SUPPLEMENTARY INFORMATION A genome-wide ......SUPPLEMENTARY INFORMATION A genome-wide association...

SUPPLEMENTARY INFORMATION

A genome-wide association study in Chinese men identifies three risk loci for non-obstructive azoospermia

Zhibin Hu 1, 2, 10, Yankai Xia 1, 3, 10, Xuejiang Guo 1, 4, 10, Juncheng Dai 2, HongGang Li 5, Hongliang Hu 6, Yue Jiang 2, Feng Lu 2, Xiaoyu Yang 1,

7, Huizhang Li 2, Bing Yao 8, Yaoting Gui 9, Yibo Wu 1, 4, Chenliang Xiong 5, Zheng Li 6, Jiayin Liu 1, 7, Chuncheng Lu 3, Zuomin Zhou 1, 4,

Hongbing Shen 1, 2, Xinru Wang 1, 3, Jiahao Sha 1

Nature Genetics: doi:10.1038/ng.1040

Supplementary Fig. 1: Plot of the first two components derived from a principle component based analysis implemented in the software package EIGENSTRAT. (A) Our samples & HapMap Phase-II data; (B) Our samples. (A) (B)


Supplementary Fig. 2: Regional plot of the 4 identified marker SNPs based on the imputation results, taking Hapmap Phase II database as reference map (CHB+JPT, released at July-2006). Results (-log10 P) are shown for SNPs for the region of flanking 400 kb on either side of the marker SNPs. The marker SNPs are shown in purple and the r 2 values of the rest of the SNPs are indicated by different colors. The genes within the interested region are annotated and shown as arrows. (A)

(B)


(C)

(D)


Supplementary Table 1: Associations of the 32 fast-track replication SNPs with NOA from the GWAS scan MAF b Chr.

(cytoband) SNP Associated Gene Allele a Cases Controls

OR c P c OR d P d

1p13.3 rs12097821 PRMT6 G/T 0.32 0.25 1.41 7.24×10-8 1.42 5.07×10-8 1p31.3 rs7523497 USP1 G/A 0.14 0.09 1.61 6.76×10-8 1.53 2.47×10-8 1p36.32 rs2477686 PEX10-PLCH2 G/C 0.18 0.11 1.62 8.86×10-10 1.60 5.04×10-9 1q21.3 rs11204885 TDRKH A/G 0.26 0.19 1.63 8.71×10-12 1.65 9.02×10-12 2p13.3 rs6546680 ZNF638 G/C 0.23 0.17 1.60 5.10×10-10 1.59 1.41×10-9 3p24.2 rs2363527 RARB A/G 0.15 0.09 1.74 1.60×10-10 1.79 4.81×10-11 3q26.1 rs11918142 IFT80 T/C 0.22 0.16 1.48 1.14×10-7 1.45 9.48×10-8 4q24 rs11937871 CXXC4 C/T 0.19 0.13 1.60 2.72×10-9 1.58 1.30×10-8 4q31.21 rs967004 INPP4B T/C 0.25 0.18 1.53 1.77×10-9 1.50 1.72×10-8 4q34.1 rs6830691 FBXO8 A/G 0.12 0.07 1.73 3.51×10-9 1.74 5.96×10-9 4q35.2 rs2584345 FAT1 T/C 0.16 0.09 1.75 3.02×10-11 1.72 2.20×10-10 5p15.1 rs163065 ZNF622 C/G 0.35 0.27 1.47 1.81×10-9 1.46 7.34×10-9 5q32 rs312620 YIPF5 T/A 0.11 0.17 0.58 2.08×10-10 0.59 1.20×10-9 5q33.1 rs253296 SYNPO C/T 0.21 0.15 1.60 7.15×10-10 1.56 8.98×10-9 6p25.3 rs4959523 FOXQ1 T/C 0.18 0.12 1.76 1.08×10-11 1.75 4.11×10-11 7q21.2 rs10248566 MTERF G/C 0.20 0.14 1.57 7.59×10-9 1.57 1.27×10-8 7q31.32 rs2501443 TAS2R16 A/G 0.27 0.20 1.55 7.01×10-10 1.55 1.30×10-9 8p11.21 rs11987150 AGPAT6 C/T 0.18 0.12 1.70 1.42×10-10 1.69 3.83×10-10 8q24.3 rs6578134 PTK2 G/A 0.12 0.06 2.13 7.63×10-14 2.07 1.92×10-12 9p22.3 rs7023221 BNC2 T/C 0.26 0.19 1.57 2.06×10-10 1.63 1.86×10-11 9q22.33 rs2295924 GALNT12 C/T 0.28 0.21 1.44 9.11×10-8 1.45 9.13×10-8 9p23 rs10978121 PTPRD T/C 0.28 0.21 1.45 3.48×10-8 1.47 2.00×10-8

