Genome-wide association study identifies …...1 Genome-wide association study identifies susceptibility loci for schizophrenia in Han Chinese at 6p21-p22.1 and 11p11.2 Wei-Hua Yue,

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Genome-wide association study identifies susceptibility loci for

schizophrenia in Han Chinese at 6p21-p22.1 and 11p11.2

Wei-Hua Yue, Hai-Feng Wang, Liang-Dan Sun, Fu-Lei Tang, Zhong-Hua Liu,

Hong-Xing Zhang, Wen-Qiang Li, Yan-Ling Zhang, Yang Zhang, Cui-Cui Ma, Bo

Du, Li-Fang Wang, Yun-Qing Ren, Yong-Feng Yang, Xiao-Feng Hu, Yi Wang, Wei

Deng, Li-Wen Tan, Yun-Long Tan, Qi Chen, Guang-Ming Xu, Gui-Gang Yang,

Xian-bo Zuo, Hao Yan, Yan-Yan Ruan, Tian-Lan Lu, Xue Han, Xiao-Hong Ma, Yan

Wang, Li-Wei Cai, Chao Jin, Hong-Yan Zhang, Jun Yan, Wei-Feng Mi, Xian-Yong

Yin, Wen-Bin Ma, Qi Liu, Lan Kang, Wei Sun, Cheng-Ying Pan, Mei Shuang, Fu-De

Yang, Chuan-Yue Wang, Jian-Li Yang, Ke-Qing Li, Xin Ma, Ling-Jiang Li, Xin Yu,

Qi-Zhai Li, Xun Huang, Lu-Xian Lv, Tao Li, Guo-Ping Zhao, Wei Huang, Xue-Jun

Zhang, & Dai Zhang

Nature Genetics: doi:10.1038/ng.979

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SUPPLEMENTARY MATERIALS INDEX

Supplementary Table 1. Results of heterogeneity test between two Chinese

groups for top six associated SNPs.

Supplementary Table 2. Association results of the remaining 40 SNPs other than

the top six associated SNPs.

Supplementary Table 3. The results of seven SNPs reported by previous studies

in the current GWAS sample.

Supplementary Table 4. Association and conditional logistic analysis for rs1635

and rs6913660 in our 746 cases and 1,599 controls.

Supplementary Table 5. The effects of Chromosomes 6p21-p22.1 and 11p11.2

SNPs on nearby genes expression on public eQTL dataset of HapMap.

Supplementary Table 6. PCR and extension primer sequences from Sequenom

SNP genotyping.

Supplementary Figure 1. The principal components analysis (PCA) of 2,345

samples and 206 reference samples from the HapMap.

Supplementary Figure 2. Genome-wide association results from the initial

GWAS analysis (Manhantton plot and Q-Q plot).

Supplementary Figure 3. Association results the LD plot across MHC region

(chr6:25-34Mb) in our GWAS sample.

Supplementary Figures 4a and 4b. NKAPL and ZKSCAN4 mRNAs expression

in postnatal mice brain using the in situ hybridization methods.

Supplementary Figure 5. Knockdown of NKAPL leads to neuronal migration

defects in the developing cerebral cortex.


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Supplementary Figure 6. Knockdown of CG6066 (NKAPL) in Drosophila

melanogaster influences the physical phenotypes of drosophilia, as well as the

synapse dysfunction.

Supplementary Note. Knockdown of NKAPL leads to neuronal migration defects

in the developing cerebral cortex of mice and synaptic abnormalities in

neuromuscular junction (NMJ) of Drosophila melanogaster.











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Supplementary Table 1. Results of heterogeneity test between two Chinese groups for top six associated SNPs Heterogeneity Q test

SNP Chr. Gene Position Location-

to-gene* Allele

P value I2(%) Model PGWAS PReplication PCombined

rs1233710 6p21 ZKSCAN4 28323425 intron -399 T/C 0.19 41 Fixed 4.14×10-6 4.09×10-7 4.76×10-11

rs1635 6p22.1 NKAPL 28335583 coding [454/754] T/G 0.28 15 Fixed 4.15×10-6 5.53×10-8 6.91×10-12

rs2142731 6p22.1 PGBD1 28358892 intron -640 A/G 0.20 39 Fixed 2.19×10-5 9.15×10-7 5.14×10-10

rs11038167 11P11.2 TSPAN18 44799710 5’UTR -37918 A/C 0.09 65 Random 1.12×10-7 3.28×10-6 1.09×10-11

rs11038172 11P11.2 TSPAN18 44812173 5’UTR -25455 A/G 0.21 37 Fixed 2.57×10-6 1.11×10-5 7.21×10-10

rs835784 11P11.2 TSPAN18 44820394 5’UTR -17234 A/G 0.25 26 Fixed 4.02×10-6 2.37×10-7 2.73×10-11

Chr., chromosome. PCombined is based on the results of GWAS and Replication study.

