EP2861735A1 - Snp markers associated with polycystic ovary syndrome - Google Patents

Abstract

SNP markers associated with PCOS and probes, chips, primers, kits and methods for detecting the SNP markers are provided. Furthermore, the use of the probes, chips and primers in predicting or diagnosing the risk of PCOS is also provided.

Description

SNP MARKERS ASSOCIATED WITH POLYCYSTIC OVARY SYNDROME Technical Field

The present invention relates to SNP (Single Nucleotide Polymorphism) markers associated with Polycystic Ovary Syndrome (PCOS). The present invention further relates to probes, chips, primers and methods for detecting the SNPs. Also, the present invention relates to the use of the probes, chips and primers in predicting and diagnosing the risk of PCOS.

Background

PCOS is a clinical condition characterized by the presence of two or more of these features: chronic oligo-ovulation or anovulation, androgen excess and polycystic ovaries.¹ As the most common cause of anovulatory infertility , PCOS affects 6-8% childbearing-aged women.²'³ Additionally, PCOS is associated with important endocrine-metabolic derangements and a broad range of adverse sequelae, including dyslipidemia, atherosclerosis, insulin resistance and type 2 diabetes.^4"6 Insulin resistance is present in perhaps 50% of women with PCOS.⁷ Among women with impaired glucose tolerance (IGT) and diabetes mellitus, about 20% were recognized at younger age to have PCOS.^8"10

The pathogenesis of PCOS is not fully understood. Heritable tendencies have long been recognized, but complex interactions exist between genetic and environmental factors. Association studies have been conducted on at least 70 candidate genes, principally related to reproductive hormones, insulin resistance, and chronic inflammation, e.g., follicle stimulating hormone receptor^FSHR), cytochrome P450, family 11A (CYPllA), insulin receptor (INSR) and interleukin 6 (IL-6) ^n"15; however, none correlates consistently with PCOS.

Summary of Invention

The present invention relates to SNPs associated with PCOS. Particularly, the present invention provides SNP markers associated with PCOS. Furthermore, the present invention provides probes, chips, primers and methods for detecting the SNPs. Also, the present invention relates to the use of them in predicting and diagnosing the risk of PCOS.

One aspect of the invention provides SNP markers, the nucleotide sequences of which are shown as: SEQ ID NO.l, wherein N is C or T; SEQ ID NO.2, wherein N is A or G; SEQ ID NO.3, wherein N is C or T; SEQ ID NO.4, wherein N is A or C; SEQ ID NO.5, wherein N is C or T; SEQ ID NO.6, wherein N is A or C; SEQ ID NO.7, wherein N is C or T; SEQ ID NO.8, wherein N is C or T; SEQ ID NO.9, wherein N is A or G; SEQ ID NO.10, wherein N is C or T; SEQ ID NO.11, wherein N is C or T; SEQ ID N0.12, wherein N is C or T; SEQ ID N0.13, wherein N is A or G; SEQ ID N0.14, wherein N is C or T; SEQ ID N0.15, wherein N is A or G; SEQ ID NO.16, wherein N is C or T; SEQ ID NO.17, wherein N is A or T; SEQ ID NO.18, wherein N is C or G; SEQ ID NO.19, wherein N is C or T; SEQ ID NO.20, wherein N is C or T; SEQ ID N0.21, wherein N is C or T; SEQ ID N0.22, wherein N is A or G; SEQ ID N0.23, wherein N is A or G; SEQ ID N0.24, wherein N is C or T; SEQ ID N0.25, wherein N is A or G; SEQ ID N0.26, wherein N is C or T; SEQ ID N0.27, wherein N is A or T; SEQ ID N0.28, wherein N is G or T; SEQ ID N0.29, wherein N is A or G; SEQ ID NO.30, wherein N is C or T; SEQ ID N0.31, wherein N is A or G; SEQ ID N0.32, wherein N is C or T; SEQ ID N0.33, wherein N is C or T; SEQ ID N0.34, wherein N is C or T; SEQ ID N0.35, wherein N is C or T; SEQ ID N0.36, wherein N is A or G; SEQ ID N0.37, wherein N is C or T; SEQ ID N0.38, wherein N is C or T; SEQ ID N0.39, wherein N is C or T; SEQ ID NO.40, wherein N is A or C; SEQ ID N0.41, wherein N is G or T; SEQ ID N0.42, wherein N is G or T; SEQ ID N0.43, wherein N is C or T; SEQ ID N0.44, wherein N is A or G; or SEQ ID N0.45, wherein N is C or T.

Another aspect of the invention provides probes for detecting the genotypes at the site N of the SNP markers of the present invention.

Still another aspect of the invention provides a chip for detecting the genotypes at the site N of the SNP markers of the present invention, wherein the chip comprises one or more probes of the present invention.

Still another aspect of the invention provides primers for determining the genotypes at the site N of the SNP markers of the present invention.

Still another aspect of the invention provides a kit comprising the probes, chip or primers of the present invention for detecting the genotypes at the site N of the SNP markers.

Still another aspect of the invention provides the use of the primers, probes, chip and kit of the present invention in the preparation of an agent for predicting or diagnosing PCOS.

Still another aspect of the invention provides the use of the primers, probes, chip and kit of the present invention in predicting or diagnosing PCOS.

Still another aspect of the invention provides a method of predicting or diagnosing PCOS based on the SNP markers, wherein the method comprises determining genotypes at the site N of the SNP markers of the present invention.

Brief Description of Drawings

Figure 1. Genome-wide Manhattan plots for the GWAS meta-analysis. Negative logio P-values are shown for SNP markers that passed quality control. The solid horizontal line indicates a P value of 10^~5. Markers within 50 kb of an SNP associated with PCOS are marked in red for those identified in a previous GWAS and replicated here, and in green for those first identified in the current study. Figure 2. Regional plots of the 3 PCOS loci from GWAS I (2pl6.3, 2p21, and 9q33.3). (a - c) Genotyped SNPs passing quality control measures in GWAS are plotted with the P values (as -logio values) as a function of genomic position (hgl8) (a) 2pl6.3, (b) 2p21, and (c) 9q33.3. In each panel, the index association SNP is represented by a diamond. Estimated recombination rates (taken from HapMap) are plotted to reflect the local LD structure. Gene annotations were taken from the University of California Santa Cruz genome browser. LD blocks were obtained from the Hapmap project (release 22, CHB+JPT).

Figure 3A-3H. Regional plots of the 8 newly discovered PCOS loci. Genotyped and imputed SNPs passing quality control are plotted with their meta-analysis P values (as -logio values) as a function of genomic position (NCBI Build 37). In each panel, SNPs genotyped are plotted as circles, and SNPs imputed as crosses. The index association SNP is represented in purple, Pgwas meta is for the combined results of the initial datasets, and PowAS-REP-Meta is for the combined results of the initial and follow-up datasets, represented by the diamond (for the index SNP) or a square (for another independent SNP of this region). Estimated recombination rates (taken from lOOOGenome ASI) are plotted to reflect the local LD structure. Gene annotations were taken from the University of California Santa Cruz genome browser.

Figure 4A-4B. PCR electrophoretograms for the 45 SNP markers. Detailed Description

As used herein, the terms "single nucleotide polymorphism" or "SNP" is a DNA sequence variation or a genetic variant that occurs when a nucleotide, e.g., adenine (A), thymine (T), cytosine (C), or guanine (G), in the genome sequence is altered to another nucleotide.

SNPs are identified herein using the rs identifier numbers in accordance with the NCBI dbSNP database.

The term "genotype" refers to a description of the alleles of a gene or genes contained in an individual or a sample. As used herein, no distinction is made between the genotype of an individual and the genotype of a sample originating from the individual. The term "odd ratio" or "OR" refers to the ratio of the odds of the disease for individuals with the marker (polymorphism) relative to the odds of the disease in individuals without the marker (polymorphism).

