a genome-wide association study in han chinese identifies ... · 23. the school of life sciences,...

A genome-wide association study in Han Chinese identifies new susceptibility loci for

ankylosing spondylitis

Supplementary Materials

Zhiming Lin1,24, Jin-Xin Bei2,24, Meixin Shen3, Qiuxia Li1, Zetao Liao1, Yanli Zhang1, Qing

Lv1, Qiujing Wei1, Hui-Qi Low4, Yun-Miao Guo2, Shuangyan Cao1, Mingcan Yang1,

Zaiying Hu1, Manlong Xu1, Xinwei Wang1, Yanlin Wei1, Li Li1, Chao Li1, Tianwang Li1,

Jianlin Huang1, Yunfeng Pan1, Ou Jin1, Yuqiong Wu1, Jing Wu1, Zishi Guo1, Peigen He5,

Shaoxian Hu5, Husheng Wu6, Hui Song6, Feng Zhan7, Shengyun Liu8, Guanmin Gao8,

Zhangsuo Liu8, Yinong Li9, Changhong Xiao10, Juan Li11, Zhizhong Ye12, Weizhen He12,

Dongzhou Liu13, Lingxun Shen14, Anbin Huang14, Henglian Wu15, Yi Tao16, Xieping Pan17,

Buyun Yu1, E Shyong Tai18,19, Yi-Xin Zeng2, Ee Chee Ren20,21, Yan Shen22, Jianjun Liu4,23,25

& Jieruo Gu1,25

1Department of Rheumatology, The Third Affiliated Hospital of Sun Yat-sen University,

Guangzhou, China.

2State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center,

Guangzhou, China.

3Department of Microbiology, National University of Singapore, Singapore.

4Human Genetics, Genome Institute of Singapore, Agency for Science, Technology and Research

(A*STAR), Singapore.

5Department of Immunology and Rheumatology, Tongji Hospital, Tongji Medical College,

Huazhong University of Science & Technology, Hubei, China.

6Department of Rheumatology & Immunology, Beijing Jishuitan Hospital, Beijing, China.

7Department of Rheumatology, Hainan Provincial People’s Hospital, Hainan, China.

8Department of Urology and Rheumatology, the First Affiliated Hospital of Zhengzhou

Nature Genetics: doi:10.1038/ng.1005

University, Henan, China.

9Department of Rheumatology, Fuzhou General Hospital of Nanjing Military Command, Fuzhou,

China.

10Rheumatology & Immunology Department, Hospital of Integrated Traditional Chinese

Medicine & Western Medicine of Southern Medical University, Guangzhou, China.

11Department of Rheumatology, Nanfang Hospital, Southern Medical University, Guangzhou,

China.

12Xiangmihu Rheumatology Branch, Fourth People’s Hospital of Shenzhen, Shenzhen

Rheumatology Institute of Guangdong Medical College, Shenzhen, China.

13Department of Rheumatology and Immunology, Jinan University Second Clinical Medical

College, Shenzhen People’s Hospital, Shenzhen, China.

14Department of Immunology and Rheumatology, Union Hospital, Tongji Medical College,

Huazhong University of Science & Technology, Hubei, China.

15Department of Urology, Dongguan People’s Hospital, Guangdong, China.

16Department of Rheumatology and Immunology, Second Affiliated Hospital of Guangzhou

Medical College, Guangzhou, China.

17Department of Rheumatology and Immunology, The First People′s Hospital of Changzhou,

The Third Affiliated Hospital of Soochow University, Jiangsu, China.

18Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore,

Singapore.

19Department of Epidemiology and Public Health, Yong Loo Lin School of Medicine, National

University of Singapore, Singapore.

20Singapore Immunology Network, A*STAR, Singapore.

21Department of Microbiology, National University of Singapore, Singapore.

22National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences,

Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University,

Beijing, China.


23The School of Life Sciences, Anhui Medical University, Hefei, China.

24These authors contributed equally to this work.

25These authors jointly directed this work.

Correspondence should be addressed to J.G. ([email protected]) or J. Liu

([email protected]).


Supplementary Table 1. Samples and numbers of individuals genotyped by different Illumina platforms.