*According to the information from the Illumina 610K annotation text


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Supplementary Table 2. Association results of the remaining 40 SNPs other than the top six associated SNPs.

GWASb Replication studyb Heterogeneity Q test Combined analysis c

ID Chr. SNP Position Adjacent Gene

Location -to -genea

A1/ A2 MAF.

Case MAF.

Control PGWAS OR

MAF. Case

MAF. Control PReplication OR

P value I2

PFixed ORF PRandom ORR

1 1 rs1801133d 11778965 MTHFR coding [115/78] C/T 0.423 0.488 0.00003 0.77 0.437 0.469 0.0004 0.88 0.07 68 2.36E-07 0.77 0.005 0.83

2 2 rs7581854 203314999 ALS2CR13 intron -13507 C/T 0.190 0.243 0.00006 0.73 0.219 0.215 0.614 1.02 0.0002 92.71 0.124 0.94 0.405 0.87

3 2 rs11544338 203340755 ALS2CR13 3'UTR [2022/1970] C/T 0.190 0.251 5.48E-06 0.70 0.215 0.213 0.762 1.01 <0.001 94.02 0.048 0.93 0.368 0.85

4 3 rs1381094 153956466 P2RY1 5'UTR -78960 G/A 0.232 0.288 5.32E-04 3.12 0.086 0.084 0.771 1.02 <0.001 99.05 1.78E-15 1.52 0.302 1.78

5 3 rs6792822 175180402 NLGN1 intron -172086 T/C 0.298 0.243 0.00007 1.32 0.279 0.280 0.916 0.99 0.0005 91.75 0.057 1.07 0.352 1.14

6 4 rs3111810 13780585 LOC152742 3'UTR -29470 T/C 0.132 0.082 1.10E-07 1.70 0.148 0.108 0.0004 1.41 0.09 64.13 3.19E-07 1.66 0.002 1.44

7 4 rs755535e 185905369 ACSL1 3'UTR -8374 G/A 0.375 0.309 6.68E-06 1.34 0.213 0.230 0.138 0.91 <0.001 94.36 0.030 1.11 0.616 1.10

8 6 rs2191037 28574967 GPX6 3'UTR -4085 C/T 0.452 0.514 0.00009 0.78 0.471 0.484 0.153 0.95 0.008 85.77 0.001 0.90 0.140 0.87

9 6 rs634651 165885469 PDE10A intron -8423 T/C 0.293 0.352 0.00006 0.76 0.338 0.329 0.326 1.04 0.0001 93.57 0.266 0.96 0.471 0.90

10 7 rs17459722 69040697 AUTS2 intron -38277 C/T 0.091 0.133 0.00004 0.65 0.118 0.125 0.192 0.93 0.0029 88.75 0.002 0.86 0.176 0.79

11 7 rs2237585 94887754 PON2 intron -4009 T/C 0.135 0.181 0.00006 0.70 0.155 0.152 0.669 1.02 0.0002 92.58 0.110 0.93 0.397 0.85

12 7 rs6978425 d 94906620 PON2 5'UTR -4300 T/G 0.421 0.491 6.94E-06 0.75 0.316 0.354 8.96E-04 0.84 0.13 56.0 9.79E-06 0.81 0.0007 0.81

13 7 rs7778623 94916232 PON2 5'UTR -13912 A/G 0.441 0.507 0.00002 0.77 0.480 0.490 0.289 0.96 0.0021 89.44 0.002 0.91 0.193 0.86

14 7 rs10226151e 142832579 EPHA1 5'UTR -16472 G/A 0.439 0.374 0.00007 1.31 0.390 0.401 0.243 0.96 0.0001 93.79 0.336 1.03 0.497 1.11

15 9 rs12352279 668837 ANKRD15 intron -1828 C/T 0.287 0.345 0.00007 0.76 0.349 0.341 0.374 1.04 0.0001 93.41 0.247 0.96 0.459 0.89

16 9 rs10738881 32153679 LOC392301 3'UTR -169776 C/T 0.357 0.449 5.30E-09 0.68 0.351 0.340 0.190 1.05 <0.001 96.91 0.072 0.94 0.449 0.85

17 9 rs10761076 106514987 OR13D1 3'UTR -17423 A/C 0.414 0.479 0.00003 0.77 0.468 0.458 0.279 1.04 <0.001 94.08 0.257 0.96 0.477 0.90

18 9 rs2065412 106638561 ABCA1 intron -521 C/T 0.093 0.061 0.00005 1.59 0.075 0.064 0.020 1.18 0.0296 78.87 4.74E-05 1.28 0.041 1.35

19 9 rs7863451 129112065 GARNL3 intron -1671 T/C 0.297 0.242 0.00007 1.32 0.268 0.277 0.299 0.96 0.0001 93.57 0.270 1.04 0.481 1.12