In the first aspect, the invention provides SNP markers, the nucleotide sequences of which are shown as: SEQ ID NO. l, wherein N is C or T; SEQ ID NO.2, wherein N is A or G; SEQ ID NO.3, wherein N is C or T; SEQ ID NO.4, wherein N is A or C; SEQ ID NO.5, wherein N is C or T; SEQ ID NO.6, wherein N is A or C; SEQ ID NO.7, wherein N is C or T; SEQ ID NO.8, wherein N is C or T; SEQ ID NO.9, wherein N is A or G; SEQ ID NO.10, wherein N is C or T; SEQ ID NO.11, wherein N is C or T; SEQ ID N0.12, wherein N is C or T; SEQ ID N0.13, wherein N is A or G; SEQ ID N0.14, wherein N is C or T; SEQ ID N0.15, wherein N is A or G; SEQ ID NO.16, wherein N is C or T; SEQ ID NO.17, wherein N is A or T; SEQ ID NO.18, wherein N is C or G; SEQ ID NO.19, wherein N is C or T; SEQ ID NO.20, wherein N is C or T; SEQ ID N0.21, wherein N is C or T; SEQ ID N0.22, wherein N is A or G; SEQ ID N0.23, wherein N is A or G; SEQ ID N0.24, wherein N is C or T; SEQ ID N0.25, wherein N is A or G; SEQ ID N0.26, wherein N is C or T; SEQ ID N0.27, wherein N is A or T; SEQ ID N0.28, wherein N is G or T; SEQ ID N0.29, wherein N is A or G; SEQ ID NO.30, wherein N is C or T; SEQ ID N0.31, wherein N is A or G; SEQ ID N0.32, wherein N is C or T; SEQ ID N0.33, wherein N is C or T; SEQ ID N0.34, wherein N is C or T; SEQ ID N0.35, wherein N is C or T; SEQ ID N0.36, wherein N is A or G; SEQ ID N0.37, wherein N is C or T; SEQ ID N0.38, wherein N is C or T; SEQ ID N0.39, wherein N is C or T; SEQ ID NO.40, wherein N is A or C; SEQ ID N0.41, wherein N is G or T; SEQ ID N0.42, wherein N is G or T; SEQ ID N0.43, wherein N is C or T; SEQ ID N0.44, wherein N is A or G; or SEQ ID N0.45, wherein N is C or T.

One embodiment of this aspect provides more than one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 SNP markers selected from the ones above.

In the present invention, each SNP marker refers to a SNP which is found to be associated with PCOS. As used herein, SNP marker and corresponding SNP relate to the same site in the nucleotide fragment. Especially when referring to the detection of the genotype at the site N of SNP marker, it should be understood that it implies the detection of the genotype at the corresponding site of the corresponding SNP, vice versa. The SNP for each SNP marker is listed in Table 1 below.

In another aspect, the invention provides probes for detecting the genotypes at the site N of one or more SNP markers of the present invention.

One embodiment of this aspect provides probes for each SNP marker listed in Table 1.

Table 1. SNP and Probes for each SNP marker

SNP marker SNP marker

SNP Probes

NO. SEQ ID NO.

ATATAATTTTTTTAAC[A/G]GAGAAATTGCATAACA

1 1 rsl l891936

(SEQ ID N0.46/ SEQ ID N0.47)

TTCAAGTCCACAGATA[C/T]AGCTTTTCATATGTGA

2 2 rsl7030684

(SEQ ID N0.48/ SEQ ID N0.49)

TCCCTGGGGGAGGTGTGACGGCAGAG[C/T]TGCATTTT

3 3 rs4340576

TATGGTATGCCCCAACA (SEQ ID NO.50/ SEQ ID N0.51)

CTGTTGTAAAGCAAAATAGAATCCTA[A/C]ACCAGAA

4 4 rsl2468394 CTTCTGCAGTTAGCCACA (SEQ ID N0.52/ SEQ ID

N0.53)

AAACTTTTACAACCAGAATTAATGTT[C/T]CCTTGTGC

5 5 rs7567607

TCTTTTAAAAAATCAAA (SEQ ID N0.54/ SEQ ID N0.55)

GGGTATAGGTGTATGTAATCAGTTT[G/T]GTTTCATCT

6 6 rsl3429458

TCTAACTTTGCACAGCA (SEQ ID N0.56/ SEQ ID N0.57) AGATGAAACAAAACTGATTACATACA[C/T]CTATACC

7 rs7568365 CTGCCACTAATTAAAAAT (SEQ ID N0.58/ SEQ ID

N0.59)

TCTTTGTTCAGAAGCACGGTACATTA[C/T]TATACAGC

8 rs7582497

TGAAGCCCTCTAGCATT (SEQ ID NO.60/ SEQ ID N0.61)

GATCCTCCCTATATAAGGCCTAAAAC[A/G]CCACCATT

9 rsl0176241

AGAGTTTTACTGCTTTA (SEQ ID N0.62/ SEQ ID N0.63)

AGGTATCCACACACACCCATTTCTTA[C/T]ACACACAT

10 rs6744642

CCCATATCATTCTCGAT (SEQ ID N0.64/ SEQ ID N0.65)

AGTAAAGCCCGGGTCCTAACATTTTATTGA[A/G]TGGT

11 rs 12478601 ACTAACCAAGACCAGCAGGAATGAAA (SEQ ID N0.66/

SEQ ID N0.67)

ACCTCTATAATTCCAGCTTCTTTTCTTCTT [G/A] GGTAG

12 rsl038822 CTAAATCACCAAAAAAAAATTTTTG (SEQ ID N0.68/

SEQ ID N0.69)

CTATGAACATTATTTTGCCTTGACACTTTT [T/C]ACATA

13 rs7559891 GCACCCAAATCTTATGTATTTAATT (SEQ ID NO.70/

SEQ ID N0.71)

CTC ATTTCTAGGCAGAACTGAGTGTC [C/T] TTCCCTAA

14 rsl873555

ACTGCCTGTATCCATTA (SEQ ID N0.72/ SEQ ID N0.73)

TGATGATGTGATGCAATAC AAGTCTC [A/G] GAATTTGT

15 rs7596052

TGGTGAGAGTGTAATTT (SEQ ID N0.74/ SEQ ID N0.75)

TCTTTTTCATGGCTGTTTCTACCATC[C/T]TGGAAATAA

16 rsl0165527

TAATTTTTAACTCTCT (SEQ ID N0.76/ SEQ ID N0.77)

TATATGTACTTATTCAACATAAATCC [ A/T] CTGTTTAG

17 rs6726014

AAAAAAGTATTATAGCT (SEQ ID N0.78/ SEQ ID N0.79)

TACCTTGTAAAAAATAATCCAGAAAG[C/G]AGTTCAA

18 rs2374551 GATCAGCCTAGGCAATAT (SEQ ID NO.80/ SEQ ID

N0.81)

TGTTCAGTATTATCAAGCTGTATATA[C/T]GTTTCGAC

19 rs6731009

ATTTCATATACATGATC (SEQ ID N0.82/ SEQ ID N0.83)

AAACACAAACAATGAGATGCTATTGT[C/T]TTCCAATC

20 rsl0179648 AGCCTAGCAAAACCAGA (SEQ ID NO.84/ SEQ ID

N0.85)

TAACTGCAACAACTCAGTGTGGATAC[C/T]ATCATCAT

21 rs7558302 GTGAAAGTCACCCATGAC (SEQ ID N0.86/ SEQ ID

N0.87)

TGCTCTGGCAGAAGAGGCACATGTTG[A/G]ACAAATG

22 rsl3405728 GCTGCATTATGGTGAGAT (SEQ ID NO.88/ SEQ ID

N0.89)

ACACATCTTCTCCCCTATTATACTCA [A/G] CCAGCAAG

23 rsl0818854

CATTCCCACCTTTAAGC (SEQ ID NO.90/ SEQ ID N0.91)

TTATTGCCCTTATTTACTTCTCCAAACATT[A/G]ATCTG

24 rs7857605

GTCTCATCGTTTGCAAAGGTGTTGC (SEQ ID N0.92/ SEQ ID N0.93)

GGCAGAGATTCTGGGGACTGGAAAGA [A/G] CTAGTTA

25 rs2479106 TGATCAAGGAACCAAAAG (SEQ ID N0.94/ SEQ ID

N0.95)

TTTATTTTCTATAGCAGGTTTATTGA[C/T]ACTTTTTTT

26 rsl778890

CTAGTAAAGTTTGAAA (SEQ ID N0.96/ SEQ ID N0.97)

TGCTGAAAAAAATGATTGGATGATAG[A/T]TCGGATT

27 rsl627536 AAGAAGGGAAGAAATAGC (SEQ ID N0.98/ SEQ ID

N0.99)

CTAAAAAGGAACAAAA[C/A]TATGTTGCATAACTCA

28 rsl0986105

(SEQ ID NO.100/ SEQ ID NO.101)

CTTTGATGCTGTGAGACGAAGGCATCTTGT[C/T]AGTG

29 rs2268361 CCCTGGGATTGAGATCTTTCATTGGT (SEQ ID NO.102/

SEQ ID NO.103)

AAAAACAGGTGTCAGGCTGGATTTGA[C/T]CCATTGG

30 rs2349415 CTGTAGTTCAGTGACACT (SEQ ID NO.104/ SEQ ID

NO.105)

