRP polypeptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. In other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a TRP polypeptide as, for example, set forth in SEQ ID NO: 2 or 4, and has the activity of that TRP polypeptide.

TRP polypeptides may be prepared by any suitable procedure known to those of skill in the art. For example, the polypeptides may be prepared by a procedure including the steps of: (a) preparing a chimeric construct comprising a nucleotide sequence that encodes at least a portion of a TRP polypeptide and that is operably linked to one or more regulatory elements; (b) introducing the chimeric construct into a host cell; (c) culturing the host cell to express the TRP polypeptide; and (d) isolating the TRP polypeptide from the host cell. In illustrative examples, the nucleotide sequence encodes at least a portion of the sequence set forth in SEQ ID NO: 2 or 4, or a variant thereof. Recombinant TRP polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook, et al., (1989, supra), in particular Sections 16 and 17; Ausubel et al., (1994, supra), in particular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Alternatively, the TRP polypeptides may be synthesised by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al., (Science, 269:202, 1995). The terms "derivative" or the plural "derivatives" and "variant" or "variants" are used interchangeable and, whether in relation to genetic or proteinaceous molecules, include as appropriate parts, mutants, fragments, and analogues as well as hybrid, chimeric or fusion molecules and glycosylation variants. Particularly useful derivatives retain the functional activity of the parent molecule and comprise single or multiple amino acid substitutions, deletions and/or additions to the TRP amino acid sequence. Preferably, the derivatives have functional activity or alternatively, modulate TRP functional activity. The term "modulate" includes up modulate or up regulate and down modulate or down regulate.

As used herein reference to a part, portion or fragment of TRP is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10 to 35 as well as greater than 35 nucleotides. Thus, this definition includes nucleic acids of 12,15, 20, 25, 40, 60, 100, 200, 500 nucleotides of nucleic acid molecules having any number of nucleotides between 500 and the number shown in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary form thereof. The same considerations apply mutatis mutandis to any reference herein to a part, portion or fragment of TRP.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage without the loss of other functions or properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine.

Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with the TRP polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and its underlying DNA coding sequence and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. MoI. Biol. 157: 105-132, 1982). Alternatively, the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The importance of hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Patent No. 4,554,101). The use of the hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Patent No. 5,691,198. The 3-D structure of various TTR proteins have been determined and TRP protein models may be developed therefrom as described in Eneqvist et ah, (supra).

The term "homolog" or "homologs" refers herein broadly to functionally or structurally related molecules including those from other species.

Reference herein to "mimetics" includes carbohydrate, nucleic acid or peptide mimetics and it intended to refer to a substance which has conformational features allowing the substance to perform as a functional analog. A peptide mimetic may be a peptide containing molecule that mimic elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics" in Biotechnology and Pharmacy, Pezzuto et ah, eds Chapman and Hall, New York, 1993). Peptide mimetics may be identified by screening random peptides libraries such as phage display libraries for peptide molecules which mimic the functional activity of TRP. Alternatively, mimetic design, synthesis and testing is employed. The recognition of carbohydrates by proteins is an important event in many biological systems and the development of chemotherapeutics based on carbohydrate-mimics which can disrupt specific recognition processes is a rapidly emerging field. For example, the synthesis of glycosyl phosphate mimics is described in a recent review by F. Nicotra in "Carbohydrate mimics" Eds Wiley (2005): Chapter 4, p 67-85.

Nucleic acid mimetics include, for example, RNA analogs containing N3'~P5' phosphoramidate internucleotide linkages which replace the naturally occurring RNA O3'~P5' phosphodiester groups. Enzyme mimetics include catalytic antibodies or their encoding sequences, which may also be humanised. Peptide or non-peptide mimetics can be developed as functional analogues of TRP by identifying those residues of the target molecule which are important for function. Modelling can be used to design molecules which interact with the target molecule and which have improved pharmacological properties. Rational drug design permits the production of structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology 9: 19-21, 1991). In one approach, one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al, Science, 249:527-533, 1990). In addition, target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol. 202: 2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

As briefly described, it is possible to design or screen for mimetics which have enhanced activity or stability or are more readily and/or more economically obtained. Analogues preferably have enhanced stability and activity. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to a subject. Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 4.

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂),, spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids and the introduction of double bonds between Q_x and C_β atoms of amino acids.

The small or large chemicals, polypeptides, nucleic acids, antibodies, peptides, chemical analogs, or mimetics of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18^th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.

For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.

For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecal Iy, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeutically effective amount. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, (supra).

Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be produced in the target cell, e.g. in a viral vector such as those described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. The vector could be targeted to the target cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages. The cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent. Alternatively, the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 73 IA and International Patent Publication No. WO 90/07936.

In accordance with this aspect of the present invention, the cells of a subject exhibiting modified TRP genetic sequences may be treated with a genetic composition comprising TRP. The provision of wild type or enhanced TRP function to a cell which carries a mutant or altered form of TRP should in this situation complement the deficiency and result in reduced cancer or hepatomegaly development in the subject. The TRP allele may be introduced into a cell in a vector such that the gene remains extrachromosomally. Alternatively, artificial chromosomes may be used. Typically, the vector may combine with the host genome and be expressed therefrom.

Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (In: Therapy for Genetic Disease, T. Friedman, Ed., Oxford University Press, pp. 105-121, 1991) or Culver {Gene Therapy: A Primer for Physicians, 2^nd Ed., Mary Ann Liebert, 1996). Suitable vectors are known, such as disclosed in U.S. Patent No. 5,252,479, International Patent Publication No. WO 93/07282 and U.S. Patent No. 5,691,198. Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non-viral transfer methods. A number of viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses (e.g. SV40, Madzak et al, J, Gen. Virol, 73:1533-1536, 1992), adenovirus (Berkner, Curr. Top. Microbiol. Immunol, 158:39- 66, 1992; Berkner et al, BioTechniques, 6:616-629, 1988; Gorziglia and Kapikian, J. Virol, 66:4407-4412, 1992; Quantin et al, Proc. Natl. Acad. Sci. USA, 59:2581-2584, 1992; Rosenfeld et al, Cell, 65:143-155, 1992; Wilkinson et al, Nucleic Acids Res., 20:2233-2239, 1992; Stratford-Perricaudet et al, Hum. Gene Ther., 1:241-256, 1990; Schneider et al, Nature Genetics, 75:180-183, 1998), vaccinia virus (Moss, Curr. Top. Microbiol. Immunol, 158:25- 38, 1992; Moss, Proc. Natl Acad. ScL USA, 93:11341-11348, 1996), adeno-associated virus (Muzyczka, Curr. Top. Microbiol Immunol, 158:97-129, 1992; Ohi et al, Gene, 59:279-282, 1990; Russell and Hirata, Nature Genetics, 75:323-328, 1998), herpesviruses including HSV and EBV (Margolskee, Curr. Top., Microbiol Immunol, 158:67-95, 1992; Johnson et al, J. Virol, 66:2952-2965, 1992; Fink et al, Hum. Gene Ther., 3:11-19, 1992; Breakefield and Geller, MoI Neurobiol, 7:339-371, 1987; Freese et al, Biochem. Pharmacol, ¥0:2189-2199, 1990; Fink et al, Ann. Rev. Neuroscl, 79:265-287, 1996), lentiviruses (Naldini et al, Science, 272:263-267, 1996), Sindbis and Semliki Forest virus (Berglund et al, Biotechnology, 77:916- 920, 1993) and retroviruses of avian (Bandyopadhyay and Temin, MoI Cell. Biol, 4:749-754, 1984; Petropoulos et al, J. Viol, 66:3391-3397, 1992), murine (Miller, Curr. Top. Microbiol. Immunol, 158:1-24, 1992; Miller et al, MoI. Cell Biol, 5:431-437, 1985; Sorge et al, MoI Cell Biol, 4:1730-1737, 1984; Mann and Baltimore, J. Virol, 54:401-407, 1985; Miller et al, J. Virol, 62:4337-4345, 1988) and human (Shimada et al, J. Clin. Invest., 55:1043-1047, 1991; Helseth et al, J. Virol, 64:2416-2420, 1990; Page et al, J. Virol, 64:5270-5276, 1990; Buchschacher and Panganiban, J. Virol, 66:2731-2739, 1982) origin. Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer. Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery.

In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged. For other techniques for the delivery of adenovirus based vectors, see U.S. Patent No. 5,691,198. Liposome/DNA complexes are also capable of mediating direct in vivo gene transfer.

Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein. In viral expression vectors, the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes TRP, expression will produce TRP. If the polynucleotide encodes a sense or antisense polynucleotide or a ribozyme or DNAzyme, expression will produce the sense or antisense polynucleotide or ribozyme or DNAzyme. Thus, in this context, expression does not require that a protein product be synthesized. In addition to the polynucleotide cloned into the expression vector, the vector also contains a promoter functional in eukaryotic cells. The cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters are routinely determined.

Receptor-mediated gene transfer may be achieved by conjugation of DNA to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. Receptors on the surface of liver cells may be advantageously targeted. These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, co-infection with adenovirus can be included to disrupt endosome function.

Accordingly, in some embodiments patients who carry an aberrant TRP allele are treated with a gene delivery vehicle such that some or all of their cells receive at least one additional copy of a functional normal TRP allele. Preferably only specific cells, such as liver cells are targeted.

Alternatively, peptides or mimetics or other functional analogues which have TRP activity (ie capable of exhibiting a TRP function) can be supplied to cells which carry aberrant TRP alleles. Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. In addition, synthetic chemistry techniques can be employed to synthesize the instant active molecules. Active molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Supply of molecules with TRP activity should lead to platelet homeostasis and a reduced risk of developing cancer and particularly liver cancer.

In a further related aspect of the present invention it has been determined that alternations in the level or activity of TRP or TRP have profound effects on cellular activities such as haematopoiesis and cancer development. Accordingly, these diseases or a susceptibility to these conditions can now be diagnosed by monitoring subjects for modification in the level or activity of TRP or specific mutations or aberrations (such a methylation events) in TRP.

One particular mutation results in Cysteine for Tyrosine in the α helix of TRP.

A wide range of mutation detection screening methods are available as would be known to those skilled in the art. Any method which allows an accurate comparison between a test and control nucleic acid sequence may be employed. Scanning methods include sequencing, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP and rSSCP, REF-SSCP), chemical cleavage methods such as CCM, ECM, DHPLC and MALDI-TOF MS and DNA chip technology. Specific methods to screen for pre-determined mutations include allele specific oligonucleotides (ASO), allele specific amplification, competitive oligonucleotide priming, oligonucleotide ligation assay, base- specific primer extension, dot blot assays and RFLP-PCR. The strengths and weaknesses of these and further approaches are reviewed in Sambrook, Chapter 13, Molecular Cloning, 2001. By identifying TRP as subject to mutations which affect the level or activity of TPO or cancer development, the present invention provides methods of diagnosis of conditions associated with modified TPO level or activity or cancer in a subject and further provides genetic or protein based methods of determining the susceptibility of a subject to develop these conditions.

The diagnostic and prognostic methods of the present invention detect or assess an aberration in the wild-type TRP gene or locus to determine if TRP will be produced or if it will be over-produced or under-produced. The term "aberration" in the TRP gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions of TRP. It also includes changes in methylation patterns of TRP or of an allele of TRP. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g. in the tumor tissue and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. A TRP allele which is not deleted (e.g. that found on the sister chromosome to a chromosome carrying a TRP deletion) can be screened for other mutations such as insertions, small deletions, point mutations and changes in methylation pattern. It is considered in accordance with the present invention that many mutations found in cells such as hepatic cells are those leading to decreased or increased expression of the TRP gene.

Useful diagnostic techniques to detect aberrations in the TRP gene include but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded coformational analysis (SSCA), Rnase protection assay, allele-specific oligonucleotide (ASO hybridization), dot blot analysis and PCR-SSCP (see below). Also useful is DNA microchip technology.

Predisposition to cancer can be ascertained by testing any tissue of a human or other mammal for mutations in a TRP gene. This can be determined by testing DNA from any tissue of a subject's body. In addition, pre-natal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of the TRP gene. Alteration of a wild-type allele whether, for example, by point mutation or by deletion or by methylation, can be detected by any number of means.

There are several methods that can be used to detect DNA sequence variation. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing, can detect sequence variation. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita et al, Proc. Nat. Acad. Sci. USA, 86:2716-2770, 1989). This method can be optimized to detect most DNA sequence variation. The increased throughput possible with SSCP makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The fragments which have shifted mobility on SSCP gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al, Am. J. Hum. Genet., 49:699-106, 1991), heteroduplex analysis (HA) (White et al, Genomics, 72:301-306, 1992) and chemical mismatch cleavage (CMC) (Grompe et al, Proc. Natl. Acad. ScL USA, §5:5855-5892, 1989). Other methods which might detect mutations in regulatory regions or which might comprise large deletions, duplications or insertions include the protein truncation assay or the asymmetric assay. A review of methods of detecting DNA sequence variation can be found in Grompe {Proc. Natl Acad. ScL USA, 5(5:5855-5892, 1993). Once a mutation is known, an allele specific detection approach such as allele specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labeled with gold nanoparticles to yield a visual color result (Elghanian et al, Science, 277:1078-1081, 1997). Other tests for confirming the presence or absence of a wild-type or mutant TRP allele include single-stranded conformation analysis (SSCA) (Orita et al, (1989; supra)); denaturing gradient gel electrophoresis (DGGE) (Wartell et al, Nucl Acids Res., 18:2699-2705, 1990; Sheffield et al, Proc. Natl Acad. ScL USA, 56:232-236, 1989); RNase protection assays (Finkelstein et al, Genomics, 7:167-172, 1990; Kinszler et al, Science, 257:1366-1370, 1991); denaturing HPLC; allele-specific oligonucleotide (ASO hybridization) (Conner et al, Proc. Natl. Acad. ScL USA, 50:278-282, 1983); the use of proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, Ann. Rev. Genet., 25:229-253, 1991) and allele-specific PCR (Ruano and Kidd, Nucl Acids. Res. 77:8392, 1989). For allele-specific PCR, primers are used which hybridize at their 3' ends to a particular TRP mutation or to junctions of DNA caused by a deletion of TRP. If the particular TRP mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Publication No. 0 332 435 and in Newtown et al (Nucl. Acids. Res. 17: 2503-2516, 1989). Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. DNA sequences of the TRP gene which have been amplified by use of PCR or other amplification reactions may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the TRP gene sequence harboring a known mutation. For example, one oligomer may be about 20-40 nucleotides in length, corresponding to a portion of the TRP gene sequence as described in Example 11. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the TRP gene. Hybridization of allele- specific probes with amplified TRP sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.