Cohort Platform Number of Genotyped Number for GWAS

AS cases 610 Quad 968 905

AS cases OmniExpress 997 932

Guangdong Controls 610 Quad 972 971

Anhui Controls 610 Quad 1230 1188

Singapore Chinese controls I 1M Duo 953 940

Singapore Chinese controls II 610 Quad 1146 1132

Supplementary Table 2. P values and Odds Ratios for 8 SNPs in MHC region derived from forward logistic regression analyses.

SNP Original results Condition results Order ID P(OR) Controlling SNP(s) P (OR)

1 rs13202464 <5.0E-324 (41.59) - - 2 rs9468898 7.68E-257 (11.35) 1st 2.54E-58 (4.08) 3 rs2523522 9.60E-196 (4.75) 1st~2nd 5.97E-24 (2.02) 4 rs9265882 1.23E-50 (0.46) 1st~3rd 2.76E-26 (0.44) 5 rs4418214 6.70E-08 (1.44) 1st~4th 2.34E-24 (3.34) 6 rs2523454 3.20E-93 (2.54) 1st~5th 1.49E-24 (2.29) 7 rs9295986 3.60E-103 (0.37) 1st~6th 1.27E-20 (0.45) 8 rs2596464 5.27E-37 (0.58) 1st~7th 4.58E-14 (2.27)

-, not tested. Supplementary Table 3. Association results of HLA-B*27 subtypes and ankylosing spondylitis.

HLA-B*27 subtypes

Case % (n=752)

Control % (n=891)

OR (95% CI) P

B*27 85.90 4.49 164.94 (108.04 ~ 251.81) 1.16E-123a

B*27:04 74.87 4.04 128.32 (84.13 ~ 195.72) 1.8E-112a

B*27:05 9.04 0.00 - 1.04E-14b

B*27:15 1.73 0.011 10.71 (1.35 ~ 84.74) 2.46E-02a

aAssociation test was carried out under logistic regression model by adjustment of first five principal components; bP value was obtained using chi square test.


Supplementary Table 4. Association results of top MHC SNP with ankylosing spondylitis after controlling HLA-B*27 alleles.

SNP controlling allele OR (95% CI) P

rs13202464 - 79.22 (55.06 ~ 114) 1.11E-122

rs13202464 B*27:04 13.15 (8.34 ~ 20.75) 1.58E-28

rs13202464 B*27:05 75.42 (52.38 ~ 108.6) 1.69E-119

rs13202464 B*27:15 78.89 (54.77~113.6) 1.02E-121

rs13202464 B*27 5.71 (3.28 ~ 9.94) 7.13E-10

Supplementary Table 5. Association results of 8 independent top MHC SNPs with ankylosing spondylitis after controlling HLA-B*27.

Order SNP OR (95% CI) P

1 rs13202464 5.71(3.28-9.94) 7.13E-10

2 rs9468898 2.20(1.45-3.36) 2.38E-04

3 rs2523522 1.69(1.25-2.29) 6.82E-04

4 rs9265882 1.18(0.86-1.62) 3.09E-01

5 rs4418214 1.32(0.83-2.11) 2.38E-01

6 rs2523454 1.00(0.75-1.33) 9.96E-01

7 rs9295986 0.72(0.54-0.95) 2.19E-02

8 rs2596464 0.98(0.74-1.29) 8.78E-01


Supplementary Table 6. Independence of association revealed by conditional regression analyses.

Controlling - rs13202464 rs4552569 rs13210693 rs17095830

Chr 6 5 6 12

SNP P(OR) P(OR) P(OR) P(OR) P(OR)

rs13202464 <5.0E-324

(41.59)

- <5.0E-324

(41.62)

<5.0E-324

(41.46)

<5.0E-324

(41.59)

rs4552569 6.91E-05

(1.20)

3.65E-03

(1.21)

- 6.54E-05

(1.20)

1.79E-04

(1.18)

rs13210693 4.25E-05

(1.19)

2.18E-03

(1.20)

4.47E-05

(1.19)

- 6.95E-05

(1.18)

rs17095830 1.91E-05

(1.32)

1.85E-02

(1.25)

2.63E-05

(1.31)

1.33E-05

(1.33)

-


Supplementary Table 7. Association results of 30 SNPs selected for validation.