20 11 rs16938094 44805851 TSPAN18 5'UTR -31777 C/T 0.176 0.132 0.00007 1.40 0.147 0.140 0.322 1.05 0.0041 87.87 0.003 1.14 0.191 1.21

21 11 rs7936879 47933458 PTPRJ 5'UTR G/A 0.188 0.240 0.00007 0.73 0.200 0.218 0.017 0.90 0.0239 80.39 5.5E-05 0.85 0.050 0.82


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-25231

22 11 rs916606 64372813 EHD1 3'UTR -3971 T/C 0.395 0.458 0.00006 0.77 0.420 0.423 0.771 0.99 0.0009 90.93 0.023 0.93 0.296 0.88

23 11 rs17134219 99071992 CNTN5 intron -123493 G/A 0.192 0.247 0.00002 0.72 0.213 0.203 0.180 1.06 <0.001 94.52 0.350 0.96 0.509 0.88

24 11 rs608426 99083719 CNTN5 intron -111766 T/G 0.353 0.418 0.00002 0.76 0.390 0.389 0.880 1.01 0.0002 92.83 0.055 0.94 0.356 0.88

25 12 rs2652007 32136040 LOC729457 3'UTR -14294 G/A 0.305 0.223 2.37E-09 1.53 0.379 0.372 0.449 1.03 <0.001 95.84 0.0005 1.12 0.262 1.25

26 12 rs11178971 70548022 TBC1D15 5'UTR -4095 C/T 0.109 0.158 6.63E-06 0.65 0.136 0.134 0.670 1.02 <0.001 94.12 0.071 0.92 0.384 0.82

27 12 rs11178993 70615409 TPH2 5'UTR -3484 T/C 0.118 0.163 0.00005 0.69 0.141 0.145 0.522 0.97 0.0015 90.13 0.012 0.89 0.252 0.82

28 12 rs10777708 94497111 USP44 5'UTR -27753 A/G 0.247 0.196 0.00007 1.34 0.226 0.231 0.53 0.97 0.0002 92.8 0.151 1.06 0.426 1.14

29 12 rs3782886 110594872 BRAP coding [24/89] G/A 0.158 0.205 0.00009 0.72 0.134 0.150 0.011 0.88 0.0512 73.7 2.62E-05 0.83 0.022 0.80

30 14 rs2192420 77932472 NRXN3 5'UTR -7374 A/G 0.243 0.300 0.00004 0.75 0.270 0.264 0.456 1.03 0.0001 93.37 0.169 0.95 0.437 0.88

31 14 rs7155022 77945623 NRXN3 intron -5649 G/A 0.247 0.306 0.00003 0.74 0.274 0.263 0.150 1.06 <0.001 94.6 0.413 0.97 0.523 0.89

32 15 rs2305252 25940933 OCA2 intron -309 A/G 0.341 0.265 1.06E-07 1.44 0.269 0.265 0.650 1.02 <0.001 94.61 0.001 1.12 0.278 1.20

33 17 rs11657292 52877854 MSI2 intron -44023 T/C 0.237 0.183 0.00001 1.39 0.222 0.208 0.056 1.09 0.0059 86.83 0.0001 1.16 0.102 1.22

34 17 rs11077721e 69554332 RPL38 5'UTR -157058 A/G 0.280 0.227 0.00008 1.32 0.353 0.383 0.0008 0.88 0.066 70.41 1.47E-06 0.85 0.008 0.82

35 17 rs2011631 78206350 RAB40B 3'UTR -1888 C/A 0.150 0.203 0.00001 0.69 0.184 0.188 0.634 0.98 0.0004 92.05 0.012 0.90 0.276 0.83

36 17 rs3744165 78383731 ZNF750 UTR [111/70] A/C 0.105 0.068 0.00002 1.59 0.086 0.077 0.069 1.13 0.0078 85.89 0.0001 1.24 0.104 1.32

37 17 rs8073471 78490781 TBCD intron -140 G/A 0.101 0.064 7.87E-06 1.64 0.080 0.072 0.089 1.12 0.048 78.03 0.0001 1.25 0.118 1.35

38 18 rs4939924 45757577 MYO5B intron -2594 C/T 0.402 0.331 2.17E-06 1.36 0.389 0.368 0.020 1.09 0.044 78.37 1.19E-05 1.15 0.082 1.21

39 20 rs656111 9978931 ANKRD5 intron -71 A/C 0.389 0.315 8.62E-07 1.39 0.312 0.293 0.020 1.10 0.0024 89.14 6.68E-06 1.17 0.083 1.23

40 22 rs2076158 41619682 PACSIN2 intron -119 C/T 0.359 0.420 0.00007 0.77 0.405 0.402 0.802 1.0 0.0004 92.11 0.077 0.95 0.371 0.889