10q25.1 rs821693 SORCS1 G/A 0.28 0.21 1.54 4.48×10-10 1.52 3.48×10-9 12p12.1 rs10842262 SOX5 G/C 0.39 0.31 1.50 4.10×10-10 1.46 8.16×10-9 13q13.2 rs12429186 RFC3 T/C 0.33 0.26 1.48 5.21×10-9 1.44 9.14×10-8 13q33.3 rs7317862 FAM155A T/G 0.12 0.07 1.85 3.81×10-10 1.81 2.96×10-9


14q21.3 rs2282277 FSCB C/T 0.34 0.27 1.50 6.99×10-10 1.52 6.60×10-10 15q26.3 rs7166401 CHSY1 A/G 0.14 0.07 2.01 2.08×10-13 2.01 6.68×10-13 16p13.2 rs7192993 C16orf68 T/C 0.26 0.20 1.48 2.84×10-8 1.51 1.13×10-8 16p13.2 rs8060684 GRIN2A C/T 0.37 0.30 1.38 2.74×10-7 1.38 9.04×10-8 18p11.23 rs9958292 PTPRM T/G 0.28 0.20 1.57 4.57×10-11 1.56 2.25×10-10 20p13 rs6080550 SIRPA-SIRPG G/A 0.25 0.18 1.54 1.21×10-9 1.55 1.35×10-9

a Major/minor alleles; b MAF, Minor allele-frequency. c OR and P values of additive model by logistic regression without adjustment; d OR and P values of additive model by logistic regression adjusted by the first principal component and age.


Supplementary Table 2: Associations of 32 SNPs with NOA in GWAS scan and validations

MAF c SNPs Chr.