GWAS (n = 1,837 cases and 4,231 controls)

validation (n = 2,100 cases and 3,496 controls)

Combined (n = 3,937 cases and 7,727

controls)

CHR SNP BP A1 F_A F_U OR

(95% CI) P F_A F_U

OR (95% CI)

P OR

(95% CI) P

3 rs4491835 38980893 T 0.43 0.46 0.84

(0.77~0.91) 2.07E-05 0.44 0.45

0.97 (0.90~1.05)

4.31E-01 0.91

(0.86~0.96) 4.78E-04

3 rs883349 39042929 T 0.45 0.48 0.86

(0.79~0.93) 1.74E-04 0.46 0.47

0.96 (0.89~1.04)

3.41E-01 0.91

(0.86~0.96) 1.05E-03

3 rs6804637 86278127 A 0.20 0.23 0.82

(0.74~0.91) 8.98E-05 0.21 0.22

0.98 (0.89~1.07)

6.16E-01 0.90

(0.84~0.96) 2.11E-03

5 rs4552569 83209349 C 0.32 0.28 1.20

(1.10~1.31) 6.91E-05 0.31 0.27

1.22 (1.12~1.33)

2.92E-06 1.21

(1.14~1.29) 8.77E-10

6 rs7760960 90367932 T 0.05 0.03 1.51

(1.24~1.85) 4.56E-05 0.03 0.04

0.93 (0.76~1.15)

5.06E-01 1.19

(1.03~1.37) 1.69E-02

6 rs6919423 109665164 A 0.24 0.22 1.18

(1.07~1.30) 9.47E-04 0.26 0.24

1.10 (1.01~1.20)

3.84E-02 1.13

(1.06~1.21) 1.86E-04

6 rs13210693 109705657 A 0.49 0.46 1.19

(1.09~1.29) 4.25E-05 0.47 0.44

1.12 (1.04~1.21)

4.00E-03 1.15

(1.09~1.22) 9.31E-07

8 rs1705691 23823812 C 0.29 0.26 1.22

(1.12~1.34) 1.58E-05 0.27 0.28

0.92 (0.84~1.00)

5.62E-02 1.05

(0.99~1.12) 1.16E-01

10 rs7068603 80442547 T 0.23 0.27 0.81

(0.74~0.89) 8.99E-06 0.24 0.26

0.92 (0.84~1.01)

7.21E-02 0.87

(0.81~0.92) 1.12E-05

10 rs7921660 80449852 C 0.33 0.37 0.84

(0.77~0.92) 7.28E-05 0.35 0.35

0.97 (0.90~1.05)

4.65E-01 0.91

(0.86~0.96) 1.09E-03

12 rs17095830 44061175 G 0.13 0.10 1.32 1.91E-05 0.12 0.10 1.27 1.96E-04 1.29 1.63E-08


(1.16~1.50) (1.12~1.43) (1.18~1.41)

13 rs9317518 65035343 T 0.26 0.28 0.83

(0.75~0.90) 3.10E-05 0.26 0.26

1.01 (0.93~1.11)

7.53E-01 0.92

(0.86~0.98) 6.96E-03

13 rs7991583 65040364 A 0.25 0.28 0.82

(0.75~0.90) 1.71E-05 0.27 0.26

1.01 (0.93~1.10)

8.29E-01 0.91

(0.86~0.97) 4.32E-03

13 rs9634642 94686672 T 0.42 0.45 0.83

(0.76~0.91) 1.98E-05 0.45 0.45

0.98 (0.91~1.06)

6.46E-01 0.91

(0.86~0.96) 1.26E-03

14 rs12146962 32450849 C 0.39 0.44 0.83

(0.77~0.90) 1.10E-05 0.41 0.43

0.93 (0.86~1.00)

5.16E-02 0.88

(0.83~0.93) 8.68E-06

14 rs10872871 32456510 T 0.39 0.43 0.83

(0.76~0.90) 6.15E-06 0.41 0.42

0.93 (0.86~1.01)

