WO2009034661A1

WO2009034661A1 - Method for diagnosis and induction of resistance to virus

Info

Publication number: WO2009034661A1
Application number: PCT/JP2007/068591
Authority: WO
Inventors: Yasuyoshi Kanari; Masaaki Miyazawa; Shinji Irie; Mario S. Clerici
Original assignee: ImmunoClin Ltd; Osaka Industrial Promotion Organization; Toppan Printing Co Ltd
Current assignee: ImmunoClin Ltd; Osaka Industrial Promotion Organization; Toppan Inc
Priority date: 2007-09-12
Filing date: 2007-09-12
Publication date: 2009-03-19
Anticipated expiration: 2010-03-12
Also published as: WO2009035170A1

Abstract

The present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject for resistance to virus. The present method, chip and kit utilize specific SNPs and nucleotide polymorphisms accumulated in ESN (HIV-I -exposed but uninfected) individuals as markers of resistance to virus. Further, the present invention provides preventative and therapeutic means to confer resistance to virus infection.

Description

IMSSCRIPTK)Jr

METHOD FOR DIAGNOSIS AND INDUCTION OF RESISTANCE TO

VIRUS

FIELD OF THE INVENTION

[0l] The present invention is related generally to the analytical testing of the samples obtained from a subject, and more particularly the method for detecting genetic predisposition of a subject to resistance to virus. The invention also provides a chip and kit for detecting such genetic predisposition. The invention further provides an application of identified genetic predisposition to induce resistance to virus infection.

BACKGROUND OF THE INVENTION

[02] It is known in the art that it is possible to diagnose a predisposition to certain diseases with the use of biomarkers such as single nucleotide polymorphisms (SNPs), nucleotide insertion mutations, nucleotide deletion mutations, microsatellite markers or other DNA sequence variations. For example, inherited polymorphisms and somatic mutations in oncogenes or tumor suppressor genes are widely regarded as being indicative of a susceptibility to certain cancers, especially in view of the associations between mutated oncogenes or deleted tumor suppressor genes and certain cancers. It is also known that some individuals are highly susceptible or resistant to infection, especially viral infection. Prediction of disease susceptibility is beneficial for those possessing predisposing genes in order to avoid unnecessary contacts with known etiological agents, chemicals, or viruses, and to take known and developing preventative means. It is also useful in the design of a vaccine against viral disease or for gene therapy. In addition, prediction of the speed of disease progression may allow an opportunity for individualized, more efficient management or therapeutic intervention. [03] Additionally, the development of an effective vaccine or therapeutic agent against major viral diseases such as human immunodeficiency virus (HIV) infection is a pressing matter with global socio-economic ramifications. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). One of the keys to the development of such a vaccine is the understanding of the mechanisms of natural resistance against HIV infection. And in the development of such a therapeutic agent, especially in the clinical trial, it is necessary to assess genetic predisposition of the subject to resistance or susceptibility to HIV infection and its disease progression, because the measured effects of the candidate agent in question can be significantly biased by the presence of genetically determined susceptibility or resistance in the subject.

[04] Several human genes are associated with resistance against human immunodeficiency virus type 1 (HIV- 1) infection or delayed development of AIDS after HIV-I seroconversion (Reference 1, O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591; Reference 2, O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574). These include genes encoding chemokine receptors and their ligands, cytokines, receptors expressed on antigen-presenting or natural killer cells, and those mapped within the major histocompatibility complex (MHC) (Reference 3, Dean, M. et al., "Genetic restriction of HIV-I infection and progression to AIDS by a deletion allele of the CCR5 structural gene", Science, 1996, vol.273, pp.1856- 1862; Reference 4, Liu, R. et al., "Homozygous defect in HIV-I coreceptor accounts for resistance of some multiply-exposed individuals to HIV-I infection", Cell, 1996, vol.86, pp.367-377; Reference 5, Samson, M. et al., "Resistance to HIV-I infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene", Nature, 1996, vol.382, pp.722-725; Reference 6, Winkler, C. et al., "Genetic restriction of AIDS pathogenesis by an SDF-I chemokine gene variant", Science, 1998, vol.279, pp.389-393; Reference 7, Gonzalez, E. et al., "The influence of CCL3L1 gene-containing segmental duplications on HIV- I/AIDS susceptibility", Science, vol.307, pp.1434- 1440; Reference 8, Carrington, M. et al., "HLA and HIV Heterozygote advantage and B*35'Cw*04 disadvantage", Science, vol.283, pp.1748-1752; Reference 9, Martin, M.P. et al., "Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS", Nature Genet., 2002, vol.31, pp.429-434; Reference 10, Fellay, J. et al., "A whole -genome association study of major determinants for host control of HIV-I", Science, Published online July 19 2007, 10.1126/science.1143767; Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59"68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule -3- grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055" 1058). [05] However, the known AIDS restriction genes (ARG) can explain only up to 15 % of the host determinants that control HIV- I/AIDS (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574; Reference 10, Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIV-I", Science, Published online July 19 2007, 10.1126/science.1143767), and the presence of other resistance genes has been predicted (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574). [06] Among existing human clusters showing natural resistance to HIV-I acquisition, there is a distinct group known as HIVl -exposed but uninfected or exposed seronegative (ESN) individuals who have evidence of multiple and repeated exposures to HIV-I through unprotected sexual contacts, but nevertheless possess no serum IgG reactive to the viral antigens (Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule- 3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058; Reference 13, Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV- seropositive individuals", Nature Med., 1997, vol.3, pp.1250- 1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374; Reference 15, Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HlVl-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412- 1422; Reference 16, Locaputo, S. et al., "Mucosal and systemic HIV-specific immunity in HIV-exposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).

[07] The detection of HIV specific T- lymphocyte responses and HlVreactive IgA antibodies in mucosal secretions (Reference 13, Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV-seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365- 1374; Reference 15, Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HlVl-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412- 1422; Reference 16, Locaputo, S. et al., "Mucosal and systemic HIV-specifϊc immunity in HIV-exposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlV l-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024) indicates that ESN individuals have been exposed to HIV-I and have mounted HIV-specific immune responses, but the exposures have not resulted in productive infection.

[08] Attempts to associate the ESN status with previously described ARG have so far been unsuccessful (Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort^" of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule -3- grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058; Reference 13, Mazzoli S. et al., "HlV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV- seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257; Reference 14, Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374; Reference 15, Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIV-1-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412-1422; Reference 16, Locaputo, S. et al., "Mucosal and systemic HIVspecific immunity in HlVexposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358; Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2 13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024⁾.

[09] The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 30, WO2004/035825, International publication of PCT application, 29 April 2004). To further narrow down the chromosomal region where the putative immune resistance gene is located, the inventors of the present invention genotyped 74 HIV-exposed but uninfected and 77 HIV-infected individuals enrolled from the same geographical region. Several SNPs are significantly associated with ESN individuals under a dominant gene hypothesis at the CardlO, CDC42EP1 and GRAP2\oci (Reference 31, WO2006/067506, International publication of PCT application, 29 June 2006). However, exact molecular genetic mechanisms that explain the observed resistance of ESN individuals to HIV-I acquisition had not been provided. [lθ] References

(l). O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591.

(2) O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574.

(3) Dean, M. et al., "Genetic restriction of HIVl infection and progression to AIDS by a deletion allele of the CCR5 structural gene", Science, 1996, vol.273, pp.1856- 1862.

(4) Liu, R. et al., "Homozygous defect in HIVl coreceptor accounts for resistance of some multiply-exposed individuals to HIVl infection", Cell, 1996, vol.86, pp.367-377.

(5) Samson, M. et al., "Resistance to HIVl infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene", Nature, 1996, vol.382, pp.722-725.

(6) Winkler, C. et al., "Genetic restriction of AIDS pathogenesis by an SDF-I chemokine gene variant", Science, 1998, vol.279, pp.389-393.

(7) Gonzalez, E. et al., "The influence of CCL3L1 gene-containing segmental duplications on HIV- I/AIDS susceptibility", Science, vol.307, pp.1434- 1440.

(8) Carrington, M. et al., "HLA and HW- Heterozygote advantage and B*35-Cw*04 disadvantage", Science, vol.283, pp.1748-1752.

(9) Martin, M.P. et al., "Epistatic interaction between KIR3DS1 and HLA B delays the progression to AIDS", Nature Genet., 2002, vol.31, pp.429-434.

(10) Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIV- 1", Science, Published online July 19 2007, 10.1126/science.1143767.

[11]

(11) Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68.

(12) Liu H. et al., "Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor- 1, RANTES, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-I", J. Infec. Dis., 2004, vol.190, pp.1055-1058.

(13) Mazzoli S. et al., "HIV-specific mucosal and cellular immunity in HIV- seronegative partners of HIV- seropositive individuals", Nature Med., 1997, vol.3, pp.1250-1257.

(14) Biasin, M. et al., "Mucosal and systemic immune activation is present in human immunodeficiency virus-exposed seronegative women", J. Infect. Dis., 2000, vol.182, pp.1365-1374.