TCAATTCTGGAATTGGAAGGGAATCC[A/G]AGGAGAT

31 rsl0865238 CTATACCAGGCAATGCAT (SEQ ID NO.106/ SEQ ID

NO.107)

CCCACCAAAGACAGTTTTGCTTGGGT [C/T] CTCTCAAA

32 rs4744370 GCTATGCTGTTGGGTTT (SEQ ID NO.108/ SEQ ID

NO.109)

GTGTGCTGTGTTGGGTGTGTGAACATTCCT[A/G]AGAC

33 rs4385527 GTCCATAAGCTGATTTATAAAAACTT (SEQ ID NO. l 10/

SEQ ID NO. I l l)

CTCCAGGAAGCAGCCATGCCTGATGTGTGC[A/G]ATG

34 rs3802457 AATATGCCTTATCCTCCCGAAACTGGC (SEQ ID

NO.112/ SEQ ID NO. l 13)

GGATTGACCACTGTCAAGTCACAGAGTCAC[G/A]AAT

35 rsl894116 TGTCTAGAATCAATATTATGTAGACTA (SEQ ID

NO.114/ SEQ ID NO.115)

CATATGTAATGTGCATTTATCCCCCC[A/G]GTGCATTA

36 rs2069408 CCTTACAATTGTCCGTA (SEQ ID NO. l 16/ SEQ ID

NO.117)

AGATAAACAGGGTAGTTGTAGTTGCAACAG[G/A]GTA

37 rs705702 GATAGAGGTAGGTCTACCCTGGGTTTA (SEQ ID

NO.118/ SEQ ID NO.119)

CAAGGAAACCAAGGAAGATTTTTCTC [C/T] TTCAGAAC

38 rsl l l71739 TCGGACCCTGAATACCA (SEQ ID NO.120/ SEQ ID

N0.121)

CTC AGGTCCCTGACTCAGCAGCCCACCAGG [G/A] C AG

39 rs877636 ACCATTCCAGTCTCCTGGAATCTAAAC (SEQ ID

NO.122/ SEQ ID NO.123) CAACAAATAGTGAAGAGACTTTTGAATCTA[T/G]AGG

40 40 rs2292239 GCAGCACTTAAGGGATCTAGGGTGGCA (SEQ ID

NO.124/ SEQ ID NO.125)

GGCCTTGGGACATTTG[C/A]AAACAAAGCTGTTGAT

41 41 rs2272046

(SEQ ID NO.126/ SEQ ID NO.127)

GTTATTTTCCCTATTAAAGAACATCC[G/T]CTCATAGT

42 42 rs4784165 TTTTCAAGTTATTATGT (SEQ ID NO.128/ SEQ ID

NO.129)

GCATTTTATACAACCTCACTGCATCAGCCT [G/A] TTAA

43 43 rs2059807 AAGCAAGAGGTCTGATTCACATACGA (SEQ ID NO.130/

SEQ ID NO.131)

ATTCGTTGACTATTTTAGCTGGTGAC [ A/G] CAATGAAA

44 44 rs6022786 AAACAGAGTCTAAGCAA (SEQ ID NO.132/ SEQ ID

N0.133)

AGGCCTGCCAGTTTTAGGGGCC ATTTGGCT [C/T] CTGA

45 45 rsl l225161 GAAGAACTGTTAATAAAAGTATTAAT (SEQ ID NO.134/

SEQ ID NO.135)

In still another aspect, the invention provides a chip for detecting the genotypes at the site N of one or more SNP markers of the present invention, wherein the chip comprises the probes of the present invention.

In one embodiment of this aspect, the chip is used to detect the genotypes at the site N of 45 SNP markers of the present invention. More preferably, the chip comprises the probes shown as SEQ ID NO. 46-135.

In another embodiment of this aspect, the chip is used to detect the genotypes at the site N of SNP markers shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44. More preferably, the chip comprises the probes shown as SEQ ID NO. 56, 57, 66, 67, 88, 89, 90, 91, 94, 95, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 118, 119, 126, 127, 128, 129, 130, 131, 132 and 133.

In still another aspect, the invention provides primers for detecting the genotypes at the site N of one or more SNP markers of the present invention.

In one embodiment of this aspect, the primers for each SNP marker are listed in Table 2.

Table 2. Primers for 45 SNP markers

SNP marker Product length (i.e.

Primer Sequence (S'-3')

NO. SNP marker length)

CGGGTTCAAGTGGTTCTGCT (forward) (SEQ ID NO.136)

1 452bp

GTTGTTGTTGTTCCTATGGTTTCC (reverse) (SEQ ID NO.137)

AGATAACAACTCTATGCTCTGGCTTC (forward) (SEQ ID NO.138)

2 445bp

AAGGCCCTTCAGTGCTGTTCT (reverse) (SEQ ID NO.139) ATCTGCCATTCCGATTTCCA (forward) (SEQ ID NO.140)

318bp

CAAGAAAGGCAGGATGGATGTT(reverse) (SEQ ID NO.141)

TCTGCCTGGGAAGTGTAAGTCTC (forward) (SEQ ID NO.142)

328bp

ATACTCCAGTCACTTTCCTGTCTCC(reverse) (SEQ ID NO.143)

TCTGTCTTGCTTTCTTAGCCTCC (forward) (SEQ ID NO.144)

401bp

TGTGCTATTGTTGTTCACTTCTATGG(reverse) (SEQ ID NO.145)

CAGCGGTATGATTTCGTAGTG (forward) (SEQ ID NO.146)

560bp

GCTAAAATCTCATCACCTGGAC (reverse) (SEQ ID NO.147)

CAGCGGTATGATTTCGTAGTG (forward) (SEQ ID NO.146)

560bp

GCTAAAATCTCATCACCTGGAC (reverse) (SEQ ID NO.147)

CAGCGGTATGATTTCGTAGTG (forward) (SEQ ID NO.146)

560bp

GCTAAAATCTCATCACCTGGAC (reverse) (SEQ ID NO.147)

AAGTAGCTGCCCAAACAATGTG (forward) (SEQ ID NO.148)

266bp

CAGGCTTGGGACCAGATTGT(reverse) (SEQ ID NO.149)

TAAACCAAGCTCCAATTTCTCATAG (forward) (SEQ ID NO.150)

342bp

CACACCTTTACTACTGTTTCCTATGC(reverse) (SEQ ID NO.151)

AGACTCAGATGAGATGCCACAT (forward) (SEQ ID NO.152)

465bp

TTACCTGTCCAACTCCAGAATG (reverse) (SEQ ID NO.153)

AGGCTGAAGCAGGAGAATCG (forward) (SEQ ID NO.154)

321bp

GGAGACGACCTTAGACTGTAGCAT (reverse) (SEQ ID NO.155)

TCATCGCTCATTCAGTCATCAGTT (forward) (SEQ ID NO.156)

553bp

GCCAACATCTTTGCTGAGGAAT(reverse) (SEQ ID NO.157)

ATTAATATGGCCAACTCAAATGAACT (forward) (SEQ ID NO.158)

460bp

GCTGGAGAAGGGTAGAGGTGC(reverse) (SEQ ID NO.159)

AAAGGACATCGACAGGCATTG(forward) (SEQ ID NO.160)

542bp

GCATCCGTAATCCAACACCTG(reverse) (SEQ ID N0.161)

CCTATTCACCTCAATTGCAGTCC (forward) (SEQ ID NO.162)

426bp

CTTCCCAAATAGCCAGTTCCA(reverse) (SEQ ID NO.163)

GGTTTTGGAACTGGCTATTTGG(forward) (SEQ ID NO.164)

521bp

CCGTCATCCTTGTCTGCCTACT(reverse) (SEQ ID NO.165)

CCATGAGCCATTATTGTAAACTGAT (forward) (SEQ ID NO.166)

297bp

TAGCTGGGACTGTAGGTGTGTGT (reverse) (SEQ ID NO.167)

TTAGAAATGCTGGTGGTTGTACAA(forward) (SEQ ID NO.168)

382bp

CTAATGTGATCCTCAAATGGCTACT(reverse) (SEQ ID NO.169)

AACCCAGGCAAAAAGAGAAATAG (forward) (SEQ ID NO.170) 446bp ACTGACTCTGGTTTTGCTAGGCT(reverse) (SEQ ID N0.171)

CCAAGTGTCACCTCTGCCATC(forward) (SEQ ID NO.172)

434bp

CCACTGTTGCAAATTCATTCCA(reverse) (SEQ ID NO.173)

GTGGTTCTTACTCTAGCACAATGAT (forward) (SEQ ID NO.174)

341bp

CCATCCACATACTCACTTCAATATC (reverse) (SEQ ID NO.175)

