DOI 10.1007/s00702-005-0420-3 J Neural Transm (2006)

Transmission disequilibrium test provides evidence of association between promoter polymorphisms in 22q11 gene DGCR14 and schizophrenia H. Wang1;2;3;
, S. Duan1;2;
, J. Du1;2 , X. Li1;2 , Y. Xu4 , Z. Zhang1 , Y. Wang1;3 , G. Huang4 , G. Feng5 , and L. He2;6 1

2

Bio-X Center, Shanghai Jiao Tong University, and Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, 3 Life Science School, Ningxia University, Yinchuan, 4 Shanghai Jiao Tong University Medical School, 5 Shanghai Institute of Mental Health, and 6 NHGG, Bio-X Center, Shanghai Jiao Tong University, Shanghai, China Received May 26, 2005; accepted November 1, 2005 Published online January 25, 2006; # Springer-Verlag 2006

Summary. Recent research has suggested that the DiGeorge syndrome critical region gene 14 (DGCR14) exhibits activity differences of more than 1.5 fold between the haplotypes of the variants in the promoter region. DGCR14 is located at 22q11.21, an acknowledged region for susceptibility to schizophrenia. To test the hypothesis that DGCR14 may be involved in the etiology of the disease, we carried out a family-based association study between the reported functional markers and schizophrenia in 235 Chinese Han trios. We found significant evidence of preferential transmission of the promoter variants of DGCR14 across all the trios (Best p-value ¼ 0.00038, Global p-value ¼ 0.0008). The positive results have suggested that DGCR14 is likely to play an important role in the etiology of schizophrenia in the Chinese Han population.
These authors have contributed equally to this work

Keywords: DGCR14, family-based, schizophrenia, polymorphisms, promoter. Introduction Schizophrenia is a common psychiatric disorder with a lifetime prevalence of approximately 1% (Jablensky et al., 1992). It is highly heritable but the genetics are complex. Positive results of linkages have been reported in several genomic regions, among which 22q11 is one of the most promising candidate regions for schizophrenia (Karayiorgou et al., 2004). The DiGeorge syndrome critical region gene 14 (DGCR14, MIM 188400) is located at 22q11.21 within the minimal DiGeorge syndrome critical region (MDGCR). The MDGCR has been thought to contain the gene(s) responsible for a group of chromosome 22 deletion syndromes such as DiGeorge syndrome (DGS) and velocardiofacial syndrome

H. Wang et al.

(VCFS). And reports have also shown that 22q11 deletion syndrome is associated with high rates of psychiatric morbidity, particularly schizophrenia (Williams et al., 2004). Therefore, the 22q genes, especially those within the common critical deleted region such as PRODH, COMT and ZDHHC8, are good candidates for the association study with the susceptibility of schizophrenia. The human DGCR14 gene was first identified and cloned by Rizzu and his colleagues in 1996 (Rizzu et al., 1996), and it is thought to be essential for early embryonic development (Lindsay et al., 1998). The descriptions in AceView (www.ncbi.nih.gov=IEB= Research=Acembly=av.cgi?db ¼ human&c ¼ Gene&l ¼ DGCR14) suggest the DGCR14 gene is highly expressed, about 3.9 times of the average genomic level. Moreover, a recent study has reported that this gene has activity differences of more than 1.5-fold between the haplotypes of the variants in the promoter region (Hoogendoorn et al., 2004). Thus, we hypothesized that the fluctuating dose of DGCR14 gene might confer susceptibility to schizophrenia, although we still had little knowledge of this 22q11 gene. In the present study, we focused on three common promoter variants which might play an important role in the biological function of the DGCR14 gene. The association study between DGCR14 and schizophrenia was carried out in 235 Chinese Han trios in order to avoid the potential misleading effect of population stratification. Materials and methods Subjects The 235 trios in our study were composed of healthy parents and their affected offspring. There were 88 female and 147 male probands with a mean age of onset of 28.3  7.1 years and a mean age of 32.3  7.5 years. The unrelated schizophrenic probands who had a clinical diagnosis of schizophrenia and were reported to be clinically stable were tested to verify that they met the following inclusion criteria: (1) a DSM-IV (Diagnostic and Statistical Manual of Mental