Microchip technology is also applicable to the present invention. In this technique, thousands of distinct oligonucleotide or cDNA probes are built up in an array on a silicon chip or other solid support such as polymer films and glass slides. Nucleic acid to be analyzed is labelled with a reporter molecule (e.g. fluorescent label) and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique, one can determine the presence of mutations or sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest or multiple genes of interest such as genes encoding products in a biochemical pathway. The technique is described in a range of publications including Hacia et al. {Nature Genetics, 14:441-447, 1996), Shoemaker et al. (Nature Genetics, 14:450-456, 1996), Chee et al. (Science, 274:610-614, 1996), Lockhart et al. (Nature Biotechnology, 14:1675-1680, 1996), DiRisi et al. (Nature Genetics, 14:457-460, 1996) and Lipshutz et al. (Biotechniques, 19:442- 447, 1995). Alteration of wild-type TRP genes can also be detected by screening for alteration of wild-type TRP proteins. For example, monoclonal antibodies immunoreactive with TRP can be used to screen a tissue. Lack of cognate antigen would indicate a TRP mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant TRP gene product. Such immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA and RAPID assays.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production is derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation (i.e. comprising TRP) or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature, 256:495-499, 1975; Kohler and Milstein, European Journal of Immunology, (5/511-519, 1976). Examples of primers used to amplify regions of TRP are set forth in the Examples. The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Materials and Methods

Generation and Screening of Mutant Mice Male C57BL/6 mice were treated with 2 doses of 70 mg/kg ENU intraperitoneally

(Bode V. C₅ Genetics 108:457-470, 1984) and mated with isogenic C57BL/6 female mice to produce first-generation progeny (Gl). Gl males and females were then inter-bred and their offspring brother-sister mated to produce families of G3 animals. A G3 pedigree (called pltl) was recognised to have thrombocytosis and this pedigree was propagated by inbreeding animals with the highest platelet counts. PU2 heterozygotes (p!t2/+) were produced by breeding aplt2/plt2 mouse with a wild type C57BL/6 partner. To determine whether the plt2 mutation was operating in the same pathway as thrombopoietin-dependent signalling through the c-Mpl receptor, plt2/plt2 male mice were mated with MpI^''^" females on a C57BL/6 background (Alexander, W. S. et αl, Blood, §7:2162-2170, 1996) to produce offspring that were obligate heterozygotes for the plt2 mutation and the MpI knock-out allele. These mice were then brother-sister mated and the platelet count of their offspring (the F2 generation) determined. The MpI genotype of the F2s was determined by Southern blot as previously described (Alexander, W. S. et αl., 1996, supra).

Hematological Analysis

The platelet count was determined at 7 weeks of age by collection of peripheral blood from the retroorbital plexus and deposition into tubes containing potassium EDTA (Sarstedt Nuembrecht, Germany). The platelet count was determined by using an Advia 120 automated hematological analyser (Bayer, Tarrytown, NY). All hematological data are presented as mean+1 standard deviation and all wild-type experimental animals were on a C57BL/6 genetic background. Spleen colony-forming units (CFU-S) were enumerated by intravenous (iv) injection of 7.5x10⁴ bone marrow cells from either a plt2/pU2 or C57BL/6 donor into five C57BL/6 recipients after they had received 11 Gy in two equal doses given three hours apart. Spleens were removed after 12 days, fixed in Carnoy's solution (60% ethanol/30% chloroform/ 10% acetic acid) and the number of macroscopic colonies counted. CFU-S values were recorded as the mean score of 5 recipients per donor (Kimura S. et al, PNASU, 95: 1195- 1200, 1998). Clonal culture of hemopoietic progenitor cells was performed using 1 ml cultures of 2.5xlO⁴ bone marrow cells or 5xlO⁴ spleen cells suspended in 0.3% agar in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum. Cells were incubated for 7 days at 37⁰C in 10%Cθ₂ with various cytokines at optimal concentrations (Roberts A. W. et al, Blood 89:212>β-21 '44, 1997). At 7 days, colonies were fixed in glutaraldehyde, stained and enumerated (Metcalf D., Clonal culture of Hemopoietic cells: techniques and applications. Amsterdam: Elsevier; 1984). Megakaryocyte numbers in the bone marrow and spleen were estimated by manual counting of 10 random high power fields of sections of sternum and spleen after staining with hematoxylin and eosin. Statistical significance of differences observed between plt2/plt2 and wild-type mice was assessed using two-sided Student's t-tests.

Flow Cytometry

Megakaryocyte number and ploidy were assessed in unfractionated bone marrow harvested into 2 ml ice cold CATCH solution (calcium and magnesium free Hank's balanced salt solution with 1 mM adenosine, 2 mM theophylline, 0.38% sodium citrate, 3.5% BSA) (Jackson C. W. et al, Blood 53:768-778, 1984). A small aliquot of this suspension was placed in a hemocytometer and a total nucleated cell count performed. The cell suspension was stained with FITC conjugated CD41 monoclonal antibody or FITC IgGl kappa as the isotype control (BD Pharmingen, San Diego CA) and then incubated in a hypotonic propidium iodide solution (0.05 mg/ml PI in 0.1% sodium citrate) for a minimum of 2 hours on ice. The stained cell suspension was then washed with CATCH solution filtered through a lOOμm cell filter to remove cell aggregates and added to TruCOUNT tubes (BD Biosciences) containing a specified number of beads. RNase was added to the suspension and the sample was analyzed on a Becton Dickinson FACScan.

Bone Marrow Transplantation and Assessment of Megakaryocyte Engraftment

5x10⁶ bone marrow cells from a plt2/plt2 or C57BL/6 male donor were injected into plt2/plt2 and C57BL/6 recipient mice after they received a myeloablative dose of radiation (2 doses of 5.5 Gy, 3 hours apart). Megakaryocyte engraftment after this procedure was studied by identifying the presence of sex-mismatch between the male bone marrow donor and female recipient mice. Specifically, bone marrow from recipient mice was harvested and cultured in IMDM supplemented with 1% Nutridoma-SP (Roche Diagnostics, Indianapolis, IN) and 50ng/mL of thrombopoietin for 5 days at 37°C. Megakaryocytes were then purified on an albumin density gradient and an aliquot cytocentrifuged onto glass slides and stained with acetylcholinesterase (Jackson C. W., Blood 42:413-421, 1973). The remaining megakaryocyte suspension was lysed in non-ionic detergent and PCR was performed using primers specific for the murine sex-determining region of the Y chromosome (Sry) (Gubbay J. et ah, Nature 346:245-250, 1990) (5'-CTCTGCCTGTGCTGGTTG-S', and 5'-

TTGTGCTTTTTGTCCTCTTGT-3') and platelet derived growth factor receptor (Pdgfr) (5'- CTGGGCCAGAGTTTGTTCTC-3', and 5'-CAAGGCTGCATCTCACAGAG-S ') as a positive control. A Southern blot was then performed on the PCR product using a hybridisation probe for the Sry (5'-TCCAGTGCAGTGCTTTATGC-S') and Pdgfr (5'- CACTGACCAATGTCACTGGG-3') loci.

Thrombopoietin Enzyme-linked immunosorbent assay (ELISA)

Peripheral blood was collected from the retroorbital plexus and allowed to clot at room temperature for 2 hours before centrifugation for 20 minutes at 200Og. The supernatant was removed and then stored at -20°C until the ELISA was performed. Serum samples were prepared in a similar manner from mice 11 days after 5FU injection (0.15 g/kg 5FU iv). Protein lysates were prepared from whole liver specimens that were weighed and then snap- frozen in liquid nitrogen. Samples were mechanically disrupted and then homogenised in KALB lysis buffer (150 mM NaCl, 50 mM Tris [pH 7.5], 1% [vol/vol] Triton X-100, 1 mM EDTA) with protease inhibitors (Complete cocktail tablets, Roche) in a Dounce Homogeniser. The protein content of the lysate was calculated using the BCA protein assay kit (Pierce, Rockford IL). The thrombopoietin concentration of the serum and liver lysates was then determined by Quantikine murine thrombopoietin ELISA (R&D Systems, Minneapolis, MN).

Measurement of thrombopoietin mRNA by quantitative real-time PCR

Total cellular RNA was isolated from organ samples after they were snap-frozen into liquid nitrogen and homogenised in TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was then purified using the RNeasy kit (Qiagen GmbH, Germany) according to the manufacturer's protocol. Bone marrow samples were prepared in the same manner after the femur was first flushed with 3 ml of cold TRIzol. First strand cDNA synthesis was performed using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR reactions were set up for Thrombopoietin (Tpo), Hydroxymethylbilane synthase (Hmbs) and RNA polymerase II {Polr2d) using the Taqman gene expression assay protocols. Specifically, 1 μL of cDNA was used in a 20 μL PCR reaction with the pre-developed Taqman assay for Tpo, Hmbs or Polr2a, 10 μL Taqman universal master mix and water. Cycle conditions were 95°C for 10 min then 15 sec at 95°C and 1 min at 6O⁰C repeated for 40 cycles. Amplification, data acquisition and automatic Ct derivation were performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Analysis of relative gene expression was performed using the 2-^ΔΔCT method (Livak K. J. et al, Methods 25:402-408, 2001).

Genetic Mapping plt2/plt2 male mice on a C57BL/6 background were intercrossed with wild type Balb/c females to produce Fl mice that, in turn, were either mated with plt2/plt2 males to produce N2 backcross mice or brother-sister mated to produce F2 intercross mice. Platelet counts of the N2 backcross mice were measured at seven weeks of age and then the mice were killed and DNA prepared from their liver. A genome wide scan was performed using 162 simple sequence length polymorphisms (SSLPs) spaced evenly throughout the genome with markers derived from the Mouse Genetic Mapping Project of the Broad Institute (www.broad.mit.edu) (Dietrich W. et al, Genetics 737:423-447, 1992). SSLPs (Table 6) were amplified by PCR using fluorescent dye labeled oligonucleotides and the sequence length of the PCR product determined on an ABI 3700 DNA sequence analyser as per the manufacturers instructions. Linkage was assessed using quantitative trait analysis in the statistical package R/qtl (Broman K. W. et al, Bioinformatics 7P:889-890, 2003). A normal model was used for the log transformed platelet count and the sex of the mice was included in the analysis. Once the region of interest was established, informative recombinants were sought among 353 F2 intercross mice. A novel SSLP marker which discriminated between C57BL/6 and Balb/c in the interval of interest was generated (D7Wehi28: 5'-GTGTGCTCTGTTGCACAGGT-S ', 5'- GGCTTCAGCTTCAAGGTCAG-3') and included in the analysis. Progeny testing was used to confirm all phenotype-based assignments of genotype in critically-informative recombinants. EXAMPLE 2 Plt2 mice display thrombocytosis and hepatomegaly

The founders of the plt2 pedigree were recognised in the third generation of an ENU mutagenesis screen by virtue of a sustained thrombocytosis. A plt2/plt2 homozygote pedigree was then established by breeding animals with the highest platelet counts and their plt2/plt2 genotype was confirmed by examining the platelet count distribution of their offspring (progeny testing). The platelet counts of the plt2/plύ animals (2038+347x10⁹/L, n=107) were increased by 47% compared to both wild-type C57BL/6 mice (1386±223,n=107; p<0.001) and obligate heterozygous animals (1381+271, n=89). The animals designated plt2/plt2 in this cohort (Figure IA) were bred from parents that both displayed thrombocytosis with platelet counts greater than 2000 xlO⁹/L (a level of thrombocytosis not observed in the wild type population in this series) and animals designated heterozygous (plt2/+) had one parent with a platelet count greater than 2000 xlO⁹/L bred with a wild-type animal. Platelet counts in the obligate heterozygotes produced from this breeding strategy were indistinguishable from the wild-type population demonstrating the recessive nature of this phenotype. Other platelet parameters, such as Mean Platelet Volume and Platelet Distribution Width were similar between the groups (Table 5). Male C57BL/6 mice had 11% higher platelet counts than female C57BL/6 mice (male 1452±232 n=57; female 1311+188 n=50), and this gender disparity was also apparent for the plt2 pedigree. The platelet counts of male plt2/plt2 mice (2177+338, n=56) were 15% greater than observed for female plt2/plt2 mice (1885+290, n=51; pO.001). In contrast to the difference seen in platelet numbers, red cell and white cell blood parameters were similar between the two groups (Table 5).

Mutant plt2/plt2 mice appeared overtly normal and were fertile. When observed for up to one year, they remained healthy with no observable complications related to the thrombocytosis. plt2/plt2 mice were of similar size to the C57BL/6 wild-type animals when they were examined at 7-10 weeks of age (body weight plt2/plt2 22.04+2.49g,n=34 compared to wild-type 22.97+3.17,n=23; p=0.21) however, plt2/plt2 mice invariably displayed enlargement of the liver that was not accompanied by an increase in any other organ. Liver weight, expressed as a proportion of the total weight of the animal, as illustrated in Figure IB, was 0.074+0.005 for the plt2/plt2 pedigree (n=35) compared to 0.054±0.004 for the wild-type (n=24; p<0.001) and 0.051±0.006 for the heterozygous animals (n=14). No histological basis for the enlarged plt2/plt2 liver was detected. Specifically, there was no cellular infiltration, no degeneration of hepatocytes, no abnormal frequency of mitoses and no suggestion of enlargement of plt2/plt2 hepatocytes. Hepatic architecture was normal.