7.80E-02 0.88

(0.83~0.93) 1.07E-05

14 rs1754298 45618062 T 0.36 0.39 0.84

(0.77~0.91) 4.63E-05 0.38 0.37

1.04 (0.96~1.12)

3.84E-01 0.94

(0.89~0.99) 3.09E-02

14 rs1742655 45622604 G 0.35 0.39 0.83

(0.77~0.91) 2.19E-05 0.38 0.37

1.06 (0.98~1.14)

1.66E-01 0.95

(0.89~1.00) 5.52E-02

14 rs893639 45682068 G 0.36 0.40 0.82

(0.76~0.90) 1.55E-05 0.39 0.38

1.04 (0.96~1.12)

3.40E-01 0.94

(0.88~0.99) 2.72E-02

15 rs12439549 25337291 G 0.38 0.41 0.84

(0.77~0.91) 3.11E-05 0.42 0.40

1.10 (1.01~1.18)

2.19E-02 0.97

(0.91~1.02) 2.32E-01

15 rs4438262 25347771 C 0.38 0.40 0.83

(0.76~0.90) 1.99E-05 0.42 0.40

1.10 (1.02~1.19)

2.00E-02 0.97

(0.91~1.02) 2.42E-01

15 rs12903540 25360745 G 0.38 0.41 0.84

(0.77~0.91) 2.29E-05 0.41 0.39

1.10 (1.02~1.19)

1.25E-02 0.97

(0.92~1.03) 2.66E-01

16 rs6564385 75675377 A 0.12 0.16 0.78

(0.69~0.88) 3.15E-05 0.14 0.13

1.13 (1.01~1.26)

3.54E-02 0.94

(0.87~1.02) 1.49E-01

16 rs11642883 81629115 G 0.26 0.30 0.83

(0.76~0.91) 6.64E-05 0.29 0.30

0.94 (0.87~1.02)

1.61E-01 0.89

(0.84~0.95) 1.88E-04


17 rs11080055 23673851 A 0.24 0.27 0.82

(0.75~0.90) 3.87E-05 0.26 0.27

0.94 (0.86~1.03)

1.58E-01 0.88

(0.83~0.94) 1.11E-04

17 rs2239908 23749392 G 0.26 0.29 0.83

(0.76~0.91) 8.80E-05 0.27 0.29

0.93 (0.85~1.01)

7.79E-02 0.88

(0.83~0.94) 6.80E-05

17 rs12450413 40628970 T 0.26 0.29 0.82

(0.75~0.90) 3.19E-05 0.29 0.29

1.02 (0.93~1.11)

6.93E-01 0.92

(0.87~0.98) 1.10E-02

18 rs8092774 5152142 G 0.15 0.12 1.28

(1.14~1.44) 4.29E-05 0.13 0.14

0.93 (0.83~1.05)

2.32E-01 1.08

(1.00~1.18) 5.62E-02

20 rs6512654 48603564 G 0.04 0.07 0.64

(0.53~0.77) 3.58E-06 0.05 0.05

0.91 (0.76~1.09)

3.25E-01 0.77

(0.67~0.87) 6.30E-05

22 rs2075726 35639992 G 0.48 0.45 1.17

(1.07~1.26) 2.44E-04 0.48 0.45

1.11 (1.03~1.20)

8.27E-03 1.14

(1.07~1.20) 9.09E-06

CHR=chromosome; BP=base pair position; A1=reference allele for testing; F_A and F_U = frequencies of referenced allele in affected cases and unaffected controls, respectively; L95 and U95 = lower and upper ranges of 95 % confidence interval.


Supplementary Table 8. Statistics of quality control for samples in different platform. SNP CHR Alleles (A/B) Platform Number of