CAAAACCAGGCTGATGACAAT (forward) (SEQ ID NO.176)

842bp

GTTTGAGAATCATAGACCAGCAC (reverse) (SEQ ID NO.177)

CTCCAGGGACTGCCTCTTTCT(forward) (SEQ ID NO.178)

472bp

TGTTTATGCATGTAACTGTAGGTGG(reverse) (SEQ ID NO.179)

GAGCAGCCACTCAAGAAACAG (forward) (SEQ ID NO.180)

429bp

AAGCCACCATCCAGTCTCAC (reverse) (SEQ ID NO.181)

AAACAAGATAGGGCTAGGCTGATT(forward) (SEQ ID NO.182)

648bp

CATGATTGACTGCCTGGTACTCC(reverse) (SEQ ID NO.183)

AGAGGCTATTCTCAGTGAGCTTCTC (forward) (SEQ ID NO.184)

273bp

GCACAGTGCATGGCAATAGTAAG(reverse) (SEQ ID NO.185)

AGCATACCTCAAGCATGAACAGAT (forward) (SEQ ID NO.186)

255bp

AAGCAATGTAGAAACATGGCACA(reverse) (SEQ ID NO.187)

GCTCCCTCCTTCAACATCCAC (forward) (SEQ ID NO.188)

304bp

GCAATGCCAACAAGAAGACAGA(reverse) (SEQ ID NO.189)

CTGTGGCTCACCTTGGAGATTAT (forward) (SEQ ID NO.190)

481bp

TGGCTTTCTGTTCCTACGTTAGAC(reverse) (SEQ ID NO.191)

TGTTATTTGATTGATGGTCCTAGAGG (forward) (SEQ ID NO.192)

300bp

CTTTAGGCTACTATCATTGCACCATT(reverse) (SEQ ID NO.193)

AATCCTGTCCGTTTCCAACACT (forward) (SEQ ID NO.194)

186bp

GCACAAACCCAACAGCATAGC(reverse) (SEQ ID NO.195)

ATCACAAGTTTGCCTTCTTAAATATG (forward) (SEQ ID NO.196)

560bp

GTGCCAGAAGATCGCAGAGTT(reverse) (SEQ ID NO.197)

CCTCTTCACCCACAGCAACAT (forward) (SEQ ID NO.198)

323bp

AGACAGTGGAAGTGGTCCTCATT(reverse) (SEQ ID NO.199)

TTTTCTGTTGTATGGGATGAATGG (forward) (SEQ ID NO.200)

427bp

TACAAGGATTGACCACTGTCAAGTC(reverse) (SEQ ID NO.201)

TGCAGTAGGCTGTCTTCAAATCA (forward) (SEQ ID NO.202)

284bp

ACCTTGTGATGCAGCCACTTC(reverse) (SEQ ID NO.203)

CGAGACAGGCAGGTTGCTAAG (forward) (SEQ ID NO.204)

488bp

AAAGACGGCTATTCAGTGTTGTTG(reverse) (SEQ ID NO.205) CAGGCTGAGGCAGGAGAATC (forward) (SEQ ID NO.206)

38 418bp

TGGCCTTACTTAGGATTTCTTACTG (reverse) (SEQ ID NO.207)

GAGCCACTACGCCTGTCTGATT (forward) (SEQ ID NO.208)

39 392bp

CGAGATGCTGAGATAGTGGTGAAG(reverse) (SEQ ID NO.209)

ACTTCTTACCATCTCCTACCCACC (forward) (SEQ ID N0.210)

40 360bp

GTCCTCCCATGACTTCAGCTATC(reverse) (SEQ ID N0.211)

GGTTTGAAATTGAAGTGATGGCT (forward) (SEQ ID N0.212)

41 180bp

TTGCTGCTTGGAGTTTCTTGAC(reverse) (SEQ ID N0.213)

AGTCCCTACTCACTGATCCTCTGC (forward) (SEQ ID N0.214)

42 202bp

TGCCCATCTTAGCACTGATACTCT(reverse) (SEQ ID N0.215)

ACAGTTGGACGGTGGTAGACATT (forward) (SEQ ID N0.216)

43 848bp

TCAAGTGGCTTGTTGCTACTGC(reverse) (SEQ ID N0.217)

TGTGCCTAAATAAGATGGTTCTCTG (forward) (SEQ ID N0.218)

44 314bp

CACGAGAATCGCTTGAACCTG(reverse) (SEQ ID N0.219)

GTAGTGCTAGAGGCCTGCCAGT (forward) (SEQ ID NO.220)

45 527bp

TAACTGTGTATCTTTCCCCTCATCTT (reverse) (SEQ ID N0.221)

In still another aspect, the invention provides a kit for detecting the genotypes at the site N of one or more SNP markers of the present invention, wherein the kit comprises the probes, chip or the primers of the present invention.

In one embodiment of this aspect, the kit is used to detect the genotypes at the site N of at least 15 SNP markers of the present invention. Preferably, the kit is used to detect the genotypes at the site N of 45 SNPs of the present invention. More preferably, the kit comprises probes shown as SEQ ID NO. 46-135.

In another embodiment of this aspect, the kit is used to detect the genotypes at the site N of 15 SNP markers shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44. More preferably, the kit comprises the probes consisted of probes shown as SEQ ID NO. 56, 57, 66, 67, 88, 89, 90, 91, 94, 95, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 118, 119, 126, 127, 128, 129, 130, 131, 132 and 133.

In another embodiment of this aspect, the kit comprises primers for detecting the genotypes at the site N of 45 SNP markers of the present invention. More preferably, the kit comprises primers consisted of the primers shown as SEQ ID NO. 136-221.

In still another embodiment of this aspect, the kit comprises primers for determining the genotypes at the site N of 15 SNP markers of the present invention, wherein the 15 SNP markers are shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44. Preferably, the kit comprises primers consisted of the primers shown as SEQ ID NO.146, 147, 152, 153, 174, 175, 176, 177, 180, 181, 188, 189, 190, 191, 196, 197, 198, 199, 200, 201, 204, 205, 212, 213, 214, 215, 216, 217, 218 and 219.

In still another aspect, the invention provides the use of the primers, probes, chip or kit of the present invention in the preparation of an agent for predicting or diagnosing PCOS, wherein the primers, probes, chip or kit is used to detect the genotypes at the site N of the SNP markers of the present invention. In one embodiment, the genotypes at the site N of at least 15 SNP markers, preferably all 45 SNP markers of the present invention are detected. In another embodiment, the genotypes at the site N of 15 SNP markers are detected, wherein the 15 SNPs are shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44.

In still another aspect, the invention provides the use of the primers, probes, chip or kit of the present invention in predicting or diagnosing PCOS, wherein the primers, probes, chip or kit is used to detect the genotypes at the site N of the SNP markers of the present invention.

Still another aspect of the invention provides a method of predicting or diagnosing PCOS, wherein the method comprises determining genotypes at the site N of one or more SNP markers of the present invention.

In one embodiment of this aspect, the method comprises determining genotypes at the site N of at least 15 SNP markers, preferably all 45 SNP markers of the present invention.

In another embodiment of this aspect, the method comprises determining genotypes at the site N of 15 SNP markers, wherein the 15 SNP markers are shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44.

In yet another embodiment of this aspect, determining genotypes at the site N of the SNP markers is performed by hybridization, for example, using the probes or chips of the present invention.

In yet another embodiment of this aspect, determining genotypes at the site N of the SNP markers is performed by sequencing, for example, PCR, Real-time Quantitative PCR, or MassARRAY (Sequenom), using primers of the present invention.

In yet another embodiment of this aspect, the present method comprises the following steps: extracting DNA from peripheral blood or saliva of a subject, determining genotypes at the site N of one or more SNP markers, and analyzing the results to predict the risk of PCOS or diagnose PCOS.

Embodiments

Subjects

All Han Chinese samples evaluated were obtained in multiple collaborating hospitals from China. The discovery sets (GWAS I and II) of 2254 Han Chinese PCOS samples and 3001 controls were recruited mainly from northern China. Subsequent replication samples (REP I and II) of 8226 cases and 7578 controls were collected from 29 provinces (Shandong, Heilongjiang, Jilin, Liaoning, Inner Mongolia, Hebei, Henan, Tianjin, Beijing, Shanxi, Shaanxi, Gansu, Ningxia, Jiangsu, Anhui, Shanghai, Guangdong, Guangxi, Fujian, Zhejiang, Hubei, Hunan, Jiangxi, Sichuan, Chongqing, Xinjiang, Yunnan, Guizhou and Hainan) throughout China. The PCOS patients were diagnosed according to the Rotterdam Consensus proposed in 200345. Clinical data of the patients were obtained from medical records. 01igo-/aovulation was assessed by menstrual cycles more than 35 days in length or a history of <8 menstrual cycles in a year. Polycystic ovarian morphology was determined when >12 follicles measuring 2-9 mm in diameter were scanned in either ovary or the ovarian volume was above 10 ml. Hyperandrogenism was confirmed if there were evidences about hyperandrogenemia and/or hirsutism. Patients with other causes of oligomenorrhea or hyperandrogenism were excluded. Clinical information was collected from the cases through a full clinical checkup by physician specialists. Additional demographic information was collected from both cases and controls through a structured questionnaire. All participants provided written informed consents. The study was approved by the Institutional Ethical Committee of each hospital and was conducted according to Declaration of Helsinki principles.