(Cytoband) Study Cases b Controls b Cases Cases

ORadd d

(95% CI) Padd d

rs12097821 1p13.3 GWAS Scan 98/424/459 106/606/945 0.32 0.25 1.42(1.25-1.61) 5.07E-08 G/T a Validation I 104/469/604 142/808/1127 0.29 0.26 1.13(1.01-1.27) 3.21E-02 Validation II 68/301/393 122/738/1133 0.29 0.25 1.23(1.08-1.40) 2.37E-03 Combined All 270/1194/1456 370/2152/3205 0.30 0.25 1.25(1.17-1.34) 5.67E-10 rs7523497 1p31.3 GWAS Scan 26/220/732 17/263/1377 0.14 0.09 1.53(1.28-1.72) 2.47E-08 G/A a Validation I 7/220/915 25/353/1690 0.10 0.10 1.06(0.89-1.25) 5.20E-01 rs2477686 1p36.32 GWAS Scan 57/220/661 21/334/1301 0.18 0.11 1.60(1.37-1.87) 5.04E-09 G/C a Validation I 14/287/878 23/400/1656 0.13 0.11 1.29(1.10-1.51) 1.34E-03 Validation II 17/154/594 32/339/1623 0.12 0.10 1.23(1.03-1.47) 2.33E-02 Combined All 88/661/2133 76/1073/4580 0.15 0.11 1.39(1.26-1.52) 5.65E-12 rs11204885 1q21.3 GWAS Scan 41/436/503 47/518/1075 0.26 0.19 1.65(1.43-1.90) 9.02E-12 A/G a Validation I 43/378/749 86/656/1234 0.20 0.21 0.93(0.82-1.06) 2.85E-01 rs6546680 2p13.3 GWAS Scan 23/413/542 38/478/1138 0.23 0.17 1.59(1.37-1.85) 1.41E-09 G/C a Validation I 50/313/779 69/636/1327 0.18 0.19 0.94(0.82-1.07) 3.49E-01 rs2363527 3p24.2 GWAS Scan 36/220/722 13/268/1368 0.15 0.09 1.79(1.50-2.13) 4.81E-11 A/G a Validation I 5/233/905 19/356/1683 0.11 0.10 1.13(0.95-1.34) 1.70E-01 rs11918142 3q26.1 GWAS Scan 46/328/601 36/432/1140 0.22 0.16 1.45(1.31-1.71) 9.48E-08 T/C a Validation I 57/294/814 59/555/1428 0.18 0.16 1.07(0.94-1.22) 3.02E-01 rs11937871 4q24 GWAS Scan 31/312/637 22/386/1245 0.19 0.13 1.58(1.35-1.85) 1.30E-08 C/T a Validation I 23/292/847 31/532/1446 0.15 0.15 0.98(0.85-1.14) 7.91E-01 rs967004 4q31.21 GWAS Scan 45/396/539 58/476/1121 0.25 0.18 1.50(1.30-1.73) 1.72E-08 T/C a Validation I 51/363/744 76/657/1321 0.20 0.20 1.02(0.90-1.16) 7.11E-01 rs6830691 4q34.1 GWAS Scan 37/160/780 14/197/1446 0.12 0.07 1.74(1.44-2.09) 5.96E-09 A/G a Validation I 6/149/1007 11/250/1808 0.07 0.07 1.06(0.87-1.29) 5.87E-01 rs2584345 4q35.2 GWAS Scan 43/225/709 15/283/1357 0.16 0.09 1.72(1.45-2.03) 2.20E-10 T/C a Validation I 7/211/943 17/347/1684 0.10 0.09 1.05(0.88-1.25) 6.06E-01