samples phenotype call rate Genotype

counts a HWE AF b

rs10865331 2 G/A OmniExpress 932 case 100.00 221/438/273 0.09 0.472 610quad 905 case 100.00 179/446/280 0.95 0.444 610quad 3291 control 99.97 920/1584/786 0.04 0.520 1MDuo 940 control 100.00 252/450/238 0.19 0.507 rs13202464 6 G/A OmniExpress 932 case 100.00 64/769/99 1.73E-95 0.481 610quad 905 case 99.89 51/751/102 1.70E-97 0.472 610quad 3291 control 99.97 11/490/2789 0.03 0.078 1MDuo 940 control 99.89 6/90/843 0.05 0.054 rs13210693 6 A/G OmniExpress 932 case 99.89 222/469/240 0.84 0.490 610quad 905 case 100.00 233/430/242 0.14 0.495 610quad 3291 control 99.88 699/1654/934 0.53 0.464 1MDuo 940 control 99.89 189/453/297 0.51 0.443 rs4552569 5 C/T OmniExpress 932 case 100.00 109/420/403 1.00 0.342 610quad 905 case 100.00 81/380/444 1.00 0.299 610quad 3291 control 99.97 269/1316/1705 0.52 0.282 1MDuo 940 control 100.00 79/373/488 0.52 0.282 rs17095830 * 12 G/A OmniExpress 932 case 99.57 16/206/706 0.77 0.128 610quad 905 case 99.23 12/196/690 0.76 0.123 610quad 3291 control 99.45 34/583/2656 0.77 0.099 1MDuo 940 control 99.79 3/164/771 0.07 0.091

rs6681012 1 A/G OmniExpress 932 case 100.00 77/376/502 0.57 0.278

610quad 905 case 99.46 65/405/453 0.05 0.290

610quad 3291 control 99.91 213/1274/1843 0.75 0.255

1MDuo 940 control 99.90 76/356/520 0.18 0.267


rs12568393 * 1 A/G OmniExpress 932 case 96.78 8/131/763 0.37 0.081 610quad 905 case 98.12 6/114/768 0.44 0.071 610quad 3291 control 97.30 22/403/2777 0.10 0.070 1MDuo 940 control 98.19 2/115/806 0.42 0.064 rs2297909 * 1 A/G OmniExpress 932 case 95.49 35/284/571 1.00 0.199 610quad 905 case 95.25 43/288/531 0.62 0.217 610quad 3291 control 96.38 156/1031/1985 0.15 0.212 1MDuo 940 control 95.75 39/280/581 0.46 0.199 rs4389526 * 4 T/A OmniExpress 932 case 98.93 2/92/828 1.00 0.052 610quad 905 case 98.34 3/97/790 1.00 0.058 610quad 3291 control 98.12 13/393/2823 1.00 0.065 1MDuo 940 control 98.62 2/138/787 0.16 0.077 rs27434 5 G/A OmniExpress 932 case 100.00 163/502/267 0.01 0.444 610quad 905 case 100.00 168/478/259 0.05 0.450 610quad 3291 control 99.97 746/1631/913 0.73 0.475 1MDuo 940 control 100.00 195/495/250 0.09 0.471 rs30187 5 C/T OmniExpress 932 case 100.00 162/501/269 0.01 0.443 610quad 905 case 100.00 168/478/259 0.05 0.450 610quad 3291 control 100.00 742/1635/914 0.83 0.474 1MDuo 940 control 100.00 195/495/250 0.09 0.471 rs6556416 * 5 A/C OmniExpress 932 case 90.45 6/100/737 0.26 0.066 610quad 905 case 92.38 7/95/734 0.08 0.065 610quad 3291 control 91.55 28/424/2561 0.03 0.080 1MDuo 940 control 91.81 6/89/768 0.06 0.059 rs4149577 12 A/G OmniExpress 932 case 100.00 95/406/431 1.00 0.320 610quad 905 case 100.00 88/401/416 0.59 0.319 610quad 3291 control 100.00 397/1481/1413 0.79 0.346


1MDuo 940 control 100.00 122/403/415 0.13 0.344 rs7210738 * 17 G/A OmniExpress 932 case 98.61 136/446/337 0.58 0.391 610quad 905 case 99.12 112/418/367 0.72 0.358 610quad 3291 control 98.82 392/1442/1418 0.39 0.342 1MDuo 940 control 98.94 108/411/411 0.77 0.337 rs2242944 21 A/G OmniExpress 932 case 100.00 144/433/355 0.53 0.387 610quad 905 case 100.00 124/410/371 0.52 0.364 610quad 3291 control 99.97 534/1583/1173 1.00 0.403 1MDuo 940 control 100.00 130/468/342 0.15 0.387 CHR=chromosome; HWE, p value of Hardy-Weinberg Equilibrium test; AF=allele frequency. * Imputed SNP. a Genotype counts are shown as numbers of AA/AB/BB. b AF, allele frequency of allele A. Supplementary Table 9. Summary of genotyping quality for validated SNPs.