DNA Extraction

EDTA anti-coagulated venous blood samples were collected from all participants. Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using Flexi Gene DNA kits (Qiagen), and was diluted to working concentrations of 50 ng/μΕ for genome -wide genotyping and 15-20 ng/μΕ for the validation study.

GWAS Genotyping and Quality Control

Affymetrix Genome-Wide Arrays were used for discovery phase: GWAS Data Set 1 was performed using the Affymetrix Genome-Wide Human SNP Array 6.0, and Sampes of GWAS Data Set 2 were genotyped using Axiom Genome- Wide Arrays. Quality control filtering of the GWAS data was performed as follows: for the SNP 6.0 arrays whose Contrast QC was 0.4 or greater being left out of further data analysis, and for the Axiom arrarys, a Dish QC (DQC) of 0.82 or better is considered a pass. Genotype data were generated using the birdseed algorithm for SNP 6.0, and the Axiom GT1 algorithm for Axiom arrays. For sample filtering, array with generated genotypes of fewer than 95% of loci were excluded. For SNP filtering (after sample filtering), SNP with call rates < 95% in either case or control samples were removed. SNPs whose MAF (minor allele frequency) was < 1%, or deviated significantly from Hardy Weinberg Equilibrium (HWE, P < 1E-5) in controls were excluded.

Imputation Analysis of Untyped SNPs

To conduct meta-analysis across array types, imputations were conducted for both GWAS date sets using MACH¹⁷'¹⁸, separately. Phased haplotypes for 90 CHB+JPT subjects (180 haplotypes) were used as the reference for imputing genotypes. Any SNP imputed with information content r²<0.3 was excluded from association analysis because of lack of power. In addition, a second imputation step was performed using IMPUTEv2¹⁹'²⁰ for the 8 new identified regions (0.5MB either side of any SNP achieved a PGWAS _META < 10^"5), using the 1,000 Genomes haplotypes Phase I interim release (Jun2011) as reference. Any SNPs imputed with proper info <0.4 were treated as poor imputation. The criteria for SNP QC filtering were the same as the genotyped ones.

Analysis of Population Substructure

Population substructure was evaluated using principal components analysis (PCA) as implemented in the software EIGENSTRAT²¹. 20 principal components (PCs) were generated for each subject. PCA were conducted twice, and the first one was for the analysis of study data (1,510 cases and 2,106 controls) combined with HapMap data. The first two principal components were plotted, and, 43 cases and 9 controlswere excluded. The second one was conducted for the remaining test samples. The PCs were generated for association analysis.

Association Analysis

Logistic regression was used to determine whether there was a significant difference in PC scores between cases and controls; significant PCs were used as covariates in the association analysis to correct for population stratification. After adjustment, little stratification was observed (λ = 1.07, λιοοο = 1.04, standardized to a sample size of 1000²²).

Meta-Analysis of GWAS Data Sets

The GWAS data sets were combined using meta-analysis. The meta-analysis was conducted using PLINK²³. The heterogeneity across the three stages was evaluated using Q-statistic P-value. The Mantel-Haenszel method is used to calculate the fixed effect estimate.

SNP Selection and Replication

The following criteria were used for the selection of SNPs for validation: Strong significant SNPs (PGWAS-_META≤ 10^"5) from the GWAS-meta analysis were selected for Replication I. Generally, those SNPs showed norminal significance (P < 0.05) in Replication I or were not significant in Replication I but with a GWAS-REP1 meta-analysis P value less than 5x 10^"6 were also kept for Replication II. The Sequenom MassARRAY system was used for most of the replication studies, except for rs2059807, which was genotyped using TaqMan assays (Applied Biosystems).

Statistical Analysis

Genome -wide association analysis at the single marker level and the HWE analysis in the case-control samples were performed using PLINK²³; R package was used for the genome wide P value plot. The regional plots were generated using LocusZoom²⁴. In the replication studies, allelic association analysis was conducted using SHEsis²⁵. The GWAS and replication data were also combined using meta-analysis using PLINK²³. Conditional logistic regression was used to test for independent effects of an individual SNP²⁶'²⁷.

Results

Totally, 45 SNPs were found to be associated with PCOS. The detailed analysis information is listed in Tables 3-5. In fact, the SNPs represent regions, which associate with PCOS and may comprise many SNPs. Among these regions, significant evidence was found for the first identified loci, 2pl6.3, 2p21, and 9q33.3²⁶, and the SNPs representing these regions are rsl3405728 (2pl6.3; PowAS-meta = 3.77 x 10^"9), rsl3429458 (2p21, P_GWAS-me,a = 4.17 10 ¹³), rsl2478601 (2p21, P_GWAS-me,a =3.37x 10^"10), rs2479106 (9q33.3, P_GwAS-meta = 5.14 x 10^"10) and rsl0818854 (9q33.3, P_GwAS-meta =2.50E-04). SNPs in 19 other regions beyond the reported 3 showed association at PowAS-meta value < 10^~5 with PCOS susceptibility in the GWAS-meta analysis. However, variants in the FSHR gene, which locates in 2p 16.3 but not directly supported in the previous GWAS, also show PowAS-meta values < 10^~5. And conditional logistic analysis supports that signals in FSHR is independent from the previous report. Therefore, the inventor selected the most significant SNPs from totally 20 regions for validation (the Replication I study).

Among these 20 regions, 7 were validated in the Replication I stage (P < 0.05 with the same allelic odds ratio direction), and other 3 regions had SNPs with P < 5x l0^"6 in the GWAS-REP1 meta-analysis. SNPs from these 10 regions were genotyped again in an independent sample set (Replication II). As a result, common variants in 8 regions, 9q22.32, l lq22.1, 12ql3.2, 12ql4.3, 16ql2.1, 19pl3.3, 20ql3.2 and the FSHR gene (2pl6.3), showed overall combined evidence of association at P value < 5 ^x 10^"8, by a meta-analysis of all stages under fixed-effects model. The results strongly support the associations between those regions and PCOS.

On 9q22.32, the most significant SNP is rs3802457 (P_GwAS-REP-Meta=5.28x lO^~14, OR_GwAS-REP-Meta=0.77), which locates in the intron region of the C9orf3 gene (Figure 3). Controlling for rs3802457, rs4385527 (P_GWAS -REP-Meta 87x 10^"9, ORGWAS -REP-Meta- 0.84) shoWS independent association in conditional logistic regression analysis, and it also locates in C9orf3. C9orf3 is a member of the Ml zinc aminopeptidase family. It is a zinc-dependent metallopeptidase that catalyzes the removal of an amino acid from the amino terminus of a protein or peptide, and may play a role in the generation of angiotensin IV. SNP rs3802458 within C9orf3 is reported associated with the development of erectile dysfunction (ED) in African- American men following radiotherapy for prostate cancer²⁸. ED in men and PCOS in women occurred when people develop conditions with inadequate or excessive amounts of sexual hormones. Interestingly, FSHR gene (rs2268363) has been identified as the most significantly associated with ED²⁸, and strong association evidence between FSHR and PCOS was also identified (discussed below).

On l lq22.1, rsl894116 (PGWAS-REP-Meta=1.08x lO^~22, ORGWAS-REP-Meta=1.27) locates in the intron region of YAPl (MIM: 606608) (Figure 3). Controlling for rs 1894116, conditional logistic regression analysis reveals that there is no additional association signal. YAPl, containing a WW domain, is a transcriptional regulator which can act both as a coactivator and a corepressor and is the critical downstream regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. YAP overexpression alters the expression of genes associated with cell proliferation, apoptosis, migration, adhesion, and epithelial-to-mesenchymal transition²⁹. Mice embryos with Yapl null mutation die between embryonic days E9.5 and El 0.5 due to yolk sac avasculogenesis and failure of attachment between the allantois and the chorion³⁰.

