Sep 2, 2011 - promoter region of chitinase 3-like 1 (CHI3L1) and susceptibil- ity to schizophrenia. Am J Hum Genet 2007; 80:12â18. 32. Chen X, Lee G, ...
AJP in Advance. Published September 2, 2011 (doi: 10.1176/appi.ajp.2011.11030381)
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A lle lic D iffe re n c e s B e tw e e n H a n C h in e se a n d E u ro p e a n s fo r F u n c tio n a l V a ria n ts in Z N F 8 0 4 A a n d T h e ir A sso c ia tio n W ith S c h iz o p h re n ia Ming Li, M.Sc. Xiong-jian Luo, Ph.D. Xiao Xiao, M.Sc. Lei Shi, M.Sc. Xing-yan Liu, M.D. Li-de Yin, M.D. Hong-bo Diao, M.D. Bing Su, Ph.D.
O b je c tiv e : ZNF804A is a schizophrenia risk gene that w as recently identified by genom e-w ide association studies as w ell as subsequent replications. Although the results are consistent am ong studies in European populations, there have been conflicting reports in Chinese populations. The authors conducted both association and functional analyses to test w hether ZNF804A is a risk gene for schizophrenia in Chinese populations. M e th o d : The authors recruited tw o casecontrol sam ples of independent Han Chinese (a total of 2,207 participants) from southw estern China. A total of six singlenucleotide polym orphism s (SNPs), including the key SNP (rs1344706) that show ed significant association w ith schizophrenia in European populations and the other five prom oter SNPs of ZNF804A, w ere tested. Based on the results of the association analysis, the authors perform ed tw o functional assays to test the im pact of the
risk SNP on transcriptional factor binding affinity and prom oter activity. R e s u lts : The SNP rs1344706 w as not associated w ith schizophrenia in either of the tw o Han Chinese groups, and this result w as confirm ed by m eta-analyses in five Han Chinese sam ples. How ever, the authors identified tw o ZNF804A prom oter SNPs that w ere significantly associated w ith schizophrenia in both sam ples, and the significance w as strengthened in the com bined sam ples and further supported by haplotype analysis. The functional assays dem onstrated that the risk SNP (rs359895) can influence Sp1 binding affinity, resulting in a higher prom oter activity of the risk allele. C o n c lu s io n s : O ur results suggest that ZNF804A is a com m on risk gene for schizophrenia in w orld populations and that the new ly identified functional SNP (rs359895) is likely a risk SNP for schizophrenia. (A m J P sy c h ia try Li e t a l.; A iA :1 –8 )
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chizophrenia is a severe and common neuropsychiatric disorder with an estimated lifetime prevalence approaching 1% worldwide (1). The essential characteristics of this disorder include various psychotic symptoms such as delusions and hallucinations, affective response, social withdrawal, apathy, and cognitive impairment (2). Family, twin, and adoption studies have unequivocally shown a strong genetic component in schizophrenia, with heritability estimates of about 80% (3). Despite such high heritability of schizophrenia, the underlying genetic risk factors have yet to be identified. Numerous genetic association studies of schizophrenia have been reported, and numerous candidate genes have been proposed through linkage analyses, candidate gene association studies, and genome-wide association studies (GWAS) (4–7). Many of them, however, await satisfactory replications in different populations and functional verification of the relevant genetic variants. ZNF804A, a novel schizophrenia susceptibility gene on chromosome 2q32.1, was first identified by GWAS in
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U.K. populations with large-scale follow-up replications (8). The risk single-nucleotide polymorphism (SNP), rs1344706, achieved genome-wide significance, and it was further confirmed by subsequent independent replication studies as well as meta-analyses in major populations (9–14). Through fine mapping analysis, Williams et al. (10) observed multiple common SNPs within ZNF804A that were strongly associated with schizophrenia. Meanwhile, Steinberg et al. (9) searched for large copy number variations in 5,408 psychiatric patients (including those with schizophrenia, bipolar disorder, anxiety, and depression) and 39,481 healthy comparison subjects and identified three copy number variations covering part of ZNF804A in psychiatric patients but not in comparison subjects (p=0.0016). Thus, ZNF804A is a promising risk gene for schizophrenia, especially in European populations. Studies of Chinese populations, however, have been inconsistent. O’Donovan et al. (8) and Steinberg et al. (9) failed to replicate the association of rs1344706 with schizophrenia in Han Chinese (p=0.166 and p=0.62, re-
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Copyright 2011 American Psychiatric Association. All rights reserved.