EXAMPLE 3

Compound plt2/plt2 MpT^1' animals demonstrate that thrombocytosis in plt2/plt2 mutant mice is dependent on thrombopoietin signalling and theplt2 mutation is acting upstream of MPL to regulate platelet production

To determine if the plt2 mutation was acting in the same pathway as thrombopoietin signalling through the c-Mpl receptor, plt2/plt2 animals were intercrossed with Mpϊ^f~ animals. As previously observed (Gurney A. L. et al., 1994, supra; Alexander, W. S. et ah, 1996, supra), MpT^1' mice are severely thrombocytopenic with a mean platelet count in our series of 155±84xlO⁹/L (n=112). MpI heterozygote C57BL/6 animals (Λφ/^+Λ) were observed to display a mild thrombocytosis, with a platelet count of 1641±220 (n=86), increased 18% compared to the wild-type C57BL/6 cohort (pO.001). The platelet counts of the F2 generation arising from the intercross of plt2/plt2 animals with the MpT*^' animals displayed a bimodal distribution (Figure 1C). One cluster of platelet counts were present around the Mpϊ'^~ mean of 150. The second cluster covered the normal platelet range for a C57BL/6 mouse, but with a tail extending towards higher platelet counts. The c-Mpl status of these F2 animals was determined by Southern blotting and the plt2 mutation status of the plt2/plt2 homozygotes inferred from determination of their corrected liver size (Figure IB). MpI^''^' mice with large liver size (inferred to be compound plt27plt2 Mpϊ'^~ mice) displayed a platelet phenotype (138±76, n=12)(Figure ID) that was indistinguishable from the MpT^1' cohort with normal liver size (p=0.94). The thrombocytosis observed with mice homozygous for the plt2 mutation on a C57BL/6 background was not apparent when the plt2/plt2 mutant mice were also MpT^1', suggesting that the plt2 mutation is acting upstream of c-Mpl-dependent pathways of platelet production. The Mpl^+/' animals that displayed hepatomegaly (designated plt2/plt2 Mpl^+/") exhibited marked thrombocytosis and accounted for most of the high platelet tail of the F2 intercross (Figure 1C). plt2/plt2 Mpf'^~ animals had a platelet count of 2656+459 (n=25), a 92% increase compared to wild-type C57BL/6 animals and also significantly increased over plt2/plt2 animals (p<0.001), suggesting an additive effect of the c-Mpl heterozygosity and the plt2 mutation on platelet count.

EXAMPLE 4

Megakaryocyte Progenitors and Megakaryocytes are increased inplt2/plt2 mice

Hemopoietic cell populations in the megakaryocyte lineage were quantitated to elucidate how the observed thrombocytosis was sustained. Colony forming units in the spleen (CFU-S) numbers were similar between wild-type C57BL/6 animals and mutants plt2/plt2 animals, as were committed hemopoietic progenitors of non-megakaryocytic lineage when assessed by clonal culture in vitro (Supplementary Table 5). In contrast, megakaryocyte progenitors generated by a variety of cytokine stimuli were consistently elevated in both bone marrow and spleen (Table 5). This increase was concordant with the degree of thrombocytosis.

Megakaryocyte populations present in the bone marrow and spleen of the mice were quantitated by both light microscopy and flow cytometry. Histology of both spleen and sternum demonstrated a consistent increase of approximately 50% in megakaryocyte number. To quantitate megakaryocytes in the bone marrow by flow cytometry, the number of CD41- positive cells were related to the total bone marrow nucleated cell count. Consistent with the histological assessment, a 50% increase in CD41 -positive megakaryocytes was observed in the bone marrow oϊplt2/plt2 animals. Flow cytometry also allowed megakaryocyte maturation to be assessed by nuclear ploidy. Wild-type and plt2/plt2 animals displayed a similar distribution of megakaryocyte ploidy in the bone marrow (Table 5) with a modal ploidy of 16N (Figure IE). Megakaryocytes of high nuclear ploidy (>8N) were more frequent in the plt2/plt2 group (58%±5%, n=6) compared to wild-type megakaryocytes (50%±6%, n=6; p=0.01). The mild lineage specific increase observed in megakaryocytes and megakaryocyte progenitors was concordant with the level of thrombocytosis observed in peripheral blood, suggesting that elevated megakaryocytopoiesis accounted for the plt2 thrombocytosis. EXAMPLE 5

Reciprocal bone marrow transplantation experiments demonstrate that thepltl mutation acts extrinsically on the hemopoietic system

To investigate how the plt2 mutation was acting on the hemopoietic system, reciprocal bone marrow transplant experiments were performed after myeloablative radiation. Platelet counts measured from recipient animals 8 weeks after transplantation clearly demonstrated a significant increase in platelet count in the plt2/plt2 recipient animals compared to the wild- type recipients (pO.OOl) regardless of whether the animal received hemopoietic cells from the plt2/plt2 or wild-type donor. No thrombocytosis was observed in wild-type recipients of plt2/plt2 bone marrow (Figure 2). This result was unchanged when animals were analyzed one year post transplant (data not shown). To ensure that the lack of thrombocytosis in wild-type recipients of plt2/plt2 marrow was not an artefact of poor or failed engraftment of plt2/plt2 cells, megakaryocyte engraftment after transplantation was studied in 10 female recipient mice that received bone marrow from one of the two male donor animals. Megakaryocytes were grown in culture from the bone marrow of the recipient animals and then purified. DNA from these purified megakaryocytes was amplified by PCR and a Southern blot performed to identify the presence of Y chromosome DNA (the Sry allele) in a semi-quantitative fashion. A control blot was also performed to identify the presence of DNA from an autosome (Pdgfr). In all female recipient animals studied, the megakaryocytes present in the bone marrow were predominantly donor as determined by the presence of the Sry allele (Figure 2). Together, these results indicate that the action of the plt2 mutation is extrinsic to the hemopoietic system.

EXAMPLE 6 Thrombopoietin assays - TPO production is specifically increased in plt2/plt2 mammals

To determine if excess thrombopoietin was responsible for the observed thrombocytosis in plt2/plt2 mice, serum thrombopoietin concentration was measured by ELISA in plt2/plt2 and wild-type mice (Figure 3A). Serum TPO was elevated by 76% in plt2/plt2 animals (mean 4930±1309 pg/mL, n=18) compared to wild-type mice (2802±1031, n=15, t test p<0.001). To control for any spurious elevation in serum TPO caused by release of TPO by the platelet mass during clotting, serum TPO was measured in wild-type mice with rebound thrombocytosis following 5FU injection (Radley J. M. et al, Blood 55:164-166, 1980). Serum collected from 6 wild-type mice, 11 days post 5FU (with a mean platelet count of 3143xlO⁹/L) demonstrated a mean serum TPO concentration 43% lower than steady state C57BL/6 mice. These data indicate that the concentration of serum TPO m plt2/plt2 mice is markedly and inappropriately elevated for the observed platelet count. The prominent hepatomegaly demonstrated by the plt2/pU2 mice suggested that hepatic

TPO production might be increased in these mice. To investigate whether TPO transcription was upregulated in plt2/plt2 mice, Tpo PvNA extracted from liver samples of 4 plt2/plt2 and 4 wild-type mice was quantitated in quadruplicate. Using the 2^'MCT method of analysis of realtime PCR data, no significant difference in Tpo transcript relative to global transcription was observed between wild-type and plt2/plt2 gene expression when normalised to Hmbs (p=0.54) or Polr2a (p=0.23)(Figure 3B).

To further investigate if a physiologically minor tissue source of TPO production displayed differentially up-regulated TPO gene expression, Tpo transcripts from a panel of tissues from 3 wild-type and 3 plt2/plt2 mice were also quantified in duplicate. A wide range of Tpo transcription across the organs sampled was observed, with kidney demonstrating the most prominent levels of transcription after the liver. However, no physiologically significant up-regulation of Tpo transcript was demonstrated in the kidneys of plt2/plt2 mice (Fig 3C), or in any organ examined. Gene expression normalised to Polr2a relative to a control kidney sample are presented in Figure 3C and similar results were found when Tpo expression was normalised to Hmbs (data not shown).

Next, hepatic thrombopoietin protein was examined in whole liver lysates by ELISA. On a weight-for-weight basis, total protein concentration of the lysate was not significantly different between plt2/plt2 (104±12mg protein/g liver weight, n=8) and wild-type livers (134±60mg/g, n=8; p=0.19). However, TPO protein per Ig of liver weight was increased in the liver lysate by 51% in the plt2/plt2 animals (259+87, n=8) compared to wild-type animals (173+56, n=8, p=0.03)(Figure 3D). This increase in the TPO content of plt2/plt2 liver lysates was also apparent when the TPO concentration was corrected for the total protein content of the lysate (plt2/pU2 2.5+1.0 absorbance units/total protein concentration, n=8, compared to wild-type 1.4±0.5, n=8; p=0.01). EXAMPLE 7

Genetic mapping localises theplt2 mutation to an 8.6 Mb region on the telomeric end of chromosome 7

With experimental data implicating TPO as the driver of the thrombocytosis in plt2/plt2 mice, it was important to exclude the thrombopoietin gene as the locus mutated in these mice. The chromosomal location of the plt2 mutation was determined by using strain specific SSLPs (see Table 6) to link the homozygous C57BL/6 genotype with thrombocytosis in an N2 backcross between plt2/plt2 animals on a C57BL/6 genetic background and wild type mice on a Balb/c genetic background. Platelet counts from 89 N2 mice ranged from 1039 to 2424x10⁹/L and displayed a bimodal distribution consistent with the autosomal recessive inheritance of the plt2 allele (data not shown). Genetic linkage for thrombocytosis was observed on chromosome 7 at D7Mitl89 with a peak LOD score of 22.85 (Figure 4A).

This linkage was confirmed in 281 N2 backcross mice genotyped at 10 SSLP markers positioned from the telomeric end of chromosome 7. In these mice, a continuous range of platelet counts was observed with a mean of 1756x10⁹/L and a standard deviation of 430 xlO⁹/L (Figure 4B). The 50 N2 mice with the highest and the 50 mice with the lowest platelet counts (both outside one standard deviation from the mean of the N2 population) were analysed, and the thrombocytosis phenotype was best linked to C57BL/6 homozygosity on the telomeric side of D7Mit71 locus (Figure 4C). This position was confirmed for the entire N2 population with the genotype at D7Mit46 being predictive of the thrombocytosis phenotype (Figure 4B) and the genetic linkage for this marker increased to a LOD score of 62.

To further refine the interval, 353 F2 intercross mice were bred and screened with SSLP markers. 15 informative mice were identified with a genetic recombination that localised the plt2 mutation to an interval of 8.6Mb between D7Wehi28 and D7Mit46, positioned at 121763753bp and 130357844bp respectively, on the UCSC May 2004 genome assembly (www.genome.ucsc.edu) (Figure 4C). One mouse that defined the centromeric end and two mice that defined the telomeric end of this genetic interval were progeny tested to confirm their non-thrombocytotic phenotypic designation (Figure 4C). EXAMPLE 8

ENU mutagenesis has been utilized to identify a novel mouse pedigree with heritable thrombocytosis and hepatomegaly. Using well-characterised genetic polymorphisms between two inbred strains of mice this mutated locus has been mapped to an 8.6Mb region on chromosome 7 which contains at least 80 recognised genes. There is no specific gene in the defined interval previously implicated in platelet homeostasis, suggesting that the plt2 mutation is acting on a novel gene, or acting via a novel function of a known gene.

The plt2/plt2 mutant mice display a relatively mild thrombocytosis that appears to be driven by excessive thrombopoietin production. The serum thrombopoietin level is elevated in these mice and the mutation is unable to rescue any of the thrombocytopenia displayed by MpY ^A mice indicating that the action of the plt2 mutation is dependent upon signalling through the c-Mpl receptor.

Thrombopoietin is primarily produced in the liver and in one embodiment the hepatomegaly alone displayed by the plt2/plt2 mice may cause the observed thrombocytosis. However, in another embodiment this mutation specifically up regulates thrombopoietin production. While no specific increase in TPO transcription was observed, TPO protein content per gram of liver weight was increased. These results may reflect either a subtle increase in TPO transcription in the liver below the level of detection using current technology or an alteration of TPO at the translational level. The translation of human TPO mRNA is reduced by the presence of inhibitory elements in the 5 '-untranslated region and these inhibitory elements are conserved between humans and mice (Ghilardi N. et al, Blood 92:4023-4030, 1998; Cazzola M., Blood 95:3280-3288, 2000). Indeed, familial forms of human essential thrombocythemia are described with mutations in the TPO gene that remove this translational inhibition and cause thrombocytosis associated with increased thrombopoietin (Cazzola M., 2000, (supra)). However, mutations in the TPO gene are not responsible for this mouse phenotype as the plt2 mutation has been mapped to chromosome 7 and the murine thrombopoietin gene is located on chromosome 16. Interestingly, there are human cases reported with thrombocytosis associated with increased thrombopoietin but no mutation in the thrombopoietin gene (Wiestner A. et al, Br. J. Haematol. 770:104-109, 2000; Hankins J. et al, J. Pediatr. Hematol Oncol. 26:142-145, 2004) and some reactive thrombocytoses also display increased circulating TPO suggesting that alternative or additional pathways regulate TPO production. Intercrossingp//2/^Λ2 and MpI^"^ mice produced animals with a range of platelet counts. This breeding strategy allowed molecular dissection of the interaction between different components in the thrombopoietin-dependent pathway of platelet production on an isogenic C57BL/6 background. As previously documented, the MpT^1' mice in this series displayed severe thrombocytopenia (Gurney A. L. et ah, 1994, supra; Alexander, W. S. et ah, 1996, supra), a phenotype that is identical to the TPO knock-out mouse. A gene dosage effect has been reported for mice heterozygous for the TPO knock-out allele (7po^+/") with these animals displaying a mild thrombocytopenia with platelet counts 67% of wild-type mice (de Sauvage F. et al., 1996, (supra)). In contrast, in this study, mice heterozygous for the c-Mpl knock-out allele displayed a mild thrombocytosis compared to wild-type C57B1/6 animals with platelets elevated by 18% compared to wild-type. The highest platelet counts of the F2 intercross were observed in plt2/plt2 Mpt'^~ mice which have increased serum TPO and decreased c-Mpl. This effect of c-Mpl gene copy on platelet count is somewhat counter-intuitive, but there is precedent for reduced c-Mpl expression being associated with increased platelet counts in humans. The recently described c-Mpl polymorphism, MpI Baltimore, results in an amino acid substitution at position 39, decreased c-Mpl protein expression on platelets, and was associated with thrombocytosis in the carriers of the polymorphism (Moliterno A. R. et al., Proc. Natl. Acad. ScL USA. 707:11444-11447, 2004).