(15) Belec, L. et al., "Cervicovaginal secretory antibodies to HIV type 1 that block viral transcytosis through epithelial barriers in highly exposed HIV-1-seronegative African women", J. Infect. Dis., 2001, vol.184, pp.1412-1422.

(16) Locaputo, S. et al., "Mucosal and systemic HIV-specific immunity in HIV-exposed but uninfected heterosexual males", AIDS, 2003, vol.17, pp.351-358.

(17) Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-I -exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024.

(18) Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HIV-seropositive individuals", Nature, 1989, vol.339, pp.383-385.

(19) Meskauskas, A. & Dinman, J. D., "Ribosomal protein L3: Gatekeeper to the A site", MoI. Cell., 2007, vol.23, pp.877-888.

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(21) Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation", Science, 2000, vol.288, pp.2219-2222.

(22) Yu, H., Leitenberg et al., "Deficiency of small GTPase Rac2 affects T cell activation", J.Exp .Med., 2001, vol.194, pp.915-925.

(23) Jacobelli, J. et al., "A single class II myosin modulates T cell motility and stopping, but not synapse formation", Nat.Immunol., 2004, vol.5, pp.531-538.

(24) Ouellet, M. et al., "Galectin-1 acts as a soluble host factor that promotes HIV-I infectivity through stabilization of virus attachment to host cells", J.Immunol., 2005, vol.174, pp.4120-4126.

(25) Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333.

(26) Douek, D. C. et al., "T cell dynamics in HIV l infection", Annu.Rev.Immunol., 2003, vol.21, pp.265-304.

(27) Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376-3386.

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(29) Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide -like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964.

(30) WO2004/035825, International publication of PCT application, published on 29 April 2004.

[13]

(31) WO2006/067506, International publication of PCT application, published on 29 June 2006.

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(33) Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879.

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(37) Holland, P.M. et al., "Detection of specific polymerase chain reaction product by utilizing the 5' — 3' exonuclease activity of Thermus aquaticus DNA polymerase", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1991, vol.88(l6), pp.7276-7280.

(38) WO 92/20702, International publication of PCT application, published on 26 November 1992

(39) Lyamichev, V. et al., "Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes", Nature Biotechnology, 1999, vol.l7(3), pp.292-296.

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SUMMARY OF THE INVENTION

[14] The inventors of the present invention performed expression analyses of all the genes and open reading frames located in the candidate segment of human chromosome 22 and extensive genome sequencing, and identified previously unforeseen association between certain sequence polymorphisms in the Rac2 gene locus and the ESN (HIV- 1 -exposed but uninfected or exposed seronegative) status. The sequence polymorphism associated with ESN confers a higher enhancer activity, and is associated with poor replication of CCR5-tropic HIV in peripheral blood mononuclear cells.

More specifically, some SNPs and specific haplotypes of them are representative of the above sequence polymorphism and thus predictive of a resistance to HIV-I infection, and these are designated by the following SNP ID Nos: rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.

Further the inventors of the present invention found that these SNPs and haplotypes have an effect on the Rac2 gene expression level and HIV replication. Thus these SNPs and haplotypes are useful for detecting genetic predisposition to resistance to virus infection, and also to provide preventative and therapeutic means to confer resistance to virus infection. [15] Accordingly, the present invention provides:

(l) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising^: a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos: (i) rs739041, (ii) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : (i) rs739041 : T; (ii) rs739042 : T; (iii) rs2284037 : T; (iv) rs933223 : C; (v) rs6000619 : A; (vi) ss73405466 : G; and (vii) rs5756570 : C.

[16] (2) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs2284037, (ii) rs739042 and (iii) rs73904i; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs2284037 : T; (ii) rs739042 : T; (iii) rs739041 : T.

[17] (3) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) ss73405466, (ii) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxii) rs933321, (xxϋi) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) ss73405466 : G; (ii) rs9798725 : T; (iϋ) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG; (iv) rs5995400 : C; (v) rs6000619 : A; (vi) rs5756570 : C; (vϋ) rs36110509 : gap; (viii) rs2899284 : T; Gx) rs6000618 : T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiii) rs9610676 : A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss73405476 : T; (xvii) ss73405477 : T; (xviii) ss73405479 : T; (xix) rs5756568 : G; (xx) rs933223 : C; (xxi) rs933222 : A; (xxii) rs933321 : A; (xxiϋ) ss73405482 : A; (xxiv) ss73405484 : gap; and (xxv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATTGAA [18] (4) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs9610683 and (ϋ) rs9610682; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) _rs9610683 : C; and (ϋ) rs9610682 : A.

[19] (5) An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iϋ) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vϋ) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxii) ss73405482, (xxiii) ss73405484 and (xxiv) ss73405485; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. ^: allele) : (i) rs9010683 : C; (ii) rs9610682 : A; (iii) rs2284036 : T; (iv) rs9798725 : T; (v) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG;

(vi) rs5995400 : C; (vϋ) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 :

T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiii) rs9610676 :

A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss73405476 : T; (xvii) ss73405477 : T; (xviii) ss73405479 : T; (xix) rs5756568 : G; (xx) rs933222 : A;

(xxi) rs933321 : A; (xxii) ss73405482 : A; (xxiii) ss73405484 : gap; and (xxiv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATT

GAA.

[20] (6) A method for inducing or enhancing resistance to virus in a subject, comprising enhancing the Rac2 gene expression in the subject.

[21] (7) The method according to the above method (6), wherein the Rac2 gene expression is enhanced by using a DNA-binding protein, peptide, oligonucleotide, or nucleotide analogue that binds to the enhancer element located in the Rac2gene region 1 (SEQ ID No:32).

[22] (8) The method according to the above method (6), wherein the Rac2 gene expression is enhanced by inhibiting the expression of an Rac2 repressor by RNAi technique.

[23] (9) Use of Rac2 protein in the preparation of a medicament for the treatment or prophylaxis of virus-induced disease.

[24] (lO) A nucleic acid exhibiting the Rac2 enhancer activity, wherein the nucleic acid is selected from the following (A) and (B):

(A) a nucleic acid having the nucleotide sequence of SEQ ID No÷32; and (B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.

[25] (11) The nucleic acid according to the above (10), wherein the nucleic acid has the following alleles at the SNP sites in the nucleotide sequence of

SEQ ID No:32 (base number in the SEQ ID No:32 : allele):

(i) 132 : C; (ii) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 : T; and

(vi) 2379 : T.

[26] (12) Use of the nucleic acid according to the above (10) or (ll) as a Rac2 gene enhancer.

[27] (13) Use of the nucleic acid according to the above (10) or (ll) for a gene therapy to induce resistance to virus in a subject.

[28] (14) A method for screening a candidate therapeutic agent against virus, comprising: a) contacting an agent with a nucleic acid fragment having a partial or entire nucleotide sequence of the nucleic acid according to the above (10) or (ll); b) measuring an interaction between the agent and the nucleic acid fragment; and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.

[29] (15) A method for screening a candidate therapeutic agent against virus, comprising: a) administering an agent to a biological material; b) measuring an expression level of the Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.

[30] (16) A method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus, comprising: a) detecting genetic predisposition of subjects by the method according to any one of the above methods (l) - (5); b) stratifying the subject by the result of the detected genetic predisposition; c) giving a treatment to the stratified subject! and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.

[31] (17) The method according to any one of the above methods (l) - (8), wherein the virus is one selected from the group consisting of HIV, HCV and

HPV

[32] (18) The method according to the above method (17), wherein the virus is HIV or HCV.

[33] (19) The method according to the above method (18), wherein the virus is HIV.

[34] (20) The use according to the above use (9) or (13), wherein the virus is one selected from the group consisting of HIV, HCV and HPV.

[35] (21) The use according to the above use (20), wherein the virus is HIV or

HCV.

[36] (22) The use according to the above use (21), wherein the virus is HIV.

[37] (23) The method according to any one of the above methods (14) - (16), wherein the virus is one selected from the group consisting of HIV, HCV and

HPV.

[38] (24) The method according to the above method (23), wherein the virus is HIV or HCV.

[39] (25) The method according to the above method (24), wherein the virus is HIV.

[40] (26) The method according to any one of the above methods (l) ^■ (5), wherein the genotyping is performed by the Invader assay.

[41] (27) A chip used in the method according to the above method (l), comprising a base plate and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and

(vii) rs5756570, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO^' 6, 4, 3, 26, 11, 7 and 12.

[42] (28) A chip used in the method according to the above method (2), comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO^3, 4 and 6. [43] (29) A chip used in the method according to the above method (3), comprising a base plate and probe sets detecting SNPs and nucleotide polymorphisms designated by the SNP ID Nos (i) ss73405466, (ϋ) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, Gx) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxii) rs933321, (xxiii) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31.

[44] (30) A chip used in the method according to the above method (4), comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO^l and 2.