rs163065 5p15.1 GWAS Scan 85/494/367 128/647/881 0.35 0.27 1.46(1.28-1.66) 7.34E-09 C/G a Validation I 89/487/589 189/829/1053 0.29 0.29 0.97(0.87-1.09) 6.12E-01 rs312620 5q32 GWAS Scan 18/174/788 48/476/1128 0.11 0.17 0.59(0.49-0.70) 1.20E-09 T/A a Validation I 37/295/844 57/601/1415 0.16 0.17 0.89(0.78-1.03) 1.07E-01 rs253296 5q33.1 GWAS Scan 30/360/590 35/421/1197 0.21 0.15 1.56(1.34-1.81) 8.98E-09 C/T a Validation I 23/306/828 45/493/1517 0.15 0.14 1.09(0.94-1.25) 2.63E-01 rs4959523 6p25.3 GWAS Scan 9/339/632 25/332/1290 0.18 0.12 1.75(1.48-2.07) 4.11E-11 T/C a Validation I 13/247/909 31/381/1616 0.12 0.11 1.08(0.92-1.26) 3.60E-01 rs10248566 7q21.2 GWAS Scan 29/324/626 30/388/1239 0.20 0.14 1.57(1.34-1.83) 1.27E-08 G/C a Validation I 26/300/838 43/456/1512 0.15 0.13 1.14(0.99-1.32) 7.19E-02 rs2501443 7q31.32 GWAS Scan 39/450/491 60/536/1058 0.27 0.20 1.55(1.34-1.78) 1.30E-09 A/G a Validation I 66/385/722 90/677/1284 0.22 0.21 1.07(0.95-1.21) 2.84E-01 rs11987150 8p11.21 GWAS Scan 22/308/649 17/352/1282 0.18 0.12 1.69(1.44-2.00) 3.83E-10 C/T a Validation I 21/250/876 22/468/1535 0.13 0.13 1.01(0.86-1.18) 9.20E-01 rs6578134 8q24.3 GWAS Scan 18/202/760 3/195/1457 0.12 0.06 2.07(1.69-2.53) 1.92E-12 G/A a Validation I 7/162/1010 19/252/1810 0.07 0.07 1.07(0.89-1.30) 4.65E-01 rs7023221 9p22.3 GWAS Scan 43/426/510 51/523/1081 0.26 0.19 1.63(1.41-1.88) 1.86E-11 T/C a Validation I 49/370/745 77/612/1331 0.20 0.19 1.07(0.95-1.22) 2.70E-01 rs2295924 9q22.33 GWAS Scan 51/442/486 73/564/1019 0.28 0.21 1.45(1.24-1.62) 9.13E-08 C/T a Validation I 60/417/677 115/703/1247 0.23 0.23 1.04(0.92-1.17) 5.38E-01 rs10978121 9p23 GWAS Scan 60/423/494 75/546/1025 0.28 0.21 1.47(1.29-1.68) 2.00E-08 T/C a Validation I 69/388/704 104/739/1170 0.23 0.24 0.95(0.84-1.08) 4.30E-01 rs821693 10q25.1 GWAS Scan 41/473/465 71/547/1022 0.28 0.21 1.52(1.32-1.74) 3.48E-09 G/A a Validation I 72/449/658 105/776/1191 0.25 0.24 1.08(0.96-1.21) 2.15E-01 rs10842262 12p12.1 GWAS Scan 94/552/297 147/725/768 0.39 0.31 1.46(1.28-1.66) 8.16E-09 G/C a Validation I 152/540/488 237/894/946 0.36 0.33 1.13(1.02-1.26) 2.17E-02 Validation II 92/353/319 197/875/922 0.35 0.32 1.16(1.03-1.32) 1.80E-02 Combined All 338/1445/1104 581/2494/2636 0.37 0.32 1.23(1.15-1.32) 2.32E-09 rs12429186 13q13.2 GWAS Scan 58/516/385 107/635/911 0.33 0.26 1.44(1.26-1.64) 9.14E-08


T/C a Validation I 90/449/624 205/751/1125 0.27 0.28 0.96(0.86-1.07) 4.77E-01 rs7317862 13q33.3 GWAS Scan 14/214/752 4/228/1412 0.12 0.07 1.81(1.49-2.20) 2.96E-09 T/G a Validation I 7/252/920 11/412/1653 0.11 0.10 1.10(0.93-1.30) 2.84E-01 rs2282277 14q21.3 GWAS Scan 58/554/368 116/641/889 0.34 0.27 1.52(1.33-1.73) 6.60E-10 C/T a Validation I 105/464/587 141/831/1037 0.29 0.28 1.08(0.96-1.20) 2.14E-01 rs7166401 15q26.3 GWAS Scan 23/221/735 6/222/1390 0.14 0.07 2.01(1.66-2.43) 6.68E-13 A/G a Validation I 9/192/938 14/334/1689 0.09 0.09 1.04(0.87-1.25) 6.55E-01 rs7192993 16p13.2 GWAS Scan 40/427/512 58/531/1068 0.26 0.20 1.51(1.31-1.74) 1.13E-08 T/C a Validation I 41/396/706 88/679/1264 0.21 0.21 0.99(0.87-1.13) 8.95E-01 rs8060684 16p13.2 GWAS Scan 102/522/355 156/693/808 0.37 0.30 1.38(1.26-1.58) 9.04E-08 C/T a Validation I 136/531/495 205/924/887 0.35 0.33 1.07(0.96-1.19) 2.27E-01 rs9958292 18p11.23 GWAS Scan 56/431/492 70/516/1064 0.28 0.20 1.56(1.36-1.78) 2.25E-10 T/G a Validation I 52/394/702 81/711/1234 0.22 0.22 1.01(0.89-1.14) 8.91E-01 rs6080550 20p13 GWAS Scan 48/401/532 54/500/1103 0.25 0.18 1.55(1.34-1.78) 1.35E-09 G/A a Validation I 62/395/720 63/680/1337 0.22 0.19 1.18(1.04-1.34) 9.45E-03