Disorders – Fourth Edition) diagnosis of schizophrenia (Flaum et al., 1997), confirmed by the Structured Clinical Interview for DSM-IV (SCID-I); (2) willingness to participate in the study procedures, expressed by providing written informed consent after complete description of the study. All the subjects were recruited from Shanghai Mental Health Center. Consensual diagnosis of each patient was made independently by two senior psychiatrists with over 15-year career experience. A standard informed consent in the protocol, which was reviewed and approved by the Shanghai Ethical Committee of Human Genetic Resources, was given by the participating subjects after the nature of study had been fully explained. All subjects were Han Chinese in origin.

Genotyping Genomic DNA was prepared from peripheral blood using the standard phenol-chloroform extraction. Five sample pools, each consisted of 24 healthy subjects, were used to examine the frequencies of tested SNPs. The genotyping of five pooled samples and the 235 trios was performed by the direct sequencing. Touch down PCR was carried out in a 10 ml reaction mixture containing 10 ng genome DNA, and 0.5 U Taq polymerase, 0.25 ml of each primer (10 mM), 1 ml PCR buffer (Qiagen), 1 ml Q-solution (Qiagen) and 1 ml dNTPs (each 2 mM) on a gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). Cycling began with a first stage of 94 C for 5 min, followed by 14 cycles of 94 C for 30 s, 60 C for 30 s (declines 0.5 C after each cycle), and 72 C for 1 min, then 32 cycles of 94 C for 30 s, 56 C for 30 s, and 72 C for 1 min, and was completed with a final stage of 72 C for 7 min. The PCR products were then purified by incubation with 0.1 U of Shrimp alkaline phosphatase (Roche, Basel, Switzerland) and 0.5 U of exonulease I (New England Biolabs, Beverly, MA, USA) at 37 C for 45 min, followed by heat inactivation at 85 C for 20 min. The products were then sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3100 DNA sequencer. The sequences of the PCR primers are as follows: forward-50 -GCG TCT CCA TCG CTA TCC-30 ; reverse-50 -CGT GAA GAA CAC GCC CTA T-30 .

Statistical analysis The Family-Based Association Test program (FBAT version 1.5.3) (Laird et al., 2000) was used to assess the genotype data for pedigree inconsistencies. The genotypes of families with pedigree errors were set to zero. The presence of Hardy-Weinberg equilibrium (HWE) was examined using the X2 test for goodness

Whole Whole Whole Male Male Male Female Female Female

Sample

FBAT(-e)

0.653 0.83 0.843 0.682 0.839 0.854 0.61 0.819 0.829

Allele Freqencies 151 151 140 96 92 84 57 61 58

Sc 138 131 121 85 77 71.5 54.5 55.5 51

E(S)d 47.5 35.5 32 28.5 20.5 17.75 19.25 15.25 14.5

Var(S)e 1.886 3.357 3.359 2.06 3.313 2.967 0.57 1.408 1.838

Z 0.18 0.0024 0.0023 0.12 0.0028 0.009 n.s. 0.48 0.2

Corrected P-valuesf 146 88 83 89 54 49 57 34 34

Transmitted 119 47 44 67 24 24 52 23 20

Non-transmitted

tdtphase-permutation 10000

0.097 0.00038 0.00049 0.078 0.00058 0.0031 0.63 0.14 0.055

P-values

0.13

0.0017g

0.0008

Global P-values

1.23 1.87 1.89 1.33 2.25 2.04 1.1 1.48 1.7

OR

V1 stands for rs737923 (G > A); V2 stands for rs1936950 (A >T); V3 stands for rs1936951 (G > A); V4 (rs3810598T > C) is a rare SNP, and not analyzed in the table. bSignificant P-values (<0.05) are in boldface. cS represents the test statistic for observed number of alleles. dE(S) represents the expected value of S under null hypothesis. eVar(S) represents variance between the observed and expected transmission. fThe p-values is corrected with three tested markers. gThe corrected global p-value with gender is 0.0034

a

V1-G V2-A V3-G V1-G V2-A V3-G V1-G V2-A V3-G

Allele

Table 1. Allele frequencies of the common variants in the affected probands and results of allelic association with schizophreniaa,b