Together these observation are consistent with the c-Mpl receptor having two independent but related functions in regulating circulating platelet mass. As originally described, TPO signals through the c-Mpl receptor to stimulate megakaryocyte and megakaryocyte progenitor proliferation and maturation, driving platelet production. However, as shown herein c-Mpl also acts as the primary mechanism by which circulating TPO is removed from the circulation. In times of severe thrombocytopenia, reduced platelet-mediated clearance leads to increased serum TPO concentration and enhanced megakaryopoiesis. This requires that intra-cellular signalling through the c-Mpl receptor is achieved at relatively low levels of receptor density, but that increasing levels of c-Mpl receptor above this level can still incrementally participate in TPO clearance. EXAMPLE 9

The gene affected by theplt2 mutation is an organ specific tumor suppressor gene

The incidence of hepatoma development in wild type and plt2 mice was compared (see Figure 5).

In older plt2/plt2 mice (which are derived from C57BL/6, the incidence of hepatoma was 67% (n=9). In wild-type C57BL/6 mice after total body radiation the comparative incidence was 11% (n=9). In plt2/plt2 mice after total body radiation, the incidence of hepatoma was 100% (n=9).

EXAMPLE 10

Further fine mapping of the 8.6MB interval on mouse chromosome 7 was undertaken to localise the specific gene affected by the plt2 mutation. This gene and its encoded products regulate the production of TPO and may provide a mechanistic explanation for some of the clinical observations related to increased circulating TPO in the setting of reactive and pathological thrombocytosis.

Fine mapping was undertaken as described in Example 8. The details of informative mice are tabulated in Table 7. Table 8 provides the primers and detection method used for each marker in the further fine mapping study. Genomic DNA was prepared from the liver of experimental mice and amplified by PCR with the oligonucleotide primers listed in Table 8. The PCR product was then sequenced in a reaction using Big Dye Terminator and analysed on an Applied Biosystems automatic sequencer according to the manufacturers instructions. A set of nested primers were used to sequence JaxSNP4 set forth in Table 8. As a result, the interval was reduced to 0.66Mb between Celera SNP 12 and Celera SNP 17.

EXAMPLE 11

Individual genes in region between Celera SNP12 and Celera SNPl 7 sequenced to identify theplt2 mutation within the nucleotide sequence of NCBI Accession No. AK00447

The known genes in the interval between Celera SNP 12 and Celera SNP 17 (see Table 9) were sequenced to identify the plt2 mutation. Specifically, exon and exon-intron boundries were sequenced in two animals that were homozygous for C57BL/6 markers across the region of interest, one intercross animal that was Balb/C across the region of interest and one control C57BL/6 mouse. Genomic DNA corresponding to gene exons identified in Table 9 was prepared from the liver of experimental mice and amplified by PCR with the oligonucleotide primers listed in Table 10. The PCR product was then sequenced in a reaction using Big Dye Terminator and analysed on an Applied Biosystems automatic sequencer according to the manufacturers instructions.

Direct sequencing of PCR products identified a point mutation in Exon 2 of AK00447 (1190003J15 Riken cDNA). The point mutation was found in both Backcross animals that were C57BL/6 in the region of interest that was absent in the intercross animal that was Balb/C in the region of interest and the C57BL/6 control. The sequencing profiles in the region of the mutation are shown in Figure 6. This mutation causes a predicted amino acid change from Tyrosine in the wild-type (encoded by nucleotides denoted TAC) to Cysteine (encoded by nucleotides denoted TGC) in the plt2/pH2 animals.

An allelic discrimination assay was developed which confirmed this mutation in a further 20 plt2/plt2 mice (see Figure 7). Specifically, Genomic DNA was prepared from a tail biopsy taken from experimental mice at approximately 3 weeks of age. DNA was amplified using primers specific for exon 2 of AK004470 (5'- GGCACCTATAAGCTGTTCTTCGA-3 ' and 5'-ACCCTGACACTCACCTCTACATAG-S'). The PCR product was then identified as mutant or wild type by measuring specific fluorescence associated with the mutant or wild- type fluorescent-tagged oligonucleotide probe (wild-type: VIC- CAGAGCGCTACTGGAAA, plt2 mutant: FAM- AGCGCTGCTGGAAA). The PCR reaction and alleleic discrimination detection were performed on an ABI Prism 7900HT Sequence Detection System according to the manufacturers instructions.

EXAMPLE 12

Genomic DNA comprising regions encoding TRP (short form) and TRP-2 (long form)

The nucleotide sequence of NCBI Accession No. AK00447 contains three exons as shown in Figure 8. The plt2 mutation is contained in the second exon of the structure. Sequencing of the cDNA from the liver of both wild-type and plt2/plt2 animals identified a gene structure as predicted in AK00447 (herein referred to as the short form) as well as a larger gene containing an additional previously unidentified 5' exon (herein referred to as the long form or TRP-PLT2/77?P-Pir2). The long form is also represented in Figure 8 which also indicates the position of the mutation in the penultimate exon.

EXAMPLE 13

Genes are differentially expressed in plt2/plt2 animals

Eight microarray chips were used to measure the liver gene transcription profile from 4 plt2/plt2 and 4 sex-matched wild-type mice at 5 weeks of age (Figure 12). Microarray data were normalised between individual gene chips with robust multiarray averaging. Linear modelling was then performed to examine transcriptional differences between replicate arrays using software designed by the Bioinformatics Division at the Walter and Eliza Hall Institute (http://bioinf.wehi.edu.au/affylmGUI). Using this approach, there was no significant differential gene expression within the 4 wild-type biological replicates, nor within the 4 plt2/plt2 biological replicates indicating relatively homogeneous gene transcription profiles for each genotype. However, analysis comparing wild-type and plt2/plt2 arrays demonstrated significant differential gene expression between the two genotypes.

Different methods are available to quantitate the statistical significance of differentially expressed genes in a microarray experiment. The Holm method (Holm S., Scandinavian Journal of Statistics, 6:65-70, 1979) uses a sequential Bonferroni procedure to adjust for multiple testing error and gives a p value for significance. Using this method, the twenty most differentially expressed genes were significantly different between plt2/plt2 and wild-type genotypes at a corrected p=0.01 for the twentieth ranked gene, Abccό. This is a rigorous method for correction of multiple testing error in microarray experiments. A more commonly used statistical analysis for assessing differential gene expression is based on the False Discovery Rate (FDR). This method suggests that for very large data sets analysed in gene microarray experiments, it is more appropriate to report the proportion of statistical errors, a q value, among a group of differentially expressed genes (Benjamini et ah, Journal of the Royal Statistical Society B, 57:289-300, 1995; Reiner et ah, Bioinformatics, 19:368-375, 2003). Using this statistical method, the rank of individual genes was unchanged, but 156 genes were differentially expressed between plt2/pU2 and wild-type genotypes at q<0.01. The threshold of statistical significance is arbitrary in this experiment. However, it is apparent using either statistical method that there are multiple differentially expressed genes between plt2/plt2 and wild-type liver. Biological correlation of changes in gene transcription between plt2/plt2 and wildtype livers was examined by grouping the most common biological functions in the 50 most differentially expressed genes using Affymetrix gene ontology software (http://www.affymetrix.com/analysis/index.affx). Changes in genes associated with metabolic function were most commonly observed with some of these genes being induced and others repressed in plt2/plt2 mice. Four genes {Ran, Ect2, CdknJc, Igfbp2) were recognised to have a role in cell cycle and growth consistent with the hepatomegaly observed in these mutant mice. Repression of 4 genes (CP, Hc, F5, Saa4) associated with the acute phase response and blood coagulation was noted in the top 50 genes and a further 5 similarly grouped genes (Pigr, polymeric immunoglobulin receptor; Serpingl, serine protease inhibitor Gl; Defl>l, defensin Bl; Proc, protein C; Fl 2, coagulation factor XII) were also repressed in the 100 most differentially expressed genes. Consistent with previous quantitative real-time PCR data there was no difference in TPO transcription between plt2/plt2 and wild-type mice measured by gene microarray.

EXAMPLE 14 Cloning of the gene mutated by the plt2 mutation

To confirm the genetic sequence of the uncharacterised gene identified by positional cloning as the mutated plt2 gene, different predicted versions of murine TRP were isolated and cloned. cDNA from liver was amplified with oligonucleotide primers specific for the predicted 5' UTR of the short (GenBank accession number BC051545) and long (Genbank BE370046) mRNAs based on EST data from the UCSC database (http://www.genome.ucsc.edu). Amplified PCR products derived from wild-type and plt2/plt2 mutant liver were ligated into the pEF-BOS expression vector (Mizushima et al, Nucleic Acids Res., 18:5322, 1990) containing a FLAG epitope tag sequence and then cloned in the DHlOB strain of E. coli. Sequencing of these cloned nucleotide products confirmed that the predicted TRP sequence from the UCSC database was correct and verified the presence of the A to G point mutation in liver cDNA from plt2/plt2 mice (Figure 13). Open reading frames from TRP short and TRP long transcripts predicted the same protein with the longer version of the gene having an additional exon at the 5' end of the transcript potentially translated into an additional 23 N- terminal amino acids (Figure 13). Both mutant clones predict a tyrosine to cytosine substitution at residue number 98 of the long peptide (Y98C). Cloning demonstrated that both short and long forms of wild-type and plt2 mutant transcript were present in the liver from wild-type and plt2/plt2 mutant mice respectively. EXAMPLE 15

TRP transcript is expressed in a variety of organs inplt2/pH2 and wild-type mice

To begin to investigate the physiological role of this uncharacterised gene, the expression of TRP mRNA was measured in a variety of tissues by semi-quantitative reverse- transcriptase (RT) PCR. Using primers specific for the 5' untranslated region of TRP short and long, mRNA expression was examined in a tissue panel from wildtype and plt2/plt2 mice (Figure 14A). In both wild-type and mutant mice, the liver was the most abundant source of both TRP short and long transcript. At high PCR cycle number, transcript was also detected in lung, kidney, spleen, thymus and brain, identifying these as sites of low TRP transcript expression. There was no measured difference in expression pattern of short or long versions of TRP transcript between wild-type and plt2/plt2 mutant mice. Consistent with the plt2 mutation acting extrinsically on the haemopoietic system to produce thrombocytosis, no TRP was expressed in bone marrow from either wild-type or mutant mice.

EXAMPLE 16

Transthyretin-related protein is expressed in hepatocytes from wild-type mice but is markedly reduced or absent in plt2/plt2 liver

Translation of the protein product from the wild-type and mutant TRP gene was next examined. To develop an antisera that would detect both mutant and wild-type protein products of the TRP gene, peptides derived from the predicted translation of sequence from the first (MSSRTAPRLMTLQC) and second (TTHVLDTASGLPAC) exons of TRP long were injected into one rabbit. As the plt2 mutation is present in the third exon of TRP long, any antibody response elicited from the rabbit would be predicted to recognise both wild-type and mutant protein. To determine the specificity of the antisera produced by immunisation, cells from the 3T3 cell line were transiently transfected with short or long wild-type constructs of murine TRP that contained a FLAG tag. Western blotting of cell lysates after transfection revealed a single band that was detected by both anti-FLAG antibody and rabbit antisera but not detected with sera from the same rabbit prior to immunisation. This single band of 15 kDa was consistent in size with that predicted from the amino acid sequence. The ability of the antisera to detect endogenous TRP protein was next examined in protein lysates derived from wild-type livers. In all wild-type liver samples tested, rabbit antisera detected a single band of approximately 15 kDa in size. When these endogenous liver lysates were processed in parallel with both short and long wild- type TRP constructs it was not possible to determine if this single endogenous band represented the protein product from TRP short or long, particularly given both constructs were of different size to endogenous protein because of the addition of a FLAG tag (Figure 14B).