[45] (31) A chip used in the method according to the above method (5), comprising a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos^ (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xϋ) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxϋ) ss73405482, (xxiii) ss73405484 and (xxiv) ss73405485, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO÷l, 2, 5, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30 or 31.

[46] (32) A kit used in the method according to the above method (l), comprising a reagent and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, (in) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: 6, 4, 3, 26, 11, 7 and 12.

[47] (33) A kit used in the method according to the above method (5), comprising a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxϋ) ss73405482, (xxiii) ss73405484 and (xxiv) ss73405485, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO^;1, 2, 5, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30 or 31. BRIEF DESCRIPTION OF THE DRAWINGS [48]

Figure 1 is an illustration of the distribution of SNPs and nucleotide polymorphisms in the Rac2 locus.

Figure 2 shows a result of DNA microarray analysis comparing the changes in gene expression levels during the HIV-I antigenic stimulation in peripheral blood mononuclear cells (PBMC).

Figure 3 is a coefficient map of linkage disequilibrium (LD) between each pair of SNP alleles.

Figure 4 is a graph showing luciferase activities induced by the Rac2 promoter and the polymorphic region 1 or 2 indicated in Figure 1.

Figure 5 is a graph showing changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.

Figure 6 is a graph showing HIV-I p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the Rac2 region 1 genotypes.

Figure 7 is an illustration of locations and spans of known genes and open reading frames in the candidate region in human chromosome 22.

Figure 8 is a table showing allele distribution at known SNP loci in chromosome 22 among the ESN and HIV-1-infected individuals. [49]

Figure 9-Al, Figure 9-A2, Figure 9Α3, Figure 9-A4, Figure 9-Bl, Figure 9-B2, Figure 9-B3, Figure 9-B4, Figure 9-Cl, Figure 9-C2, Figure 9-C3, Figure 9-C4, Figure 9"Dl, Figure 9-D2, Figure 9-D3 and Figure 9-D4 are tables showing genotypes at all detected polymorphic loci between the CardlO and U2rb loci and the results of case-control analyses between the ESN and HIV-1-infected individuals. AIC: Akaike Information Criterion.

In Figure 9"Al, Figure 9-A2, Figure 9-A3 and Figure 9-A4, same nucleotide polymorphisms (rs8142629 ~ ss74804657) are listed and case-control statistical analysis are performed under dominant gene, recessive gene, and codominant gene models, as well as for allele frequencies, in the presented order.

In Figure 9-Bl, Figure 9-B2, Figure 9-B3 and Figure 9-B4, same nucleotide polymorphisms (rsllO89844 ~ rsll22084) are listed and case control statistical analysis are performed under dominant gene, recessive gene, and codominant gene models, as well as for allele frequencies, in the presented order.

In Figure 9-Cl, Figure 9-C2, Figure 9-C3 and Figure 9-C4, same nucleotide polymorphisms (ss73405526 ~ ss73405426) are listed and case -control statistical analysis are performed under dominant gene, recessive gene, and codominant gene models, as well as for allele frequencies, in the presented order.

In Figure 9-Dl, Figure 9-D2, Figure 9-D3 and Figure 9-D4, same nucleotide polymorphisms (rs2213430 ~ rs9610678) are listed and case-control statistical analysis are performed under dominant gene, recessive gene, and codominant gene models, as well as for allele frequencies, in the presented order.

In Figure 9-El, Figure 9Ε2, Figure 9"E3 and Figure 9-E4, same nucleotide polymorphisms (rs5756570 ~ rs2284034) are listed and case-control statistical analysis are performed under under dominant gene, recessive gene, and codominant gene models, as well as for allele frequencies, in the presented order.

DETAILED DESCRIPTION OF THE INVENTION

[50] The present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject to resistance to virus. Further, the present invention provides a method, biopharmaceutical and nucleic acid for inducing or enhancing resistance to virus in a subject.

Examples of the virus include, but are not limited to, Human immunodeficiency virus (HIV), Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV) and Influenza (Flu) virus. Preferably, the virus is HIV, HPV or HCV. More preferably, the virus is HIV. HIV includes HIV-I and HIV-2.

[51] In the present specification and claims, the term "genetic predisposition" refers to genetic factors which influence the phenotypes of an organism. However, the effect of the genetic predisposition can be modified by environmental conditions.

The term "subject", as used herein, refers to an object for the method for detecting genetic predisposition. Preferably, the subject is a primate such as human, monkey and ape. More preferably, the subject is human. Most preferably, the human is Caucasian.

The term "resistance to virus", as used herein, refers to an act or power to prevent virus infection or virus-induced disease progression. [52] The present invention is based on the research demonstrating that particular SNPs and haplotypes located in the Rac2 gene region are significantly associated with ESN (HIV- 1-exposed but uninfected or exposed seronegative) individuals. Summary of the research is described below. [53] The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423.

To further narrow down the location of the putative HIV-I resistance gene, the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Figure 8 for complete listing of the observed genotypes).

[54] Expression levels of all the genes located in the above candidate region were compared between the ESN and HIV-infected individuals by using DNA microarrays. Among all the chromosome 22 genes tested, only a small number were constitutively expressed at high levels or induced upon the stimulation with HIV-I antigens (Figure 2). One of the induced genes is the Rac2, which encodes a Rho-subfamily small GTPase that is selectively expressed in and involved in the activation of T- helper type-1 (ThI) cells. The Myh9 and Lgalsl were also induced upon the antigenic stimulation. [55] The chromosomal segments spanning the Myh9, I12rb, Rac2-Pscd4, CardlO, and Lgalsl loci were sequenced (see Figure 7 for the location of the sequenced segments), and the frequencies of ESN and HIV-1-infected individuals possessing a particular allele at each of the observed polymorphic loci were compared. Notable accumulation of sequence polymorphisms with significantly different allele frequencies between the two groups was observed in a segment between the CardlO and Rac2 (Figure 3 and Figure 9). The observed case-control differences apparently increased toward the downstream (centromeric) portion of the Rac2 locus, and there were two regions, one between the 5th and 6th Rac2 exons (region l) and the other downstream of the 7th exon (region 2), where multiple polymorphic loci with statistically significant case-control differences between the ESN and HIV-infected groups were accumulated (Figure 1 and Figure 3). [56] SNPs and nucleotide polymorphisms located in the regions 1 and 2 and significantly accumulated in ESN individuals are listed in Table 1.

[Table l]

[57] Based on the research described above, that is the significant accumulation of a particular allele at the indicated loci in ESN individuals who show resistance to HIV acquisition despite repeated sexual exposures to HIV-I, the present invention provides use of the above listed SNPs and nucleotide sequence polymorphisms for diagnosis of resistance to HIV infection.

Among these SNPs and nucleotide polymorphisms, (i) rs739041, (ii) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570 are more significant for detecting genetic predisposition to resistance to virus.

The SNP ID Nos are identification numbers used in dbSNP which is world's largest database for nucleotide variations and is part of the US National Center for Biotechnology Information (NCBI). The "rs" ID number is an identification tag assigned by NCBI to a group of SNPs that map to an identical location. The rs ID number is assigned after submission. The "ss" ID number is simply a unique identifier assigned by NCBI when SNP is submitted to dbSNP. Anyone can download the information about SNPs having SNP ID Nos from the following siteJ httpV/www.ncbi.nlm. nih.gov/projects/SNP/index.html

[58] The above described SNP genotypes are highly linked to each other within each of the above Rac2 regions, with the levels of LD being higher among the ESN than in HIV-infected individuals in the region 2 (Figure 3). Thus, the polymorphic loci within the regions 1 and 2 constitute small numbers of haplotypes. In particular, the region 1 downstream haplotype TTT is highly accumulated among the ESN individuals (P = 0.0087), while the haplotype CCC is more frequently found in the HIV-infected group (P = 0.0080). In the region 2, a haplotype indicated in red in Figure 1 with characteristic base deletions is also observed more frequently among the ESN than among the HIV-I -infected individuals (P= 0.011). Therefore, in the following analyses the region 1 haplotype CA-TTT and the region 2 haplotype with the base deletions are designated R and those shown in blue S for simplicity.

[59] Figure 1 illustrates the observed haplotypes divided in three blocks. Haplotypes of the left block consist of SNPs designated by SNP ID Nos. rs9610683 and rs9610682. Haplotypes of the central block consist of SNPs designated by SNP ID Nos. rs2284037, rs739042 and rs739041. Hapotypes of the right block consist of SNPs and nucleotide polymorphisms designated by SNP ID Nos. ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.

The haplotype CA in the left block, TTT in the central block, and the haplotype (GT+CAC— -TTTACATCTTTGCAAA") in the right block were significantly accumulated in ESN individuals. Thus, the present invention provides use of the above three haplotypes for diagnosis of resistance to HIV infection.