DGCR14 and schizophrenia

0.453 0.35 0.153

(V1–V2–V3) GAG AAG GTA 40 50.8 29.8

55 50

0.85 0.14

female

73 62.6 44.2 13.2

93 84 13

0.83 0.15 0.02

0.56 0.29 0.12 0.03

113 111.5 76.1 19.3

0.52 0.32 0.14 0.028

72 61.7 40.8 10.5

male

112 111.8 70.5 14.7

0.514 0.318 0.126 0.021

0.555 0.294 0.109 0.026

whole

(V1–V2–V3) GAG AAG GTA ATA (V1–V2) GA AA GT AT (V2–V3) AG TA TG

59.88 52.12 16.12

96 17

121.95 72.05 21.05 0.95

119.85 71.15 19.15 0.85

156.98 32.98 4.02

182.57 123.44 41.44 2.57

178.14 122.83 34.79 2.17

57.45 50.04 17.29

81 29.5

112.62 69.39 26.39 7.62

109.97 69.03 23.25 6.26

134.99 49.99 7.01

172.17 119.84 47.34 10.67

166.11 118.87 40.13 8.35

E(S)f

17.42 17.54 8.45

20.5 17.75

27.51 18.41 12.22 4.12

27.35 18.18 10.6 3.47

34.49 30.5 3.99

44.76 36.19 21.74 5.17

43.84 35.37 18.71 4.19

Var(S)g

0.58 0.5 0.41

3.31 2.97

1.78 0.62 1.53 3.28

1.89 0.5 1.26 2.9

3.74 3.08 1.5

1.56 0.6 1.27 3.56

1.82 0.67 1.23 3.02

Z-score

0.56 0.62 0.69

0.0009 0.003

0.075 0.53 0.13 0.00103

0.06 0.62 0.21 0.0037

0.00018 0.0021 0.13

0.12 0.55 0.21 0.00037

0.07 0.51 0.22 0.0025

P-value

6.16(3)

11.46(2)

12.12(4)

12.37(4)

18.65(3)

13.04(4)

16.58(4)

0.1(0.2)

0.0032(0.0064)

0.007(0.014)

0.015(0.03)

0.00032

0.0046

0.0023

Global P-values (P-values corrected by gender)

X2(df)

Se

Afreqc

fam#d

tdtphase-permutation 10000

FBAT(-e)

(V1–V2–V3) GAG AAG GTA ATA (V1–V2) GA AA GT AT (V2–V3) AG TA

Sample

Haplotype Group

Table 2. Estimated haplotype frequencies and association significancea,b

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V1 stands for rs737923 (G> A); V2 stands for rs1936950 (A >T); V3 stands for rs1936951 (G > A); V4 (rs3810598T > C) is a rare SNP, and not analyzed in the table. bIt only lists P-values of haplotype with minimum families >10 and significant P-values (<0.05) are in boldface. cAfreq represents marker allele frequencies. dfam# represents informative family count. eS represents the test statistic for observed number of alleles. fE(S) represents the expected value of S under null hypothesis. gVar(S) represents variance between the observed and expected transmission

a

0.037(0.074) 8.48(2) 0.047 0.17 1.99 1.39 39 34 0.8 0.17

63 16

55.5 21

14.25 13

0.71 0.83 0.85 41 49.8 33.3

(V1–V2) GA AA GT (V2–V3) AG TA

0.46 0.36 0.17

62.07 51.93 20.93

60.46 51.04 21.54

18.39 18 10

0.38 0.21 0.19

3.42(3)

0.33(0.66)