The organ distribution of endogenous transthyretin-related protein in mice was then examined in protein lysates derived from a variety of tissues by Western blotting (Figure 15). In wild-type mice, a single band of approximately 15 kDa in size was detected in the liver. No band was appreciable in tissue lysates from any other organ examined or from protein derived from serum. In marked contrast to wild-type mice, no transthyretin-related protein was detected in lysates from any organ from plt2/plt2 mice by the same method (Figure 15A). These data suggested translation of TRP by liver cells is aberrant or the protein unstable in plt2/plt2 mice. To confirm this finding and determine the specific cell type expressing TRP in the mouse, hepatocytes were isolated from wild-type and plt2/plt2 mutant mice and examined for TRP expression by confocal microscopy (Figure 15B). In wild type animals, TRP protein was clearly present in isolated hepatocytes, distributed predominantly in the cytoplasm. In contrast, hepatocytes isolated from plt2/plt2 mice demonstrated no fluorescent signal, indicating the translated product of the mutant TRP gene was either markedly reduced or absent in these cells. These data indicate that under normal physiological conditions TRP is produced in the hepatocytes of wild-type mice. Plt2/plt2 mice transcribe the product of the TRP gene in liver, but transthyretin-related protein is not detected in hepatocytes suggesting the plt2 mutation is responsible for a defect that alters the normal expression of this protein in liver cells.

EXAMPLE 17 Predicted protein structure of TRP in wild-type andplt2/pU2 mice

The absence of a detectable protein product from the mutant TRP gene of plt2/pU2 mice indicates that the tyrosine mutated in these animals is important for normal function of the protein. To examine whether this specific amino acid was conserved throughout evolution, predicted protein homology was studied in different organisms. A BLAST search

(http://www.ncbi.nlm.nih.gov/) was performed with murine TRP sequence to derive homologous sequences from bacteria (E. coli), plants (A. thalianά), and invertebrates (D. melanogaster and C. elegans) as well as transthyretin sequence from mice and humans. Homologous nucleotide sequence was then used to align predicted proteins with bioinformatic software (http://www.ebi.ac.uk/clusthalw) (Figure 16). Consistent with a previous alignment of the transthyretin protein family (Eneqvist T. et ah, 2003 (supra)), TRP amino acid sequence is highly conserved throughout evolution. TRP is also closely related to transthyretin, with 29% of amino acids identical between mouse TRP and transthyretin. Conservation of the tyrosine mutated by the plt2 mutation in TRP, from bacteria to mouse shows that this residue is important for normal protein function. This tyrosine is also conserved between TRP and transthyretin (Figure 16).

The protein structure of transthyretin is well characterised (Blake et ah, J. MoI. Biol, 88:1-12, 1974; Hamilton et ah, J. Biol. Chem., 268:2416-2424, 1993). Given the high degree of amino acid homology between TRP and transthyretin, a model of mouse TRP was generated from the structural information determined from fish transthyretin (Sparus aurata) using Swiss model software (Guex et al, 1997 (supra)). This modelling predicts that murine TRP adopts a barrel structure similar to transthyretin. In this model, the tyrosine mutated in plt2/plt2 mice (Y98C), is placed in the highly structured helix area of the molecule (Figure 17). The replacement of the normal tyrosine residue with an aromatic side chain with a sulphur- containing side chain of cysteine in the plt2 mutant is likely to disrupt this highly ordered helix structure.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Table 1

Summary of sequence identifiers

Table 2

Amino acid sub-classification

Table 3

Exemplary and Preferred Amino Acid Substitutions

Table 4

Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nm leu

D-arginine Darg L-N-methyllysine Nm Iy s

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-am inobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanlne Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-m ethylthreon ine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyI)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 , 3 -dipheny lpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3 -guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtφ N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-fø?-hydroxyphenyl)glycine Nhtyr Iw-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-/-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N~(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-rhethylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylρropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Nm be ethylamino)cyclopropane

Table 5 Hematological Profile of plt2/plt2 mice

wild-type plt2/plt2

Peripheral Blood

Platelets (x10⁹/L) 1386 ± 223 2038 ± 347 **

MPV (fL) 7.3 ± 0.5 7.2 ± 1

PDW (%) 51.7 + 2.7 48.7 ± 4

Hematocrit (%) 51.2 ± 2.8 50.2 ± 2.9

White Cells (x10⁹/L) 9.8 ± 1.7 10.6 ± 2.3

Neutrophils 0.92 ± 0.34 1.12 + 0.58

Lymphocytes 8.3 + 1.5 8.7 ± 2.1

Bone Marrow

CFU-S 7.4 + 1.5 6.7 ± 2.3

Megakaryocyte-forming progenitor cells

Stimulus

BM IL-3 4.5 ± 1.3 9.8 ± 3.5 *

TPO 1.7 ± 0.6 3.5 + 0.6 **

SCF+IL3+EP0 18.3 ± 5.5 28.8 + 4.6 *

Spleen SCF+IL3+EP0 3.8 ± 0.5 14 ± 4.3 **

Other Colony-forming progenitor cells (#)

Stimulus

BM GM-CSF

Neutrophil 16 ± 2 17 ± 3

Granulocyte/Macrophage 4 + 2 9 ± 5

Macrophage 24 ± 5 22 ± 7

Eosinophil 3 ± 1 3 + 2

Megakaryocytes

BM - Quantification

Histology (per 10 hpf) 70 ± 13 108 ± 15 **

Flow Cytometry (per 1000 cells) 0.84 + 0.18 1.2 + 0.1 **

Spleen - Quantification

Histology (per 10 hpf) 5.7 ± 2.1 15.7 + 9.3 *

BM - Ploidy (% frequency)

2N 30 + 5 24 ± 4

4N 12 ± 2 10 ± 1

8N 7.5 + 2.6 8.2 + 2.6

16N 38 + 5 42 ± 4

32N 12 ± 4 16 + 4

64N 0.4 ± 0.3 0.3 ± 0.2

For peripheral blood parameters, n=107 for each genotype. For bone marrow assays, n=3-4 for CFU-S and progenitor assays and 6-7 per genotype for megakaryocyte assays.

*p<0.05 **p<0.01

(# Supplementary table 5 contains the complete data set to a wide range of stimuli.) Supplementary Table 5 Colony-forming progenitor cells to a wide range of stimuli.

Granulocyte/

Blast Neutrophil Macrophage Macrophage Eosinophil Megakaryocyte

Bone Marrow

GM-CSF wild-type 16 ± 2 4± 2 24 + 5 3± 1 plt2/plt2 17± 3 9+ 5 22 + 7 3± 2

G-CSF wild-type 10+ 4 plt2/plt2 12 ± 2

M-CSF wild-type 3+ 2 2± 1 39 ± 7 plt2/plt2 2± 1 4± 1 52 ± 5

IL-3 wild-type 5± 2 12 ± 1 10 ± 3 12 + 5 2 + 1 5± 1 plt2/plt2 12 ± 7 22+ 3 15 ± 6 17 + 5 2 ± 1 10+ 4

SCF wild-type 6± 1 16 ± 5 1 ± 1 plt2/plt2 7 + 3 12 ± 3 1 ± 1

IL-6 wild-type 8± 2 1 + 1 0 plt2/plt2 10+ 4 1 + 1 1 + 1

FL+LIF wild-type 4± 1 0 + plt2/plt2 5± 1 1 + 1

EPO wild-type 5+ 3 plt2/plt2 7± 2

TPO wild-type 2± 1 plt2/plt2 4± 1

SCF+IL-3+EP0 wild-type 6± 3 19+ 3 13+ 2 15 ± 6 2 + 1 18 ± 6 plt2/plt2 16± 3 17± 2 12 ± 3 15 + 6 2± 1 29 ± 5

