WO2000056856A2 - Polymorphic microsatellite repeats in the costimulatory receptor locus and uses thereof - Google Patents

Abstract

The invention relates to polymorphic markers within the costimulatory receptor gene locus. These markers are characterized by sets of oligonucleotide primers according to the invention useful in PCR amplification and DNA segment resolution.

Description

POLYMORPHIC MICROSATELLITE REPEATS IN THE COSTIMULATORY RECEPTOR LOCUS AND USES THEREOF

Cross-Reference to Related Applications This application claims priority to provisional application serial number

60/126,215, entitled "Polymorphism of CTLA-4 and Uses Thereof.^*' filed on March 25, 1999. The entire contents of that application are incorporated herein by reference.

Background of the Invention In order for T cells to respond to foreign proteins, two signals must be provided by antigen-presenting cells (APCs) to resting T lymphocytes (Jenkins. M. and Schwartz, R. (1987) J. Exp. Med. 165. 302-319; Mueller. D.L., et al. (1990) J. Immunol. 144, 3701- 3709). The first signal, which confers specificity to the immune response, is transduced via the T cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histoconipatibility complex (MHC). The second signal, termed costimulation, induces T cells to proliferate and become functional (Lenschow et al. 1996. Annu. Rev. Immunol. 14:233). Costimulation is neither antigen- specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, M.K., et al. 1988 J. Immunol. 140, 3324- 3330; Linsley. P.S.. et al. 1991 J. Exp. Med. L73, 721-730; Gimmi. CD., et al.. 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J.W.. et al. 1992 J. Clin. Invest. 90, 229-237; Koulova, L.. et al. 1991 J Exp. Med. 113, 759-762; Reiser. H.. et al. 1992 Proc. Natl. Acad. Sci. USA. 89, 271-275; van-Seventer. G.A.. et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, J.M.. et al., 1991 J Immunol. 141. 774-80; Dustin. M.I., et al., 1989 J. Exp. Med. 169. 503; Armitage. R.J., et al. 1992 Nature 357. 80-82; Liu, Y., et al. 1992 J Exp. Med. 115. 437-445).

The CD80 (B7-1) and CD86 (B7) proteins, expressed on APCs. are critical costimulatory molecules (Freeman et al. 1991. J. Exp. Med. 174:625; Freeman et al. 1989 J Immunol. 143:2714; Azuma et al. 1993 Nature 366:76: Freeman et al. 1993. Science 262:909). B7 appears to play a predominant role during primary immune responses, while B7-1. which is upregulated later in the course of an immune response. may be important in prolonging primary T cell responses or costimulating secondary T cell responses (Bluestone. 1995. Immunity. 2:555).

One receptor to which B7-1 and B7 bind. CD28. is constitutively expressed on resting T cells and increases in expression after activation. After signaling through the T cell receptor, ligation of CD28 and transduction of a costimulatory signal induces T cells to proliferate and secrete IL-2 (Linsley, P.S.. et al. 1991 J. Exp. Med. 173. 721-730; Gimmi. CD., et al. 1991 Proc. Natl. Acad. Sci. USA. 88. 6575-6579; June. C.H., et al. 1990 Immunol. Today. U. 21 1-6: Harding, F.A.. et al. 1992 Nature. 356. 607-609). A second receptor, termed CTLA4 (CD 152) is homologous to CD28 but is not expressed on resting T cells and appears following T cell activation (Brunet. J.F.. et al.. 1987 Nature 328. 267-270). CTLA4 appears to be critical in negative regulation of T cell responses (Waterhouse et al. 1995. Science 270:985). Blockade of CTLA4 has been found to remove inhibitory signals, while aggregation of CTLA4 has been found to provide inhibitory signals that downregulate T cell responses (Allison and Krummel. 1995. Science 270:932). In addition, lymphoproliferative disease has been associated with CTLA-4 gene-deficient mice (Bluestone, J.A.. et al. (1997). J. Immunol 158: 1989- 93; June et al, (1994) Immunol Today 15: 321-31 ; Tivol et al, (1996). Curr Opin Immunol 8:822-30: Tivol et al. (1995) Immunity 3: 541-7). although data conflicting this interpretation also exist (Liu. Y. (1997). Immunol Today 18: 569-72: Wu. Y. et al. (1997) J Exp Med 185: 1327-35; Zheng, Y.. et al. (1998) Proc Natl Acad Sci USA 95: 6284-9). A new molecule related to CD28 and CTLA4, ICOS. has been identified and seems to be important in IL-10 production (Hutloff et al. 1999. Nature. 397:263; WO 98/38216).

The genetic organization of CTLA-4 has been previously described (Brunet, J. F., et al, (1987). Nature 328: 267-70: Dariavach. P.. et al. (1988). Eur J Immunol 18: 1901-5.) as being comprised of 4 exons which encode separate functional domains: a leader sequence, an extracellular domain, a transmembrane domain, and cytoplasmic domain. Within the extracellular domain, the B7 binding motif is centered on the amino acids MYPPPY, a sequence also found in the extracellular domain of CD28, the primary B7 receptor responsible for T-cell activation (Balzano, C. et al,

(1992). Int J Cancer Siφpl 1: 28-32). The cytoplasmic domain of CTLA-4 encodes the motif YVK-M in which the phosphorylation state of tyrosine has been implicated in both signal transduction through SYP/SHP2 phosphatase (Marengere, L. E.. et al. (1996). Science 111: 1170-3. [published errata appear in Science 1996 Dec 6:274(5293)1597 and 1997 Apr 4:276(5309):21]; Shiratori. T.. et al (1997). Immunity 6: 583-9), and the intracellular accumulation of CTLA-4 via AP50 clatharin-mediated endocytosis

(Chuang, E.. et al, (1997). J Immunol 159: 144-51 : Zhang, Y., and Allison. J. P. (1997) Proc Natl Acad Sci U S A 94: 9273-8). CTLA-4 has also been reported to be involved with T-cell receptor signaling by interfering with ERK and JNK activation (Calvo, C. R., et al. (1991). J Exp Med \S6: 1645-53). Recently, polymorphisms in the non-coding region 3' of human CTLA-4 DNA have been correlated with a number of autoimmune diseases, including: Grave^'s disease (Dormer. H., et al, (1997a). J Clin Endocrinol Metab 82: 4130-2 Dormer, H.. et al, (1997b). J Clin Endocrinol Metab 82: 143-6; Kotsa. K.. et al, (1997). Clin Endocrinol (Oxfi 46: 551-4; Nistico. L., et al, (1996). Hwm Mol Genet 5: 1075-80). Hashimoto's disease (Braun, J.. et al. (1998). Tissue Antigens 51 : 563-6; Tomer. Y.. et al. (1997). J Clin Endocrinol Metab 82: 1645-8, myasthenia gravis with thymoma (Huang. D., et al, (1998). J Neuroimmunol 88: 192-8), and IDDM (Marron. M. P., et al, (1997). Hum Mol Genet 6: 1275-82; Nistico, L.. et al, (1996). Hum Mol Genet 5: 1075-80) in patients.

The minimal promoter of mouse CTLA-4 suggests that transcriptional initiation control is localized approximately 335 bp upstream from the initiation codon. However, the contribution from other regions of the CTLA-4 locus to the regulation of gene expression has not been examined (Finn. P. W.. et al, (1997). J Immunol 158: 4074-81 : Perkins. D.. et al. (1996). J Immunol 156: 4154-9). Despite the tightly regulated control of CTLA-4 expression and the importance of this key immunoregulatory protein, the published genomic sequences of the human CTLA-4 are incomplete. Further, no data are available for the intron sequences of mouse CTLA-4. In addition, the genomic structure of other costimulatory receptors is not well understood.

Areas of simple repetitive DNA (i.e.. microsatellite DNA) interspersed throughout the genome have been used extensively to map chromosomes. It has been found that these simple repeats often vary in length among individuals, thus, they have facilitated genetic linkage studies of diseases within populations. Unlike long and short interspersed repeats, the mechanism by which simple repeats are generated and inserted into the genome is not known, and their potential role in modulating biochemical processes is not clear (Epplen, C. et al. (1997). Electrophoresis 18: 1577-85; Epplen. J. T.. et al. (1994). Biol Chem Hoppe Seyler 375: 795-801. Certain polymorphisms of a particular sequence in particular regions have been correlated with the development of. or susceptibility, to a disease or other condition. Because the genes responsible for disorders or conditions associated with the immune response have not all been cloned, it is useful to utilize such markers for a variety of diagnostic and prognistic assays. The utility of such markers depends upon how tightly the marker and the disease locus are linked. Accordingly, the identification of novel DNA polymorphisms that are associated with disease states is desirable and aids in the diagnosis or prognosis of diseases or conditions to which they are linked.

Summary of the Invention This application relates, at least in part, to the identification of polymorphic microsatellite repeat ("PMR") sequences in the costimulatory receptor gene locus. These sequences are useful as markers e.g., identifying genetic material from a given individual and/or in identifying individuals at risk for developing a particular disease or condition or at risk for giving birth to an offspring likely to develop a particular disease or condition. In particular, the subject markers are linked to a variety of autoimmune diseases or conditions. Because PMR sequences vary in length among individuals, these sequences can be amplified to generate products that differ in size and can be detected by rapid and convenient high resolution processes. The invention further relates to primer sequences based on the disclosed PMR sequences that can be used to amplify PMR sequences of the costimulatory receptor gene locus.

In one aspect, the invention pertains to a method for determining the predisposition of a human subject to autoimmune disease, said method comprising detecting a polymorphic microsatellite repeat (PMR) sequence in the CD28 or the ICOS gene of the human costimulatory gene locus to thereby determine the predisposition of a human subject to autoimmune disease. In another aspect, the invention pertains to a method for determining the predisposition of a human subject to autoimmune disease, said method comprising detecting a polymorphic microsatellite repeat (PMR) in the CTLA4 gene of the human costimulatory gene locus, wherein the PMR sequence is selected from the group consisting of: nucleotides 5722-5746 of SEQ ID NO: 1 : nucleotides 23904-23957 of SEQ ID NO: l :nucleotides 27689-27780 of SEQ ID NOT ; nucleotides 30766-30801 of SEQ ID NOT ; nucleotides 6550-6597 of SEQ ID NO: l :nucleotides 19911-19956 of SEQ ID NOT : nucleotides 19767-19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152-48199 of SEQ ID NO:2:nucleotides 49894-49925 of SEQ ID NO:2; nucleotides 10141-10177 of SEQ ID NO:3;nucleotides 11459-11520 of SEQ ID NO:3; nucleotides 12329-12419 of SEQ ID NO:3: nucleotides 15527-15567 of SEQ ID NO:3; nucleotides 24050-24075 of SEQ ID NO:3;nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317-27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3; nucleotides30535-30574 of SEQ ID NO:3;nucleotides 33714-33758 of SEQ ID NO:3: nucleotides 43819-43925 of SEQ ID NO:3;nucleotides 46547-46572 of SEQ ID NO:3; and nucleotides 46828-46875 of SEQ ID NO:3 to thereby determine the predisposition of a human subject to autoimmune disease.