Chr., chromosome; SNP, single neucleotide polymoriphism; Position, chromosome position; MAF, minor allele frequency; OR, odds ratio; ORF, OR value of fixed effect model; ORR, OR value of random effect model; A1/ A2, minor allele / major allele. a Information of the location to the transcription initiation site to adjacent genes came from the annotation text of the Illumina HumanHap 610-Quad BeadChips (Illumina, San Diego, CA). b The sample of GWAS enrolled 746 cases and 1,599 controls. The sample of replication study included 3,024 case and 3,045 controls. c For SNPs with severe heterogeneity (I2<50%), the fixed effect model (Mantel-Haenszel) was used; otherwise, the random effect model (DerSimonian-Laird) was used.4-5 d The SNPs were genotyped by using the TaqMan and SNaPshot gentotyping methods. e The genotype distribution in replication study did not fitted the Hardy-Weinberg equilibrium test (P < 0.05).


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Supplementary Table 3. The results of seven SNPs reported by previous studies in the current GWAS sample.

Our GWAS ISC SGENE MGS

SNP BPA A1/A2 MAF.

Case

MAF.

Control

P

value OR MAF P value

P value

(COMB) MAF P value

P value

(COMB) MAF P value

P value

(COMB)

rs6904071 27155235 A/G 0.02145 0.03565 0.0091 0.593 0.142 3.00E-04 1.80E-08 0.186 1.21E-02 1.78E-08 N.A. N.A. N.A.

rs6913660 27199404 A/C 0.02145 0.03565 0.0091 0.593 0.142 3.00E-04 2.40E-08 0.15 4.70E-06 1.10E-09 0.184 1.71E-02 2.36E-08

rs6938200 27339129 G/A 0.03686 0.03755 0.9084 0.981 N.A. N.A. N.A. N.A. N.A. N.A. 0.205 5.28E-02 3.02E-07

rs6932590 27356910 G/A 0.07306 0.07036 0.7378 1.041 0.238 2.20E-03 7.10E-08 0.22 4.40E-09 1.40E-12 0.24 3.37E-03 7.13E-08

rs7746199 27369303 A/G 0.03217 0.02099 0.0212 1.550 0.18 8.80E-04 5.00E-08 0.185 6.85E-04 5.03E-08 N.A. N.A. N.A.

rs7776351 27834710 A/G 0.2107 0.2248 0.2808 0.921 N.A. N.A. N.A. 0.255 2.83E-02 3.22E-07 N.A. N.A. N.A.

rs3131296 32280971 A/G 0.07708 0.08741 0.2354 0.872 N.A. N.A. N.A. N.A. N.A. N.A. 0.13 2.10E-08 2.30E-10

SNP, single neucleotide polymoriphism; BP, chromosome position; MAF, minor allele frequency. A1/ A2, minor allele / major allele. P value (COMB), P value of combined analysis. OR,

odds ratio. N.A., not applicable. a Information of the location to the transcription initiation site to adjacent genes came from the annotation text of the Illumina HumanHap 610-Quad

BeadChips (Illumina, San Diego, CA).


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Supplementary Table 4. Association and conditional logistic analysis for rs1635 and rs6913660 in our 746 cases and 1,599 controls.

GWAS Conditional

rs6913660b

Conditional

rs1635b SNP BP

Adjacent

Gene

Location-

to-genea A1/A2

MAF

Case

MAF

Control P-value OR

P-value OR P-value OR

rs6913660 27199404 HIST1H2BJ 3’UTR

-8670 A/C 0.0215 0.0357 9.11E-03 0.59 N.A. N.A. 0.043 0.66

rs1635 28335583 NKAPL coding

[454/754] A/C 0.2634 0.3301 4.15E-06 0.73 1.28E-05 0.73 N.A.

N.A

.

SNP, single neucleotide polymoriphism; BP, chromosome position; A1/ A2, minor allele / major allele; MAF, minor allele frequency; OR, odds ratio; NA, not applicable.

a Information of the location to the transcription initiation site to adjacent genes came from the annotation text of the Illumina HumanHap 610-Quad BeadChips (Illumina, San Diego, CA).

b Using the SNPs as a covariates to implement the conditional logistic analyses.


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Supplementary Table 5. The effects of Chromosomes 6p21-p22.1 and 11p11.2

SNPs on nearby genes expression on public eQTL dataset of HapMap.