[60] To examine the functions of the above polymorphic regions 1 and 2 in the Rac2 gene, the genomic fragments harbouring each of the polymorphic regions 1 and 2 isolated from representative healthy control individuals possessing homozygous R/R or S/S haplotype were cloned and inserted into the downstream of the luc gene along with the known Rac2 promoter in the upstream. When the genomic DNA fragment corresponding to the region 1 was placed downstream of the luc gene, the expression levels of the luciferase was higher than that induced with the promoter alone, indicating that the Rac2 region 1 is an enhancer of the gene expression. The luc constructs harbouring the region 1 fragment of the R haplotype induced significantly higher luciferase activities than that of the S halotype (Figure 4), revealing a functional difference between the two haplotypes. Thus, the present invention provides novel enhancer having improved activity enhancing the Rac2 gene expression.

[61] Peripheral blood mononuclear cells (PBMC) from healthy control individuals possessing the homozygous S/S haplotype or harbouring the R haplotype in the region 1 were infected in vitro with HIV-I. In supernatants of the cells harbouring the R haplotype, p24 concentrations were significantly lower as compared to what observed in supernatants of homozygous S/S cells upon infection with either of the two CCR5-tropic HIV-I isolates (Figure 6). On the other hand, p24 concentrations in the supernatants of cells infected with CXCRΦtropic HIV clone or isolate were not different between the S/S and R-harbouring cells. Thus, the Rac2 region 1 haplotype R is functionally more active, and is associated with a restricted replication of CCR5"tropic HIV-I.

[62] It is known that Rac2 is required for the migration of Bl lymphocytes, a specific subpopulation of B-lymphocytes, from the peritoneal cavity to gut-associated lymphoid tissues (Reference 27, Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376"3386), and Bl-derived plasma cells are the predominant sources of pathogen-reactive mucosal IgA (Reference 28, Fagarasan, S. et al., "T-independent immune response: new aspects of B cell biology", Science, 2000, vol.290, pp.89-92). Therefore, the observed association between the Rac2 enhancer haplotype and the ESN status might also explain the presence of HIVl -reactive IgA in mucosal secretions of ESN individuals. Further, since Rac2 is pivotal in inducing the differentiation of CD4-positibe effctor T cells toward ThI cells (Reference 21, Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation", Science, 2000, vol.288, pp.2219-2222), the observed association between the Rac2 enhancer polymorphism and ESN status also explains the higher production of interferon (IFN) -γ upon stimulation with HIVl antigens in PBMC from ESN than those from HIV-1-infected individuals (References 13, Mazzoli S. et al., "HlV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals", Nature Med., 1997, vol.3, pp.1250- 1257; and 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024).

[63] Therefore, this virus resistance mechanism mediated by the Rac2 gene expression also has effect against all other viruses infected via mucosal route such as Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV), Influenza (Flu) virus and the like. It is noteworthy that a SNP associated with differences in the course of HCV infection is located in the region close to the Rac2 gene (Reference 32, Saito, T., et al., "Genetic variations in humans associated with differences in the course of hepatitis C", Biochemical and Biophysical Research Communication (BBRC), 2004, vol.317, pp.335-341).

[64] Accordingly, based on the above described research, the present invention provides an ex vivo method, a chip and a kit for detecting genetic predisposition of a subject to resistance to virus.

In addition, the present invention provides a method and a biopharmaceutical (Rac2 protein or the Rac2 gene inducer) for inducing or enhancing resistance to virus in a subject by utilizing virus resistance mechanism mediated by Rac2.

Further, the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing Rac2 or the Rac2 regulatory region as a target molecule. And the present invention provides use of the Rac2 enhancer region for a gene therapy to induce resistance to virus in a subject.

[65] In one embodiment, the ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprises following steps (a) and (b): the step (a) is genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and SS73405485;

[65] And the step (b) is assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : rs9010683 : C; rs9610682 : A; rs2284037 : T; rs739042 : T; rs2284036 : T; rs739041 : C; ss73405466 : G; rs9798725 : T; ss73405467 : GGCCTCATCCTTCCAAGTTC

AAGTTCAG; rs5995400 : C; rs6000619 : C; rs5756570 : G; rs36110509 : gap,^' rs2899284 : T; rs6000618 : T; rs6000617 : T; rs9610677 : A; rs6000616 : C; rs9610676 : A; rs9610675 : T; rs9622582 : C; ss73405476 : T; ss73405477 : T; ss73405479 : T; rs5756568 : G; rs933223 : T; rs933222 : A; rs933321. : A; ss73405482 : A; ss73405484 : gap; and ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATTGAA.

[66] In the present specification and claims, the term "genotyping" refers to a process of determining the genetic constitutions such as nucleotide sequence of an individual with a biological assay.

The term "sample", as used herein, refers to a biological sample or material obtained from a subject, for example a cell, tissue, organ, blood, hair or the like.

The term "site", as used herein, refers to a position or location of specific nucleotide or polynucleotide in the genome.

The term "nucleotide polymorphism", as used herein, refers to difference of nucleotide sequence between members of a species (or between paired chromosomes in an individual).

[67] The term "SNP" or "single nucleotide polymorphism", as used herein, refers to DNA sequence variation occurring when a single nucleotide A, T, C or G in the genome differs between members of a species (or between paired chromosomes in an idividuals).

The term "allele", as used herein, refers to a particular form of a gene or DNA sequence at a specific chromosomal location (locus).

The term "resistant allele", used herein for convenience to claim the invention, refers to the alleles accumulated in ESN individuals. [68] In another embodiment, the ex vivo method of the present invention comprises (a) genotypying a sample of the subject at sites of SNPs and nucleotide polymorphism constituting a haplotype; and (b) assessing the subject as having genetic predisposition of resistance to virus if the haplotype which is significantly accumulated in ESN individuals is present.

In the present specification and claims, the term "haplotype" refers to a set of closely linked genetic markers present on one chromosome which tends to be inherited together.

[69] In one embodiment, the haplotypes significantly accumulated in ESN individuals are following three haplotypes :

(1) A haplotype comprising: (SNP ID No. : allele) (i) rs9610683 : C; and (ii) rs9610682 : A;

(2) A haplotype comprising: (SNP ID No. : allele) (i) rs2284037 : T; (ii) rs739042 : T; and (iii) rs739041 : T; and

(3) A haplotype comprising: (SNP ID No. : allele) (i) rs9010683 : C; (ii) rs9610682 : A; (iii) rs2284036 : T; (iv) rs9798725 : T; (v) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG; (vi) rs5995400 : C; (vii) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 : T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiii) rs9610676 : A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss73405476 : T; (xvii) ss73405477 : T; (xviii) ss73405479 : T; (xix) rs5756568 : G; (xx) rs933222 : A; (xxi) rs933321 : A; (xxii) ss73405482 : A; (xxiϋ) ss73405484 : gap,^" and (xxiv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATTGAA. [70] For genotyping a sample of the subject at sites of SNPs and nucleotide polymorphisms, any techniques publicly known in the art can be used, including single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC), direct DNA sequencing, Invader assay, and TaqMan assay.

In one embodiment, SNP or nucleotide polymorphism can be detected by the single-strand conformation polymorphism (SSCP) analysis in which electrophoretic separation of single -stranded nucleic acids based on subtle differences in sequence (often a single base pair) results in a different secondary structure and a measurable difference in mobility through a gel (Reference 33, Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879).

[71] In one embodiment, nucleotide polymorphism can be detected by the heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC) that is based on DNA heteroduplex formation and separation of heteroduplex from homoduplex molecular species by means of ion-pair reverse phase HPLC (Reference 34, Oefner, P.J. et al., "DNA mutation detection using denaturing high performance liquid chromatography", Current protocols in human genetics, 1998, p.7 10:1-7 10 12). [72] In one embodiment, SNP or nucleotide polymorphism can be detected by direct DNA sequencing, such as the chain termination method (Reference 35, Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(l2), pp.5463-5467), which are well known in the art.

In one embodiment of direct DNA sequence, a subsequence of the Rac2 gene region encompassing the SNPs and nucleotide polymorphisms is amplified and either cloned into a suitable plasmid and then sequenced, or sequenced directly.

[73] In one embodiment, SNP can be detected by Invader assay. In this assay, three single -stranded DNA chains form a ternary complex with one base-pair overlap. This complex is composed of a DNA target oligonucleotide, which contains the SNP sequence of interest, and two other oligonucleotides. The upstream oligonucleotide is designated as invader oligo and downstream oligonucleotide is designated as a probe. These three oligonucleotide strands hybridize to one another, forming a one base-pair junction causing the 5' end of the probe to form a unhybridized 5' flap. This 5' flap is then cleaved by a Cleavase enzyme, resulting in the release of the 5' flap of the probe.