Validation II 44/236/485 98/567/1329 0.21 0.19 1.13(0.98-1.29) 1.00E-01 Combined All 154/1032/1737 215/1747/3769 0.23 0.19 1.27(1.17-1.37) 2.17E-09 a Major/minor alleles; b Variant homozygote/Heterozygote/Wild type homozygote; c MAF, Minor allele frequency d Adjusted by the first principal component and age.


Supplementary Table 3: Meta-analysis of the 4 SNPs on validation results across regions

MAF c SNPs Areas Cases b Controls b Cases Controls

OR (95% CI) d P d P e I2(%)

rs12097821 Nanjing 86/365/482 139/783/1134 0.29 0.26 1.16(1.03-1.31) 1.72E-02 0.891 0.0 1p13.3 G/T a Wuhan 42/202/267 60/386/572 0.28 0.25 1.18(0.99-1.39) 6.23E-02

Shanghai 44/203/248 65/376/554 0.29 0.25 1.22(1.03-1.44) 2.20E-02 Meta-analysis 1.18(1.08-1.28) 1.28E-04

rs2477686 Nanjing 11/205/717 26/368/1662 0.12 0.10 1.22(1.02-1.44) 2.52E-02 0.725 0.0 1p36.32 G/C a Wuhan 9/116/388 12/191/816 0.13 0.11 1.27(1.01-1.60) 4.02E-02


rs10842262 Nanjing 114/430/389 227/886/941 0.35 0.33 1.12(1.00-1.26) 4.60E-02 0.788 0.0 12p12.1 G/C a Wuhan 70/232/211 108/439/471 0.36 0.32 1.20(1.02-1.40) 2.52E-02


rs6080550 Nanjing 55/294/584 74/629/1353 0.22 0.19 1.18(1.03-1.35) 1.45E-02 0.329 10.0 20p13 G/A a Wuhan 28/173/311 43/293/683 0.22 0.19 1.25(1.04-1.50) 1.61E-02


a Major/minor alleles; b Variant homozygote/Heterozygote/Wild type homozygote; c MAF, Minor allele frequency; d Adjusted by the first principal component and age; e P for heterogeneity

test.


Supplementary Table 4: SNPs identified by GWAS genotyping or imputation analyses at chromosomes1p36.32, 12p12.1, 20p13 and their associations with NOA (P <1.0×10-5)

MAF Chr. SNP Position Minor

Allele Major Allele Cases Controls P a ORadd Padd Identification by

1p36.32 rs10910078 2380448 T C 0.15 0.11 0.902 1.41 3.13E-07 Imputing 1p36.32 chr1:2380575 2380575 C G 0.16 0.11 0.902 1.43 1.50E-07 Imputing 1p36.32 rs2477686 2382508 C G 0.16 0.11 1.000 1.42 1.98E-07 Genotyping 12p12.1 rs1909351 24074115 C T 0.39 0.32 0.084 1.38 8.89E-08 Imputing 12p12.1 rs10842262 24075811 C G 0.37 0.31 0.452 1.37 1.44E-08 Genotyping 12p12.1 rs1039229 24076787 T C 0.37 0.31 0.452 1.36 2.72E-08 Imputing 20p13 rs6080550 1707590 A G 0.26 0.18 0.558 1.67 3.30E-10 Genotyping 20p13 chr20:1707957 1707957 A G 0.18 0.11 0.032 1.86 1.16E-09 Imputing 20p13 rs6080552 1708811 C G 0.23 0.16 0.923 1.59 4.28E-10 Imputing a P values for Hardy-Weinberg Equilibrium in controls. Note: None SNP was detected around 1p13.3:rs12097821 at the P <1.0×10-5

level after Imputation.