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Va r iants in Z N F 8 0 4 A and S ch iz o p h r en ia in H an C h inese and E u r o p eans F IG UR E 1 . L in k a g e D ise q u ilib riu m (L D ) M a p s o f th e S ix S in g le N u c le o tid e P o ly m o rp h ism s (S N P s) in th e Yu x i S a m p le a
19
25 72
30 65
23
14 7
13 5
2
10 49
21
21
Block 1 (1 kb) 3 4 26
74
81 23
40
5
6
22 6
21 4
16
a
27
rs1344706
1
rs359895
rs11888068
6
rs10497655
rs1021042
Block 1 (1 kb) 4 5
rs1344706
rs13026173 3
rs359895
rs11888068 2
rs10497655
rs1021042 1
rs13026173
LD for Comparison Subjects
LD for Patients
18 53
14 47
13
The linkage disequilibrium of the tested SNPs was calculated using the r2 algorithm by the Haploview program.
spectively), but Zhang et al. (11) did replicate the association (p=0.00083). This suggests that either ZNF804A may not be a risk gene for schizophrenia in the Chinese population, or there are other risk SNPs. Additionally, it was reported that when considering disease status, the expression of ZNF804A was higher in patients than it was in comparison subjects but was not significantly different (p=0.107). When considering rs1344706 allele status in comparison samples only, expression was significantly higher from the associated allele (p=0.033) (13). We speculate that those SNPs located in the promoter region of ZNF804A may affect ZNF804A expression and eventually contribute to the pathogenesis of schizophrenia. To test whether ZNF804A is a risk gene for schizophrenia in the Chinese population, we first examined rs1344706 in two independent case-control samples collected from southwestern China. We then tested five ZNF 804A promoter SNPs (rs359895, rs10497655, rs13026173, rs11888068, and rs1021042) in these samples, in which we observed two SNPS (rs1021042 and rs359895) that were significantly associated with schizophrenia. Finally, we investigated the functional impact of rs359895 on transcription factor binding affinity and promoter activity.
M e th o d P a rtic ip a n ts We recruited two case-control samples independently from the southwestern Chinese cities of Yuxi (502 patients with a mean age of 38.5 years [SD=10.4]; 694 comparison subjects with a mean age of 37.1 years [SD=6.8]) and Kunming (404 patients with a mean age of 36.3 years [SD=8.7]; 607 comparison subjects with a mean age of 36.6 years [SD=7.0]). The patients were diagnosed with
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schizophrenia according to ICD-10 (Yuxi) or DSM-IV (Kunming) criteria. We collected detailed information on the course of the clinical disorder, age at onset, symptoms, and family history of psychiatric illnesses. Potential participants with a history of alcoholism, epilepsy, neurological disorders, or drug abuse were excluded from the study. Meanwhile, unrelated healthy volunteers were recruited from the local communities as comparison subjects. These individuals were asked to provide detailed information about their medical and family psychiatric histories. Those who had a history of psychiatric disorders, psychiatric treatment, alcohol dependence, or drug abuse or a family history of psychiatric disorders were excluded. The patients and comparison subjects were of Han Chinese origin from the Yunnan province of southwestern China, and they were all unrelated. All participants provided written informed consent, and the research protocol was approved by the internal review board of the Kunming Institute of Zoology at the Chinese Academy of Sciences.