195 HapMap samples Chr./SNP Nearby genes

SNP Genotype

LCLs mRNA expression

P value

Chr. 6p21-p22.1 rs1233710 ZKSCAN4 CC 8.09±0.22 0.013 CT 8.00±0.18 TT 7.98±0.17 NKAPL CC 5.93±0.11 0.665 CT 5.93±0.11 TT 5.95±0.10 PGBD1 CC 7.27±0.33 0.053 CT 7.17±0.25 TT 7.12±0.32 ZNF323 CC 6.78±0.20 0.001 CT 6.68±0.14 TT 6.67±0.19 rs1635 ZKSCAN4 GG 8.09±0.22 0.017 GT 8.01±0.18 TT 7.99±0.17 NKAPL GG 5.93±0.10 0.692 GT 5.93±0.11 TT 5.96±0.10 PGBD1 GG 7.28±0.33 0.012 GT 7.17±0.24 TT 7.10±0.32 ZNF323 GG 6.79±0.20 <0.001 GT 6.68±0.13 TT 6.66±0.20 rs2142731 ZKSCAN4 CC 7.97±0.15 0.174 CT 8.01±0.17 TT 8.06±0.21 NKAPL CC 5.92±0.08 0.894 CT 5.93±0.09 TT 5.93±0.11 PGBD1 CC 7.10±0.26 0.080 CT 7.13±0.25 TT 7.25±0.31 ZNF323 TT 6.62±0.14 0.003 CC 6.66±0.14 CT 6.76±0.19


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Chr. 11p11.2 rs11038167 TSPAN18 AA 6.41±0.13 0.293 AC 6.38±0.19 CC 6.38±0.13 TP53I11 AA 6.03±0.08 <0.001 AC 5.97±0.08 CC 6.01±0.08 PRDM11 AA 6.09±0.08 <0.001 AC 5.99±0.08 CC 5.97±0.08 rs11038172 TSPAN18 AA 6.41±0.13 0.206 AG 6.37±0.13 GG 6.41±0.21 TP53I11 AA 6.03±0.08 <0.001 AG 5.98±0.07 GG 5.97±0.06 PRDM11 AA 6.08±0.09 <0.001 AG 6.00±0.07 GG 5.97±0.09 rs835784 TSPAN18 AA 9.10±0.29 0.073 AG 8.99±0.38 GG 8.94±0.36 TP53I11 AA 6.04±0.08 0.035 AG 6.00±0.09 GG 5.99±0.08 PRDM11 AA 6.07±0.09 0.087 AG 6.05±0.09 GG 6.04±0.09

Abbreviation: expression quantitative trait loci (eQTL); Chromosome (Chr.);, single nucleotide

polymorphism (SNP); human blood lymphoblastoid cell lines (LCLs); zinc finger protein 323 (ZNF323);

NFKB activating protein-like (NKAPL), zinc finger with KRAB and SCAN domains 4 (ZKSCAN4),

piggyBac transposable element derived 1 (PGBD1); zinc finger protein 323 (ZNF323); tumor protein p53

inducible protein 11 (TP53I11); PR domain containing 11 (PRDM11); The 195 HapMap stage II samples

consisted by 55 Caucasian(CEU), 42 Chinese Han (CHB), 42 Japanese (JPT), 56 Yoruba (YRI) samples.


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Supplementary Table 6. PCR and extension primer sequences from Sequenom SNP genotyping.