SNP CHR Call rate (%)a HWE b

GWAS Validation GWAS Validation

rs4552569 5 100.0 / 99.98 100.0 / 99.91 0.96 / 0.38 0.34 / 0.64

rs17095830 12 99.40 / 99.53 99.05 / 99.23 1.00 / 0.66 0.67 / 1.00

rs13210693 6 99.95 / 99.88 99.95 / 99.86 0.38 / 0.83 0.17 / 0.11

-, not tested. a Shown as case/control. b Hardy-Weinberg Equilibrium P values for case/control groups.


Supplementary Figure 1. Plots of principal components from the PCA for population

stratification. SNPs within five regions with long-range LD were excluded from analysis,

including Chr5:44-51.5Mb, Chr6:25-37Mb, Chr8:0-12.7Mb, Chr11:45-57Mb and

Chr11:84-86Mb.

a, b: plots of the first two PCs, and the 1st and 5th PCs from the PCA of 1,837 ankylosing

spondylitis cases, 4,233 controls and 206 HapMap individuals, including 57 Yoruba in Ibadan,

Nigeria (YRI), 44 Japanese in Tokyo, Japan (JPT), 45 Han Chinese in Beijing, China (CHB) and

60 CEPH (Utah residents with ancestry from northern and western Europe) (CEU); Case (AS),

Guangdong control (GD-Controls), northern Chinese controls (NC-Controls), Singapore Chinese

control (Sg-Controls), CHB, JPT and YRI are indicated in different colors, and 2 Sg-controls

showing closer to Japanese population and were excluded for further analysis; c - g: plots of

paired PCs, which derived from PCA of 1,837 cases (red), 971 Guangdong Chinese (yellow),

1,188 Northern Chinese (deep blue) and 2,072 Singaporean Chinese (light blue). The case-control

matching and the low lambda GC values (1.049 and 1.036, after and before removal of MHC

region) suggested minimal evidence of population stratification.


Supplementary Figure 2. Quantile-Quantile plot of observed P values for association. The

blue points represented the distribution of P values for the association of all the 1,356,350 SNPs

in 1,837 cases and 4,231 controls, with open triangles for those P values less than 10-7, and the

red points represented that for the association of SNPs after excluding the SNPs within MHC

region (chr. 6: 25–37 Mb).

λGC=1.036

λGC=1.049


Supplementary Figure 3. Manhattan plot of the genome-wide P values of associations.

Association was assessed using logistic regression model with adjustment of the first five

principal components of population stratification. The -log10 P values (y axis) of 1,356,350 SNPs

in 1,837 AS cases and 4,231 controls were shown according to their chromosomal positions (x

axis). The points within MHC region with P value <10-10 were truncated. The top MHC SNP is

rs13202464 with the smallest P value <5.00×10-324. The replicated non-HLA SNPs in the shown

loci were indicated as triangles edged in red.


Supplementary Figure 4. Regional plots of the association results within chromosome 6q21

(a) and MHC region (b). The P values of the genotyped and imputed SNPs are presented as

open circles and triangles, based on their chromosomal positions (x axis) and –log10 P values (y

axis). The SNPs of interested were highlighted in read shape and blue background, with

indication of SNP ID. The genetic recombination rates (cM/Mb) were presented in blue vertical

lines. The genes within interested regions were also presented as green arrow/arrowheads and

some genes were removed for brevity, which included psuedogenes, hypothetical genes and

non-protein coding genes. b: 8 SNPs with independent association were restricted in MHC class I

region and indicated in red.

a


b


Supplementary Figure 5. Distributions of Percent heterozygosity and missingness for the AS

GWAS samples. A total of 87 samples deviated from 3 times standard deviation of mean

heterozygosity (indicated by Red and green horizontal lines) were removed for further analysis.


a genome-wide association study in han chinese identifies ... · 23. the school of life sciences,...

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