On 12ql3.2, the most significant SNP rs705702 (PGWAS-REP-Meta=8.64x lO^~26, OPvGWAS-REP-Meta=1.27) locates in the intergenic region between RAB5B (MIM: 179514) and SUOX (MIM: 606887) (Figure 3). Controlling for rs705702, conditional logistic regression analysis reveals that there is no additional association signal. RAB5B is a member of the RAS superfamily, and it is associated with the plasma membrane and early endosomes. SUOX encodes a homodimeric protein localized to the intermembrane space of mitochondria. There are several SNPs showing evidence of association with PCOS risk. Of them, rs2292239 (PGWAS-REP-Meta=2.72>< 10^~22, .25) appears to be a most interesting one, which is

31 33 34

reported associated with Type 1 diabetes ^" and Type 1 diabetes autoantibodies . Rs2292239 locates in intron 7 of ERBB3. ERBB3, an activator of the phosphatidylinositol-3 -kinase/ Akt pathway, is a member of the epidermal growth factor tyrosine kinase receptor family which regulates cell survival and vesicle trafficking. ERBB3 plays a critical role in determining antigen presenting cells function³⁵.

On 12ql4.3, rs2272046 (PGWAS-REP-Meta=1.95x lO^~21, ORGWAS-REP-Meta=0.70) locates in an intronic region of HMGA2 (MIM: 600698), which encodes a protein with structural DNA-binding domains and acts as a transcriptional regulating factor (Figure 3). Controlling for rs2272046, there is no additional association signals in this region. HMGA2 has previously been identified to be associated with adult stature³⁶, vascular tumors including angiomyxomas and pulmonary hamartomas³⁷, and Type 2 Diabetes³⁸. Interestingly, a mutation in the gene can result in the "pygmy" mouse, with a significant reduction in body weight, reduced amounts of fat tissue, and infertility in both sexes³⁹, which suggests its vital role in growth and reproduction.

On 16ql2.1, the most significant SNP is rs4784165 (PGWAS-REP-Meta=3.64x lO^~n, (Figure 3). Controlling for rs4784165, conditional logistic regression analysis reveals that there is no additional association signal. TOX3 (MIM: 611416) is the nearest gene to this top signal. TOX3 belongs to the large and diverse family of HMG-box proteins that function as architectural factors in the modification of chromatin structure by bending and unwinding DNA⁴⁰.

On 19pl3.3, rs2059807 (P_GWAS -REP-Meta- 1.09^X 10^"8, ORGWAS -REP-Meta-1.14) locates in the intron region of the INSR gene (MIM: 147670) (Figure 3). Controlling for rs2059807, conditional logistic regression analysis reveals that there is no additional association signals. INSR plays an important role in insulin metabolism. The tyrosine kinase domain mutations of the insulin receptor have been shown to cause severe hyperinsulinemia and insulin resistance^41"43. In previous studies, common SNP in INSR gene has been reported to be associated with PCOS in Han Chinese and Caucasian⁴⁴'⁴⁵. Insr null mice grow slowly and die by 7 days of age with ketoacidosis, high serum insulin and triglycerides, low glycogen stores and fatty livers⁴⁶.

On 20ql3.2, the top signal is rs6022786 .83^X 10^"9, ORGWAS -REP-Meta- 1 - 13), locates in an intergenic region between genes SUMO IP 1 and ZNF217 (MIM: 602967) (Figure 3). Controlling for rs6022786, conditional logistic regression analysis reveals that there is no additional association signals. SUMO IP 1 is the SUMOl pseudogene 1. ZNF217, zinc finger protein 217, can attenuate apoptotic signals resulting from telomere dysfunction as well as from doxorubicin-induced DNA damage, may promote neoplastic transformation by increasing cell survival during telomeric crisis, and may promote later stages of malignancy by increasing cell survival during chemotherapy⁴⁷.

And, 2pl6.3 has been reported in the previous GWAS of PCOS²⁶. In that study, global significant findings in this region only locates in the LHCGR gene (MIM: 152790), and the top signal was not directly linked with the FSHR gene (MIM: 136435), mainly due to a recombination hot spot. However, FSHR has been considered to be one of the most compelling candidate genes for PCOS for a long time⁴⁸. FSHR null mutant females are sterile with small ovaries, blocked follicular development, atrophic uterus and imperforate vagina, and null mutant males are fertile despite reduction in testis weight, oligozoospermia and reduced testosterone levels⁴⁹ In the current study, SNPs in the FSHR gene meet the selection criteria for validation in the initial stage, and global significant findings were obtained in the combined analysis (top signal is rs2268361, PGWAS-REP-Meta⁼9.89x 10^"13, OPVGWAS -REP-Meta-0.87) (Figure 3,). Conditional logistical regression analysis supports that the association of FSHR is independent from those previous signals in LHCGR.

Finally, independent 15 SNPs are selected to represent these regions, which are most associated with PCOS. The 15 SNPs refer to SNP marker Nos. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44.

Table 3. Analysis results for the No.1-28 SNP markers in GWAS-I

Table 4. Analysis results for the No. 29-44 SNP markers in GWAS-II

aMinor allele/major allele.

b N represents the nucleotide more correlative to PCOS in the site.

Table 5. Analysis result for the No. 45 SNP marker .

MAF, minor allele frequency.

In meta-analysis, the P is calculated by fixed effect model and P(R) is calculated by random effect model.

Based on the study above and practice use, detecting genotypes at the site N of at least 15 SNP markers, for example, all 45 SNP markers in the present invention, is useful for predicting or diagnosing PCOS. However, detecting genotypes at the site N of 15 independent SNP markers of 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44 can also work, with less expense.

Detecting genotypes at the site N of the SNP markers

There are many processes for detecting genotypes at the site N of the SNP markers, for example, by hybridization or sequencing.

As to hybridization, probes should be designed to specifically hybridize with the locus of SNP, and then the hybridization could be analyzed whether SNP is present. An example of probes for all 45 SNPs is given just for the purpose of exemplifying, which is not intended to limit the scope of the invention. A person skilled in the art could easily design similar probes to hybridize with the SNPs, which all fall into the scope of the invention.

Generally, probes should be presented in a carrier, for example, a chip, so that more than one SNP markers can be detected at a time. The present invention also provides a chip comprising probes detecting the SNP markers shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44 (i.e. SNPs of rsl3429458, rsl2478601, rsl3405728, rsl0818854, rs2479106, rs2268361, rs2349415, rs4385527, rs3802457, rsl894116, rs705702, rs2272046, rs4784165, rs2059807 and rs6022786). A person skilled in the art well knows how to produce such chip when the probes are selected.

As to sequencing, primers should be designed forward and afterward the interested locus. An example of primers for all 45 SNPs (listed in Table 2) is given just for the purpose of exemplifying, which is not intended to limit the scope of the invention. A person skilled in the art can easily design similar primers to sequence the SNP markers, which also fall into the scope of the present invention. Furthermore, the process and agents used in the sequencing are also well known in the art.

Another useful method for genotyping SNP markers uses iPLEX of Sequenom platform (Sequenom, Inc., San Diego, CA). Polymerase chain reaction (PCR) and extension primers for the SNPs were designed using the MassARRAY Assay Design 3.0 software. PCR and extension reactions are performed according to the manufacturer's instructions, and extension product sizes were determined by mass spectrometry using the Sequenom iPLEX system.

Method of predicting or diagnosing PCOS

The 45 SNP markers based on the present invention can be used to predict or diagnose PCOS. Firstly, the DNA from peripheral blood or saliva of a subject is extracted, and then, the genotypes at the site N of the SNPs are detected, for example, by hybridization with probes or chips above, or by sequencing. At last, the results will be analyzed to predict the risk of PCOS. Examples

The following examples are just for the purpose of exemplifying and should not be considered to limit the scope of the present invention.

Example 1

All the 45 SNP markers are amplified by PCR using the primers listed in Table 2. The following processes are followed for the PCR reaction.

A. Reaction system

Reagent Volume (|jL)

ddH₂0 1.8

10* buffer 0.5

Mg²⁺ 0.4

dNTP 0.1

Hotstar 0.2

F Primer/ R Primer 1

DNA sample 1

Total 5

B. Reaction process

The products are tested by electrophoresis and sequencing and are confirmed. Figure 4 shows the electrophoretogram for all the 45 SNP markers.

Example 2

1. Extracting DNA from peripheral blood or saliva of a subject, purifying DNA and adjusting DNA concentration to 20 ng/mL.

2. Detecting genotypes at the site N of 15 SNP markers by sequencing and the 15 SNP markers are shown as SEQ ID NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44. The primers used are listed in Table 2.