In another aspect, the invention pertains to a method for determining the predisposition of a human subject to autoimmune disease, said method comprising detecting a polymorphic microsatellite repeat (PMR) in the human costimulatory gene locus, wherein the PMR sequence is not an hR2 sequence, to thereby determine the predisposition of a human subject to autoimmune disease.

In one embodiment, the autoimmune disease is selected from the group consisting of: insulin-dependent diabetes mellitus (IDDM), Addison^'s disease. Graves' disease, autoimmune hypothyroidism. myasthenia gravis. thymoma, lupus, thyroiditis, postpartum thyroiditis. rheumatoid arthritis, Hashimoto's disease, coeliac disease and leprosy.

In another embodiment, the step of detecting is performed using a polymerase chain reaction (PCR) employing a first and second primer. In one embodiment, the first or second primer comprises the sequence selected from the group consisting of SEQ ID NO: SEQ ID No: 51. SEQ ID No: 52. SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26. SEQ ID No: 49. SEQ ID No: 50. SEQ ID No: 47, SEQ ID No: 48. SEQ ID No: 31. SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 43. SEQ ID No: 44, SEQ ID No: 17, SEQ ID No: 18. SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 27. SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 15, SEQ ID No: 16. SEQ ID No: 37. SEQ ID No: 38. SEQ ID No: 11. SEQ ID No: 12. SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33. SEQ ID No: 34. SEQ ID No: 35, SEQ ID No: 36. SEQ ID No: 53. and 54.

In another aspect, the invention pertains to a method for determining the predisposition of a human subject to autoimmune disease, said method comprising detecting an hRl PMR sequence to thereby determine the predisposition of a human subject to autoimmune disease.

In one embodiment, the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus (IDDM). Addison^'s disease. Graves^' disease, autoimmune hypothyroidism, myasthenia gravis. thymoma. lupus, thyroiditis, postpartum thyroiditis. rheumatoid arthritis. Hashimoto^'s disease, coeliac disease and leprosy.

In one embodiment, the step of detecting is performed using PCR employing a first and second primer.

In another embodiment, the first or second primer comprises a sequence selected from the group consisting of SEQ ID NO: SEQ ID No: 51. SEQ ID No: 52. SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25, SEQ ID No: 26. SEQ ID No: 49, SEQ ID No: 50. SEQ ID No: 47. SEQ ID No: 48, SEQ ID No: 31. SEQ ID No: 32, SEQ ID No: 7. SEQ ID No: 8, SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 27. SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 15. SEQ ID No: 16. SEQ ID No: 37. SEQ ID No: 38. SEQ ID No: 11. SEQ ID No: 12. SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33. SEQ ID No: 34. SEQ ID No: 35. SEQ ID No: 36. SEQ ID No: 53. and 54.

In one aspect, the invention pertains to a method for determining the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject, said method comprising detecting a polymorphic microsatellite repeat sequence in the CD28 or the ICOS gene of the human costimulatory gene locus to thereby determine the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject.

In another embodiment, the invention pertains to a method for determining the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject, said method comprising detecting a polymorphic microsatellite repeat (PMR) in the CTLA4 gene of the human costimulatory gene locus, wherein the PMR sequence is selected from the group consisting of: nucleotides 5722-5746 of SEQ ID NOT; nucleotides 23904-23957 of SEQ ID NO:l;nucleotides 27689-27780 of SEQ ID NO: 1 : nucleotides 30766-30801 of SEQ ID NO: 1 ; nucleotides 6550-6597 of SEQ ID NO: 1 mucleotides 19911 - 19956 of SEQ ID NO: 1 ; nucleotides 19767- 19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2: nucleotides 48152-48199 of SEQ ID NO:2:nucleotides 49894-49925 of SEQ ID NO:2: nucleotides 10141-10177 of SEQ ID NO:3;nucleotides 1 1459-11520 of SEQ ID NO:3; nucleotides 12329-12419 of SEQ ID NO:3: nucleotides 15527-15567 of SEQ ID NO:3: nucleotides 24050-24075 of SEQ ID NO:3:nucleotides 26009-26056 of SEQ ID NO:3: nucleotides 27317-27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3: nucleotides30535-30574 of SEQ ID NO:3:nucleotides 33714-33758 of SEQ ID NO:3; nucleotides 43819-43925 of SEQ ID NO:3;nucleotides 46547-46572 of SEQ ID NO:3: and nucleotides 46828-46875 of SEQ ID NO: 3 to thereby determine the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject

In another embodiment, the invention pertains to a method for determining the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject, said method comprising detecting a polymorphic microsatellite repeat (PMR) in the human costimulatory gene locus, wherein the PMR sequence is not an hR2 sequence to thereby determine the polymorphic variant or subtype of a PMR sequence in the costimulatory locus in a human subject.

In one embodiment, the step of detecting is performed using PCR employing a first and second primer. In one embodiment, the first or second primer comprises a sequence selected from the group consisting of SEQ ID NO: consisting of SEQ ID NO: SEQ ID No: 51. SEQ ID No: 52. SEQ ID No: 23. SEQ ID No: 24, SEQ ID No: 25. SEQ ID No: 26, SEQ ID No: 49. SEQ ID No: 50. SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 31. SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10, SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 43. SEQ ID No: 44. SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20. SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 1 1. SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. SEQ ID No: 33, SEQ ID No: 34. SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 53, and 54.

In another aspect, the invention pertains to a PCR primer pair capable of amplifying a PMR sequence in the ICOS or CD28 region of the costimulatory locus of a human subject.

In yet another aspect, the invention pertains to a PCR primer pair capable of amplifying a PMR sequence selected from the group consisting of: nucleotides 5722- 5746 of SEQ ID NO: 1 ; nucleotides 23904-23957 of SEQ ID NO: 1 mucleotides 27689- 27780 of SEQ ID NOT ; nucleotides 30766-30801 of SEQ ID NOT ; nucleotides 6550- 6597 of SEQ ID NO: l ;nucleotides 1991 1-19956 of SEQ ID NOT ; nucleotides 19767- 19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152- 48199 of SEQ ID NO:2:nucleotides 49894-49925 of SEQ ID NO:2; nucleotides 10141- 10177 of SEQ ID NO:3:nucleotides 11459-11520 of SEQ ID NO:3: nucleotides 12329- 12419 of SEQ ID NO:3; nucleotides 15527-15567 of SEQ ID NO:3; nucleotides 24050- 24075 of SEQ ID NO:3:nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317- 27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3; nucleotides30535- 30574 of SEQ ID NO:3:nucleotιdes 33714-33758 of SEQ ID NO:3; nucleotides 43819- 46828-46875 of SEQ ID NO:3.

In still another aspect, the invention pertains to a PCR primer capable of amplifying a PMR sequence in the costimulatory locus of a human subject, wherein the primer is less than or equal to about 200 base pairs in length and comprises a nucleotide sequence selected from the group consisting of: SEQ ID No: 51. SEQ ID No: 52, SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25, SEQ ID No: 26. SEQ ID No: 49. SEQ ID No: 50, SEQ ID No: 47. SEQ ID No: 48. SEQ ID No: 31. SEQ ID No: 32, SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 45. SEQ ID No: 46, SEQ ID No: 39. SEQ ID No: 40. SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 43. SEQ ID No: 44. SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19. SEQ ID No: 20, SEQ ID No: 21. SEQ ID No: 22. SEQ ID No: 27, SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 37, SEQ ID No: 38. SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 33. SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36. SEQ ID No: 53, and 54.

In another aspect, the invention pertains to a PCR primer capable of amplifying a PMR sequence in the costimulatory locus of a human subject, wherein the primer consists of a nucleotide sequence selected from the group consisting of: SEQ ID No: 51, SEQ ID No: 52. SEQ ID No: 23. SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26, SEQ ID No: 49. SEQ ID No: 50. SEQ ID No: 47. SEQ ID No: 48. SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 45, SEQ ID No: 46. SEQ ID No: 39. SEQ ID No: 40. SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 43, SEQ ID No: 44. SEQ ID No: 17. SEQ ID No: 18. SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 27, SEQ ID No: 28. SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 15. SEQ ID No: 16, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 1 1. SEQ ID No: 12, SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35. SEQ ID No: 36. SEQ ID No: 53. and 54.

Brief Description of the Drawings Figure 1 is a diagrammatic map of mouse and human CTLA-4 loci. The four filled rectangles in each map represent the location of CTLA-4 coding sequences 1-4. Restriction sites of EcoRI, Hindlll. Pstl. BamHI, Xhol. and Xbal are displayed. Boxed entries represent either clone # or Genbank accession # used to assemble the contiguous map of the designated CTLA-4 locus. A bar representing 1000 bp is shown. Panel A is the mouse CTLA-4 locus. Panel B is the human CTLA-4 locus. Figure 2 is a graphic analysis of CTLA-4 sequence conservation. Panel A is a dotplot comparison of the human and mouse CTLA-4 loci. Analysis was performed using Geneworks 2.5 software, with a window of 30 nucleotides and threshold of 65% identity. Filled rectangles on line along side edges of graph represent CTLA-4 coding sequences. Panel B is a sequence similarity index based on aligned mouse and human CTLA-4. Bestfit alignment was used to determine the consensus sequence between mouse and human CTLA-4. Analysis of sequential 50 bp intervals of the consensus sequence for sequence similarity between the two species was performed and values were plotted against the Y axis where a similarity index of one corresponds to 100 % sequence identity. Vertical lines reflect a major gap in sequence similarity between the two loci. Triangles represent simple repeats present in mouse sequences while circles represent simple repeats found in human sequences. Rectangles representing CTLA-4 coding regions are as indicated.

Figure 3 is a multiple alignment of the mouse (SEQ ID NO:55) and human (SEQ ID NO:56) CTLA-4 loci. Bestfit alignment output is presented with boxed nucleotides representing coding sequences and underlined nucleotides indicating simple repetitive elements.

Figure 4 shows a multiple tissue northern blots of mouse and human CTLA-4 and T-cell receptor. Panel A is a mouse MTN blot of poly A+ RNA derived from major physiological organs was hybridized to either mouse CTLA-4 or T-cell receptor constant region probe (TCR const). Hybridization signal to both probes was detected in multiple tissue types. Panel B shows two human MTN blots were analogously hybridized to radiolabelled human CTLA-4 probe or T-cell receptor constant region as described. Likewise, multiple tissue types generated hybridization signals for each probe used. Figure 5 shows that different polymorphisms are observed using the sara31/32 primer set to amplify the PMR shown in nucleotides 19767-19792 of SEQ ID NO:2 as compared to those observed when the hR2 PMR (shown in nucleotides 39887-39926 of SEQ ID NO:2) is amplified.