DGCR14 and schizophrenia of fit. TDT was used to detect linkage disequilibrium (LD) with illness. It was implemented using FBAT and the tdtphase module of UNPHASED software (Dudbridge, 2003), respectively. Both programs are suitable for use with both family trio and sibling pair sample structures. They are able to deal with the transmission of multi-locus haplotypes, even when phase is unknown and parental genotypes may be missing. We employed the empirical-variance estimator (the -e flag option in the FBAT program) and assumed an additive model for each SNP. The additive model can be expected to perform well even when the true model is nonadditive. Using Bonferroni’s correction, the significance level of the FBAT statistic was adjusted for the number of FBATs computed. We employed the hbat -e option and assumed a multi-allelic mode to obtain the global significance of the haplotypes. Markers were also tested for transmission=non-transmission counts, using the tdtphase program of the UNPHASED software package. Power analysis was performed using the Genetic Power Calculator (http:==statgen.iop.kcl.ac. uk=gpc=) (Purcell et al., 2003).

Results We detected three common SNPs (V1: rs737923 (G>A); V2: rs1936950 (A>T); V3: rs1936951 (G>A)) and a rare SNP (V4: rs3810598 (T>C)) in the PCR product. The genotypic distribution of three common SNPs was in HWE in both the patient group and the parent group. Linkage disequilibrium (expressed in D0 ) between V1 and V2, V1 and V3, V2 and V3, was 0.99, 0.98 and 0.98, respectively. TDT analysis of individual SNPs showed positive results in V2 and V3 with corrected P-values 0.0024 and 0.0023, respectively. Sliding window haplotype analysis of two loci was carried out using the FBAT1.5.3 program in multi-allelic mode. We found evidence of association between haplotypes V1–V2 and V2–V3 and schizophrenia (V1–V2: X2 ¼ 13.04, Global P ¼ 0.0046, df ¼ 3; V2–V3: X2 ¼ 18.65, Global P ¼ 0.00032, df ¼ 3). The same result was observed in the three-locus system (V1–V2–V3: X2 ¼ 16.58, Global P ¼ 0.0023, df ¼ 4). Eight haplotypes were observed but only four of them showed frequencies greater than 1% and accounted for the majority of the haplotype diversity (98%). The global

H. Wang et al.

P-value for these four haplotypes is 0.0023 (X2 ¼ 16.58, df ¼ 4). In the exploratory analysis, where the sample was sub-grouped according to gender of probands, significant evidence of preferential transmission of the V2, V3 alleles of the DGCR14 gene was also detected in the male group (V2: Transmitted: Non-transmitted ¼ 54:24; P ¼ 0.00058; V3: Transmitted: Non-transmitted ¼ 49:24; P ¼ 0.0031; Global significance ¼ 0.0017). In contrast, no significant transmission disequilibrium was observed in the female group (V2: Transmitted: Non-transmitted ¼ 34:23; P ¼ 0.14; V3: Transmitted: Non-transmitted ¼ 34:20; P ¼ 0.055; Global significance ¼ 0.13). The global significance in the male group still remains (global significance ¼ 0.0034) after allowing for Bonferroni’s correction for gender. Power analysis showed that our sample of 235 trios has powers of 79.3% and 96.9% (alpha<0.05) in an additive model with a genotype relative risk of 1.5 and allele frequencies of 0.15 and 0.3, respectively. Discussion The long arm of chromosome 22 is a promising region for genetic research into schizophrenia. A rank-based genome scan meta-analysis (GSMA) has ranked 22q the eighth (in weighted analysis) most susceptible region for schizophrenia among a total of 120 30-cM bins (Lewis et al., 2003). Clinical and epidemiological findings of the high rates of schizophrenia among the patients of some 22q11 deletion syndromes have confirmed that the critical deleted 2 Mb region might harbor genes relevant to the etiology of schizophrenia (Karayiorgou et al., 2004; Murphy, 2002). Of genes mapped to 22q11, the CatecholO-methytransferase (COMT) (Karayiorgou et al., 2004; Williams et al., 2004) and proline dehydrogenase (PRODH) (Karayiorgou et al., 2004) have been the most extensively studied candidates. Apart from the above