SCF+G-CSF wild-type 8 + 2 20 ± 2 2+ 2 2± 2 plt2/plt2 11 ± 4 19 ± 5 2+ 2 3± 1

Spleen

M-CSF wild-type 1 + 1 plt2/plt2 2 + 1

IL-3 wild-type 1 ± 1 1 ± 1 0 0 0 plt2/plt2 1 ± 1 2+ 0 1 ± 1 1 ± 1 1 ± 1

SCF+IL-3+EP0 wild-type 0 1 ± 1 0 4+ 1 plt2/plt2 2+ 1 2+ 2 2± 3 14 ± 4

Table 6

Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

DlMit231 ACCCACAATTGCCTGTGG GTCTTTGCAAGCCACCAAAT 1 8.7

DlMit212 TCTCATGAGGTGTGTGAGTTTG GGATCCCCTTGCTTCACTAA 1 19.7

DlMit22 TCTGTTCCCTCTACACACATGC CTACCATGCTTACCTAGGTCCTG 1 32.8

DlMitl32 TATTGTTTATGGAAATTGGACCC CATCTCTGAAGGAAAAAGTGCA 1 43.7

DlMit84 TGTCTCCCCAAAGTAGCAGG GTGATGCAGGAGTTTCTGCA 1 56.8

DlMit26 GAGGAATCTTGAATGGGCAA CTGACAACACCCTCTGGCTT 1 64.5

DlMitl4 GCCAGACAGGGCTACATTGT AGACTGAACTCTGGCCTCCA 1 82

DlMit206 TGAGGCACCTTTGTATTCAGC CCAGATGTCTTTGAACATTCTCC 1 94

DlMit291 TGCCCGTGATAACCCTATGT TTGTGCACAAGCAGGAGC 1 103.8

DlMitl55 ATGCATGCATGCACACGT ACCGTGAAATGTTCACCCAT 1 115.8 ≤

D2Mitl L I I I I I CGTATGTGGTGGGG AACATTGGGCCTCTATGCAC 2 2.2

D2Mit83 TCTCTCTCCCACCATTCCC TATTAGCCTGCCCCACACAC 2 21.9

D2Mit271 TTCTGAATTGATGCTGTGATCC TGCTTCAATCTCATTGCCTG 2 44.8

D2Mit420 ACATATCTGTATGTGAATGTTTGCG GTTCCTTCTTCACAGGGAAGC 2 54.6

D2Mit255 GCAAGTGTGATCTGGGTGC TGAGCACACTTACACTGTGGTG 2 56.8

D2Mit224 GGAACTACTAACTAGGTGAATTCACTG CTTGAGAATGTTATGCTAGGAAGG 2 61.2

D2Mit285 TCAATCCCTGTCTGTGGTAGG TATGACACTTACAAGG I I I I I GGTG 2 72.1

D2Mit48 GCTCTGCAGAAGATGCTGC GCTGAGACGCAGAGTCGC 2 73.2

D2Mit229 TCAGATGCCATTGGTCTCAG ACCACACAAATAACACTGTATGCA 2 85.2

D2Mit265 AATAATAATCAAGGTTGTCATTGAACC TAGTCAAAATTC I I I I GTGTGTTGC 2 91.8

D2Mit230 GCCTGCTTGCTTCTCAGC CAGGCTTCCTCCCTGAAAC 2 97.3

D3Mitl64 GCTCCTGGGAAAGGAAGAAT GATACTTGGGGTTGTGCATACA 3 2.2

D3Mitl78 GTGTACATGCACATGAGTTTGC CAGAGAGTGCTAGGCTCAAACA 3 12

D3Mitl37 CTGGTATGTGCATGTAACCTTAGC ATGTAAAAGTGCTTTATCATTATCACG 3 26.2

Table 6 continued

Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

D3Mit40 CAGCTGGTCTAACTATCCCCC CCTTATTAAGTGCATGACCTTGC 3 29.5

D3Mltll CCAACCACAGTAACACATGT TGGAGACCAATGCGAACAAC 3 37.2

D3MitlO6 ACTTGTGCATGGTGTGTATGC TGTGATGGCACCTTTGGTAA 3 40.4

D3Mitl4 ATTGCGGTTAAAGTTTGCTT TCCTGCAAATTGTCCTCTGA 3 44.8

D3Mit38 CTGAACCAGAAAGTTG I I I I I CTG ACCATGGCCAGCTTCTAATG 3 49.2

D3Mit86 TGCTCAACATAAAATGTCTGGC AAGCACAGAAACATCTCTCACG 3 54.6

D3Mitl47 TCTGCCTCTGTTAGATAGATATCCG TTGTTCATCTATCCTCTGAAGTTCC 3 59

D3Mitl63 TGGATACATACATATACATGGAAATGC TTTCTCCAGACCCATGAACC 3 66.7

D4Mitl01 GAATGTCACTCGAGACTACTTTGG AGTTTCATCTAGGTTTTCCTGCA 4 5.5

D4Mit236 TCTTAGCATGCTTACCGCCT GGCCCGTAGGATGACTGTC 4 16.4

D4Mitlll TGAGATGTTGGCATACTGTGTG TTGTAAGCCTAACTTTATCCACCC 4 25.1

D4Mit87 ACAGGTAGGAATGGAGCCCT TCATCCCTTTGCCAAAGC 4 30.6

D4Mit27 GCACGGTAG I I I I I CCAGGA TGGTGGGCAGGCAATAGT 4 35

D4Mit249 AAAAGCAAACACTAACACTTGGG TTGCTTTGGTTTGATCTTTGG 4 54.6

D4Mit204 CTGCTGCAGCGATTCTCTC TCAGGCACCTAAGTACATGTGC 4 61.2

D4Mit251 AAAAATCGTTCTTTGACTTCTACATG TTTAAAAGGG I I I C I I I ATCCTGTG 4 66.7

D5Mitl45 TATCAGCAATACAGACTCAGTAGGC TGCCCCTTAAATTCATGGTC 5 0

D5Mit387 CCCCATGTATCTCTAGATTAACAATG GCACTCGTGTACATAACCAAATAC 5 10.9

D5Mit255 CCCTGTGCTCTGGATTAGTTG TCAAGACCAGCATCAAACCA 5 25.1

D5Mitl8 CTGTAGTGGGTGGTTTTAAAATTG ATGCCACTGGTGCTCTCTG 5 32.8

D5Mit24 CACTTGCCACACAGCAGG CGTGCATGCACTAGTGTGTG 5 45.9

D5Mit95 TGTTCTTGTCCATGTCTGATCC AACCAAAGCATGAAACAGCC 5 57.9

D5Mit99 CAGAAAAGAGAAAACGGAGGG TTCCTGCTGCCTGAAGTΠT 5 73.2

D5Mit287 TAAAGACCTCATTGCCCCAC GTGATCGGAGGCAAGATAGC 5 82

D6Mit74 CATGTGCAGTGTAAGTAAGACCTC TCTCCTCCATCCTTCTCCAT 6 10.9

D6Mitll8 TTCAGACCTTG I I I I I AAAAAGTGG GAGCCCTTTAAAATAGTAAGTATGGC 6 12

D6Mitl86 GGTiTATAACTCCAGTTCCTGGG ATGAGAAAACAGACACTCATTGTAGG 6 20.8

Table 6 continued

Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

D6MitlO2 CCATGTGGATATCTTCCCTTG GTATACCCAGTTGTAAATCTTGTGTG 6 31.7

D6MitlO5 CTGTCTCCACTACTTCTATTCCTGG CAAAAGCCTTATATATTACACCTCACC 6 41.5

D6Mitl35 CCTAACAGTTCAATTTGTCAGCC CCAGCCCCCAATTTGATATA 6 51.4

D6Mitl4 ATGCAGAAACATGAGTGGGG CACAAGGCCTGATGACCTCT 6 63.4

D6Mitl5 CACTGACCCTAGCACAGCAG TCCTGGCTTCCACAGGTACT 6 66.7

D7Mitl78 ACCTCTGATTTCAGAACCCTTG TAGAGAGCCACTAGCATATCATAACC 7 0

D7Mit25 AGGGGCACATGTTCAACTATG GGTTGTTTCCAGCTTTGGG 7 13.1

D7Nlitl58 CTTCATCTGAGCCTGGGAAG ACTGTAGACCCATGTTCTGATTAGG 7 18.6

D7Mit84 AACTTGCCAGCCATGGTAAG AGTTAGAAGACCCACCCTAAATCC 7 26.2

D7Mitl81 TACAGTTTAAAA I I I I C I I I GCGTG AGTTTGGTCTCTTGAATCTACATGG 7 28.4

D7Mitl26 AGTGCTGGAATTAGTTATATAACACCA AAGGGAAATGTCTCTCTCTCCC 7 37.2

D7Mit281 TTCCTCTACCTCCTGAGCCA GCCACAAGGAAGACACCATT 7 38.3

D7Mitl01 TACAGTGTGAACATGTAGGGGTG TCCCAACATGGATGTGCTAA 7 45.9

D7MitlO5 AGCAAAGTAAGGCAGACTTTGG AGGAGAGGCAGAACATGGAA 7 49.2

D7Mit71 CCACCTGGAATACATGTAACCC TAAGATCCAAGAGATGGGTTAAGC 7 53.6

D7Mitl89 CCTGGTGAGTAGAGAGGGAGG AGGAACATATGTGCACATGCA 7 67.8

D8Mitl55 TTGGACAGGGAAAATTCTGC TGAGGACTTGCTTTAAGAGTACTCC 8 0

D8Mitl24 CAACTGTGTATCATAAACTGGGAA GAAGAATCACTCAGCAGTGTATGG 8 4.4

D8Mit4 CCAACTCATCCCCAAAGGTA GTATGTTCAAGGCTGGGCAT 8 12

D8Mitl90 CTTTGTTGCTGTiTCATTCTGG AGTCATATACAAGGTCAACCTGAGC 8 21.9

D8Mitl29 GTCAAAGGTAAAACATATGGACACA ACCGTGCAGGTTCTCTGC 8 32.8

D8Mit75 TGGTGACTATGGTTGCCTGA GCU I I I GGAGAGCAACACT 8 38.3

D8Mit267 CTTGACCATAAAGAGAAAAAAATGC GAGGTACATGGTATATTTGCCTACG 8 42.6

D8Mit242 TGTGCAACCAA I I I C I I CCA CCCATGATTTATTCAGACTGAGG 8 47

D8 M itl 12 ATATCAGGCATGCATTATGATCC TCTCTCTAGTGGGATTATCAACACA 8 55.7

D8Mitl4 TTTTCACACTCACGTGTGCG GTCTCTCCTTCCTGGCGCTG 8 68.9

D8Mit56 ACACTCAGAGACCATGAGTACACC GAGTTCACTACCCACAAGTCTCC 8 75.4

Table 6 continued Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

D9Mit90 AGGAGTCTCCCTGTACCTACACC AAGTAGAGGGGAGGAATGAACC 9 7.7

D9Mit67 ATCTCCTCCCCGAACTGC AGAACTGCCTTTAACTTCAGTTGC 9 13.1

D9Mit2 GTGGTCTGCCCTCTTCACAT CAAAGCCAGTCCAACTCCAA 9 13.1

D9Mitl91 GTAAGTATGCCTGCGGAGGA CAGAAGACCGC I I I I CAAGG 9 20.8

D9Mitl30 TCAATTTATTGTTGCAGTTGGC AACACAGCATGAGATGGATACG 9 21.9

D9Mitl33 CGCTGTACACATCAACATTATCTC TCTCTTTCAGAAAGTGC I I I I GG 9 41.5

D9Mit275 GGAAAGAACAGTTTCAAAAAAACG TGTAACAAAAGATCCAAAAATAGCC 9 43.7

D9Mitll5 TCCAGACTCCTGGGAACTACA TTTCCCAGCCAGTAAAGGC 9 54.6

D9Mit51 CCAACAGGCAGTTAGGGCTC GCCAACTGTTTCCCAGAGAA 9 59

D9Mit278 CTAAACAGATGGAAAATAGAAACACA GAGACCAACAAACCTCAGAACC 9 61.2

D9Mitl9 CCAAACACAACCCCTCAGAA TCATGGCTTCAAGACTGCTT 9 68.9

DIOMitδO CAAAAAAAACCCTGATTCTACCA GTGTGCATATGGCAGTAACTTTG 10 2.2

D10Mitl06 TGTGCTGGCATTCACTCTTC ACAGTGACCTTCTGTGTAAAATGC 10 12

D10Mit20 CACCCTCACACAGATATGCG GCATTGGGAAGTCCATGAGT 10 25.1

D10Mit42 GCATTCAGAAGCTGGAAAGG TGCCCAGCATATGTTTAAAGG 10 41.5

D10Mit68 GGCATCAGCAACCCTAGAAC AATAAAGGAGGAAGGTAATGACCA 10 51.4

D10Mitl34 AATCCTAGAAGATACATGCTGATGC AGTTAAGCACCAAAATTGAAATCA 10 57.9

D10Mitl4 AGAGGGGACAAGGAGAGACC AAGGTTTGGGTTCAGTTCCC 10 69.9

DllMit62 GAATAACCCATGTTTATATCGGTG CTCTGGACTTGTGTTCTATGCC 11 2.2

DllMit20 CCTGTCCAGGTTTGAGAGGA CTTGGGAGCCTCTTCGGT 11 19.7

DllMit208 TTTTAAAAAAGGATGAACATAAATGTG AGGACAACCAGGGCTATGTG 11 29.5

DllMit29 TTGAGGCATGAGGGGATTAG TTTCCGTCATTGCTAAAGGG 11 37.2

DllMit39 TTTCATGACCCCTAATTTCCC GTGGGTGTGCCTGTCAATC 11 44.8

DllMit212 CTCTGGTCTCTCTGTATACATGTGC AGCAACTGGGGCATTTAATG 11 48.1

DllMitlδl TCATCTGTCCCTGCCTCC TACTGTATTTGATACATATACGTGCCC 11 69.9

DllMitl68 CAGGGATTTGAC I I I I AACCTCC GAAATGGCTCCTACAACCTCC 11 75.4

DllMitlO4 CACATGATCATACACTGTTTCTCC GCCACGTGTTCTAACCTTCC 11 82

Name Sequence Sequence Chromosome Location (cM)

D12Mit38 TCTGAAGTTTGAATGGTTGTGG CGTGTTCATTTTGCCATTGT 12 1.1

D12Mitl82 GTACATACAATACATCACACAAACGG GGCAAGAAAACAGACCAATAGG 12 2.2

D12Mitl36 TTTAATTTTGAGTGGGTTTGGC TTGCTACATGTACACTGATCTCCA 12 9.8

D12Mitl71 TGCCCACACATAAAAATGTAGC TCAGTCTGCTCCTGTCATGG 12 9.8

D12Mitl72 AACTGAAATCGCATTACAAAACC TAATATTGCGAGTTAGAAATGACCA 12 17.5

D12Mit4 ACATCCCCAGCTCTTGTTTG AAACCAAACCAAAGAAGCTTAGG 12 28.4

D12Mlt5 CACATAGACCAGACAGGCATGCGT CAAGGTCACGTTGCTAGCTAGGAA 12 31.7

D12Mitl32 CCATATACATTTCTAACACCCTTGC AGAACTTACTTCTAGTGGAGACAATGC 12 47

D12Mitl50 CTTGTCAAAATTTCTGTTGTTTTACA AAAGGATTTTGTCACTAAGACATGG 12 56.8

D13Mit55 TCAATATTAACTGCTAGCATGGTT GCTΠTCCTCCCCAAACATT 13 0 OO

D13Mit64 CCTCAGCACCAAAAAAGGAC ACATCAGTGACCAGGCATCA 13 15.3

D13Mit66 CTGCCCTGCTTGTTTGGG CCAACTTCAGCCATAAGACAG 13 24

D13Mit99 CAACAGGCAGATTTGGTGG TATAGTGGCAACTTTCAGATGGA 13 25.1

D13Mitl47 CATCCAGGAAGGCAATAAGG CAAATGCACAGTGCCGAG 13 37.2

D13Mit37 AACTGTCTCCTTCCTG I i I 1 I CC AGACCACTGACTTTGCAGTAAGC 13 37.2

D13Mitl51 TCCTGCAAAAGTGGAGCC TGGAAACAAGCTCTTGGAGG 13 40.4

D13Mit76 ATGCACCTGTCTAAATGTGTGC AGAGGGACTGTGGGACTGTG 13 42.6

D13Mit78 ACAGCACGGGTTTATCATCC TATGCCTGCCAGGCTTCTAT 13 59

D14Mit48 TTTCTAGCCCTGACCCCC TCTGTTCACTCTGTGTAATTCTCC 14 0

D14Mitl33 TTGTCAAATAATTGCATGAGGC AACTATGACTCAGATTCCAAGTTGG 14 16.4

D14Mitl8 AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 14 25.1

D14Mit257 TTGTATAGGCATGTGCTCACG TTTAAAATGATGAGTGTCTTTGCC 14 25.1

D14MitlO2 CACAGAACTCCAGTCTAACTATCACA GAGGGTTATGAAAGTCAGCACC 14 37.2

D14Mitl93 CTCTGGCTTCTAAACAAAACACTG CATGTGGACGTGTGTATACATCC 14 47

D14Mitl25 GTTGAGGTCCCACTGCAAAT TTGAAGGAGATATATCACTCTGTGTG 14 52.5

D14Mitl65 TGTACATTAAATiTGGAAACCTGG ACTACACTTTCAGCATAAGACACACA 14 62.3

D14MitlO7 AAATGGTCATCCCTGAAAAGA CAGGCCTCTCCAAAGTACCA 14 69.9

Table 6 continued

Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

D15Mit53 CTCCCTTACCTTCGGCTCTT AGGGTAATTTCAATTAAACTCGTG 15 7.7

D15Mitl54 AGCACTGGGTACACAAACTGG ATGAAAGCATGTGTAGTCTTTCTCA 15 17.5

D15Mitl7 GCGTCACTGATAGTAGGGAG GTACCCCAATCCTGAACCAC 15 26.2

D15Mit63 ACCAATGATCGTTGATGCCT TAATTTCACACTAGCAAAACCAAA 15 28.4

D15Mit71 CCCAACTCATATGTATTATCCTGC TAATGACAGTGCCAAATCTTGG 15 35

D15MitlO7 CAACACTTATACACTTGTGTCAGGG TCATGGTTGGAACAGCAGAC 15 41.5

D15Mitl71 CCCATCAGTCCAAGAGAGATG AGGTGTACAGAAGTTCAGAAACAGC 15 52.5

D16Mit56 ATATTAATAATGGTTCCTCAAGAGGA CTTCATCTAAGTATGTGAAGGTCTGC 16 12 I

D16MitlO3 GGTGTGCATACACACATGCA TGACTAGACTTGACTCCTCCACC 16 21.9

D16MitlO5 CCCACACCTGATCCTGAACT TGTTCTTACTTGTATGTTTCTGCTCC 16 33.9 I

D16Mitl89 ACAGTGTTTGTTTGTTTGTTTGTG CAGTACAGGAAGTCTTTGCATCC 16 40.4

D16Mit224 CAAACAGAAAAAAAAAGGAAAAGC AATGAACTTTAAAGGGTATTTTGTTG 16 47

D16Mit86 TAATGTGGCAAGCAACCAAA GCATGTTTCCATGTGTCTGG 16 51.4

D17Mitll3 TCTGTCTCCTCCGTACTGGG GTCAATAAGTTCAATCACTGAACACA 17 2.2

D17Mit51 TCTGCCCTGTAACAGGAGCT CTTCTGGAATCAGAGGATCCC 17 14.2

D17Mit66 GGCTTCCACACATGATTGC TTCTGGGTCCATCATCACAA 17 19.7

D17Mitll7 AGTCCATTTATCGGGGGC TTTAATGGCACATCTGGCAA 17 25.1

D17Mitl39 AGACATGTGAGTACTGCACAGACA ATGATGACATACCTCCTAGTAGTCCC 17 25.1

D17Mitl52 CCAGTATTCTAGCTGCCCGA GATAAAAATGAGATCAAGATGGGG 17 32.8

D17Mit219 TTCAAAAGCCTATGTCAGACCC ATCCTGATCTATAAGCACCAGACA 17 40.4

D17Mit221 AACCAG ATC ATTAACAGTAATAAAG CA TTGTGGCAAAAACAACCAAA 17 50.3

D18Mit40 GGTAGGAGTCACTTTCCGTCC TΠTGTGAGCAI I I I IATACCATT 18 25.1

D18Mitl84 CACACATGTGTAGGTAGGTAGGTAGG CGCACAAGGACTACTGAAACA 18 26.2

D18Mit4 ACTGTTGCTGGGGAATGG CCAAGTTCAAAGCTGCTGG 18 37.2

D19Mit59 CTCTAACTATCCTCTGACCTTCACA TTTTAAGCAGAACATTGAGGACC 19 0

D19Mit63 CGCTTTCTTTGACTGGAATG GTCCTTTCACTTTCCACATGTG 19 24

Table 6 continued

Genome Wide Scan using 162 SSLP markers on 89N2 backcross mice

Name Sequence Sequence Chromosome Location (cM)

D19Mit88 TGTTGGTAGGTGTGTGTCATACA TCCATCTATCCACCTGCAAA 19 26.2

D19Mitl AATCCTTGTTCACTCTATCAAGGC CATGAAGAGTCCAGTAGAAACCTC 19 43.7

D19Mit34 CAGTGAAAGAACCTGTCGCA TTGTATGTGTGCTGAGCATCTG 19 44.8

D19Mit6 ATTAGTAAACTGACTCCCATGCG CTCATGAGTCCCCTGGGTTA 19 57.9

OO o

Table 7

Informative mice for mapping plt2 mutation in combined backcross and intercross animals. wehi28 (in- platelet sex ID no. mit71 house SSLP) JaxSNP4 CeleraSNP12 CeleraSNP33 CeleraSNP17 JaxSNPδ mit46 mit175

OO

Table 8

Primers and Method used for further fine mapping.