Detailed Description of the Invention

The instant invention provides polymorphic microsatellite repeat ("PMR") sequences in the costimulatory receptor gene locus. These sequences are useful as markers e.g., in genetic testing, for example, to identify genetic material from a given individual and/or in identifying individuals at risk for developing a particular disease or condition. In particular, the subject PMRs are useful in identifying individuals that carry or are at risk for developing diseases or conditions associated with signaling via a costimulatory receptor, such as CD28. CTLA4. or I COS. such as autoimmune diseases or conditions. In addition, the invention provides sequences suitable for use as primers in amplifying the PMRs of the invention.

I. Definitions

As used herein the term "costimulatory receptor gene locus^" includes the genetic region comprising the genes encoding the costimulatory receptors CD28. CTLA4. and ICOS. This locus spans approximately 300 kb on chromosome 2q33.

As used herein the term "polymorphic microsatellite repeat (PMR)" includes regions of a chromosome containing runs of short repeated sequences (e.g., ATATAT). These simple microsatellite DNA repeats tend to be interspersed throughout the genome and the number of such repeats is highly variable in the population. For example, individuals may have a different number of copies of the repeat at a particular locus.

As used herein the term "polymorphism^" with respect to a particular region of a DNA molecule includes naturally occurring variations in nucleotide sequence among individuals that occur in a particular region. Such polymorphisms can occur, e.g.. when DNA from one individual has an insertion of an additional nucleotide(s), a deletion of a nucleotide(s). a substitution of a nucleotide(s) when compared to DNA from another individual. Polymorphisms in microsatellite repeats frequently lead to differences in the length of the repeat that can be easily visualized, e.g., by Southern blot analysis of chromosomal DNA fragments using an oligonucleotide probe to visualize the size DNA fragment containing the particular repeat.

As used herein, the term "immune cell^" includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells: myeloid cells, such as monocytes, macrophages. eosinophils. mast cells, basophils. and granulocytes.

As used herein, the term "costimulate" with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non- activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. As used herein the term "costimulatory molecule" includes molecules which are present on antigen presenting cells (e.g., B7-1. B7, B7RP-1 (Yoshinaga et al. 1999. Nature 402:827). B7h (Swallow et al. 1999. Immunity. 11 :423) and/or related molecules (e.g., homologs)) that bind to costimulatory receptors (e.g., CD28. CTLA4. ICOS (Hutloff et al. 1999. Nature 397:263). B7h ligand (Swallow et al. 1999. Immunity. 11 :423) and/or related molecules) on T cells.

As used herein, the phrase "autoimmune disorder or condition^" includes immune responses against self antigens. As used herein, the term " immune response" includes T and/or B cell responses, i.e., cellular and/or humoral immune responses.

As used herein, the term "detect^" with respect to PMR sequences includes various methods of analyzing for a polymorphism at a particular site in the genome. The term "detect" includes both "direct detection," such as sequencing, and "indirect detection." using methods such as amplification or hybridization.

II. Isolation of Genetic Material

The subject PMR sequences are useful as markers, e.g.. to identify genetic material as being derived from a particular individual or in making assessments regarding the propensity of an individual to develop a particular disorder or condition. the ability of an individual to respond to a certain course of treatment, or in other diagnostic or prognostic assays described in more detail below.

Genetic material suitable for use in such assays can be derived from a variety of sources. For example, nucleic acid molecules (preferably genomic DNA) can be isolated from a cell from a living or deceased individual using standard methods. Cells can be obtained from biological samples, e.g., from tissue samples or from bodily fluid samples that contain cells, such as blood, urine, semen, or saliva. The term "biological sample" is intended to include tissues, cells and biological fluids containing cells which are isolated from a subject, as well as tissues, cells and fluids present within a subject. The subject detection methods of the invention can be used to detect PMR polymorphisms in DNA in a biological sample in intact cells (e.g., using in situ hybridization) or in extracted DNA, e.g., using Southern blot hybridization. In one embodiment, immune cells are used to extract genetic material for use in the subject assays.

III. PMR Sequences In the Costimulatory Receptor Locus

Any of the PMRs identified in the costimulatory receptor locus identified herein can be utilized as a marker to detect DNA polymorphisms among individuals.

Several approaches were taken to identify the subject PMR sequences. In one approach, two human bacterial artificial chromosome (BAC) clones (clones 22700 and 22608) that included and flanked the human CTLA4 locus were shotgun cloned and sequenced. The sequence data was assembled into 262.117 base pairs of contiguous sequence that is presented in SEQ ID Nos: 1- 6. The sequence information in SEQ ID Nos: 1-6 is contiguous (i.e., nucleotide number 50,000 of SEQ ID NOT is upstream from (5' and adjacent to) nucleotide number 1 of SEQ ID NO:2 and so forth). BAC clone 22700 begins at nucleotide 1 of SEQ ID NO: 1 and ends at nucleotide 31662 of SEQ ID NO: 4 and BAC clone 22608 begins at nucleotide 14576 of SEQ ID NO: 3 and ends at nucleotide 12117 of SEQ ID NO:6.

A number of PMR sequences were identified in this contiguous sequence. In addition, the ICOS gene locus was localized to this region. The position of the ICOS exons is set forth in SEQ ID NO: 4. The PMR sequences identified in the costimulatory receptor locus (as well as exemplary primers that can be used to amplify them) are set forth in the table below:

PMR SEQUENCE PRIMERS USED TO AMPLIFY nucleotides 5722-5746 of SEQ ID NO: 1 sara41/42 (SEQ ID Nos: 51 and 52) nucleotides 23904-23957 of SEQ ID NOT sara45/46 (SEQ ID Nos: 23 and 24) nucleotides 27689-27780 of SEQ ID NOT saral 7/18 (SEQ ID Nos: 25 and 26) nucleotides 30766-30801 of SEQ ID NOT saral9/20 (SEQ ID Nos: 49 and 50) nucleotides 6550-6597 of SEQ ID NO: 1 sara43/44 (SEQ ID Nos: 47 and 48) nucleotides 1991 1-19956 of SEQ ID NOT nucleotides 19767-19792 of SEQ ID NO:2 sara25/26 (SEQ ID Nos: 31 and 32) nucleotides 39887-39926 of SEQ ID NO:2 nucleotides 48152-48199 of SEQ ID NO:2 saral/2 (SEQ ID Nos: 7 and 8) nucleotides 49894-49925 of SEQ ID NO:2 sara3/4 (SEQ ID Nos: 9 and 10) nucleotides 10141-10177 of SEQ ID NO:3 sara39/40 (SEQ ID Nos: 45 and 46) nucleotides 11459-11520 of SEQ ID NO:3 sara33/34 (SEQ ID Nos: 39 and 40) nucleotides 12329-12419 of SEQ ID NO:3 sara35/36 (SEQ ID Nos: 29 and 30) nucleotides 15527-15567 of SEQ ID NO:3 sara37/38 (SEQ ID Nos: 43 and 44) nucleotides 24050-24075 of SEQ ID NO:3 saral 1/12 (SEQ ID Nos: 17 and 18) nucleotides 26009-26056 of SEQ ID NO:3 saral3/14 (SEQ ID Nos: 19 and20) nucleotides 27317-27350 of SEQ ID NO:3 saral5/16 (SEQ ID Nos: 21 and 22) nucleotides 30069-30101 of SEQ ID NO:3 sara21/22(SEQ ID Nos: 27 and 28) nucleotides30535-30574 of SEQ ID NO:3 sara23/24 (SEQ ID Nos: 29 and 30) nucleotides 33714-33758 of SEQ ID NO:3 sara9/10 (SEQ ID Nos: 15 and 16) nucleotides 43819-43925 of SEQ ID NO:3 sara31/32 (SEQ ID Nos: 37 and 38) nucleotides 46547-46572 of SEQ ID NO: 3 sara5/6 (SEQ ID Nos: 11 and 12) nucleotides 46828-46875 of SEQ ID NO:3 sara7/8 (SEQ ID Nos: 13 and 14) nucleotides 21433-21459 of SEQ ID NO:4 sara27/28 (SEQ ID Nos: 33 and 34) nucleotides 21 141 -21 177 of SEQ ID NO :4 sara29/30 (SEQ ID Nos: 35 and 36) nucleotides 25980-26031 of SEQ ID NO:4 sara47/48 (SEQ ID Nos: 53 and 54) In one embodiment. PMRs of the invention are conserved between the human and mouse costimulatory receptor locus (the mouse CTLA-4 locus is shown in SEQ ID NO:55 and the human CLTA-4 locus is shown in SEQ ID NO:56). For example, a number of PMR sequences were identified in the mouse CTLA-4 locus. Mouse B2-like repeat elements were found from nucleotides. 98-31 , 3265-3053, and 7957-7899 of SEQ ID NO: 55 while two sqr-like repeat elements (Genbank accession # X03942) were found in tandem from nt. 8810-8695 and 8760-8596 of SEQ ID NO:55. Within the area compared between human and mouse CTLA-4. multiple simple repeats were found interspersed throughout the non-coding regions of the two CTLA-4 loci, with the exception of intron 2. These repeats consisted of dinucleotide. trinucleotide and tetranucleotide motifs. Five simple repetitive sequences were found in the mouse CTLA-4 gene (mRl-mR5), while two were present in the human CTLA-4 gene (hRl-hR2).

The hRl repeat sequence is located at nucleotides 5412-5496 of SEQ ID NO:56. The hR2 repeat sequence (also referred to as the CTLA4 3 'UTR microsatellite repeat (Yanagawa et al. 1997. Thyroid 7:843)) is located at nucleotides 6561-6624 of SEQ ID NO: 56 (and is also shown in nucleotides 39887-39926 of SEQ ID NO: 2).