iPLEX SNP 2nd-PCRP 1st-PCRP UEP_SEQ

1 rs7581854 ACGTTGGATGGTACTACACAATCTACATGGG ACGTTGGATGCATTTGTACAGGTCTGCTTTG TGGGGAAAATTAAGAACACTT

2 rs11544338 ACGTTGGATGCACCATCTAAAACCTGACGG ACGTTGGATGGAGTTTAAATTCGAGATGC tATTTATTCAAATGTTGAATTTATCC

2 rs1381094 ACGTTGGATGCTTCCTACATTTACCATGGG ACGTTGGATGTTCCCCTCATGGGATCAACA GGATGACATGATTGGAGC

1 rs6792822 ACGTTGGATGGGGAGCTGAAGATTAACCAC ACGTTGGATGGGATTGTGGTAAAATTTTCC ACAGAGATATAAATACAGTTATTGATAA

2 rs3111810 ACGTTGGATGTCTATGTACTGTATTCTCC ACGTTGGATGACAATGCAGTGTCTGAAAGG CTCCTTTTTTCTTCTCTCATTG

1 rs755535e ACGTTGGATGGAGGTTTCGTTCTACAGCAG ACGTTGGATGAGCAGATGGCACCTGGCAGA tgtgTCGTTCTACAGCAGGTTCTA

1 rs2191037 ACGTTGGATGGCTTCTGTGGTCTTATGAAC ACGTTGGATGTGCCTCAATTCTTGTCTCAG cccttTTGGCTTCTGTTCTCC

2 rs634651 ACGTTGGATGATTGAGGGCAGATAGCAGCG ACGTTGGATGTTTCAGTCCTCCAGCATGCC ATAGCAGCGAAGACC

1 rs17459722 ACGTTGGATGTGAAGAGAAGACCGTGATGC ACGTTGGATGAGCACAGAAACAGCAGGTAG GATGCCAGCTGTTAGT

2 rs2237585 ACGTTGGATGAGATCGGGAAGGAATTTTTG ACGTTGGATGTATCATACAGGATGTGTGCC TTGAAAAGGACTAAATAGTGAACTTA

2 rs7778623 ACGTTGGATGATTCCTAGGTAATTTATAG ACGTTGGATGTTTTATGTCAGCCCTGGGAA AGTTTTTTTGTGATATCATGGC

2 rs10226151e ACGTTGGATGGGCGGGGTATTCCACAGTA ACGTTGGATGCCCAGCCCAGACACATTATG aggaGGGGTATTCCACAGTATAATAT

2 rs12352279 ACGTTGGATGAATGGAAGCAAATGGAGGGC ACGTTGGATGTTTCAAGTGTAGTGGGTGGG AGGAAACCACATCATCAG

1 rs10738881 ACGTTGGATGTGCAGCCTCACTGATTCCTC ACGTTGGATGTAGTGCCACAATGCCCTAAG gggacCTGATTCCTCCTGGGTCTC

1 rs10761076 ACGTTGGATGACCTTTCCTGCTGGGTGTAA ACGTTGGATGATGAGCACTCACCCAGTTTG ggcaTGCTGGGTGTAACATATC

1 rs2065412 ACGTTGGATGCTCAGGCAAAGGAGACATTC ACGTTGGATGGGGATAAGTGCTCTAGAGAC CCTGCTCCACATTTGAA

1 rs7863451 ACGTTGGATGTGAATCTTGCATGGTCTGGG ACGTTGGATGCAAAGCTGCAATAGAGTGAC tTGTTAGATGTCAGTTTTCCC

1 rs16938094 ACGTTGGATGCCATTCTCGCAAATGCACAC ACGTTGGATGACCTCTCAAGAGACCTGTTC aCACACTGAGCTTCCCAC

2 rs7936879 ACGTTGGATGCCAGGCTTAGCCCATAAAAC ACGTTGGATGTCTGTCAGTTTCACCAGGTC aacatCCCATAAAACCTTTTATGCTTA

1 rs916606 ACGTTGGATGAAGGAGCTCAAGGCTGGCAC ACGTTGGATGCTAGGAGAGAGTTATTACCC AGCTTCCATGCATTAGTCT

2 rs17134219 ACGTTGGATGCATTGGTATATGAGGTAGTG ACGTTGGATGCATTTCATATATTTAATTAGG acATGAGGTAGTGAGAAAACTA

2 rs608426 ACGTTGGATGATGAGAGATACCAGAAAAC ACGTTGGATGCAGGTATGATTGCTGAGAG cCTCCTTGTTAAAATGTTTAGC

1 rs2652007 ACGTTGGATGGCTATACTGATTGGTTTGAC ACGTTGGATGTGATGCTATGGGCATGGAAC ggggaTGATTGGTTTGACTTGGTTT


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1 rs11178971 ACGTTGGATGAATTATGTACCAATGCAAC ACGTTGGATGGCTACTAAATCTTACAAAGTC TTTTTCCTGTGCCCT