Method for determination of

Genetic

Name Sequence Sequence Chromosome Location Polymorphism

D7Mit84 AACTTGCCAGCCATGGTAAG AGTTAGAAGACCCACCCTAAATCC 7 46805126 TO 46805250 SSLP

D7Mit126 AGTGCTGGAATTAGTTATATAACACCA AAGGGAMTGTCTCTCTCTCCC 7 88547119 TO 88547293 SSLP

D7Mit71 CCACCTGGAATACATGTAACCC TAAGATCCAAGAGATGGGTTAAGC 7 118593362 TO 118593478 SSLP OO to

D7Wehi28 GTGTGCTCTGTTGCACAGGT GGCTTCAGCTTCAAGGTCAG 7 121763753 SSLP

JaxSNP4 GCTGCTTGGTCCTTAGGTTG ACTCAGGCCATGGATTGCT 7 127438668 to 127438868 SNP

CeleraSNP12 CCTTTTCCTCCACAACTTGC GGACACGTGCTCCACTATGTT 7 128327932 SSLP

CeleraSNP33 CTAACCTGGGCCGACTAATG ATTAATGGGTAGGGGGAGCA 7 128522680 to 128522876 SSLP

CeIeraSNP17 AGGGCAGTCCAGGCTACATA GGCCCTTTTCGTCTTTGAG 7 128991556 SSLP

JaxSNPδ TGCAAAAATCTGCCCTATCA TGCTCAAAGCACAAGTAGCC 7 130056432 to 130056632 SNP

D7Mit46 AATAGAACTTAATTGGCACAAGCC CAATTATGTGGGTGTGCATACC 7 130357685 TO 130357867 SSLP

D7Mit175 ACTGGAAGTTGTTCTCTGGCA GCACACATGCATATGTGTATGG 7 130914411 TO 130914541 SSLP

D7Mit223 ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 7 132314625 TO 132314731 SSLP

D7Mit189 CCTGGTGAGTAGAGAGGGAGG AGGAACATATGTGCACATGCA 7 132691081 TO 132691212 SSLP

Table 8 continued

Primers and Method used for further fine mapping.

Nested primers used in

Name Source of Polymorphism sequencing reaction

D7Mit84 Mouse Genetic Mapping Project of the Broad Institute

D7Mit126 Mouse Genetic Mapping Project of the Broad Institute

D7Mit71 Mouse Genetic Mapping Project of the Broad Institute OO

D7Wehi28 Developed "in-house" in Cancer and Haematology Division, WEHI

JaxSNP4 Mouse Phenome Database (SNP ID WI_WGS_7_129989877) CATAGAGCTTCCTCGCATGT CTCAAGCTCCATGAAACACA

CeleraSNP12 Celera Discovery System (SNP ID mCV24854430)

CeleraSNP33 Celera Discovery System (SNP ID mCV22978873)

CeleraSNP17 Celera Discovery System (SNP ID mCV25166077)

JaxSNPδ Mouse Phenome Database (SNP ID WI_WGS_7_132776503)

D7Mit46 Mouse Genetic Mapping Project of the Broad Institute

D7MR175 Mouse Genetic Mapping Project of the Broad Institute

D7Mit223 Mouse Genetic Mapping Project of the Broad Institute

D7Mit189 Mouse Genetic Mapping Project of the Broad Institute

MOUSE PHENOME DATABASE (aretha.jax.org)

CELERA DISCOVERY SYSTEM (www.celeradiscoverysystem.com)

BROAD INSTITUTE (www.broad.mit.edu)

Table 9

Known Genes in interval between CeleraSNP12 and CeleraSNP17

Based on Prediction by UCSC Genome Browser

(May 2004 genome assembly - genes based on SWISS-PROT,

TrEMBL, mRNA, RefSeq)

Numebr of exons Number of Exons Sequenced

MOR253-5

AF102529

Olfr539

Olfr45

Olfr541

Cyp2e1 8

X01026

AK016694

BC066028

AK004470 3

Odf3

Bet1!

AU 14950 4 10

Sirt3 5 7

Psmd13 9 10

Coxδb

BC031139 (NALP6) 5

BC023151

Ifitmδ 2 2

Ifitm2 2 2

Ifitml 3 3

IfitmS

AY247203

AB114827

BC038881

Pkp3

AI256711

Tp53i5

Ptdss2

Rnh1

BC061885

Hrasi 5

BC050837

AJ437023

AY373386 Table 9 continued

Known Genes in interval between CeleraSNP12 and CeleraSNP17

Based on Prediction by UCSC Genome Browser

(May 2004 genome assembly - genes based on SWISS-PROT₁

TrEMBL₁ mRNA, RefSeq)

Numebr of exons Number of Exons Sequenced

5730427C23Rik

AK005476

2400009B11Rik 5

BC055395

AK042050

Irf7 10 10

Mucdhi 15 15

Set 4 4

BC048484

Drd4

Deaf 1.

Other Genes Sequenced close to interval

Eps8l2 16 20

Table 10

Oligonucleotide primers used in PCR and sequencing reaction

CYP2e1 CCCCAGGACTGACCTATGAA GAGTGTTCCATGCCCAAGAT

BC013451 TGGTGAGTGTTGGCATTGAT ttcaaggaccctcattccag pos strand AAGTCCAAGAGGCMCCAGA GACAGCCACTTTTGCTCTCC

GCAGCTAAAGGAACCCTGTG CGGATCCTGAAGTGAGAAGG GTTGGCTCAGCATCCTTCTC AAAATGAAGGTGGGGGAAAC GCTGAGCTTGACCCCTACAG AGCACTGGGCCATAAACATC ACATCTACTGGGTGGCTTGG CTGCCATGGGATCTCAGTTT GATATCGGGCCTTCCTTTTC CTGGGCTCCTTGTGAAATCT gccctcaatgttatgcgagt aaggggacaaggctctcatt

AK00470 TTGCACAGACCAGAGCTTCA GCCTTACTGCTCCACCTCAG pos strand TCTCCTTCTGACCAGCCTGT GCAGCTCAGTGTGTCATCGT catggcacctgacctcct ccctttctcagactgcttctgt

Al 114950 ctctcccagcatccctcac gtgctccctgccaccact atggatgctgcttcagaggt GTTGGACTGCTGAGCACAAG GCTGCAGACTATCCGAATCC caaagggaagcaaactgtcc ggttgcaggtgggtattctc cccctgatgaagtcaaaggt ccatgtgtaccccaggagaa gtcagagcaactgtccacga cggtacaggcaaggatgaac CGAGTCCTCACATCCCTCAG CCTGTGCTGAACGTGTTGAC ggcagaggtcaatgtgtgaa TAGCTGCCGAGTTCCTCTTT ctcaaacaccagcctgacaa ggtgtactaaggcccagcaa tgtccaatacagcctcacca ggttaggattggggcctagt CAGCCACATCCAAGAAACAA

SIRT3 TTGGCTTCCCAACCAATAAG cgtaaccttacccaaggctgt

Negstrand GTTGGTAGTCATGCGTGGTG GCTTGGGGTTGTGAAAGAAA

AK075861 AGGTGGAGGAAGCAGTGAGA CAGAAGGTGTGCAGGAGACA aggttctgctagagcccaca ggggaggcagagatccttag CGCTCCCCACATCTAAGAAA CCGAGATCAGTCCTTTCAGC TGGATGGGAAAGAGATCACC GTTCCCAGGCTTCCTATTCC gctggctctgtgtgtgatgt tcatccaggaggagaacacc gagctgggaacaacacaagc accgggagatttcatcgttt

NALP6 GGCCAGTGAAGCGGATACT TGACCTCAGTTTTTAGCACTCC

BC031139 GTGTGCTGGATCGTGTGC CAGCAGCTCCAGTCCAAAAT

NEG STR CGGAGCTACGTGGTCATCTT TTGCAAGGAATGGGTAAAGC

CAACTACTGGCCCCAAGGTA CAGTGTCCATCAGCACTTGG TTGCTCAGCTCTTGGTGATG AGGGAGCATTTAGGGTGCTT TGAAGGTAGCTCCTGCCCTA TTTGGATAGGCCTGCAGAAG tcagacagctccaaccaaga TTGTGGGACTGAGGGAAGAC

Hras cagccaaccacaacaggtc cacaggcagagcgagtcc

BC011083 GCCTGCAGTCAGTCATGTCC acaggagcaaggcagatgat

NEG STR GAAAACCCCCTACAAGACCTG CCCTTTTCAAGCTGAAGGTG

CCATTGGCACATCATCTGAA TGGGGTATGATCCATCAGTG CTGCGGTCTGGGAGACTTAC ACCTATGGCTAGCCCGTGA CAGCACCGTTTCCTTCCTT GTGTTGGTTTTGCAGCTGAG Table 10 continued

Oligonucleotide primers used in PCR and sequencing reaction

Hras cagccaaccacaacaggtc cacaggcagagcgagtcc

BC011083 GCCTGCAGTCAGTCATGTCC acaggagcaaggcagatgat

NEG STR GAAAACCCCCTACAAGACCTG CCCTTTTCAAGCTGAAGGTG

CCATTGGCACATCATCTGAA TGGGGTATGATCCATCAGTG CTGCGGTCTGGGAGACTTAC ACCTATGGCTAGCCCGTGA CAGCACCGTTTCCTTCCTT GTGTTGGTTTTGCAGCTGAG

2400009B11 Rik cagtcctgagccagttgttg catggaagggaagatgctgt tgagtttcttggggacaagg TCTGCAAAGCCGTCTGGTAT CAGCCCATCTATTCCTGAGC cagcaggtggagagttgaga ccagggagcatactgtatatgga atggatgggggtgtgtgt TGAGTCCCGAGGgtatgttt TCTTCTCTCCATGCCTTTGG

IRF-7 CTGAGCCTGGCAGATAGACC TGTCTCCACTGTCTCCATGC

U73037 GCATGGAGACAGTGGAGACA CAGTCTCCAAACAGCACTCG POS STR TGCAATAGCTGACTGCTCCA ACCTTATGCGGATCAACTGG

CGAGTGCTGTTTGGAGACTG GTACCTCACTCAGCCCAAGG

GCCTCTGTCATTTTGGTGGT GGGCAGATGGGTGTTCTCTA

AACTGCTTGGGCATCGTAAC GCTGCATAGGGTTCCTCGTA

CCTCTTGCTTCAGGTTCTGC GGCCCTTGTACATGATGGTC

CTGCCCTACTGCTCCTTCAC TGTAGTGTGGTGACCCTTGC

TGATCTTTCCCAGTCCTGCT CCAGGTCCATGAGGAAGTGT

GAAGACCCTGATCCTGGTGA actgggggtcaccttctttc

MUCDH CTCCCCACACCTATTCTCCA GGCATAAGGAGTGGACAAGG

AK027913 TCCTTCTTCCAGGTGACAGC AGGGGCCCTATGAGTCACTT POS STR GGGAAGATTCAGGGAAGGAG GACCTCAAAAGGGGCCTAAG

GGCCCCTTTCTCTGATCTTC TCAGCTGTGTCTCAGGGATG

CGCCAAAATTCTCCTTTGAA GTGTGGCTGGGCTCTACATT

AACCGTCATCCCTGAGACAC GCCTCCTTTCTCCCCATTAC

AGGGCGTAAACTACCCTGCT GGCCTCAGAGGAGACTGTCA

GGGCAAAAATCAGTGAGGAG CCCATCCATCATCCAGTACC

TTGAGGGGCAACAAGAGAGT ACTGGAGTGGGTTTCCAGTG

ACTCCACCCCCAAATTCTTC CTCCATAGGCACAGCAGTCA

CACTCCAGTTCTCCCAGAGC CTGTCCCCAGATATGCCTGT

GGCACCAGGCCTATGTCTTA GGGGTGGGTGGTCTTAGAGT

AGAACTGCCCTCCACAGGT TGGATGAGAGGTTCCTGGTT

CACCTGGTGGCACAACTCTA cagactccttggaggtggtc

GCCTACCTCCATTTCCTTCC ACCGCAACTCCAGATGGT

ACAGTGTGCCCTTGAAGACC CATGAATGACTCCCACGATG gaagagagtgggagccaagc AACCACACAGCCTTGTAGCC