From the 5' end of the mouse sequence, the first discernable simple repetitive sequence (mRl) encountered in the mouse gene (nucleotides 363-424 of SEQ ID NO: 55) was a GT repeat located 363 nt from the 5^" end of the compiled sequence. This simple repeat contained a 63 nucleotide block in which were interspersed 14 deviating nucleotide substitutions. Despite the substitutions, the periodicity of the GT repeat was maintained throughout this block of DNA. The second (mR2) and third (mR3) repetitive sequence in mouse were localized in intron 1 (nucleotides 2237-2422 of SEQ ID NO:55 and nucleotides 3492-3527 of SEQ ID NO: 55. respectively) and comprised of repeated G A G A/G A tetranucleotide motif 191 b.p. in length, and a repeated CA A/G trinucleotide motif 50 b.p. in length. The tetranucleotide periodicity was not perfectly maintained for mR2 whereas mR3. located 1069 bp downstream of mR2. retained trinucleotide periodicity. The first human repeated sequence (hRl ; nucleotides5412- 5496 of SEQ ID NO: 56) and the fourth mouse repeated sequence (mR4: nucleotides 5827-5919 of SEQ ID NO: 55) were CT dinucleotide rich regions that shared similar positional location in intron 3 and exhibited 72% sequence identity with each other.

Among mRl-mR5 and hRl-hR2, only two sequences, hRl and mR4. were shared between the two species. Although both mR4 and hRl sequences were degenerate with numerous base mismatches and low degrees of periodicity, the CT motifs were more distinctly retained in hRl (n = 30) compared to that of mR4 (n = 22). Eleven base pairs downstream of mR4 was the fifth repetitive mouse sequence. mR5 (nucleotides 5931-5984 of SEQ ID NO: 55), comprised of 26 CA repeats with perfect dinucleotide periodicity and only one C to T nucleotide substitution. The hR2 DNA segment (nucleotides 6561 -6623 of SEQ ID NO: 56) in the human CTLA-4 locus was located 507 base pairs downstream of the termination codon and consisted of 32 (AT) repeats with one base pair substitution. This well characterized hR2 repeat has been used extensively in genetic studies in testing the linkage of CTLA-4 to numerous autoimmune diseases in humans. In a preferred embodiment, a PMR of the invention does not include the hR2 repeat.

IV. PMR Sequences In The Costimulatory Receptor Locus And Genetic Diseases

Polymorphisms in the CTLA-4 gene have been linked to various autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM) (Witas et al.. Biomedical Letters 58: 163-168. 1998); Addison's disease. Graves^' disease and autoimmune hypothyroidism (Kemp et al.. Clin. Endocrinol. 49:609-613, 1998); myasthenia gravis and thymoma (Huang et al.. J. Neuorimmunol. 88:192-198. 1998); lupus (Mehrian et al.. Arthritis Rheum. 41 :596-602, 1998); thyroiditis, particularly postpartum thyroiditis (Waterman et al.. Clin. Endocrinol.. 49:251-255. 1998); rheumatoid arthritis (Seidl et al.. Tissue Antigens 51 :62-66, 1998); Hashimoto^'s disease (Barbesino et al.. J. Clin.

Endocrnol. and Metab. 83:1580-1584. 1998): coeliac disease (Djilali-Saiah et al.. Gut 43:187-189, 1998): and leprosy (Kaur et al.. Hum. Genet. 100:43-50. 1997). Of these diseases. IDDM. Grave^'s disease and hypothyroidism (Kotsa. K.. et al. (1997). Clin Endocrinol (Oxf) 46: 551-4: Marron. M. P.. et al, (1997). Hum Mol Genet 6: 1275-82) have been found to be associated with certain alleles of the hR2 region of human CTLA-4 (position 6560 of SEQ ID NO: 56). Currently. there is no information available on whether the hR2 region confers biologically significant attenuation of CTLA-4 expression or whether this polymorphism is merely a marker for an associated gene closely linked to this CTLA-4 allele. The novel PMR sequences described herein provide additional markers that may be more closely linked with certain autoimmune disorders or conditions. As described in the appended Examples, use of the instant PMR sequences as markers can provide different results, i.e.. different distribution of polymorphisms, than those obtained using the hR2 marker, indicating that the PMR markers disclosed herein can be used to further refine genetic alleles linked to the costimulatory receptor locus.

V. Uses of PMR Sequences Of The Invention

The PMR sequences of the invention are useful as markers in a variety of different assays. The PMR sequences of the invention can be used, e.g., in diagnostic assays, prognostic assays, and in monitoring clinical trials for the purposes of predicting outcomes of possible or ongoing therapeutic approaches. The results of such assays can, e.g., be used to prescribe a prophylactic course of treatment for an individual, to prescribe a course of therapy after onset of a disease or disorder, or to alter an ongoing therapeutic regimen.

Accordingly, one aspect of the present invention relates to diagnostic assays for detecting PMRs in a biological sample (e.g., cells, fluid, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder linked to one or more PMR polymorphisms. The subject assays can also be used to determine whether an individual is at risk for passing on the propensity to develop a disease or disorder to an offspring. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a autoimmune disorder or condition. For example, polymorphisms in a PMR sequence can be assayed in a biological sample. Such assays can be used for prognostic, diagnostic, or predictive purpose to thereby phophylactically or therapeutically treat an individual prior to or after the onset of an autoimmune disorder associated with one or more PMR polymorphisms. In another embodiment, the methods further involve obtaining a control biological sample from a control subject, determining the PMR polymorphism in the sample and comparing the PMR polymorphisms present in the control sample with those in a test sample. The invention also encompasses kits for detecting the PMR polymorphism in a biological sample. For example, the kit can comprise a primer capable of detecting one or more PMR sequences in a biological sample. The kit can further comprise instructions for using the kit to detect PMR sequences in the sample.

Polymorphisms in the costimulatory receptor locus among individuals can be used to identify genetic material as being derived from a particular individual. For example, minute biological samples can be obtained from an individual and an individual's genomic DNA can be amplified using primers which amplify one or more of the disclosed PMR sequences to obtain a unique pattern of bands. A particular band pattern can be compared with a band pattern in a sample known to have come from a certain individual to determine whether the patterns match. Other exemplary methods for detection are set forth below. Panels of corresponding DNA sequences from individuals can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

The subject PMR sequences can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. For example, to make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

The PMR nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., probes which can be used in. for example, an in situ hybridization technique, to identify a specific tissue, e.g.. in cases where a forensic pathologist is presented with a tissue of unknown origin. VI. Detection of Polymorphisms

Practical applications of techniques for identifying and detecting polymorphisms relate to many fields including forensic medicine, disease diagnosis and human genome mapping. DNA polymorphisms can occur, e.g., when one nucleotide sequence comprises at least one of 1) a deletion of one or more nucleotides from a PMR sequence; 2) an addition of one or more nucleotides to a PMR sequence; 3) a substitution of one or more nucleotides of a PMR sequence, or 4) a chromosomal rearrangement of a PMR sequence as compared with another sequence. As described herein, there are a large number of assay techniques known in the art which can be used for detecting alterations in a PMR sequence.

Repeats associated with specific genetic alleles are commonly used as molecular markers in phenotyping human populations. Microsatellite repeats (simple repetitive elements) are defined as motifs of 1-6 bases in length and tandemly reiterated 5-100 times or more. The assay of repeats is amenable to automation, and thus has gained wide use in forensic science and genetic disease linkage determination. These repeats are dispersed throughout the genome and currently are not known to have any definitive biological function, although some reports suggest a role of microsatellites in binding nuclear proteins. Indeed a growing number of genetic diseases are being attributed to the presence of alleles containing unusually large repeats (Epplen, C. et al. (1997). Electrophoresis 18: 1577-85).

Analysis of repeats is amenable to highly sensitive PCR approaches using specific primers flanking the repetitive sequence of interest. In one embodiment, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4.683.195 and 4.683.202), such as anchor PCR or RACE PCR. or. alternatively, in a ligation chain reaction (LCR) (see. e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1994) PNAS 91 :360-364), the latter of which can be particularly useful for detecting polymorphisms in the PMR sequence (see Abravaya et al. (1995) Nucleic Acids Res .23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic. DNA) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically amplify a PMR sequence under conditions such that hybridization and amplification of the PMR sequence (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting polymorphisms described herein. Alternative amplification methods include: self sustained sequence replication

(Guatelli. J.C. et al. 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1 197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In one embodiment, after extraction of genomic DNA, amplification is performed using standard PCR methods, followed by molecular size analysis of the amplified product (Tautz. 1993; Vogel. 1997). Typically DNA amplification products are labeled by the incorporation of radiolabelled nucleotides or phosphate end groups followed by fractionation on sequencing gels alongside standard dideoxy DNA sequencing ladders. By autoradiography, the size of the repeated sequence can be visualized and detected heterogeneity in alleles recorded. More recent innovations include the incorporation of fluorescently labeled nucleotides in PCR reactions followed by automated sequencing. Both methods have been used in the study of a human CTLA-4 repeats (Yanagawa, T., et al, (1995). J Clin Endocrinol Metab 80: 41-5 Huang, D.. et al, (1998). J Neuroimmunol 88: 192-8.

In other embodiments, PMR polymorphism can be identified by hybridizing a sample and control nucleic acids to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin. M.T. et al. (1996) Human Mutation 1: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, polymorphisms in PMR can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin. M.T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of polymorphisms. This step is followed by a second hybridization array that allows the characterization of specific polymorphisms by using smaller, specialized probe arrays complementary to all polymorphisms detected. At the present time in this art. the most accurate and informative way to compare

DNA segments requires a method which provides the complete nucleotide sequence for each DNA segment. Particular techniques have been developed for determining actual sequences in order to study polymorphism in human genes. See. for example, Proc. Natl. Acad. Sci. U.S.A. 85. 544-548 (1988) and Nature 330. 384-386 (1987); Maxim and Gilbert. 1977. PNAS 74:560: Sanger 1977. RN4S 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see. e.g.. PCT International Publication No. WO 94/16101: Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159). However, because of the extensive amounts of time and high costs to determine, interpret, and compare sequence information, presently it is not practical to use extensive sequencing for comparing more than just a few DNA segments.

In genetic mapping, the most frequently used screening for DNA polymorphisms arising from mutations consist of digesting the DNA strand with restriction endonucleases and analyzing the resulting fragments by means of Southern blots. See Am. J. Hum. Genet. 32. 314-331 (1980) or Sci. Am. 258. 40-48 (1988). Since polymorphisms often occur randomly they may affect the recognition sequence of the endonuclease and preclude the enzymatic cleavage at that cite. Restriction fragment length polymo hism mappings (RFLPS) are based on changes at a restriction enzyme site. In one embodiment, polymorphisms in a PMR sequence from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Moreover, the use of sequence specific ribozymes (see. for example, U.S. Patent No. 5.498.531) can be used to score for the presence of a specific ribozyme cleavage site.