Secondary reaction generates quantifiable signals as follows^: FRET cassette is labeled with a fluorophore (F) and a quencher (Q) so cleavage between them generates a fluorescence signal. FRET cassette is self hybridized at 5' portion, and 5' flap is hybridized with 3' portion of the FRET cassette so as to form a ternary complex with one base-pair overlap. And then, cleavage of 5' end of FRET cassette by Cleavase enzyme causes fluorescence signal (Reference 36, U.S. Patent No. 5,846,717; Reference 39, Lyamichev, V. et al., "Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes", Nature Biotechnology, 1999, vol.l7(3), pp.292-296.). [74] In one embodiment, SNP can be detected by TaqMan assay which uses two types (wild type and mutant type) of probes having fluorescence dye and quencher and utilizes 5¹ to 3' nuclease activity of Taq polymerase. DNA fragment harboring SNP site is amplified by PCR primer. On the process of the amplification, TaqMan probe anneals to the DNA. Where the PCR primer extends to 5 ' side of the TaqMan probe and there is no mismatch between the TaqMan probe and DNA, the TaqMan probe is degraded by the 5' to 3' nuclease activity of the Taq polymerase. With the degradation of the TaqMan probe, fluorescence dye is separated by quencher and fluorescence is generated. Where there is a mismatch between the Taqman probe and DNA, the probe is not degraded, and fluorescence does not occur. SNP can be detected by measurement of this fluorescence (Reference 37, Holland, P.M. et al., "Detection of specific polymerase chain reaction product by utilizing the 5'-— 3' exonuclease activity of Thermus aquaticus DNA polymerase", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1991, vol.88(l6), pp.7276-7280).

[75] In one embodiment, the present invention provides a method for inducing or enhancing resistance to virus in a subject. This method comprises a step of enhancing the Rac2 gene expression in the subject.

The step of enhancing the Rac2 gene expression can be performed by any technique known in the art, for example, modulation of the Rac2 enhancer region by using a DNA-binding protein, peptide, oligonucleotide, nucleotide analogue or chemical compound. And RNAi technique may be used for inhibiting an expression of the Rac2 repressor in order to enhance the Rac2 gene expression.

[76] In one embodiment, the present invention provides use of Rac2 protein in the preparation of a medicament for a treatment or prophylaxis of virus-induced disease. In other words, the present invention provides a method for treatment or prophylaxis of virus-induced disease comprising administering Rac2 protein to a subject.

The term "Rac2 protein", as used herein, refers to a full length polypeptide of Rac2 protein, its homologue or functional fragment thereof. The amino acid sequence and coding sequence of Rac2 is registered in Genbank under accession number NM_002872.

[77] In one embodiment, the present invention provides a nucleic acid exhibiting the Rac2 enhancer activity (the polymorphic region l) which is selected from the following nucleic acids ^'• (A) a nucleic acid having the nucleotide sequence of SEQ ID No:32,^' and (B) a nucleic acid having more than 80%, 90% or 95% identity with the nucleotide sequence of SEQ ID No:32.

Preferably, the present invention provides a nucleic acid exhibiting improved Rac2 enhancer activity, which has specific alleles at the sites of SNPs in the nucleotide sequence of SEQ ID No.32 as follows (base number in the SEQ ID No.32 : aUele): (i) 132 : C; (ii) l44 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 :T; and (vi) 2379 : T.

The term "identity", as used herein, refers to a percentage of identical nucleic acid bases among two nucleotide sequences which are aligned in high order match. The percentage of the identity can be calculated by BLAST program under default setting (Reference 40, Altschul, S. F. et al, "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res., 1997, vol.25, pp.3389-3402). [78] The nucleic acid may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 38). Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry.

The nucleic acid of the present invention can be used as a Rac2 enhancer and can be utilized for a gene therapy in order to enhance the Rac2 gene expression and induce resistance to virus in a subject. [79] In another embodiment, the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing the Rac2 enhancer as a target molecule. This method comprises following steps: (a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide of the Rac2 region 1 nucleic acid of the present invention; (b) measuring an interaction between the agent and the nucleic acid fragment! and (c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.

In the present specification and Claims, the term "a candidate therapeutic agent" refers to a chemical compound, lead compound or biological molecule which has a potential activity to induce resistance to virus in a subject. [80] In one embodiment, the present invention provides a method for screening a candidate therapeutic agent against virus. This method comprises the following steps: (a) administering an agent to a biological material; (b) measuring an expression level of the Rac2 gene in the biological material; and (c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.

The term "biological material", as used herein, refers to the material which possesses a fragment of or the entire Rac2 gene or the Rac2 gene regulatory element, for example, DNA fragment, a cell, a tissue, an organ, an organism and individual.

The measurement of the expression level of the Rac2 gene can be performed by using Luciferase or other reporter gene which is functionally linked to the Rac2 promoter, or quantitative real-time polymerase chain reactions (PCR) with primers and probes specific for the Rac2 sequence. [81] In one embodiment, the present invention provides a method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus. This method comprises following steps: (a) detecting genetic predisposition of subjects by the detecting method of the present invention,' (b) stratifying the subject by the result of the detected genetic predisposition;

(c) giving a prevention, reduction, prophylaxis or treatment to the stratified subject; and (d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.

[82] In the present specification and claims, the term "clinical trial" refers to any research studies designed to collect clinical data on response to a particular treatment, and includes, but not limited to, phase I, phase II, and phase III clinical trials.

The term "stratifying the subject", as used herein, refers to arrangement of each individual in a clinical trial population in a hierarchical order according to any status such as resistance level to virus infection or virus disease progression.

Examples of the "prevention, reduction, prophylaxis" and "treatment", as used herein, include administering therapeutic agent, surgery and the like. [83] In another embodiment, the present invention provides a chip or a kit for detecting genetic predisposition of a subject for resistance to virus. The chip comprises base plate and at least one probe detecting SNP or nucleotide polymorphism. The kit comprises reagent and at least one probe detecting SNP or nucleotide polymorphism. The SNP or nucleotide polymorphism is selected from the group consisting of SNPs and nucleotide polymorphisms designated by the SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.

[84] The probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: l (for rs9610683), 2 (for rs9610682), 3 (for rs2284037), 4 (for rs739042), 5 (for rs2284036), 6 (for rs73904l), 7 (for ss73405466), 8 (for rs9798725), 9 (for ss73405467), 10 (for rs5995400), 11 (for rs6000619), 12 (for rs5756570), 13 (for rs36110509), 14 (for rs2899284), 15 (for rs6000618), 16 (for rs6000617), 17 (for rs9610677), 18 (for rs6000616), 19 (for rs9610676), 20 (for rs9610675), 21 (for rs9622582), 22 (for ss73405476), 23 (for ss73405477), 24 (for ss73405479), 25 (for rs5756568), 26 (for rs933223), 27 (for rs933222), 28 (for rs93332l), 29 (for ss73405482), 30 (for ss73405484) and 31 (for ss73405485). The sequences designated by SEQ ID Nos 1 - 31 represent flanking region sequences harboring each SNP or nucleotide polymorphism designated by each SNP ID Nos. [85] In the present specification and claims, the term "chip" refers to a tool or instrument for biological analysis having a portable size. The scope of the "chip" includes a plate or array used in biological experiments.

The term "base plate", as used herein, refers to a material holding a probe or probe sets.

The term "reagent", as used herein, refers to any chemical compound, solution or protein for a reaction detecting SNP or nucleotide polymorphism. Examples of the reagent include, but are not limited to, polymerase, Clevase enzyme, restriction enzyme, ligase, fluorescent dye, quencher, reaction buffer, hybridization buffer or etc.

[86] The probe sequence may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 38). Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry. Once synthesized, oligonucleotide probes may be labeled by any well-known methods.

[87] The probe comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31. Preferably, the probe has 7 to 100 bases, more preferably, 10 to 50 bases, and most preferably, 15 to 30 bases.

The probe can comprise any sequence other than partial sequence or partial complementary sequence of SEQ ID Nos 1- 31. For example, the probe may comprise linker sequence fixed to the base plate or 5' flap sequence for invader reaction.

[88] The probe is used for detecting SNP or nucleotide polymorphism in any form. For example, the probe can be used as "invader oligo" or "probe" in the invader assay described above. In the above described TaqMan assay, the probe can be used as "TaqMan probe".

EXAMPLES

[89] The following EXAMPLES are presented in order to more fully describe the base of the present invention. This EXAMPLES should in no way be construed as limiting the scope of the invention as defined by the Claims. [90] Summary of Methods in EXMAPLEs

ESN, their HIV-1-infected partner, and unexposed control individuals were enrolled with written informed consents as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and genetic analyses were performed with an approval from the ethical committee of the Kinki University School of Medicine. PBMC were stimulated with a mixture of HIV-I Gag and Env peptides as described (Reference 18, Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HIV- seropositive individuals", Nature, 1989, vol.339, pp.383-385), and total RNA extracted for both microarray analyses and real-time PCR quantification of the Rac2 expression. The microarray analyses were performed by using 12K chips (CombiMatrix Corporation,Mukilteo, U.S.A) harbouring 2 to 10 probes for each of the genes enlisted in Figure 7. Genotyping of known SNP was performed by TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, U.S.A.). Genomic DNA was amplified with primers (enlisted in the SEQUENCE LISTING under SEQ ID Nos: 33 - 72) and sequenced. Case-control analyses of the observed genotypes, calculation of the coefficients of LD, and extraction of haplotype compositions were performed by using the SNPAlyze ver. 5.1 (DYNACOM Co., Ltd., Yokohama, Japan). Putative Rac2 regulatory regions were cloned into the pGL3 plasmid (Promega Corporation, Madison, U.S.A.), transfected into human Jurkat cells, and the Dual-Luciferase Reporter Assays (Promega) were performed. The in vitro infection of PBMC with HIV-I and measurements of p24 were performed as described elsewhere (Reference 29, Biasin, M. et al., J. Infec. Dis., 2007, vol.195, pp.960-964).