Supplementary Table 5: A tabular overview of the putative biological function of the 4 loci SNP Surrounding

genes Cytogenetic location

OMIM Associated phenotypes

rs12097821 PRMT6 1p13.3 608274 Family members involved in germ cell development rs2477686 PEX10

MMEL1

1p36.32

602859

Adrenoleukodystrophy syndrome (PEX10); Zellweger syndrome (PEX10); Spermatogenesis defect (PEX10) Impaired fertilization (MMEL1, MP:0000242, Mouse); Reduced male fertility (MMEL1, MP:0001922, Mouse)

rs10842262 SOX5 12p12.1 604975 Human testicular seminomas; Family members involved in spermatogenesis

rs6080550 LOC100289473, SIRPA,SIRPG

20p13 605466 --


Supplementary Table 6: Summary of GWAS scans for the 4 SNPs with or without b2b3 deletion samples MAF c

SNP Study Cases b Controls b Cases Controls

ORhet (95% CI)

ORhomo (95% CI)

ORadd (95% CI) Padd

rs12097821 GWAS Scan 98/424/459 106/606/945 0.32 0.25 1.44(1.21-1.70) 1.98(1.46-2.68) 1.46(1.28-1.66) 5.07×10-8 1p13.3 G/T a GWAS Scan d 95/393/415 99/567/880 0.32 0.25 1.48(1.24-1.76) 2.11(1.55-2.88) 1.46(1.28-1.67) 1.20×10-8 rs2477686 GWAS Scan 57/220/661 21/334/1301 0.18 0.11 1.26(1.03-1.53) 5.53(3.29-9.32) 1.60(1.37-1.87) 5.04×10-9 1p36.32 G/C a GWAS Scan d 53/198/614 21/307/1217 0.18 0.11 1.24(1.01-1.52) 5.09(3.00-8.60) 1.57(1.33-1.85) 5.96×10-8 rs10842262 GWAS Scan 94/552/297 147/725/768 0.39 0.31 1.93(1.62-2.30) 1.56(1.16-2.10) 1.46(1.28-1.66) 8.16×10-9 12p12.1 G/C a GWAS Scan d 87/501/281 133/677/720 0.39 0.31 1.85(1.54-2.22) 1.60(1.18-2.18) 1.45(1.27-1.66) 4.02×10-8 rs6080550 GWAS Scan 48/401/532 54/500/1103 0.25 0.18 1.66(1.40-1.97) 1.93(1.28-2.91) 1.55(1.34-1.78) 1.35×10-9 20p13 G/A a GWAS Scan d 44/371/488 51/476/1019 0.25 0.19 1.61(1.35-1.92) 1.88(1.23-2.88) 1.51(1.31-1.75) 2.70×10-8 a Major/minor alleles. b Variant homozygote/Heterozygote/Wild type homozygote; c Minor allele frequency (MAF); ORhet: Heterozygote vs. Wild type homozygote; ORhomo: Variant

homozygote vs. Wild type homozygote; ORadd, Padd: calculated by additive model, and adjusted by age and the first principal component. d Without 190 individuals with b2b3 deletion (78 cases,

111 controls).


Supplementary Table 7: Table for SNP exclusion Dataset 1 Dataset 2 Dataset 3

QC process for SNPs Cases vs. all Controls SNPs remained (SNPs

removed)

Cases vs. Control set 1 SNPs remained (SNPs

removed)

Cases vs. Control set 2 SNPs remained (SNPs

removed) Step1: Non-autosome (Chr. 24, 26) 868257(38,446) 868257(38,446) 868257(38,446) Step2: Call rate (95%) 848115(20,142) 848867(19,390) 847816(20,441) Step3: HWE (10E-05 for all controls and 10E-4 for Control set 1 and 2) & MAF (0.05) & Chr. 23

588100(260,015) 588761 (260,106) 588054(259,762)

Step4: Intersection 587,693 Step5: SNPs without clear cluster 587,347 (346)


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