Another technique for detecting specific polymorphisms in particular DNA segment involves hybridizing DNA segments which are being analyzed (target DNA) with a complimentary, labeled oligonucleotide probe. See Nucl. Acids Res. 9. 879-894 (1981). Since DNA duplexes containing even a single base pair mismatch exhibit high thermal instability, the differential melting temperature can be used to distinguish target DNAs that are perfectly complimentary to the probe from target DNAs that only differ by a single nucleotide. This method has been adapted to detect the presence or absence of a specific restriction site, U.S. Pat. No. 4,683,194. The method involves using an end- labeled oligonucleotide probe spanning a restriction site which is hybridized to a target DNA. The hybridized duplex of DNA is then incubated with the restriction enzyme appropriate for that site. Reformed restriction sites will be cleaved by digestion in the pair of duplexes between the probe and target by using the restriction endonuclease. The specific restriction site is present in the target DNA if shortened probe molecules are detected.

Other methods for detecting polymorphisms in nucleic acid sequences include methods in which protection from cleavage agents is used to detect mismatched bases in RNA RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the PMR sequence with potentially polymorphic RNA or DNA obtained from a tissue sample. The double- stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with S 1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels. See. for example. Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping polymorphisms in PMR sequences obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a PMR sequence is hybridized to a DNA molecule from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See. for example, U.S. Patent No. 5,459.039. In other embodiments, alterations in electrophoretic mobility will be used to identify polymorphisms in PMR sequences. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766. see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control PMR nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA). in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of nucleic acid molecule comprising PMR sequences in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 12753).

Examples of other techniques for detecting polymorphisms include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the PMR is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different polymorphisms when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the polymorphism of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. ( 1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 1 1 :238). In addition it may be desirable to introduce a novel restriction site in the region of the PMR to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known polymorphism at a specific site by looking for the presence or absence of amplification.

Another process for studying differences in DNA structure is the primer extension process which consists of hybridizing a labeled oligonucleotide primer to a template RNA or DNA and then using a DNA polymerase and deoxynucleoside triphosphates to extend the primer to the 5' end of the template. Resolution of the labeled primer extension product is then done by fractionating on the basis of size, e.g., by electrophoresis via a denaturing polyacrylamide gel. This process is often used to compare homologous DNA segments and to detect differences due to nucleotide insertion or deletion. Differences due to nucleotide substitution are not detected since size is the sole criterion used to characterize the primer extension product.

Another process exploits the fact that the incorporation of some nucleotide analogs into DNA causes an incremental shift of mobility when the DNA is subjected to a size fractionation process, such as electrophoresis. Nucleotide analogs can be used to identify changes since they can cause an electrophoretic mobility shift. See, U.S. Pat. No. 4,879,214.

Further, subtle genetic differences among related individuals regarding nucleotides which are substituted in the DNA chains are difficult to detect. V TR's or Jeffrey's probes (which the^' FBI is using to test and identify DNA chains) are very informative but labor intensive, in contrast to microsatellites. such as the microsatellite repeats of the costimulatory locus disclosed herein, which are equally informative but much easier to detect.

The use of certain nucleotide repeat polymorphisms for identifying or comparing DNA segments have been described (e.g., by Weber & May 1989. Am Hum Genet

44:388; Litt & Luthy. 1989 Am Hum Genet 44:397). However the particular PMRs and primers used to identify the polymorphisms (for identification and comparison purposes) of the present invention have not been previously taught or suggested. Many other techniques for identifying and detecting polymorphisms are known to those skilled in the art. including those described in "DNA Markers: Protocols, Applications and Overview.^" G. Caetano-Anolles and P. Gresshoff ed., (Wiley-VCH, New York) 1997. which is incorporated herein by reference as if fully set forth. Since a polymorphic marker and an index locus occur as a "pair", attaching a primer oligonucleotide according to the present invention to one member of the pair, e.g., the polymorphic marker allows PCR amplification of the segment pair. The amplified DNA segment can then be resolved by electrophoresis and autoradiography. A resulting autoradiograph can then be analyzed for its similarity to another DNA segment by autoradiography. Following the PCR amplification procedure, electrophoretic mobility enhancing DNA analogs may optionally be used to increase the accuracy of the electrophoresis step.

The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe/primer nucleic acid or antibody reagent described herein, which may be conveniently used. e.g.. in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a PMR sequence.

VII. Primers for Amplification of PMR Sequences Given the discovery of the instant PMR sequences, primers can readily be designed to amplify the PMR sequences by one of ordinary skill in the art.

In one embodiment, a PMR primer of the invention amplifies a PMR in the ICOS region of the costimulatory receptor locus.

In another embodiment a PMR primer of the invention amplifies a PMR in the CD28 region of the costimulatory receptor locus.

In another embodiment, a PMR primer of the invention amplifies a PMR in the CTLA4 region (e.g.. the 5' UT region, in an intron. or in the 3^'UT region of the CTLA4 gene) of the costimulatory receptor locus. Preferably, were the primer amplifies a PMR in the CTLA4 region of the costimulatory receptor locus, the PMR is not in the 3' untranslated region of the CTLA4 gene. In another embodiment, a PMR primer of the invention that amplifies a PMR in the CTLA4 region of the costimulatory receptor locus does not amplify an hR2 PMR sequence.

In another embodiment, where a PMR primer of the invention that amplifies a PMR in the CTLA4 region of the costimulatory receptor locus amplifies a PMR sequence shown in a nucleotide sequence selected from the group consisting of: nucleotides 5722-5746 of SEQ ID NO: 1 ; nucleotides 23904-23957 of SEQ ID NO: 1 : nucleotides 27689-27780 of SEQ ID NOT ; nucleotides 30766-30801 of SEQ ID NOT : nucleotides 6550-6597 of SEQ ID NO: 1 ucleotides 19911-19956 of SEQ ID NOT: nucleotides 19767-19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152-48199 of SEQ ID NO:2;nucleotides 49894-49925 of SEQ ID NO:2: nucleotides 10141-10177 of SEQ ID NO:3:nucleotides 1 1459-1 1520 of SEQ ID NO:3: nucleotides 12329-12419 of SEQ ID NO:3: nucleotides 15527-15567 of SEQ ID NO:3: nucleotides 24050-24075 of SEQ ID NO:3:nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317-27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3: nucleotides30535-30574 of SEQ ID NO:3;nucleotides 33714-33758 of SEQ ID NO:3; nucleotides 43819-43925 of SEQ ID NO:3;nucleotides 46547-46572 of SEQ ID NO:3: nucleotides 46828-46875 of SEQ ID NO:3.

In one embodiment, a primer for amplification of a PMR sequence is at least about 5-10 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 15-20 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 20-30 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 30-40 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 40-50 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 50-60 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 60-70 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 70- 80 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 80-90 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 90-100 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 100-110 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 110-120 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 120-130 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 130-140 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 140-150 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 150-160 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 160-170 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 170-180 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 180-190 base pairs in length. In one embodiment, a primer for amplification of a PMR sequence is at least about 190-200 base pairs in length.

Preferred primers of the invention comprise the sara sequences set forth in SEQ ID No: 51. SEQ ID No: 52, SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26, SEQ ID No: 49. SEQ ID No: 50, SEQ ID No: 47, SEQ ID No: 48. SEQ ID No: 31. SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 43. SEQ ID No: 44. SEQ ID No: 17. SEQ ID No: 18. SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21. SEQ ID No: 22. SEQ ID No: 27. SEQ ID No: 28, SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 15, SEQ ID No: 16. SEQ ID No: 37. SEQ ID No: 38, SEQ ID No: 11. SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14. SEQ ID No: 33. SEQ ID No: 34, SEQ ID No: 35. SEQ ID No: 36. SEQ ID No: 53, and 54. In one embodiment, a primer for the amplification of a PMR sequence is less than or equal to about 200 base pairs in length (e.g., is about 200. about 175, about 150, about 125, about 100, about 75, about 50, or about 25 base pairs in length) and comprises a nucleotide sequence selected from the group consisting of: SEQ ID No: 51, SEQ ID No: 52. SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25. SEQ ID No: 26. SEQ ID No: 49. SEQ ID No: 50, SEQ ID No: 47. SEQ ID No: 48, SEQ ID No: 31, SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9, SEQ ID No: 10. SEQ ID No: 45, SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 43. SEQ ID No: 44. SEQ ID No: 17, SEQ ID No: 18. SEQ ID No: 19, SEQ ID No: 20. SEQ ID No: 21. SEQ ID No: 22. SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 15, SEQ ID No: 16. SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 1 1. SEQ ID No: 12. SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33, SEQ ID No: 34. SEQ ID No: 35. SEQ ID No: 36, SEQ ID No: 53. and 54.

In another embodiment, primers for the amplification of a PMR sequence consist of a nucleotide sequence selected from the group consisting of: SEQ ID No: 51. SEQ ID No: 52, SEQ ID No: 23. SEQ ID No: 24. SEQ ID No: 25, SEQ ID No: 26. SEQ ID No: 49. SEQ ID No: 50. SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 45, SEQ ID No: 46. SEQ ID No: 39. SEQ ID No: 40, SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21. SEQ ID No: 22, SEQ ID No: 27, SEQ ID No: 28. SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 15. SEQ ID No: 16, SEQ ID No: 37. SEQ ID No: 38, SEQ ID No: 1 1, SEQ ID No: 12. SEQ ID No: 13. SEQ ID No: 14, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35. SEQ ID No: 36, SEQ ID No: 53, and 54.

The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.

Examples

The following materials and methods were used in Examples 1-6: DNA source: Genomic clones from a mouse 129Sv PI library were isolated and subcloned as EcoRl fragments into pBS plasmid vectors as previously described (Tivol, E. A., et al, (1995 Immunity 3: 541-7). Full bidirectional sequencing of all plasmids was performed by fluorescence tagged chain termination followed by fractionation on an Applied Biosystems 373 A automated DNA sequencer (Perkin Elmer/Applied Biosystems, Foster City. CA 94404). Sequence compilation was performed using the Sequencher DNA analysis package (Genecodes Corp., Ann Arbor. MI 48108). Human CTLA-4 genomic DNA sequences (Genbank accession #: M74363, X15070. X15071. XI 5072) were used to assemble the partial genomic locus of human CTLA-4. Additional genomic DNA was isolated from 129Sv Jae mouse embryos and human leukocytes by overnight incubation at 55 °C in lysis buffer. 100 mM Tris pH 8.0. 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 100 ug/ml proteinase K (Life Technologies, Gaithersburg, MD 02894). Genomic DNA was isopropanol precipitated and used as template for high fidelity PCR using Klentaq polymerase (Clontech. Palo Alto. CA 94303) according to manufacturers^" protocols and using the primers: 5^'- CATGGGCAACGGGACGCAGATT-3' and 5'-TCACATTCTGGCTCTGTTGG-3' corresponding to the sequences in exon 2 (nt. 4618-4639) and exon 4 (nt. 6555-6536) for mouse CTLA-4. and 5'-TACCTGGGCATAGGCAACGGA-3' and 5^'- AATGGTTTCTCAATTGATGGG-3' corresponding to the sequences in exon 2 (nt 4137-4157) and exon 4 (nt. 6064-6044) for human CTLA-4. PCR was performed in a GeneAmp 9600 thermal cycler (Perkin-Elmer/Applied Biosystems) with cycling parameters: 95 °C - 2 min. 1 cycle; 95 °C - 15 sec. 60 °C - 20 sec. 72 °C - 45 sec, 35 cycles; 72 °C - 10 min 1 cycle. Amplified DNA was gel purified and subcloned into pCR2T using TOPO-TA cloning kit (Invitrogen. Carlsbad. CA. 92008) according to the manufacturer's protocol. Six individual mouse clones from 6 separate PCR reactions and 5 individual human clones from 5 separate PCR reactions were subsequently isolated. Complete bidirectional sequencing of each of the two sets of PCR clones followed by alignment yielded separate mouse and human consensus sequences. Of the PCR clones isolated, mouse clone 49-13 exhibited 100% sequence identity with the mouse consensus sequence and human clone 14- 10#4C exhibited 100% identity with the human consensus sequence. RNA blots: Mouse and human Multiple Tissue Northern (MTN) blots