2 rs11178993 ACGTTGGATGATGAAATGTCTAGAAGATG ACGTTGGATGCTACTTTCTGTGTGTGTCTC TCTAGAAGATGTAAAAAGAGATC

2 rs10777708 ACGTTGGATGGGAAGCTAGAAGCAGCAATA ACGTTGGATGCACTACCTTAATTCAGGTCC tggAATGTTATCACAGTAATCCAGG

2 rs3782886 ACGTTGGATGAGGAACAATTCACACTTAC ACGTTGGATGAGAAGATGACGTTTGCCAGC gcGATTTGAGCACTTCAGC

1 rs2192420 ACGTTGGATGCACAGTAAGCAAAAATGTGG ACGTTGGATGTATTTCAATCATCAGACAG cctgCTTCTCCTTTGAGCCT

2 rs7155022 ACGTTGGATGCCTTTGCCCTTCCTTTGCAT ACGTTGGATGGGATTTTCACCCCTTCATAG CATTCAAAATTATTATCTCTCTCTC

1 rs2305252 ACGTTGGATGACAGCAGGGACTGGAAGATG ACGTTGGATGTTTCCCTAGCTCCCCGATAC ATGGGTCACGCTGAA

1 rs11657292 ACGTTGGATGTGGTATCCTCAGCACCTTTG ACGTTGGATGCTGTCACTAACCCATCCCTC gCAGCACCTTTGGGGTCTTT

1 rs11077721e ACGTTGGATGCTGTATGGAACTTCTACTT ACGTTGGATGCCAGAGAGGCTCTTTTGGAT AAAGAAATGAAAAAATCTATGGA

2 rs2011631 ACGTTGGATGCCGTAACAATCGCCACAAAC ACGTTGGATGACTTCCGGCTTCCAGAGCTA CCTGGTGACTTAAATAACAAATAT

1 rs3744165 ACGTTGGATGGCACTTCGTGGTTTCTAAAG ACGTTGGATGCGCAGAGCTGGCTTCTGATA tccaGCTTTGCTTTCTTTCCCGA

1 rs8073471 ACGTTGGATGGAAAGAGGGTTCCCAGGTC ACGTTGGATGAGAAGAGGTTTCGGCTACAG cctTCCACATCCTCTGCTT

2 rs4939924 ACGTTGGATGAGCATCATTTAATTTTGCC ACGTTGGATGTGTTGATTGAAGGTGCTGAC ccTTTTGCCTAAATGTAATTGTAC

2 rs656111 ACGTTGGATGGTGGCCTAAATTTTCACGAG ACGTTGGATGGTAGCACTTTTCTATATTAC CAAGGAAGTGAAAAGATGAC

1 rs2076158 ACGTTGGATGATGCCAACCAGGCCCTGATG ACGTTGGATGACACGGAGTGCCTTTCAAAC cGGGTCATGTTTACCACA


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Supplementary Figures

Supplementary Figure 1. The principal components analysis (PCA) of 2,345

samples and 206 reference samples from the HapMap. Plots of first five principal

components (PCs) from the principal components analysis (PCA) using 2,345

participant samples (746 cases, 1,599 controls) alone or in combination with 206

HapMap samples. Cases are in red, controls are in green, CHB samples are in purple,

CEU samples are in light brown, and YRI samples are in dark brown. a/e. plot of the

first and second principal components; b/f. plot of the first and third principal

components; c/g. plot of the first and fourth principal components; and d/h. plot of the

first and fifth principal components. a-d. PCA of 2,551 samples (from our GWAS and

HapMap); e-h. PCA of 2,345 samples from our GWAS.


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Supplementary Figure 2. Genome-wide association results from the initial

GWAS analysis. a. The genome-wide P values of the Cochran-Armitage trend test in

746 cases and 1,599 controls. The genome-wide P values (-log10P) of the association

analysis for 493,203 SNPs are plotted against positions on each chromosome. Each

chromosome is depicted in different colors. The blue horizontal line presents a liberal

threshold of 10-5 for suggestive significance. b. Quantile - quantile (Q - Q) plots of the

observed P values versus the expected P value of association.


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Supplementary Figure 3. Association results the LD plot across MCH region

(chr6:25-34Mb) in our GWAS sample. a. Association results across the MHC

region in our GWAS sample. Results are shown as -log10 (P value) for genotyped

SNPs (left y axis). The most associated SNP is shown as a purple diamond. The color

of the remaining markers reflects r2 with rs1635, light blue, r2>0.1, red, r2>0.8. The

recombination rate from the CHB HapMap (second y axis) is plotted in light blue.

b. Linkage disequilibrium (LD) values across the extended MHC region are illustrated

for our GWAS sample. Shown are LD relationships (r2, color coded) for the 200

SNPs in the extended MHC region on chromosome 6 that yielded P-values < 10-3 in

our GWAS sample. The location is shown in blue words for the peak evidence for

association rs13194053 at 27.25 Mb in the extended Class I region that was reported

by the ISC (2009). The other locations were the peak associated SNP rs1635 (28.34

Mb), relative peak associated SNPs rs1655900 (30.02 Mb) and rs6901869 (31.32 Mb)

in HLA Class I, in turns. Locations of the classical HLA Class I, II and III regions are

also shown.1 SNP rs1635 and its adjacent region showed peak significant association,

is in extended HLA region, but not in LD with rs13194053 or rs1655900 and


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rs6901869 in HLA Class I region.

Supplementary Figure 4. NKAPL and ZKSCAN4 mRNAs expression in postnatal

mice brain using the in situ hybridization methods. In situ hybridization was

performed on mice brain sections (coronal) using oligonucleotide probes specific for

the NKAPL and ZKSCAN4. a. NKAPL mRNA highly expression in postnatal 0 day

(P0) mice cortical (Ctx), hippocampus (Hip) CA1-CA3, ventral lateral nucleus (VL),

locus cerulus (LC), and other brain areas, compared to control. b. ZKSCZ4 mRNA

highly expression in postnatal mice cortical (Ctx), paraventricular nucleus (PV),

amygdale (A) at postnatal 0 day (P0); and in hippocampus (Hip) CA1-CA3 and Dense

granule (DG) especially at postnatal 14 days (P14) mouse. Scale bars were 50µm and

20µm, respectively.