S N P S e le c tio n SNP selection was based on previous studies and our HapMap data analysis (8–13). For the initial screening in the Yuxi sample, a total of six SNPs were selected. We first selected rs1344706, which has been identified by GWAS in European populations (8). We then examined the linkage disequilibrium pattern of the ZNF804A promoter region (about 10 kb) in the Chinese population using data from HapMap. We initially selected five tagging SNPs spanning the promoter region of ZNF804A (rs1021042, rs13026173, rs359895, rs10497655, and rs359894) with the use of the tagger procedure implemented in Haploview (15). Since the minor allele frequency of rs359894 is less than 0.05 in Han Chinese (minor allele frequency=0.022, shown in HapMap), we excluded this SNP from further analysis and selected another one (rs11888068) close to rs359894. All the selected SNPs are biallelic with the minor allele frequency greater than 5%. In total, six SNPs were selected for screening the Yuxi sample (see Figure S1 in the data supplement that accompanies the online edition of this article). For the replication analysis in the Kunming sample, three SNPs were tested, A JP in A dva n ce
L i, Lu o , X ia o , et al . TA B L E 1 . A lle le Fre q u e n c y a n d S in g le N u c le o tid e P o ly m o rp h ism (S N P ) A sso c ia tio n A n a ly sis in Tw o C h in e se S a m p le s Frequency Sample Yuxi sample
Kunming sample
Combined sample
SNP ID
Position
Allele
Patients
Comparison Subjects
p (nominal)
rs1021042 rs11888068 rs13026173 rs10497655 rs359895 rs1344706 rs1021042 rs359895 rs1344706 rs1021042 rs359895 rs1344706
185453158 185460295 185460842 185462041 185463185 185778428 185453158 185463185 185778428 185453158 185463185 185778428
G T C C A G G A G G A G
0.2814 0.4327 0.2802 0.4746 0.1573 0.4939 0.3284 0.1725 0.5000 0.3019 0.1641 0.4966
0.2241 0.4654 0.2334 0.4863 0.2197 0.4971 0.2544 0.2199 0.4843 0.2378 0.2198 0.4911
1.5×10 0.11 0.010 0.58 1.4×10 –4 0.88 4.7×10 –4 9.3×10 –3 0.49 2.5×10 –6 5.0×10 –6 0.72
including rs1344706 and the two SNPs (rs359895 and rs1021042) that showed significant associations with schizophrenia in the Yuxi sample. The linkage disequilibrium map of the tested SNPs in the Yuxi sample is shown in Figure 1, and the linkage disequilibrium maps of the ZNF804A promoter SNPs downloaded from the HapMap database are shown in Figure S1 and Figure S2 in the online data supplement. The SNP information is shown in Table 1.
S N P G e n o ty p in g Venous blood was collected from all participants, and genomic DNA was extracted from the blood sample using the standard phenol-chloroform method. DNA samples of the patients and comparison subjects were randomly distributed in the DNA sample plates. All the selected SNPs were genotyped by the SNaPShot method as described in our previous study (16). Details of all primers and assay conditions are available on request. The SNP genotype calls were automatically performed using GeneMapper 4.0 (Applied Biosystems) and verified manually. To make sure of the accuracy of genotyping, we conducted bidirectional sequencing of 100 randomly selected individuals, and no genotyping errors were observed. The genotyping success rate for the six tested SNPs was 98.4%.
E le c tro p h o re tic M o b ility S h ift A ssa y For functional prediction of the candidate SNPs, we used the online software program AliBaba (www.gene-regulation.com/ pub/programs.html#alibaba2) to predict the DNA-binding motifs in the ZNF804A promoter region. A 100% match to the matrix yields a maximum score of 1.00, and a matrix similarity score greater than 0.80 is considered a good match. Electrophoretic mobility shift assay was performed using the gel shift assay system (Pierce Protein Research, Rockford, Ill.) under the guidelines provided. The single-strand oligonucleotides were 3:-end biotin labeled and then annealed to form double strands. The binding reaction contained purified recombinant Sp1 protein (Alexis Biochemicals, San Diego), 10× binding buffer, and, if needed, unlabeled competitors; the labeled probes were added after 20 minutes, and samples were incubated at room temperature for a total of 30 minutes. After incubation, samples were separated on a native 6% polyacrylamide gel and then transferred to a nylon membrane. The positions of biotin end-labeled oligonucleotides were detected by a chemiluminescent reaction with streptavidin-horseradish peroxidase. The nucleotide sequences of the double-stranded oligonucleotides with either A or T allele were: A allele: 5(-GTATCAGCCCAGTGGCTCCCAGCCATTGGCTCAGTGCAATG-3(