(Clontech) were hybridized using cDNA probes encoding CTLA-4 and T-cell receptor. For CTLA-4 probes, mouse and human cDNA fragments corresponding to the complete coding sequence (Genbank accession # X05719 and # L15006. respectively) were radiolabelled using specific primers for the 5^' and 3' end of the cDNA under conditions routinely used for random priming (Ling. V.. et al. (1998). Exp Cell Res 241 : 55-65). Mouse and human T-cell receptor alpha (Genbank accession # L34703 and # U07659. respectively) cDNA fragments corresponding to the constant region (nt. 420-827 for mouse, nt. 416-840 for human) were similarly radiolabelled using gene specific oligonucleotides. Mouse MTN and human MTN blots were prehybridized and hybridized using Express Hyb hybridization solution (Clontech) according to manufacturer^'s protocols and incubated at 55 °C overnight. After hybridization, blots were sequentially washed with 2 x SSC, 1% SDS. and 0.1 x SSC, 1 % SDS at 65 °C until low background was achieved. Autoradiographic images were generated either by exposure to Kodak X-OMAT film using dual intensifying screens at -80 for 3 days or by exposure to Fuji phosphoimaging plates overnight. Software: Primary sequence input, mapping, dotplot. and graphic output were performed using Gene Works 2.5.1 (Oxford Molecular Group Inc.. Campbell. CA 95008). Alignment calculations were performed using Bestfit module of the GCG (Wisconsin Package 9.1, Madison. WI 5371 1). using default settings. Local similarity calculations were based on successive 50 base pair intervals of the consensus sequence derived from Bestfit alignment. Local similarity scores were calculated based on the number of matches/ (matches + mismatches) across consecutive 50 bases of consensus sequence where gaps, irrespective of length, were counted as 1 mismatch. Transcription factor binding site analysis software used was Matlnspector (Quandt. K.. et al. (1995). Nucleic Acids Res 23: 4878-84).

Example 1. Structure and Molecular Cloning of CTLA-4 DNA

The structural organization of the CTLA-4 gene is shared between human and mouse, but the exon designation for human CTLA-4 in the literature is not consistent, and further, is not based on comparative genomic sequence analysis with any full length human CTLA-4 cDNA clones (Dariavach, P.. et al. (1988). Eur J Immunol 18: 1901-5: Harper. .. et al., (1991). J Immunol Harper. ... et al, (1991). J Immunol 147: 1037- 44). The authors in those studies chose to equate the term exon with the term coding sequence (CDS) despite lacking information concerning 5^" and 3' untranslated regions of the human CTLA-4 cDNA necessary for determining exon structure. That first report identifying human genomic CTLA-4 DNA lacked the leader sequence (Dariavach. P.. et al, (1988). Eur J Immunol 18: 1901-5) and thus the extracellular domain (CDS 2) was designated as exon 1. transmembrane domain (CDS 3) as exon 2. and cytoplasmic domain (CDS 4) as exon 3. Although a genomic clone containing the actual leader sequence (CDS 1) was later identified (Harper. K.. et al. (1991). J Immunol 147: 1037- 44) and necessitated revision of this exon/intron designation, many current publications continue to adhere to the earlier nomenclature (Braun. J., et al, (1998). Tissue Antigens 51 : 563-6: Kotsa. K., et al, (1997). Clin Endocrinol (Oxfi 46: 551-4; Yanagawa. T.. et al. (1995). J Clin Endocrinol Metab 80: 41-5). To avoid further confusion, the CDS designation will be used hereafter.

In order to obtain mouse CTLA-4 genomic DNA for sequencing, two plasmids. clone 30-5 and clone 13, encoding mouse CTLA-4 were derived by subcloning selected DNA fragments of a genomic PI 129Sv mouse library, as described previously (Tivol, E. A., et al, (1995 Immunity 3: 541-7). Sequence analysis of clone 30-5 identified a DNA fragment 4772 bp in length encoding regions 5^' to CDS 1, CDS 1. intron 1. CDS 2 and part of intron 2. Clone 13 encoded a portion of intron 3. CDS 4 and sequences 3^' to the CDS 4. The data derived from clone 13 extends the previously published mouse CTLA-4 cDNA 3' UTR by 2935 b.p.. Genomic sequence of the intronic regions between clone 13 and 30-5 was obtained by multiple independent high fidelity PCR reactions yielding one cloned product (clone 49-13) identical to both the consensus sequence of the 6 individual PCR clones and existing mouse CTLA-4 coding region sequences. The aligned data from clones 13. 30-5 and 49-13 resulted in 10.640 bp of primary sequence containing four coding sequences, three introns. as well as non-coding sequences upstream and downstream (Figure 1 A) of the mouse CTLA-4 gene.

To date, the human genomic sequence of CTLA-4 is incomplete, lacking portions of intron 2 and intron 3 (Dariavach, P., et al, (1988). Eur J Immunol 18: 1901- 5). To complete the genomic sequence of human CTLA-4, multiple independent PCR cloning reactions were performed spanning the region between exon 2 and exon 4. PCR Clone 14-10#4C exhibited 100% sequence similarity with published human CTLA-4 CDS 3 DNA sequences as well as the consensus sequence derived from the independent PCR clones. The compiled 7915 bp human CTLA-4 genetic locus contained 4962 bp separating the initiation codon of CDS 1 and termination codon of CDS 4 (Figure 1 B). The murine CTLA-4 gene sequence is presented as SEQ ID NO:55. The human CTLA- 4 gene sequence, as determined by applicants, is reported as SEQ ID NO:56.

Example 2. Sequence Analysis Comparison of the mouse genomic sequence with the earlier mouse CTLA-4

DNA sequences (Brunet. J. F.. et al, (1987). Nature 328: 267-70) revealed 42 discrepancies consisting of 14 base substitutions, 7 insertions, 20 deletions and the absence of an extra 49 bp repeated sequence. Only one nucleotide discrepancy, a T to A nucleotide substitution, was in a translated codon. located in exon 3 at position 5184. This discrepancy was reflected as a serine codon in the published cDNA sequence and as a threonine codon in the genomic sequence. Within the 3' UTR region, a 49 bp region (position 6913 to 6961 of the genomic sequence) was found to be directly repeated in tandem at location 1096 in the published cDNA sequence. This tandem repeat was not detected in the sequencing of both strands of the genomic sequence, nor in sequencing the products of multiple 3' RACE of spleen RNA samples of either the 129Sv or

Swiss- Webster mouse strains (data not shown). It must be noted that although there appear to be numerous nucleotide discrepancies with the published data, others have also mentioned similar findings (Perkins. D., et al, (1996). J Immunol 156: 4154-9). These 42 anomalies may be due to sequencing methodologies or due to actual genetic differences between mouse strains used in this study and the BIO.BR strain used in the previous study (Brunet. J. F., et al, (1987). Nature 328: 267-70). The complete determination of both mouse and human CTLA-4 genomic sequences enabled a comparison of the two gene loci. Graphic representation of sequence similarity by dot plot analysis demonstrated a nearly 1 : 1 sequence correspondence extending throughout the loci, including 5^' and 3^' untranslated regions and intronic sequences (Figure 2A). When the consensus sequence was analyzed in 50 nucleotide intervals and plotted against similarity (Figure 2B), a positionally based graph of sequence conservation was generated with a mean similarity of 68%. Peaks of high sequence similarity (>85% identity) were found in sequences 5^" to the initiation codon, CDS 1. intron 1, CDS 2. CDS 3, CDS 4 and sequences 3^' to the termination codon. Valleys of low conservation (<50%) were found in only introns 1 and 3. Bestfit alignment between the two sequences revealed extensive similarities between the human and mouse CTLA-4 loci from positions 343 to 7195 of human CTLA-4 and positions 163 to 7701 of mouse CTLA-4 (Figure 3). For maximal alignment of intron 1, one major gap of 214 bp was introduced into the human CTLA-4 sequence at position 2994. corresponding to positions 3192 to 3406 in the mouse sequence. Bestfit alignment over the entire length of the gene resulted in an overall similarity score of 71% identity, with individual homologous coding and non-coding regions yielding similarity values ranging from 65% to 90%. Suφrisingly. certain non- coding regions on average, had equally high or higher similarity values than some coding regions. A higher degree of average similarity was found between homologous 5^' and 3' flanking non-coding sequences than that of homologous CDS 1 or CDS 2. Of the 3 introns. intron 3, which separates transmembrane and cytoplasmic coding domains, was found to have the highest degree of sequence similarity (71%). As reported previously, the highest degree of sequence conservation among the 4 coding regions was found in CDS 4 (98%) encoding the invariant cytoplasmic domain of CTLA-4 (Dariavach. P., et al, (1988). Eur J Immunol 18: 1901-5).