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Supplementary Figure 5. Knockdown of NKAPL leads to neuronal migration

defects in the developing cerebral cortex. RNA interference (RNAi) constructs were

electroporated into the ventricular zone (VZ) at embryonic day 14.5 (E14.5) and

analyzed at postnatal day 0 (P0). In brain with control RNAi (Mock), 40% of

GFP-labeled cells exited the VZ, and 25% of GFP-labeled cells completed migration

and formed the superficial layers of the cortex. By contrast, only less than 15% of

GFP- positive cells reached the superficial layers in brain slices with NKAPL RNAi,

with the majority of GFP-positive cells remaining in the intermediate zone (IZ),


19

subventricular zone (SVZ), and VZ. Silencing of NKAPL induces delayed radial

migration (P < 0.001). Green indicates cells cotransfected with GFP and RNAi

constructs; Mock: control RNAi. Scale bar, 100 µm.

Supplementary Figure 6. Knockdown of CG6066 (NKAPL) in Drosophila

melanogaster (51.9% homologous as to NKAPL in homo sapiens) influences the

physical phenotypes of drosophila, as well as the synapse dysfunction. Compared with

the wild type, the flies with CG6066 (NKAPL) RNAi showed defected figures

(Adults displayed with misshapen wings, rough eyes, and crooked and twisted

metathoracic legs). Compared with wide type, the flies with CG6066 (NKAPL) RNAi

showed synaptic defects at the neuromuscular junction (NMJ), which including the

enlarged volume of synapses, and less number of synapses. HRP: Horseradish

peroxidase; Syt: Synaptotagmin.


20


21

Supplementary Note. Knockdown of NKAPL leads to neuronal migration defects

in the developing cerebral cortex of mice and synaptic abnormalities in

neuromuscular junction (NMJ) of Drosophila melanogaster.

To evaluate the feasibility of the in utero gene transfer technique to analyze the

modulation of gene expression in the prefrontal cotex (PFC), an expression construct

of green fluorescent protein (GFP) was injected into bilateral lateral ventricles (LV)

and incorporated by electroporation into progenitor cells in the ventricular zone (VZ)

at embryonic day 14.5 (E14.5) in rat. A green fluorescent protein (GFP) expression

vector with CAG promoter was cotransfected with NKAPL RNAi constructs at a

concentration of 1.5 µg/µL in 2µL were introduced directly into the ventricular zone

(VZ) by in utero electroporation of embryonic day 14.5 embryos as reported

previously.2-3 Sagittal slices of the developing cerebral cortex were prepared at

postnatal day 0 as described previously. The brains were fixed with 4%

paraformaldehyde and sectioned with a cryostat at 20 µm on postnatal day 0. Green

fluorescent images were captured after immunofluorescent staining with an anti-GFP

antibody (dilution, 1:500). Slice images were acquired with a fluorescence

microscope (BX51; Olympus Optical, Pennsylvania). To quantify the pattern of

migration, the numbers of GFP-positive cells in the developmental cerebral cortex,

including the ventricular zone (VZ), the subventricular zone (SVZ)/intermediate zone

(IZ), and the cortical plate (CP), were counted from 3 independent sections. Statistical

analyses were conducted with 1-way analysis of variance followed by post hoc

testing.

To understand the function of NKAPL, we examined the development of NMJ

synapses in flies in which CG6066 (NKAPL) expression had been knockdown by the

musclespecific C57-Gal4 and pan-neuronal elav-Gal4. Preparation and antibody


22

staining for dissected third instar larvae have been described elsewhere.4-5 Briefly, the

third instar larvae was dissected and fixed in 4% formaldehyde for 30 min, and then

the following antibodies were used: mouse anti-synaptotagmin [1:5, Developmental

Studies Hybridoma Bank (DSHB) at the University of Iowa] and Fluorescein

(FITC)-conjugated goat anti-Horseradish Peroxidase (1:200, Jackson

ImmunoResearch, PA, USA) and anti-mouse secondary antibodies conjugated to were

used at 1:100. Images were collected with a Nikon confocal microscope. All images

analyzed were three-dimensional projections from complete z-stacks through the

entire NMJ4 of abdominal segment A3. At least 20 NMJ4 terminals of different

genotypes were analyzed.

REFERENCES

1. Horton, R., et al. Gene map of the extended human MHC. Nat Rev Genet 5,

889-899 (2004).

2. Saito T. In vivo electroporation in the embryonic mouse central nervous system.

Nat. Protoc. 1, 1552-1558 (2006).

3. Tabata, H., & Nakajima K. Efficient in utero gene transfer system to the

developing mouse brain using electroporation: visualization of neuronal

migration in the developing cortex. Neurosci. 103, 865-872 (2001).

4. Patel, N.H. Imaging neuronal subsets and other cell types in whole-mount

Drosophila embryos and larvae using antibody probes. Methods. Cell Biol. 44,

445-487. (1994).

5. Broadie K.S. & Bate, M. Development of the embryonic neuromuscular synapse

of Drosophila melanogaster. J. Neurosci. 13, 144–166. (1993).


Genome-wide association study identifies …...1 Genome-wide association study identifies susceptibility loci for schizophrenia in Han Chinese at 6p21-p22.1 and 11p11.2 Wei-Hua Yue,

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