EXAMPLE 1

[91] COMPARISONS OF THE CHANGES IN GENE EXPRESSION

LEVELS DURING THE HIV- 1 ANTIGENIC STIMULATION

The inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 30, WO2004/035825, International publication of PCT application, 29 April 2004). To further narrow down the location of the putative HIV-I resistance gene, the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Figure 8 for complete listing of the observed genotypes).

[92] Consistent with our previous observations (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024), the number of enrollees possessing the allele 229 at the D22S423 locus was significantly higher among the ESN than among the HIV-infected individuals (P = 0.0009, Fisher's exact test). As shown in Figure 2 and Figure 8 for the details, the numbers of individuals possessing a particular allele were different between the ESN and HIV-infected individuals at the SNP loci linked to the CardlO and GRAP2, the latter located closely to the D22S423 locus. Analyses of linkage disequilibrium (LD) revealed a strong linkage (P < 10^~5) between the rs202642 and rsl39562 loci in the HIVl -infected individuals, but the lack of such linkage among the ESN individuals, in consistency with the previously reported disruption of LD at the D22S276 locus (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024).

[93] Expression levels of all the genes located in the above candidate region were compared between the ESN and HIV-infected individuals by using DNA microarrays. Probes for the representative human cytokines, chemokine ligands, and transcription factors were also included, along with the plant and phage genes as negative controls. To selectively detect the possible HlVreactive changes in the host gene expression without being biased by non-genetic factors, the inventors of the present invention stimulated peripheral blood mononuclear cells (PBMC) prepared from each examined individual with a pool of 6 and 5 synthetic peptides representing the promiscuous HIVl Gag and gpl60 envelope epitopes (Reference 18, Clerici, M. et al., "Interleukin-2 production used to detect antigenic peptide recognition by T-lymphocytes from asymptomatic HIV-seropositive individuals", Nature, 1989, vol.339, pp.383-385), respectively, and prepared RNA before and at various time-points after the antigenic stimulation. Data obtained with multiple and redundantly placed probes for each gene were normalized for the entire array between individuals and between different time -points by using the Lowess equation.

[94] The overall expression levels of the tested genes were not different between the cells prepared before and at 1 hour after the antigenic stimulation,' however, drastic changes in the expression of several host genes were observed between 1 and 6 hours after the antigenic stimulation. Therefore, the inventors of the present invention focused their comparisons on changes in host gene expressions between 1 and 6 hours after the antigenic stimulation. The induction of multiple cytokine and chemokine genes including those encoding IL-6, TNF-α, and CCL3L1 was observed upon antigenic stimulation, and the expression levels of these genes were comparable in HIV-infected and in ESN individuals at 6 hours after the antigenic stimulation (Figure 2). This indicates that differences in gene expression between the tested groups, if any, were not due to possible HIV-induced changes in T-cell subset compositions.

[95] Among all the chromosome 22 genes tested, only a small number were constitutively expressed at high levels or induced upon the stimulation with HIV- 1 antigens (Figure 2). Of these, constitutive expression of the RPL3 and apparent induction of the EIF3S7 were expected because ribosomal protein L3 is an indispensable component of the peptidyltransferase center (Reference 19, Meskauskas, A. & Dinman, J.D., "Ribosomal protein L3: Gatekeeper to the A site", MoI. Cell., 2007, vol.23, pp.877-888), and eIF3 is the largest eukaryotic initiation factor that plays a central role in protein synthesis (Reference 20, Asano, K. et al., "Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits", J.Biol.Chem., 1997, vol.272, pp.27042-27052).

[96] Three genes of particular interest in possible relation to the resistance to HIV infection were nevertheless preferentially expressed in some ESN individuals after the antigenic stimulation^ the Rac2 encoding a Rho-subfamily small GTPase that is selectively expressed in and involved in the activation of T-helper tyρe-1 (Thl) cells (Reference 21, Li, B. et al., "Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation", Science, 2000, vol.288, pp.2219-2222; Reference 22, Yu, H., Leitenberg et al., "Deficiency of small GTPase Rac2 affects T cell activation", J.Exp.Med., 2001, vol.194, pp.915-925); the Myh9 encoding the only non-muscle myosin heavy chain expressed in T cells, which regulates T-cell motility (Reference 23, Jacobelli, J. et al., "A single class II myosin modulates T cell motility and stopping, but not synapse formation", Nat. Immunol., 2004, vol.5, pp.531-538); and the Lgalsl encoding a galactoside -binding lectin which may facilitate the attachment of HIV- 1 to the cell surface (Reference 24, Ouellet, M. et al., "Galectin-1 acts as a soluble host factor that promotes HIV-I infectivity through stabilization of virus attachment to host cells", J.Immunol., 2005, vol.174, pp.4120-4126). The Rac2 and Lgalsl loci are located close to the two SNPs at which the allele frequencies were different between the ESN and HIV-infected groups (Figure 2). The augmented expression of the Rac2 after the antigenic stimulation was further confirmed by real-time PCR (Figure 5).

[97] Detailed Method of DNA Microarray Analysis Study Population

Heterosexual couples discordant for HIV-I serostatus and healthy uninfected donors were enrolled from the Santa Maria Annunziata Hospital, Firenze, and the Luigi Sacco Hospital, Milano as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024). Written informed consent was obtained from all the enrolees. All the enrolees are Caucasians from Toscany region. The criteria for HIV-I infection and the ESN status have been described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024). The ethics committees of the above hospitals have approved the research protocols. [98] DNA Microarray analysis

Total RNA was prepared from antigen-stimulated PBMC as described (Reference 29, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide -like 3G^: a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was produced in the presence of an RNase inhibitor by using the T7-oligo(dT)24 primer. The resultant cDNA was purified, and biotinylated cRNA was prepared by using Biotin-16UTP and MEGAscript transcription kit (Ambion, Inc., Austin, U.S.A.). Two to 10 oligodeoxynucleoti.de probes were designed for each of the genes enlisted in Figure 7 by using the TargetSpecifier (CombiMatrix Corporation, Mukilteo, U.S.A.) and synthesized on microarray chips. After prehybridization, denatured biotin-conjugated cRNA samples were hybridized at 45⁰C, overnight. After washing and blocking, microarray chips were incubated with Cy3-conjugated streptavidin and washed vigorously. A fluorescence image of each microarray was scanned by a GenePix (Molecular Devices Corporation, Union City, U.S.A.), and analyzed by using the Microarray Imager (CombiMatrix Corporation).

[99] Two to 10 oligodeoxynucleotide probes were designed for each of the genes enlisted in Figure 7, and detected fluorescence intensities were Lowess normalized and colour-scaled in Figure 2 with each horizontal line representing one probe. The sample columns are arranged to combine expression levels in each individual of all the tested genes at 1 and 6 hours after the antigenic stimulation. The data obtained with PBMC from 5 representative ESN and 4 representative HIV-I -infected individuals are shown. The right half of Figure 2 shows the summary of the results of SNP genotyping, the details of which are shown in Figure 8. Corresponding chromosomal and microarray locations of representative genes are also indicated. Numbers of individuals possessing a particular allele are compared between the groups by adopting a dominant gene hypothesis using Fisher's exact test. LD between SNP was analyzed by likelihood ratio test as described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024), and exact P values were obtained.

EXAMPLE 2

[100] DNA SEQUENCE POLYMORPHISMS ACCUMULATED IN ESN

INDIVIDUALS

Based on the results presented as EXAMPLE 1, the inventors of the present invention sequenced the chromosomal segments spanning the Myh9, I12rb, Rac2-Pscd4, CardlO, and Lgalsl loci (see Figure 7 for the location of the sequenced segments), and compared the frequencies of ESN and HIV-1-infected individuals possessing a particular allele at each of the observed polymorphic loci. The inventors of the present invention detected no sequence polymorphisms with a significant case -control difference at the Myh9, I12rb, and Lgalsl loci. On the other hand, notable accumulation of sequence polymorphisms with significantly different allele frequencies between the two groups was observed in a segment between the CardlO and Rac2 (Figure 1, Figure 3 and Figure 9). The observed case-control differences apparently increased toward the downstream (centromeric) portion of the Rac2 locus, and there were two regions, one between the 5th and 6th Rac2 exons (region l) and the other downstream of the 7th exon (region 2), where multiple polymorphic loci with statistically significant case-control differences between the ESN and HIV-infected groups were accumulated.