Example 3. CTLA-4 Expression Patterns of Whole Tissue RNA

In previous expression pattern studies. RNA blot analyses of CTLA-4 were performed on cell lines and selected tissues, but to date no analyses have been described using a full panel of the major organs in any species. Using full length CTLA-4 cDNA coding sequences as probes, commercially produced multiple tissue RNA blots were analyzed for the presence of CTLA-4 transcripts in both human and mouse tissues. In mouse RNA blots, CTLA-4 transcripts were detected at high levels in heart, spleen, lung, and skeletal muscle, while lower levels were detected in brain, liver and testis. No clear signal was observable for kidney samples. The predominant transcript size detected was approximately 2 kb. while a minor band of approximately 6 kb was detected in heart, spleen, lung and skeletal muscle (Figure 4A). In human multiple tissue RNA blots, high levels of hybridization to CTLA-4 were detected in spleen, thymus. peripheral blood leukocytes, while lower signals appeared for testis, uterus, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Little or no signal was present in either prostate or small intestine (Figure 4B). Multiple human transcripts sizes were detected in tissues highly expressing CTLA-4. with sizes of approximately 1 kb. 2.4 kb, 5 kb. and 7 kb. In samples with lower expression levels of CTLA-4. only the 2.4 kb and 5 kb transcripts were detected. To assess whether tissue CTLA-4 expression was correlated with T-cell gene expression, blots were stripped and reprobed with corresponding radiolabelled mouse or human T-cell receptor cDNA. As seen in the blots, all tissues with high expression of CTLA-4 also exhibited high levels of T-cell receptor gene expression, a marker for the presence of T-cells. Because RNA blots are commercially produced, the condition or treatment of the tissues prior to RNA extraction is not known, thus the close association of CTLA-4 and T-cell receptor expression patterns suggests a common cellular origin of both transcripts most probably from highly activated passenger lymphocytes lodged within those tissues. However, given the unexpectedly high degree of hybridization signal of CTLA-4 message, one could not exclude the possibility that CTLA-4 message may also originate from other non- lymphoid cell types found within those tissues.

The role of CTLA-4 as a regulator of T-cell activation and peripheral tolerance has emerged in recent years. Initial evidence suggesting that CTLA-4 would have important function in the immune system arose from comparative surveys of CTLA-4 cDNA sequences. Of those sequences, mouse, human, cow. rabbit and rat CTLA-4 cDNA exhibited high DNA sequence conservation with complete amino acid conservation of the intracellular domain of CTLA-4.

Example 4. Implications for Non-coding Sequence Similarities.

In this study, analyses of the mouse and human CTLA-4 gene demonstrated high sequence similarities extending into non-coding regions of the gene sequences. To date, only a few comparative studies of homologous human and rodent genes have been performed on gene loci, gene clusters and chromosomal regions (Hardison. R. C. et al. (1997). Genome Res 1: 959-66). Based on those studies, a mosaic of non-coding sequence conservation patterns has been described, in that the degree of sequence similarities between non-coding mouse and human genomic DNA are not uniformly distributed within the genome. DNA examined thus far include the beta-like globin gene clusters. alpha globin gene clusters (Koop. B. F., and Hood. L. (1994). Nat Genet 1: 48- 53). the BTK genomic region (including Bruton^'s tyrosine kinase-BTK. alpha-galactosidase A. L44L. FTP-3. and FCI-12); (Oeltjen. J. C, et al. (1997). Genome Res 1: 315-29). the gene cluster at human chromosome 12pl3 and its syntenic region in mouse chromosome 6 (Ansari-Lari. M. A., et al, (1998). Genome Res 8: 29-40). the immunoglobin heavy chain (Koop. B. F.. et al. (1996). Mol Phylogenet Evol 5: 33-49) and the T-cell receptor (Koop, B. F.. and Hood, L. (1994). Nat Genet 1: 48-53). Of the mouse and human sequences compared, the T- cell receptor gene alignments and myosin heavy chain gene alignments demonstrated extreme intron sequence conservation over the entire length of the compared loci (-70% identity). With regards to other immunologically related gene loci, the mouse and human immunoglobin heavy chain and Bruton^'s tyrosine kinase (BTK) loci also exhibited high sequence similarity over a more limited segment of the gene, while little sequence similarity was detected beyond exon sequences in comparisons of mouse and human CD4 gene loci. It is interesting to note that CTLA-4 is a member of the Ig superfamily member of proteins, as are T-cell receptor and immunoglobin heavy chains. Other members of the Ig superfamily genes, especially the stimulatory B7 costimulatory receptor CD28, may also have conserved intron sequence.

From the standpoint of a mechanistic evolutionary theory, it has been postulated that the existence of disparate types of similarity patterns for non-coding regions argues for a model of genomic DNA serving as an information organelle. and that certain regions of the chromosome are preferentially shielded against mutations during natural selection (Koop. B. F.. and Hood. L. (1994). Nat Genet 7: 48-53). The presence of highly conserved non-coding sequences in CTLA-4 is consistent with the notion that unidentified functional constraints and regulatory biochemical mechanisms are imposed on selected parts of the genome; perhaps relating to the transcriptional status of that region encoded. An alternate, less likely explanation was that the occasional striking degree of high similarity between large tracts of DNA within the genome arise by chance (Koop, B. F., and Hood, L. (1994). Nat Genet 1: 48-53). This hypothesis may be tested in the future when additional comparative sequence analyses are performed with corresponding gene loci from non-rodent and non-primate species. It is also possible that the rate of nucleotide mutations within the genome is truly constant with respect to time. The high sequence similarity observed between the non-coding regions of genes such as those seen for CTLA-4 and T-cell receptor may reflect a more recent introduction of these genes during the course of natural history. This model of sequential gene addition could also generate zones of sequence conservation organized as a mosaic within the genome, independent of sequence placement in any particular chromosome, and further it would dispense with the need for a localized mutation- constraining mechanism within the genome.

Example 5. Evolutionarily Conserved Flanking Sequences

Current knowledge of the molecular basis for CTLA-4 gene expression has been limited to the delineation of the promoter region. In those reports, the minimal promoter region was reported to localize within the first 335 bp. upstream of the initiation methionine. Cell activation by T-cell receptor and CD28 ligation induced reporter gene transcription from this CTLA-4 promoter, suggesting the presence of control motifs within this region of DNA (Finn, P. W., et al, (1997). J Immunol 158: 4074-81). The 5' upstream region of both human and mouse CTLA-4 are conspicuously similar in sequence within approximately 800 bp of the initiation codon. Similar findings of extensive sequence similarities at the 5' flanking regions of genes have been reported for other genes (Phinney. D. G., et al, (\996)Oncogene 13: 1875-83: Phinney. D. G., et al... (1995). Genomics 28: 228-34) and have pointed to conserved transcriptional control over those loci. Of the transcription factor binding sites analyzed (Quandt. K.. et al, (1995). Nucleic Acids Res 23: 4878-84), 68 motifs were found of which 50 were shared between mouse and human CTLA-4 5' flanking sequences. Such transcription factor binding sites shared by both human and mouse CTLA-4 include: AP-1. CEBP. GATA-1, IK1-3, and OCT-1 ; however, because DNA binding motifs are generally only four to six base pairs in length, many such motifs can arise upon matrix analysis, thus verification of actual transcription factor binding sites remains to be demonstrated by biochemical analysis. The high degree of sequence similarities of introns 2 and 3 between mouse and human CTLA-4 genes also suggest targeted studies of these regions may reveal further zones of transcriptional regulation. Example 6. CTLA-4 Expression Patterns

To date, analyses of CTLA-4 expression in both mouse and humans have been focused on mostly lymphoid cells and cell lines. From those reports. CTLA-4 expression was found in activated lymphoid cell and cell lines, but not in non-activated cells. In this report, however. CTLA-4 message was found in a number of different tissue sources, including non-lymphoid organs such as liver, skeletal muscle and testis from both mouse and human tissues. Because the expression of T-cell receptor alpha was also detected in the same samples which expressed CTLA-4. one could not exclude the possibility that activated passenger T cells could potentially contribute to the presence of CTLA-4 transcripts, and that recent advances in probe hybridization methods have enabled greater sensitivity in detecting CTLA-4 message. Given that passenger T cells to any one particular tissue should not be particularly abundant, this CTLA-4 expression pattern suggest that if the passenger cells did contribute to the CTLA-4 signal, then those lymphoid cells would necessarily be highly activated. Alternatively, CTLA-4 expression may be wider than previously thought, and is not necessarily confined to activated T cells. Indeed, we have previously found low levels of CTLA-4 gene expression as well as B7-1. B7-2, and CD28 by RT-PCR in differentiating embryonic stem cells where no defined role of immunologic costimulation exists (Ling, V., et al. (1998). Exp Cell Res 241 : 55-65). CTLA-4 may have a cryptic ontological role in embryonic development and is later re-utilized predominantly in lymphocyte signaling.

With the complete mouse and human CTLA-4 loci, future studies directed at uncovering the cross species transcriptional control of these two gene loci should now be possible. An additional target for future studies would be the sequence determination and transcriptional analysis of the mouse and human CD28 gene loci. Although CTLA-4 and CD28 appear to have opposing signaling function, these two receptors share some similarity in intron/exon structure and protein sequence. These structural similarities have led to the hypothesis that these two molecules are arose by an ancient gene duplication event (Dariavach. P.. et al. (1988). E r J Immunol 18: 1901-5). Further. CTLA-4 and CD28 are extremely closely linked on the genome, possibly separated by a distance of only 25-150 kb (Balzano, C. et al, (1992). Int J Cancer Suppl 7: 28-32). Because the function of these two receptors appears to be physiologically linked during the lymphocyte activation process, coordinate control of transcription factors associated with CD28 and CTLA-4 must be stringent. The sequence conservation seen between mouse and human T-cell receptor extended over 100 kb. a distance similar to that found between CTLA-4 and CD28; thus the CD28 loci may also be highly conserved.

Example 7. The HR2 And The PMR Shown In Nucleotides 43819-43925 Of SEQ ID No:3 Detect Different Distributions Of Polymoφhisms

Polymoφhisms in the PMR amplified by sara31/32 primers was compared with polymoφhisms in hR2. As shown in Figure 5, a different distribution of polymoφhisms was obtained when these different PMRs were amplified. Thus, the instant PMR sequences as markers can provide different results than those obtained using the hR2 marker, indicating that the PMR markers disclosed herein can be used to further refine genetic alleles linked to the costimulatory receptor locus.

Example 8. Assembling Contiguous Sequences in the Costimulatory Receptor Locus by Hybridization Screening. 2 Human Bacterial Artifical Chromosomes BAC clones were isolated that contained the CD28. CTLA4. and ICOS loci.

These three costimulatory receptors co-localize within a span of approximately 300 kb on chromosome 2q33. Shotgun sequence analysis of a human BAC clone (170 kb.3x coverage) and a mouse BAC clone (130kb. 2x coverage) generated non-contiguous sequence data containing both the CTLA4 and ICOS genomic loci. Unlike the 4 exon domain structured CTLA4 and CD28 loci, both mouse and human ICOS receptors are encoded by 5 exons containing leader sequence, extracellular domain, transmembrane domain, cytoplasmic domain 1 and cytoplasmic domain 2. Within the human BAC clone, twenty-four simple repetitive sequence elements were identified in addition to the known polymoφhic CTLA4 3' UT microsatellite repeat (Yanagawa et al. 1997. Thyroid 7:843). EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for determining the predisposition of a human subject to develop autoimmune disease, said method comprising detecting a polymoφhic microsatellite repeat (PMR) sequence in the CD28 or the ICOS gene of the human costimulatory receptor gene locus to thereby determine the predisposition of a human subject to develop autoimmune disease.