–3
p (corrected) Odds Ratio 7.0×10 0.37 0.041 0.97 7.0×10 –4 1.0 9.0×10 –4 0.023 0.84 3.0×10 –6 1.0×10 –5 0.97 –3
1.36 0.88 1.28 0.95 0.66 0.99 1.43 0.74 1.07 1.39 0.70 1.02
95% CI 1.12–1.64 0.74–1.03 1.06–1.54 0.81–1.12 0.54–0.82 0.84–1.16 1.17–1.76 0.59–0.93 0.89–1.27 1.21–1.60 0.60–0.81 0.91–1.15
T allele: 5(-GTATCAGCCCAGTGGCTCCCTGCCATTGGCTCAGTGCAATG-3(
Lu c ife ra se R e p o rte r A ssa y To construct ZNF804A promoter, we amplified fragments encompassing nucleotides from −1089 base pairs (bp) to +65 bp (relative to transcription start site +1) from two individual homozygotes with respect to the corresponding genotypes (TT and AA) for rs359895. Then the amplified fragments were cloned into the pGL3-basic plasmid vectors. We verified all recombinant clones by bidirectional DNA sequencing to make sure no de novo mutation was introduced. These plasmids were all accurately quantified with an Eppendorf (Hamburg) BioPhotometer, and equal amounts of the plasmids were used for transfection. The reporters containing either T allele or A allele were transiently cotransfected into HEK293T and HeLa cells together with pRL-TK plasmid (a standard reporter). After a 36-hour incubation, we collected the cells and measured luciferase activity using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, Wisc.). All assays were performed in at least three independent experiments with a minimum of five replications.
S ta tistic a l A n a ly sis We tested for Hardy-Weinberg equilibrium using Haploview 4.1 by examining the genotypic distributions of ZNF804A SNPs in each sample. All of the SNPs genotyped in this study were in Hardy-Weinberg equilibrium. Linkage disequilibrium between paired SNPs was estimated by Haploview using the r2 algorithm. Allelic and genotypic associations were accessed with PLINK (17). Since the SNPs may not be totally independent because of linkage disequilibrium, to avoid type II error when applying the Bonferroni correction, we corrected the p values with a max(T) permutation procedure implemented in PLINK, using the “–mperm” option (N=1,000,000), which takes a single parameter (the number of permutations to be performed). This is achieved by comparing each observed test statistic against the maximum of all permuted statistics (i.e., over all SNPs) for each single replicate. We calculated significance for the combined samples (the Yuxi and Kunming samples) using the Cochran-Mantel-Haenszel test and conditioning by site as implemented in PLINK. The 95% confidence intervals (CIs) of odds ratios were calculated with an online tool (http://faculty.vassar.edu/lowry/odds2x2.html). For meta-analysis of rs1344706 in the five Han Chinese samples independently collected from Shanghai, Xi’an, Sichuan, Yuxi, and Kunming (from O’Donovan et al. [8], Steinberg et al. [9], Zhang et al. [11], and the present study), because there is genetic heterogeneity caused by the data from Zhang et al. (11), we used the Mantel-
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Va r iants in Z N F 8 0 4 A and S ch iz o p h r en ia in H an C h inese and E u r o p eans TA B L E 2 . T h e rs3 5 9 8 9 5 -rs1 0 2 1 0 4 2 H a p lo ty p e A sso c ia tio n A n a ly sis in th e Yu x i a n d K u n m in g S a m p le s Frequency Sample Yuxi sample
Kunming sample
Combined sample
Haplo type
Schizo phrenia
Comparison Subjects
p (Nominal)
p (Corrected)
Odds Ratio
95% CI
A-C A-G T-C T-G A-C A-G T-C T-G A-C A-G T-C T-G
0.1524 0.0051 0.5670 0.2756 0.1624 0.0111 0.5083 0.3183 0.1566 0.0080 0.5417 0.2937
0.2086 0.0112 0.5674 0.2129 0.2040 0.0159 0.5427 0.2373 0.2068 0.0131 0.5560 0.2242
2.0×10 -4 0.28 0.87 4.0×10 -4 8.0×10 -3 0.70 0.22 9.0×10 -5