3. Method:

I. PCR reaction

A. Reaction system

Reagent Volume (JJL)

ddH₂0 1.8 10* buffer 0.5

Mg²⁺ 0.4

dNTP 0.1

Hotstar 0.2

F Primer/ R Primer 1

DNA sample 1

Total 5

B. Reaction process

C. Purification of PCR product

The PCR product is precipitated by 25 μΐ. PEG (22%, w/v) and 2μΕ NaCl (5 M) at room temperature. Then the plate is stored at 4 ^°C for 30 minutes. The left-over PEG was washed by 80 of 75% ethanol three times by centrifugation at 4 ^°C .

D. Cycle sequencing of purified PCR product

The purified DNA was dissolved in 5 μΕ ddH₂Q.

Reagent Volume ( μ L)

ddH₂0 3.7

BigDye Terminator 3.1 Sequencing Buffer (ABI) 1.125

Primer 0.675

Total 5.5

Then, the plates are mixed well and spun shortly. The initial denaturation procedure is performed by a rapid thermal ramp to 96 ^°C and lasts for 1 minute. 25 cycles of reactions are performed with denaturation for 10 seconds over 96 ^°C , annealing for 5 seconds over 50 ^°C and extention for 4 minutes over 60 ^°C . Rapid thermal ramp to 4 ^°C is performed. And the product is hold until ready to purify.

E. Ethanol/EDTA/Sodium Acetate Precipitation

2 μΕ of 125 mM EDTA and 2 μΕ of 3 M sodium acetate are added to each well. And then 50μΙ_^ 100% ethanol is added to each well. The plate is sealed and mixed by inverting4 times. The plate is incubated at room temperature for 15 minutes. Then the precipitated DNA is washed with 75% ethanol for 3 times.

F. Capillary electrophoresis on ABI 3730 XL genetic analyzer

Each well is added 10 μί HI-DI formamide and denatured at 95 ^°C for 5 minutes. The precipitated DNA is loaded on ABI 3730 XL genetic analyzer for capillary electrophoresis.

II. MassARRAY

A. Main apparatus and reagent

^ Amplification: ABI GeneAmp® 9700 384 Dual;

2) Mechanical arm: MassARRAY Nanodispenser RSI 000;

3) Analyze: MassARRAY Compact System;

4) Reagent: Complete Genotyping Reagent Kit for MassARRAY® Compact 384

B. Procedure

Perform 384 PCR reactions (same multiplexed assays, different DNA). These instructions cover performing PCR for a whole 384-well microtiter plate of reactions in which the same assay will be applied to different DNA.

Prepare a PCR cocktail as described in the following table

1) Add the reagents in the order in which they appear in the table for multiplexed PCR cocktail, without DNA, for 384 reactions (same multiplexed assays, different DNA).

Component Volume ( μΐ, )

ddH₂0 1.8

10* buffer 0.5

Mg²⁺ 0.4

dNTP 0.1

Hotstar 0.2

F Primer/ R Primer 1

DNA 1

Total 5

2) To each well of a 384-well microtiter plate (Marsh Biomedical Products, Inc. #SP 0401 Sequen), add ΙμΙ, of the appropriate genomic DNA (5-10 ng^L).

3) Dispense of the PCR cocktail into each well of the 384-well plate.

4) Centrifuge the microtiter plate at 1,000 RPM for 1 minute.

5) Gently mix or vortex the plate, and spin down before thermocycling.

6) Thermocycle the 384-well microtiter plate as follows:

94 ^°C 4 minutes

94 ^°C 20 seconds

56 ^°C 30 seconds 45 cycles

72 ^°C 1 minute 72 ^°C 3 minutes

4^°C forever

Prepare the SAP enzyme solution

1) Add the reagents in the order in which they appear in the following table into a 1.5 mL tube to prepare the SAP enzyme solution.

SAP Enzyme Solution Volume

Component Volume ( )

SAP*Buffer 0.17

SAP Enzyme 0.3

ddH₂0 1.53

Total 2

2) Hold the 1.5 mL tube, containing the SAP enzyme solution, to a vortex for five seconds to mix the solution.

3) Centrifuge the 1.5 mL tube of SAP enzyme solution for ten seconds at 5000 RPM.

4) of SAP enzyme solution is added to each well in the 384-well sample microtiter plate.

5) Seal the 384-well sample microtiter plate with plate sealing film.

6) Centrifuge the 384-well sample microtiter plate at 1000 RPM for 1 minute.

7) Incubate the 384-well sample microtiter plate as follows:

37^°C 40 minutes

85 ^°C 5 minutes

4^°C forever

Prepare the High Plex iPLEX Gold reaction cocktail (same multiplexed assays, different DNA)

1) Prepare the high plex iPLEX Gold reaction cocktail, as described in the following table in a 1.5 mL tube. Add the reagents in the order in which they appear in the table.

Multiplexed high plex iPLEX Gold reaction cocktail (same assays, different DNA)

Component Volume ( )

dd¾0 0.619

Primer mix 0.94

Gold*Buffer ( lOx ) 0.2

Termination mix 0.2

Enzyme 0.041

Total 2

2) Centrifuge the cocktail microtiter plate at 1000 RPM for one minute.

3) Add the High Plex iLEX Gold reaction into 384-well sample microtiter plate.

4) Seal the 384-well sample microtiter plate with plate sealing film. 5) Centrifuge the 384-well sample microtiter plate at 1000 RPM for one minute.

6) Thermocycle the 384-well sample microtiter plate as follows:

94 ^°C 30 seconds

94 ^°C 5 seconds

52 ^°C 5 seconds

For 5 cycles For 40 cycles

80 ^°C 5 seconds

72 ^°C 3 minutes

4^°C forever

Clean up the High Pie iPLEX Gold Reaction Products. The cleanup of high plex iPLEX Gold reaction products involves adding water and then Clean Resin to the sample microtiter plate. Spread Clean Resin onto the 384-well dimple plate. Add nanopure water to each well of the 384-well sample microtiter plate. Add Clean Resin to the 384-well sample microtiter plate.

Rotate and centrifuge the 384-well sample microtiter plate.

Acquiring Spectra

The ACQUIRE module controls the MassARRAY Analyzer Compact (Compact) to acquire spectra from SpectroCHIPs. As each SpectroCHIP is processed by the Compact, the spectral data is automatically processed and saved to the MassARRAY database.

The method involves 15 SNP markers which are most associated with PCOS and the credibility thereof is higher. The detecting process can be more easily carried out with less expense.

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Claims

Claims

1. A SNP marker, wherein the nucleotide sequence thereof is shown as: SEQ ID NO.l, wherein N is C or T; SEQ ID NO.2, wherein N is A or G; SEQ ID NO.3, wherein N is C or T; SEQ ID NO.4, wherein N is A or C; SEQ ID NO.5, wherein N is C or T; SEQ ID NO.6, wherein N is A or C; SEQ ID NO.7, wherein N is C or T; SEQ ID NO.8, wherein N is C or T; SEQ ID NO.9, wherein N is A or G; SEQ ID NO.10, wherein N is C or T; SEQ ID NO. l l, wherein N is C or T; SEQ ID N0.12, wherein N is C or T; SEQ ID NO.13, wherein N is A or G; SEQ ID NO.14, wherein N is C or T; SEQ ID N0.15, wherein N is A or G; SEQ ID NO.16, wherein N is C or T; SEQ ID N0.17, wherein N is A or T; SEQ ID NO.18, wherein N is C or G; SEQ ID NO.19, wherein N is C or T; SEQ ID NO.20, wherein N is C or T; SEQ ID N0.21, wherein N is C or T; SEQ ID N0.22, wherein N is A or G; SEQ ID N0.23, wherein N is A or G; SEQ ID N0.24, wherein N is C or T; SEQ ID N0.25, wherein N is A or G; SEQ ID N0.26, wherein N is C or T; SEQ ID N0.27, wherein N is A or T; SEQ ID N0.28, wherein N is G or T; SEQ ID N0.29, wherein N is A or G; SEQ ID NO.30, wherein N is C or T; SEQ ID N0.31, wherein N is A or G; SEQ ID N0.32, wherein N is C or T; SEQ ID N0.33, wherein N is C or T; SEQ ID N0.34, wherein N is C or T; SEQ ID N0.35, wherein N is C or T; SEQ ID N0.36, wherein N is A or G; SEQ ID N0.37, wherein N is C or T; SEQ ID N0.38, wherein N is C or T; SEQ ID N0.39, wherein N is C or T; SEQ ID NO.40, wherein N is A or C; SEQ ID N0.41, wherein N is G or T; SEQ ID N0.42, wherein N is G or T; SEQ ID N0.43, wherein N is C or T; SEQ ID N0.44, wherein N is A or G; or SEQ ID N0.45, wherein N is C or T.