2. A method for determining the predisposition of a human subject to develop autoimmune disease, said method comprising detecting a polymoφhic microsatellite repeat (PMR) in the CTLA4 gene of the human costimulatory receptor gene locus, wherein the PMR sequence is selected from the group consisting of: nucleotides 5722- 5746 of SEQ ID NOT; nucleotides 23904-23957 of SEQ ID NO:l;nucleotides 27689- 27780 of SEQ ID NOT ; nucleotides 30766-30801 of SEQ ID NOT; nucleotides 6550- 6597 of SEQ ID NOT ;nucleotides 19911-19956 of SEQ ID NOT; nucleotides 19767- 19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152- 48199 of SEQ ID NO:2;nucleotides 49894-49925 of SEQ ID NO:2; nucleotides 10141- 10177 of SEQ ID NO:3;nucleotides 11459-1 1520 of SEQ ID NO:3; nucleotides 12329- 12419 of SEQ ID NO:3; nucleotides 15527-15567 of SEQ ID NO:3; nucleotides 24050- 24075 of SEQ ID NO:3;nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317- 27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3; nucleotides30535- 30574 of SEQ ID NO:3;nucleotides 33714-33758 of SEQ ID NO:3; nucleotides 43819- 43925 of SEQ ID NO:3:nucleotides 46547-46572 of SEQ ID NO:3; and nucleotides 46828-46875 of SEQ ID NO:3 to thereby determine the predisposition of a human subject to develop autoimmune disease.

3. A method for determining the predisposition of a human subject to develop autoimmune disease, said method comprising detecting a polymoφhic microsatellite repeat (PMR) in the human costimulatory receptor gene locus, wherein the PMR sequence is not an hR2 sequence, to thereby determine the predisposition of a human subject to develop autoimmune disease.

4. The method of any one of claims 1 -3 wherein the autoimmune disease is selected from the group consisting of: insulin-dependent diabetes mellitus (IDDM), Addison's disease. Graves^" disease, autoimmune hypothyroidism. myasthenia gravis, thymoma, lupus, thyroiditis. postpartum thyroiditis. rheumatoid arthritis. Hashimoto^'s disease. coeliac disease and leprosy.

5. The method of any one of claims 1-3. wherein the step of detecting is performed using a polymerase chain reaction (PCR) employing a first and second primer.

6. The method of claim 5. wherein the first or second primer comprises the sequence selected from the group consisting of SEQ ID NO: SEQ ID No: 51. SEQ ID No: 52. SEQ ID No: 23. SEQ ID No: 24, SEQ ID No: 25. SEQ ID No: 26. SEQ ID No: 49, SEQ ID No: 50. SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10. SEQ ID No: 45, SEQ ID No: 46, SEQ ID No: 39. SEQ ID No: 40. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 43. SEQ ID No: 44, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21. SEQ ID No: 22. SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 15. SEQ ID No: 16, SEQ ID No: 37. SEQ ID No: 38, SEQ ID No: 11. SEQ ID No: 12. SEQ ID No: 13, SEQ ID No: 14. SEQ ID No: 33. SEQ ID No: 34. SEQ ID No: 35. SEQ ID No: 36, SEQ ID No: 53. and 54.

7. A method for determining the predisposition of a human subject to autoimmune disease, said method comprising detecting an hRl PMR sequence to thereby determine the predisposition of a human subject to autoimmune disease.

8. The method of claim 7. wherein the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus (IDDM). Addison^'s disease. Graves^* disease, autoimmune hypothyroidism, myasthenia gravis, thymoma. lupus, thyroiditis, postpartum thyroiditis, rheumatoid arthritis. Hashimoto's disease, coeliac disease and leprosy.

9. The method of claim 8 wherein said detecting is performed using PCR employing a first and second primer.

10. The method of claim 9 wherein the first or second primer comprises a sequence selected from the group consisting of SEQ ID NO: SEQ ID No: 51. SEQ ID No: 52,

SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26, SEQ ID No: 49. SEQ ID No: 50. SEQ ID No: 47, SEQ ID No: 48. SEQ ID No: 31, SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8. SEQ ID No: 9. SEQ ID No: 10. SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 43. SEQ ID No: 44. SEQ ID No: 17. SEQ ID No: 18. SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21. SEQ ID No: 22. SEQ ID No: 27. SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30. SEQ ID No: 15. SEQ ID No: 16, SEQ ID No: 37. SEQ ID No: 38, SEQ ID No: 11. SEQ ID No: 12, SEQ ID No: 13. SEQ ID No: 14, SEQ ID No: 33, SEQ ID No: 34. SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 53. and 54.

11. A method for determining the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject, said method comprising detecting a polymoφhic microsatellite repeat sequence in the CD28 or the ICOS gene of the human costimulatory gene locus to thereby determine the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject.

12. A method for determining the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject, said method comprising detecting a polymoφhic microsatellite repeat (PMR) in the CTLA4 gene of the human costimulatory gene locus, wherein the PMR sequence is selected from the group consisting of: nucleotides 5722-5746 of SEQ ID NO: 1 ; nucleotides 23904-23957 of SEQ ID NO mucleotides 27689-27780 of SEQ ID NOT; nucleotides 30766-30801 of SEQ ID NOT: nucleotides 6550-6597 of SEQ ID NOT;nucleotides 19911-19956 of SEQ ID NOT; nucleotides 19767-19792 of SEQ ID NO:2; nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152-48199 of SEQ ID NO:2;nucleotides 49894-49925 of SEQ ID NO:2: nucleotides 10141-10177 of SEQ ID NO:3;nucleotides 11459-11520 of SEQ ID NO:3: nucleotides 12329-12419 of SEQ ID NO:3; nucleotides 15527-15567 of SEQ ID NO:3; nucleotides 24050-24075 of SEQ ID NO:3;nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317-27350 of SEQ ID NO:3;nucleotides 30069-30101 of SEQ ID NO:3; nucleotides30535-30574 of SEQ ID NO:3;nucleotides 33714-33758 of SEQ ID NO:3; nucleotides 43819-43925 of SEQ ID NO:3;nucleotides 46547-46572 of SEQ ID NO:3; and nucleotides 46828-46875 of SEQ ID NO:3 to thereby determine the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject

13. A method for determining the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject, said method comprising detecting a polymoφhic microsatellite repeat (PMR) in the human costimulatory receptor gene locus, wherein the PMR sequence is not an hR2 sequence to thereby determine the polymoφhic variant or subtype of a PMR sequence in the costimulatory receptor locus in a human subject.

14. The method of any one of claims 11-13, wherein the step of detecting is performed using PCR employing a first and second primer.

15. The method of claim 14 wherein the first or second primer comprises a sequence selected from the group consisting of SEQ ID No: 51, SEQ ID No: 52, SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25. SEQ ID No: 26. SEQ ID No: 49. SEQ ID No: 50, SEQ ID No: 47, SEQ ID No: 48. SEQ ID No: 31. SEQ ID No: 32. SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9. SEQ ID No: 10, SEQ ID No: 45. SEQ ID No: 46. SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 43. SEQ ID No: 44, SEQ ID No: 17. SEQ ID No: 18. SEQ ID No: 19. SEQ ID No: 20. SEQ ID No: 21. SEQ ID No: 22, SEQ ID No: 27, SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 15, SEQ ID No: 16. SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13. SEQ ID No: 14, SEQ ID No: 33, SEQ ID No: 34. SEQ ID No: 35. SEQ ID No: 36. SEQ ID No: 53. and 54.

16. A PCR primer pair capable of amplifying a PMR sequence in the ICOS or CD28 region of the costimulatory receptor locus of a human subject.

17. A PCR primer pair capable of amplifying a PMR sequence selected from the group consisting of: nucleotides 5722-5746 of SEQ ID NOT ; nucleotides 23904-23957 of SEQ ID NO:l ;nucleotides 27689-27780 of SEQ ID NOT ; nucleotides 30766-30801 of SEQ ID NOT ; nucleotides 6550-6597 of SEQ ID NOT mucleotides 19911-19956 of SEQ ID NOT; nucleotides 19767-19792 of SEQ ID NO:2: nucleotides 39887-39926 of SEQ ID NO:2; nucleotides 48152-48199 of SEQ ID NO:2;nucleotides 49894-49925 of SEQ ID NO.2; nucleotides 10141-10177 of SEQ ID NO:3:nucleotides 11459-11520 of SEQ ID NO:3; nucleotides 12329-12419 of SEQ ID NO:3; nucleotides 15527-15567 of SEQ ID NO:3; nucleotides 24050-24075 of SEQ ID NO:3:nucleotides 26009-26056 of SEQ ID NO:3; nucleotides 27317-27350 of SEQ ID NO:3:nucleotides 30069-30101 of SEQ ID NO:3; nucleotides30535-30574 of SEQ ID NO:3;nucleotides 33714-33758 of SEQ ID NO:3; nucleotides 43819-43925 of SEQ ID NO:3:nucleotides 46547-46572 of SEQ ID NO:3; and nucleotides 46828-46875 of SEQ ID NO:3.

18. A PCR primer capable of amplifying a PMR sequence in the costimulatory receptor locus of a human subject, wherein the primer is between about 20 and about 200 base pairs in length and comprises a nucleotide sequence selected from the group consisting of: SEQ ID No: 51. SEQ ID No: 52. SEQ ID No: 23, SEQ ID No: 24. SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 49. SEQ ID No: 50, SEQ ID No: 47. SEQ ID No: 48, SEQ ID No: 31, SEQ ID No: 32. SEQ ID No: 7. SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 45, SEQ ID No: 46, SEQ ID No: 39, SEQ ID No: 40. SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 43, SEQ ID No: 44. SEQ ID No: 17. SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22. SEQ ID No: 27, SEQ ID No: 28. SEQ ID No: 29. SEQ ID No: 30, SEQ ID No: 15. SEQ ID No: 16, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 11, SEQ ID No: 12. SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 53. and 54.

19 A PCR primer capable of amplifying a PMR sequence in the costimulatory receptor locus of a human subject, wherein the primer consists of a nucleotide sequence selected from the group consisting of: SEQ ID No: 51, SEQ ID No: 52, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25. SEQ ID No: 26, SEQ ID No: 49, SEQ ID No: 50, SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 31. SEQ ID No: 32. SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9. SEQ ID No: 10, SEQ ID No: 45. SEQ ID No: 46, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19. SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30. SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 1 1, SEQ ID No: 12, SEQ ID No: 13. SEQ ID No: 14. SEQ ID No: 33. SEQ ID No: 34. SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 53, and 54.