[101] Further, the SNP genotypes were highly linked to each other within each of the above Rac2 regions, with the levels of LD being higher among the ESN than in HIV-infected individuals in the region 2 (Figure 3). Thus, the polymorphic loci within the regions 1 and 2 constitute small numbers of haplotypes. In particular, the region 1 downstream haplotype TTT was highly accumulated among the ESN individuals (P = 0.0087), while the haplotype CCC was more frequently found in the HIV-infected group (P = 0.0080). The above region 1 haplotype TTTw&s linked with the upstream haplotype CA in more than half of the chromosomes sequenced.

In the region 2, a haplotype indicated in red in Figure 1 with characteristic base deletions was also observed more frequently among the ESN than among the HIV l infected individuals (P = 0.011). Therefore, in the following analyses the region 1 haplotype CA TTT and the region 2 haplotype with the base deletions are designated R and those shown in blue S for simplicity.

[102] Detailed Method of Identifying DNA Polymorphism Accumulated in ESN Individuals

Partially duplicated 71 genomic DNA samples from 30 ESN and 21 HIV-I -infected individuals were sequenced through the segments shown in Figure 7, and frequencies of each observed sequence polymorphism enlisted in Figure 9 were compared by case-control methods. Known ARG were also genotyped with the above samples, and only 1 individual in each phenotypic group possessed the CCR5Δ32 allele heterozygously. No one possessed the CCR2-64I, and DCSIGN -336 SNP frequencies were not different between the groups.

[103] Figure 3 shows distribution of observed SNP with significant case-control differences by adopting either a dominant (red lines) or a recessive (blue lines) hypothesis. P values shown are those calculated by χ² (chi-square) test. See Figure 9 for odds ratios and l values calculated by Fisher's exact test. A representative value is shown for each cluster of SNP with significant case -control differences in the Rac2 locus, with the upper and lower brackets corresponding respectively to the regions 1 and 2 in Figure 1. Coefficients of LD between each pair of SNP alleles are shown here in /lvalues and colour-scaled by using SNPAlyze ver. 5.1. [104] Figure 1 shows distribution of sequence polymorphisms (arrows) in the Rac2 locus with those showing significant case -control differences between ESN and HIV-infected groups shown in red. Longer arrows indicate SNP with case-control differences of P < 0.006. Chromosomal regions showing >50 % sequence homologies between humans and mice are indicated with horizontal lines labelled HH for high homology. In the polymorphic region 2, there were also gaps (small red squares in the diagram) and base deletions (-)'■ Δl, a 28-bp deletion (-) relative to the database -reported genome sequence (+); Δ2, an 82-bp insertion (+) relative to the reported genome sequence (-); and Δ3, a 34"bp insertion (+). Haplotypes in each of the three blocks are those identified by the Four Gamete method using SNPAlyze ver. 5.1, and all observed linkages between each haplotype across the blocks are shown. Exact frequencies of sequenced chromosomes with each observed linkage are shown only for the major ones indicated with thick lines. The numbers of individuals possessing a particular haplotype are compared between the two groups by Fisher's exact test.

EXAMPLE 3

[105] FUNCTIONAL ANALYSIS OF THE POLYMORPHIC REGIONS 1

AND 2

The above polymorphic regions 1 and 2 colocalized with the regions that showed >50 % sequence homology between humans and mice (Figure l), indicating that these polymorphic regions may contain functional regulatory elements. To examine this directly, the genomic fragments harbouring the polymorphic region 1 or 2 isolated from representative R/R and S/S healthy control individuals were cloned and inserted into the downstream of the luc gene along with the known Rac2 promoter (Reference 25, Ladd, PD. et al., "Identification of a genomic fragment that directs hematopoietic- specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333) in the upstream. Although the Rac2 promoter is known to be strong and promiscuous (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333), the luc constructs harbouring the region 1 fragment induced significantly higher luciferase activities in transfected human Jurkat cells than that containing the promoter alone (Figure 4), indicating the presence of an enhancer element within this genomic fragment. More importantly, the region 1 fragment of the R haplotype induced significantly higher luciferase activities than that of the S halotype, revealing a functional difference between the two haplotypes. The region 2 fragments apparently reduced the expression of the luc gene regardless of the haplotype, in consistency with the previously indicated presence of a repressor in this region (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333).

[106] The region 1 haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC. Thus, when levels of the Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2.5-fold higher level of the Rac2 message was seen after 6 hours of stimulation with the HIV-I peptides in the individuals possessing the homozygous R/R haplotype in region 1. On the other hand, levels of the Rac2 expression did not change after the antigenic stimulation in cells homozygous for the S haplotype in region 1 (Figure 5). Importantly, haplotypic differences in the region 2 did not affect the Rac2 induction after antigenic stimulation, again indicating that the polymorphic region 1, but not the region 2, harbours a functional enhancer of the Rac2 expression. [107] Finally, the Rac2 region 1 haplotypes also affected HIVl replication in vitro. Thus, when PBMC from healthy control individuals possessing the homozygous S/S hap Io type or harbouring the R hap Io type in region 1 were infected in vitro with HIV-I, different behaviours were observed. In supernatants of S/S PBMC, p24 concentrations were significantly higher as compared to what observed in supernatants of cells harbouring the R haplotype both upon infection with a CCR5-tropic laboratory strain of HIV-I or with a primary CCR5-tropic HIV-I isolate (Figure 6). Median p24 levels in cultures of the R/R cells at 5 days after infection with either one of the two CCR5-tropic viruses were <5 % of those observed with the S/S cells. On the other hand, p24 concentrations did not differ significantly between the cells of different region 1 haplotypes when PBMC were inoculated with a CXCR4-tropic laboratory strain of HIV-I or a primary CXCR4"tropic HIV-I isolate. Thus, the Rac2 enhancer haplotype which is significantly accumulated among ESN individuals is functionally more active, and is associated with a restricted replication of CCR5'tropic HIV-I. [108] Detailed Method of Analysis of Polymorphic Regions 1 and 2

In Figure 4, Human Jurkat line T cells were trasfected with a luc plasmid harbouring the Rac2 promoter either with or without a genomic fragment of the putative regulatory regions. Data shown here are ratios of luciferase activities relative to those obtained by transfecting the luc gene without the promoter. The Dual-Luciferase reporter assays were performed at 18, 21, and 24 hours after the plasmid transfection, and averages (mean ± S.E.M) from 3 and 5 repeated experiments performed at 18 and 21 hours after transfection, respectively, are shown. Similar results were obtained at 24 hours after transfection. Statistical analyses were done by two-tailed paired ϊHest.

[109] Figure 5 shows changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides. Real-time PCR analyses were performed with samples obtained from 10 ESN and 6 HIV-I -infected individuals, which were prepared as described for Figure 2, and the results were re-grouped based on the separately determined region 1 and region 2 genotypes. Data are shown here as ratios of the Rac2 expression between 1 and 6 hours after the antigenic stimulation calculated as 2 ^MCT, and bars indicate S.E.M. Statistical analyses were done by two-tailed Welch's ttest. The numbers of individuals possessing each identified genotype were: 6 R/R, 7 R/S, and 3 S/S in the regionl, and 6 R/R, 4 R/S, and 6 S/S in the region 2. [110] Figure 6 shows replication of HIV-I in cultured PBMC. Uninfected healthy control individuals were genotyped with written informed consent for the Rac2 region 1 and region 2 haplotypes, and their PBMC were infected with each of the 4 different HIV-I clones or isolates, or with the dual-tropic isolate 89.6. None of the individuals included here possessed the CCR5Δ32 allele. Data shown here are p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the region 1 genotypes, with horizontal bars indicating median values. Statistical comparisons were done both between the S/S and R/R groups and between the S/S and combined R groups by Mann-Whitney's U test because p24 concentrations do not follow normal distribution and medians were compared. However, use of two-tailed Welch's *!-test also indicated significant differences (Day 5 with BaL, P = 0.021 for S/S vs R/R, P - 0.035 for S/S vs combined R; Day 5 with v.6, P = 0.013 for S/S vs R/R, P- 0.013 for S/S vs combined R). The dual-tropic 89.6 replicated efficiently in PMBC of all three genotypes, and no significant differences were observed in p24 concentrations between the S/S and R/R individuals at day 5 (not sown), [ill] Method of Luciferase Assays

For functional analyses of the putative Rac2 regulatory regions, the 2504-bp region 1 and 8904-bp region 2 genomic fragments were amplified by PCR using the following oligonucleotide primers and LA-Taq polymerase (TAKARA BIO, Inc., Ohtsu, Japan):

Rac2RlL ATCCTCGAGCACCAAGCTGGACCTGCGGGACGACAAG Rac2RlR ATCGTCGACGAGAGGATGTCACTCGCTCTGAGTCACATG Rac2R2L ATCCTCGAGCTCCCACCTAGATGGGTCTGATCCTCCAG and