GTTGAGGACCACCACGTCA GGCTAGGATGGGTGTTCTGA

AAGCTTCCGGGTCTCCTAAG gtgggaacttgcttgtggtt Table 10 continued

Oligonucleotide primers used in PCR and sequencing reaction

EPS8L2 CCATGTCCCCATCTGCTACT GCGGTTCTTGTGCTAAAAGC

BC009098 AGGAACACCCTTGCAGAGAA AAAGGCCTCTCCCCTTACAG Neg strand CTACATTTCCTGCCCCTCAC CCCAGTGCAGTTCCTTGTTT

TAAGCCACAAAGGCACACAC AGCCAGTCTAGGTTGCCAAA

GTGTTCCTATCTGGGGAGCA AGGGCAAGATCCCTCACTTT

CGTATACCCTCCCACAGGAA ATCCCTCAGCCTACCTGGTT

GTGGAGGTAAGGCAGACAGC CAGGCAAGGATGGAAAAGAG

CCTGGGGGTGTAGAAAGTGA AACTGCTTGAAAGCCTCAGC

GCTGTGCTGGCTCAGAGAAT CCCTCCAGCTTAGTGTGTGC

CTGGGGCAAGAAACGGTAG CCAAACCCATGTGGATCACT

TCAACCTGCTGGTGAGTCAG ATTCAGAGCTGTGGTCAGCA

CATAGCACGCTCTGTCTCCA TAGTCGATTCTTGGGGGTCA

ACCCGTGCTTCTACCTTCAA gaggggaaaccatgatcaga

CCAGCCTCTGAAGATCTCGT AGCCCCTACTGGCTTCTACC

GAATCCAGGCCACCTACTGA GGAAGAGGTGAGGGGTAAGG

AAGCTACGAAATCGCAGTGG CGGACCTGACTCGAAGGTAA

CCTTACCCCTCACCTCTTCC GGTGCAAGTGCCTAAGAAGG

CAGCCACATCAAGACACAGC CAGTTACACCCAGCCCATCT

CAGCCAACACACTTCACCAG ATACCAGCAGGTCACCCAAG

ACCCCCAACCCAATACTCTT GCATACTCTATACCAGCAGGTCAC

IFITM 1 TGCTTAGCAACTTGACTTCATCTA TCTACCCCAAATCCTGACCC GCCCACTGCGCAGCAGGCTC CACCTCCTGGGATTCCCTC CTAGGAAGGTGATGGGGAGC AACTCTGGTTAATTACTGCCCAG

IFITM2 AAGGGCGGGTCTACAGAACC TGAGTAGATGGCGCTTCAGG CAGGGAGCAGTTGGGGAAAT GGAGACCAGAAGCCTGACAA

IFITM5 GTGGAGCTGGTCAGCCAG CCGGACTCCACATCACCT

GCCTGTGCTCTACAGTTGCTCCG AGGTCACGGCTGGGCAGGGA

PSMD13 TGAGGCTAGTGGGACGCTCC TCCTCCACATCCCTGACCCT TGTGCAGTGGGACCTGGGAA CTTCTGGCATCCTTGGGCGC GTCTTAGTGCTGCCTCTGTC TACTGCCTCACCCCAGTGTT GTAATGATCGGAGAGAGGGA TAGAATACGAGAGTGCTTTGGCT

CCTTCATACTTCCTGGCTCACTGCAAGACCTATTCCCTTCAGCACG GAACCATTCTAAGAACGGCTTCT TGGTGTCTGCTCACAGCAGTG GGAGCTCTCAGTGAGCCTCAGC GGGCCCTGAGAGGCAGGAAG

SCT CGGCGGTCCCGGCAGCTATAA ATTAGTGGTGTACAGGGATGTGG TAGTATGGGACAGGCCCGCCA CCACGCCTTCCAGGCTCTCG Table 11 Twenty most differentially expressed genes between plt2/plt2 and wildtype animals

Induced ( I) or Fold p value q value

Repressedφ in Change (Holm) (FDR) pltllplQ mice.

SuItIaZ (sulfotransferase family 2A, member 2) \ 47.0 8.4xlCr^s 8.4X10^'8

Igfi>p2 (insulin-like growth factor binding protein 2) \ 4.7 2.4xlCr^s 1.2xlO-^s

Scd2 (stearoyl-Coenzyme A desaturase 2) T 6.3 9.9xlO-^s 3.3xlO-^s

CcM2 (cysteine conjugate-beta lyase 2) I 5.7 0.00014 3.5xlO^"5

LgaJsl (lectin, galactose binding, soluble 1) t 2.7 0.00036 6.9xlO^"5

Cyp7bl (cytochrome P450, 7bl) t 3.7 0.0004S 6.9xlO"⁵

Cbrl (carbonyl reductase 1) t 2.8 0.00053 6.9xlO-⁵

Agxt- (alanine-glyoxylate aminotransferase) I 2.5 0.00062 6.9xlO^"5

Caτ2 (carbonic anhydrase 2) t 2.9 O.OOOSS S.δxlO^"5

SuJtSaI (sulfotransferase family 5A, member 1) 1 2.3 0.0011 0.00010

Hgfac (kepatocyte growth factor activator) I 3.0 0.0016 0.00012

EST AD17230 t 2.2 0.0016 0.00013

EpIuI (epoxide hydrolase 1, microsomal) t 2.2 0.0027 0.00019

Acsl4 (acyl-CoA syntlietase long-chain 4) I 2,2 0.0029 0.00020

Itihl (inter-alpha trypsin inhibitor, heavy chain 1) 1 2.6 0.0036 0.00023

CQ (complement component 9) I 2.6 0.0060 0.00036

Lrgl (leucine-rich alρha-2-glycoprotein 1) I 2.7 0.0064 0.00036

RpU3a (ribosomal protein LOa) t 2.0 0.0069 0.00037

Cd63 (Cd63 antigen) t 5.5 0.0094 0.00047

Abccδ (ATP-bindϊng cassette, family C, 6) i 2.2 0.0Ϊ0 0.00049

Table 12 Gene ontology groups of most differentially expressed genes

Metabolism Induced (f) orRepressed(l)

Lipid Metabolism in pU2!plt2 mice.

Scd2 (stearoyl-Coenzyme A desaturase 2) t

AcsM (acyl-CoA synthetase long-chain 4) f

Abhd5 (abhydrolase domain containing 5) f

Sult2a2 (sulfotransferase family 2A, member 2) I

Hsdl7b2 (hydroxysteroid (17-beta) dehydrogenase 2) J

Slc27a5 (solute carrier family 27, member 5) {

Protein metabolism

RpIHa (ribosomal protein L13a) f

Lamrl (laminin receptor 1) t

Nola2 (nucleolar protein family A, member 2) f

Biotransformation / Electron transport

Cy/»7W(cytochrome P450 7bl) f

Gsttn (glutathione S -transferase, mu land 2) f

Gsta (glutathione S-transferase, alpha 2) f

Cyp2blQ (cytochrome P450 2b 10) J

CypSbl (cytochrome. P450 SbI) \

Cyp2d26 (cytochrome P450 2d26) J

Cyp2c40 (cytochrome P450 2c40) i

Other Metabolism

C'brJ (carbonyl reductase 1) f

Carl (carbonic anhydrase 2) f

Ccbll (cysteine conjugate-beta lyase 2) J

Agxi (alanine-glyαxylate aminotransferase) J

SuItSaI (sulfotransferase family 5 A, member 1) {

Prodh2 (proline dehydrogenase oxidase 2) \

Regulation of Cell Cycle and Growth

Ran (RAS-like, family 2, locus 9) f

Σct2 (ect2 oncogene) f

Cdknlc (cyclin-depεndent kinase inhibitor 1C, P57) f

Igβp2 (insulin-like, groλvth factor binding protein 2) {

Acute Phase Response and Blood Coagulation

C9 (complement component 9) {

Hc (hemolytic complement) {

F5 (coagulation factor V) \

Saa4 (serum amyloid A 4) I

Proteolysis and Peptidolysis

Ephxl (epoxide hydrolase 1, microsomal) f

Hgfiic (hepatocyte growth factor activator) |

Mstl (macrophage stimulating 1) {

TUhI (inter-alpna trypsin inhibitor, heavy chain 1 ) { BIBLIOGRAPHY

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Claims

Claims:

1. An isolated genetically modified cell or non-human animal comprising such cells wherein a TRP gene is modified and the cell or animal produces a substantially enhanced level or activity of TRP polypeptide, or substantially reduced level or activity of TRP polypeptide compared to a non-modified cell or animal of the same species, or is substantially incapable of producing active TRP polypeptide.

2. The genetically modified cell or organism of claim 1 which produces substantially lower levels of TRP polypeptide or is substantially incapable of producing active TRP polypeptide.

3. The cell or organism of claim 1 or 2 wherein the modification is in one or both alleles of the TRP gene.

4. The cell or organism of claim 1 which is from a non-human primate, live stock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish or bird.

5. The cell or organism of claim 1 wherein the genetically modified non-human animal is a mouse.

6. The cell of claim 1 wherein the cell is a human or bacterial cell.

7. The cell of claim 1 wherein the cell is a stem cell, embryonic cell, liver cell, lung cell, kidney cell, spleen cell, thymus cell or brain cell.

8. The cell of claim 7 wherein the cell is a liver cell.

9. The cell of claim 1 or 2 wherein the cell is an autologous or syngeneic cell suitable for transplantation.

10. The cell or organism of claim 2 wherein the modification is in the third exon of the 77^P gene.

11. The cell or organism of claim 2 wherein the modification causes a non-conservative amino acid change in the α-helix region of TRP polypeptide.

12. The cell or organism of claim 10 wherein the modification causes a Y98C mutation in a long TRP polypeptide or Y75C in a short TRP polypeptide.

13. The cell or organism of claim 1 or 2 wherein the cell or organism is further modified with a modification in the TPO or c-mpl gene.

14. The cell or organism of claim 1 or 2 wherein the genetic modification is by introduction of a genetic construct comprising a sequence of nucleotides encoding a long and/or short form of TRP polypeptide.

15. The cell or organism of claim 14 wherein the sequence of nucleotides comprises a loss of function mutation.

16. The isolated cell of any one of claims 1 to 15 when used in vitro or in vivo.

17. A method of screening for an agent capable of complementing a modified TRP polypeptide activity in a cell or organism said method comprising contacting an agent to be tested with a genetically modified cell or a non-human organism comprising such cells wherein the organism or cell comprises a modification to the TRP gene or a regulatory region required for expression of TRP gene such that the cell produces substantially modified levels of TRP polypeptide compared to a non-genetically modified cell or organism of the same species, or is incapable of producing active TRP polypeptide, and screening for a phenotype the same or equivalent to a cell which produces levels of TRP polypeptide which allow for normal function of the cell or organism in relation to pathways associated with TRP polypeptide.

18. The method of claim 17 wherein the modified TRP polypeptide activity is a substantially reduced level or activity of TRP polypeptide.

19. The method of claim 17 or 18 wherein the modification is in one or both alleles of the TRP gene.

20. The method of claim 17 wherein the genetically modified cell or organism is from a non-human primate, live stock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish or bird.

21. The method of claim 17 wherein the genetically modified non-human animal is a mouse.

22. The method of claim 17 wherein the cell is a human or bacterial cell.

23. The method of claim 17 wherein the cell is a stem cell, embryonic cell, liver cell, lung cell, kidney cell, spleen cell, thymus cell or brain cell.

24. The method of claim 23 wherein the cell is a liver cell.

25. The method of claim 18 wherein the modification is in the third exon of the TRP gene.

26. The method of claim 18 wherein the modification causes a non-conservative amino acid change in the α-helix region of TRP polypeptide.

27. The method of claim 18 wherein the modification causes a Y98C mutation in a long TRP polypeptide or Y75C in a short TRP polypeptide.

28. The method of claim 17 or 18 wherein the cell or organism is further modified with a modification in the TPO or c-mpl gene.

29. A method of treating or preventing a condition in a subject wherein the condition is characterised by reduction in TRP polypeptide level or activity or absence of TRP polypeptide wherein the method comprises administering to the subject an effective amount of an agent which is capable of complementing deficient TRP polypeptide activity or inducing up-regulation of TRP expression.

30. The method of claim 29 wherein the agent is a TRP polypeptide or an agent from which TRP polypeptide is producible.

31. The method according to claim 29 wherein the condition is characterised by megakaryocytopoiesis and/or cancer development.

32. The method according to claim 31 wherein the cancer is of the liver.

33. An isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a TRP-PLT2 polypeptide having an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof.

34. The nucleic acid molecule of claim 33 wherein the amino acid sequence comprises about 60% or greater sequence identity to about 20 to 30 contiguous amino acid residues at the N-terminal region of the polypeptide.

35. The nucleic acid molecule of claim 33 comprising a sequence of nucleotides substantially as set out in SEQ ID NO: 3 or a functional variant thereof or a complementary form of one of these.

36. The nucleic acid molecule of claim 33 having about 60% or greater sequence identity to SEQ ID NO: 3 or a complementary form thereof over at least a 5'-terminaI portion comprising about 60 to 100 contiguous nucleotides.

37. An isolated TRP-PLT2 polypeptide comprising an amino acid sequence substantially as set out in SEQ ID NO: 4 or a functional variant thereof.

38. An isolated genetic construct comprising the plt2 mutation wherein the nucleotide sequence encodes a long or short form of TRP polypeptide.

39. A TRP polypeptide or an agent from which is TRP polypeptide is producible for use in the treatment or prevention of conditions involving thrombocytoses and/or hepatoma.

40. Use of a TRP polypeptide or an agent from which TRP polypeptide is producible in the manufacture of a medicament for the treatment or prevention of thrombocytoses and/or hepatoma.