2. Probes detecting the genotypes at the site N of one or more SNP markers of claim 1.

3. The probes of claim 2, wherein the probes are shown as SEQ ID NO. 46 and 47 for SNP marker NO.l SEQ ID NO. 48 and 49 for SNP marker NO.2; SEQ ID NO. 50 and 51 for SNP marker NO.3 SEQ ID NO. 52 and 53 for SNP marker NO.4; SEQ ID NO. 54 and 55 for SNP marker NO.5 SEQ ID NO. 56 and 57 for SNP marker NO.6; SEQ ID NO. 58 and 59 for SNP marker NO.7 SEQ ID NO. 60 and 61 for SNP marker NO.8; SEQ ID NO. 62 and 63 for SNP marker NO.9; SEQ ID NO. 64 and 65 for SNP marker NO.10; SEQ ID NO. 66 and 67 for SNP marker

NO.l l; SEQ ID NO. 68 and 69 for SNP marker NO.12: SEQ ID NO 70 and 71 for SNP marker N0.13; SEQ ID NO. 72 and 73 for SNP marker N0.14 SEQ ID NO 74 and 75 for SNP marker N0.15; SEQ ID NO. 76 and 77 for SNP marker NO.16 SEQ ID NO 78 and 79 for SNP marker NO.17; SEQ ID NO. 80 and 81 for SNP marker NO.18 SEQ ID NO 82 and 83 for SNP marker NO.19; SEQ ID NO. 84 and 85 for SNP marker NO.20 SEQ ID NO 86 and 87 for SNP marker N0.21; SEQ ID NO. 88 and 89 for SNP marker N0.22 SEQ ID NO 90 and 91 for SNP marker N0.23; SEQ ID NO. 92 and 93 for SNP marker N0.24 SEQ ID NO 94 and 95 for SNP marker N0.25; SEQ ID NO. 96 and 97 for SNP marker N0.26; SEQ ID NO. 98 and 99 for SNP marker N0.27; SEQ ID NO. 100 and 101 for SNP marker N0.28; SEQ ID NO. 102 and 103 for SNP marker N0.29; SEQ ID NO. 104 and 105 for SNP marker NO.30; SEQ ID NO. 106 and 107 for SNP marker N0.31; SEQ ID NO. 108 and 109 for SNP marker N0.32; SEQ ID NO. 110 and 111 for SNP marker N0.33; SEQ ID NO. 112 and 113 for SNP marker N0.34; SEQ ID NO. 114 and 115 for SNP marker N0.35; SEQ ID NO. 116 and 117 for SNP marker N0.36; SEQ ID NO. 118 and 119 for SNP marker N0.37; SEQ ID NO. 120 and 121 for SNP marker N0.38; SEQ ID NO. 122 and 123 for SNP marker N0.39; SEQ ID NO. 124 and 125 for SNP marker NO.40; SEQ ID NO. 126 and 127 for SNP marker N0.41; SEQ ID NO. 128 and 129 for SNP marker N0.42; SEQ ID NO. 130 and 131 for SNP marker N0.43; SEQ ID NO. 132 and 133 for SNP marker N0.44; and/or SEQ ID NO. 134 and 135 for SNP marker N0.45.

4. A chip for detecting the genotypes at the site N of one or more SNP markers of claim 1, wherein the chip comprises the probes of claim 2 or 3.

5. The chip of claim 4, wherein the chip comprises probes consisted of SEQ ID NO. 46-135 or probes shown as SEQ ID NO. 56, 57, 66, 67, 88, 89, 90, 91, 94, 95, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 118, 119, 126, 127, 128, 129, 130, 131, 132 and 133.

6. Primers detecting the genotypes at the site N of one or more SNP markers of claim 1.

7. The primers of claim 6, wherein the primers are shown as SEQ ID NO. 136 and 137 for SNP marker NO. l; SEQ ID NO. 138 and 139 for SNP marker NO.2; SEQ ID NO. 140 and 141 for SNP marker NO.3; SEQ ID NO. 142 and 143 for SNP marker NO.4; SEQ ID NO. 144 and 145 for SNP marker NO.5; SEQ ID NO. 146 and 147 for SNP marker NO.6, 7 or 8; SEQ ID NO. 148 and 149 for SNP marker NO.9; SEQ ID NO. 150 and 151 for SNP marker NO.10; SEQ ID NO. 152 and 153 for SNP marker NO. l l; SEQ ID NO. 154 and 155 for SNP marker NO.12; SEQ ID NO. 156 and 157 for SNP marker N0.13; SEQ ID NO. 158 and 159 for SNP marker N0.14; SEQ ID NO. 160 and 161 for SNP marker NO.15; SEQ ID NO. 162 and 163 for SNP marker NO.16; SEQ ID NO. 164 and 165 for SNP marker NO.17; SEQ ID NO. 166 and 167 for SNP marker NO.18; SEQ ID NO. 168 and 169 for SNP marker NO.19; SEQ ID NO. 170 and 171 for SNP marker NO.20; SEQ ID NO. 172 and 173 for SNP marker N0.21; SEQ ID NO. 174 and 175 for SNP marker N0.22; SEQ ID NO. 176 and 177 for SNP marker N0.23; SEQ ID NO. 178 and 179 for SNP marker N0.24; SEQ ID NO. 180 and 181 for SNP marker N0.25; SEQ ID NO. 182 and 183 for SNP marker N0.26; SEQ ID NO. 184 and 185 for SNP marker N0.27; SEQ ID NO. 186 and 187 for SNP marker N0.28; SEQ ID NO. 188 and 189 for SNP marker N0.29; SEQ ID NO. 190 and 191 for SNP marker NO.30; SEQ ID NO. 192 and 193 for SNP marker N0.31; SEQ ID NO. 194 and 195 for SNP marker N0.32; SEQ ID NO. 196 and 197 for SNP marker N0.33; SEQ ID NO. 198 and 199 for SNP marker N0.34; SEQ ID NO. 200 and 201 for SNP marker N0.35; SEQ ID NO. 202 and 203 for SNP marker N0.36; SEQ ID NO. 204 and 205 for SNP marker N0.37; SEQ ID NO. 206 and 207 for SNP marker N0.38; SEQ ID NO. 208 and 209 for SNP marker N0.39; SEQ ID NO. 210 and 211 for SNP marker NO.40; SEQ ID NO. 212 and 213 for SNP marker N0.41; SEQ ID NO. 214 and 215 for SNP marker N0.42; SEQ ID NO. 216 and 217 for SNP marker N0.43; SEQ ID NO. 218 and 219 for SNP marker N0.44; and/or SEQ ID NO. 220 and 221 for SNP marker NO.45.

8. A kit for detecting the genotypes at the site N of one or more SNP markers of claim 1, wherein the kit comprises the probes of claim 2 or 3, the chip of claim 4 or 5, and/or the primers of claim 6 or 7.

9. The kit of claim 8 used to detect the genotypes at the site N of SNP markers NO. 6, 11, 22, 23, 25, 29, 30, 33, 34, 35, 37, 41, 42, 43 and 44, wherein the kit comprises probes consisted of SEQ ID 56, 57, 66, 67, 88, 89, 90, 91, 94, 95, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 118, 119, 126, 127, 128, 129, 130, 131, 132 and 133, or primers consisted of SEQ ID N0.146, 147, 152, 153, 174, 175, 176, 177, 180, 181, 188, 189, 190, 191, 196, 197, 198, 199, 200, 201, 204, 205, 212, 213, 214, 215, 216, 217, 218 and 219.

10. The use of the probes of claim 2 or 3, chip of claim 4 or 5, primers of claim 6 or 7, or kit of claim 8 or 9 in the preparation of an agent for predicting or diagnosing PCOS.

11. The use of the probes of claim 2 or 3, chip of claim 4 or 5, primers of claim 6 or 7, or kit of claim 8 or 9 in predicting or diagnosing PCOS.

12. A method of predicting or diagnosing PCOS, wherein the method comprises determining genotypes at the site N of one or more SNP markers of claim 1.

13. The method of claim 12, wherein the method is carried out by hybridization, for example, using the probes of claim 2 or 3, the chip of claim 4 or 5 or the kit of claim 8 or 9.

14. The method of claim 12, wherein the method is carried out by sequencing, for example, PCR, Real-time Quantitative PCR, or MassARRAY, preferably, using primers of claim 6 or 7, or the kit of claim 8 or 9.

15. The method of any one of claims 12-14, wherein the method comprises the following steps: extracting DNA from peripheral blood or saliva of a subject, determining genotypes at the site N of one or more SNP markers, and analyzing the results to predict the risk of PCOS or diagnose PCOS.