Rac2R2R ATCCTCGAGTTTTGATGTAGCATAGCTCCCAGTAACTTTCAG

The cloned Rac2 genomic fragments were inserted to the SaR restriction site located downstream of the luc gene within the pGL3 plasmid (Promega Corporation, Madison, U.S.A.) harbouring the previously described Rac2core promoter (-260 to +130 bp) (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333). Each of the resultant reporter constructs along with the pRL-TK were cotransfected into 1.0 to 1.2 x 10⁶ human Jurkat cells using an amaxa Nucleofector (amaxa AG, Cologne, Germany) with the solution V and condition C- 17 according to the manufacturer's recommendations. After 18, 21 and 24 hours, cells were lysed and expressed luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's protocol. [112] Method of RNA Extraction and Real-Time PCR Analyses

Total RNA was extracted from antigen- stimulated PBMC as described (Reference 29, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide -like 3G^: a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was generated by using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative real-time PCR assays were performed by using an ABI 7900HT Real-Time PCR System (Applied Biosystems). Reactions were preformed using the TaqMan probes for the human Rac2 and GAPDH (Applied Biosystems) with Platinum Quantitative PCR SuperMix-UDG (Invitrogen Corporation, Carlsbad, U.S.A.). The relative expression levels of the Rac2 were calculated and normalized to those of GAPDH using the software provided with the ABI 7900HT PCR Systems. [113] Method of In Vitro HIV- 1 Infection and Measurement of p24

PBMC was stimulated for 2 days in complete medium containing 20 % foetal bovine serum, 5 mg/ml phytohemagglutinin, and 10 ng/ml recombinant human interleukin (IL) -2 at a concentration of 2 x 10⁶ cells/ml. After a viability assessment, 3 x 10⁶ cells were resuspended in medium containing 0.05 ng p24 equivalent per 10⁶ cells of HIV-I, and incubated for 3 hours at 37°C. Infected cells were then washed and resuspended in 3 ml of complete medium containing IL-2, and were plated in 3 wells at 1 x 10⁶ cells/well. Cultures were re-fed on day 3 and supernatants were collected on days 2, 3, and 5. Absolute levels of HIV-I p24 were measured using the Alliance HIV-I p24 ELISA kit (PerkinElmer Inc., Waltham, U.S.A.). HIV-I BaL and IIIB were provided by Drs. S. Gartner, M. Popovic, and R. Gallo (Courtesy of the NIH AIDS Research and Reference Reagent Program). HIV-I primary isolates were kind gifts from Prof. C. -F. Perno, University of Roma, Tor Vergata, Italy.

Claims

1. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos: (i) rs739041, (ii) rs739042, (ϋi) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No. ^'■ allele) : (i) rs739041 : T; (ii) rs739042 : T, (iii) rs2284037 : T; (iv) rs933223 : C; (v) rs6000619 : A; (vi) ss73405466 : G; and (vii) rs5756570 : C.

2. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs2284037, (ii) rs739042 and (iii) rs73904i; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs2284037 : T; (ii) rs739042 : T; (iii) rs739041 : T.

3. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) ss73405466, (ii) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxii) rs933321, (xxiii) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) ss73405466 : G; (ii) rs9798725 : T; (iii) ss73405467 : GGCCTCATCCTTCCAAGTT CAAGTTCAG; (iv) rs5995400 : C; (v) rs6000619 : A; (vi) rs5756570 : C; (vii) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 : T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiii) rs9610676 : A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss73405476 : T; (xvii) ss73405477 : T; (xviii) ss73405479 : T; (xix) rs5756568 : G; (xx) rs933223 : C; (xxi) rs933222 : A; (xxii) rs933321 : A; (xxiii) ss73405482 : A; (xxiv) ss73405484 : gap; and (xxv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAG GTAATTGAA.

4. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs9610683 and (ii) rs9610682; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs9610683 : C; and (ii) rs9610682 : A.

5. An ex vivo method for detecting genetic predisposition of a subject to resistance to virus, comprising: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, Gx) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiϋ) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxii) ss73405482, (xxϋi) ss73405484 and (xxiv) ss73405485; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : aUele) : (i) rs9010683 : C; Gi) rs9610682 : A; (iii) rs2284036 : T; (iv) rs9798725 : T; (v) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG; (vi) rs5995400 : C; (vii) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 : T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiϋ) rs9610676 : A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss73405476 : T; (xvii) ss73405477 : T; (xviii) ss73405479 : T; (xix) rs5756568 : G; (xx) rs933222 : A; (xxi) rs933321 : A; (xxii) ss73405482 : A; (xxiϋ) ss73405484 : gap; and (xxiv) ss73405485 : TCCCATAATCCAGGTGGGAGGAACCCAGTGGGAGGTAATTGA A.

6. A method for inducing or enhancing resistance to virus in a subject, comprising enhancing the Rac2 gene expression in the subject.

7. The method according to Claim 6, wherein the Rac2 gene expression is enhanced by using a DNA-binding protein, peptide, oligonucleotide, or nucleotide analogue that binds to the enhancer element located in the region 1.

8. The method according to Claim 6, wherein the Rac2 gene expression is enhanced by inhibiting an expression of the Rac2 gene repressor by RNAi technique.

9. Use of Rac2 protein in the preparation of a medicament for the treatment or prophylaxis of virus -induced disease.

10. A nucleic acid exhibiting the Rac2 enhancer activity, wherein the nucleic acid is selected from the following (A) and (B):

(A) a nucleic acid having the nucleotide sequence of SEQ ID No:32,^" and

(B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.

11. The nucleic acid according to Claim 10, wherein the nucleic acid has the following alleles at the SNP sites in the nucleotide sequence of SEQ ID No:32 (base number in the SEQ ID No.32 : allele):

(i) 132 : C; (ii) 144 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 : T; and (vi) 2379 : T.

12. Use of the nucleic acid according to Claim 10 or 11 as a Rac2 gene enhancer.

13. Use of the nucleic acid according to Claim 10 or 11 for a gene therapy to induce resistance to virus in a subject.

14. A method for screening a candidate therapeutic agent against virus, comprising: a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide sequence of the nucleic acid according to Claim 10 or 11; b) measuring an interaction between the agent and the nucleic acid fragment; and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.

15. A method for screening a candidate therapeutic agent against virus, comprising: a) administering an agent to a biological material; b) measuring an expression level of Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of Rac2 gene in the biological material as a candidate therapeutic agent against virus.

16. A method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus, comprising: a) detecting genetic predisposition of subjects by the method according to any one of Claims 1 - 5; b) stratifying the subject by the result of the detected genetic predisposition; c) giving a prevention, reduction, prophylaxis or treatment to the stratified subject; and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.

17. The method according to any one of Claims 1 - 8, wherein the virus is one selected from the group consisting of HIV, HCV and HPV.

18. The method according to Claim 17, wherein the virus is HIV or HCV.

19. The method according to Claim 18, wherein the virus is HIV

20. The use according to Claims 9 or 13, wherein the virus is one selected from the group consisting of HIV, HCV and HPV

21. The use according to Claim 20, wherein the virus is HIV or HCV

22. The use according to Claim 21, wherein the virus is HIV

23. The method according to Claim 14 - 16, wherein the virus is one selected from the group consisting of HIV, HCV and HPV

24. The method according to Claim 23, wherein the virus is HIV or HCV.

25. The method according to Claim 24, wherein the virus is HIV

26. The method according to any one of Claims 1- 5, wherein the genotyping is performed by the Invader assay.

27. A chip used in the method according to Claim 1, comprising a base plate and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, (ϋi) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:6, 4, 3, 26, 11, 7 and 12.

28. A chip used in the method according to Claim 2, comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:3, 4 and 6.

29. A chip used in the method according to Claim 3, comprising a base plate and probe sets detecting SNPs and nucleotide polymorphisms designated by the SNP ID Nos (i) ss73405466, (ϋ) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xvϋi) ss73405479, (xix) rs5756568, (xx) rs933223, (xxi) rs933222, (xxϋ) rs933321, (xxiii) ss73405482, (xxiv) ss73405484 and (xxv) ss73405485, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31.

30. A chip used in the method according to Claim 4, comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO^l and 2.

31. A chip used in the method according to Claim 5, comprising a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xϋ) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxii) ss73405482, (xxiii) ss73405484 and (xxiv) ss73405485, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NO:l, 2, 5, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30 or 31.

32. A kit used in the method according to Claim 1, comprising a reagent and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ϋ) rs739042, (ϋi) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO:6, 4, 3, 26, 11, 7 and 12.

33. A kit used in the method according to Claim 5, comprising a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ϋ) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (vϋi) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix) rs5756568, (xx) rs933222, (xxi) rs933321, (xxii) ss73405482, (xxiii) ss73405484 and (xxiv) ss73405485, wherein the probe sets comprises at least one probe comprises more than 7 bases of partial sequence or partial complementary sequence of the nucleotide sequence selected from the group consisting of SEQ ID NOa, 2, 5, 8, 9, 10, 13, 14, 15, 16, 17, 18, 19, , 21, 22, 23, 24, 25, 27, 28, 29, 30 or 31.