two, the other susceptible genes in and around 22q11 region are now under intense scrutiny, including ZDHHC8 (Karayiorgou et al., 2004; Mukai et al., 2004; Chen et al., 2004; Saito et al., 2005), CLDN5 (Sun et al., 2004; Ye et al., 2005), ARVCF (Sanders et al., 2005; Chen et al., 2005), YWHAH (Duan et al., 2005; Toyooka et al., 1999; Hayakawa et al., 1998; Bell et al., 2000; Wong et al., 2003), SNAP29 (Saito et al., 2005), MLC1 (Meyer et al., 2001; Devaney et al., 2002; Verma et al., 2005), PCQAP (De Luca et al., 2003), BZRP (Kurumaji et al., 2000), RTN4R (Sinibaldi et al., 2004), UFD1L (De Luca et al., 2001), TBX1 (Maynard et al., 2003; Williams et al., 2004), APOL (Mimmack et al., 2002; McGhee et al., 2005), ZNF74 (Takase et al., 2001), PVALB (Hashimoto et al., 2003; Reynolds et al., 2004; Lewis et al., 2005) and etc (as shown in Fig. 1). Our lab has investigated several 22q genes (COMT [Fan et al., 2002, 2005], PRODH [Fan et al., 2003], ZDHHC8 [Chen et al., 2004], XBP1 [Chen et al., 2004] and YWHAH [Duan et al., 2005]). However, we have found evidence of association with schizophrenia only at ZDHHC8 and XBP1 in the long arm of chromosome 22. The difference between the initial reports and our findings might reflect differences in LD structure across different populations. In this study, we conducted an association study to investigate the relationship between the promoter variants of the DGCR14 gene and schizophrenia in 235 Chinese trios. Both TDT and haplotype analysis have shown a strong association between DGCR14 and schizophrenia. The advantage of a familybased study is that it avoids false-positive results due to population stratification. We found significant association of V2 and V3 with schizophrenia. The V2 and V3 are only 5 bps apart and are in tight linkage disequilibrium (D0 ¼ 0.99, r2 ¼ 0.97). Four haplotypes of V2 and V3 were observed but the haplotype AG and TA accounted for the

Fig. 1. Demonstration of the candidate genes of schizophrenia in 22q. The region between D22S427 and D22S636 is the common deletion region for VCFS, while the approximate 2 Mega-bases pairs between D22S427 and D22S264 contains the Minimum deletion region for VCFS; The most significant findings for schizophrenia so far are those for COMT and PRODH; A SNP in 30 -UTR of CLDN5 has been reported to be associated with schizophrenia; ARVCF is located immediately next to COMT, thus a positional candidate; TBX1 is a major candidate gene for 22q11 deletion syndrome; ZDHHC8 has been reported to be associated with schizophrenia in both case-control and family-based studies; RTN4R is a crucial regulator of neurite outgrowth, and furthermore its missense mutations have been detected only in schizophrenic patients; ZNF74 and PCQAP have been reported to be significantly associated with age-at-onset of schizophrenia; Polymorphisms in promoter regions of UFD1L and SNAP29 have been reported to be associated with schizophrenia; XBP1 has been found to be associated with both schizophrenia and bipolar disorder; YWHAH has been reported to be associated with schizophrenia by several groups; BZRP has been found to be associated with schizophrenia in Japanese; MLC1 has been observed to be associated with schizophrenia in Southern India; The expression of APOL1, APOL2, APOL4 and PVALB has been found significantly altered across the subjects with schizophrenia; DGCR14, GSTT2, SERPIND1, FBXO7 and a gene tentatively called DKFZP434P211 showed activity differences between haplotypes of greater than 1.5-fold, and we prioritize DGCR14 gene in the present study for its special position (within the Minimum deletion region and near the PRODH) and some other reasons mentioned in the Discussion section

DGCR14 and schizophrenia

H. Wang et al.

majority of the haplotype diversity (97.9%). The global P-value for these four haplotypes was 0.00032 (X2 ¼ 18.65, 3df), of which the most significant overtransmitted haplotype was the most common one namely ‘‘AG’’ (frequency ¼ 82.9%, Z ¼ 3.744, uncorrected P ¼ 0.00018). The haplotype ‘‘TA’’ also showed evidence of association with schizophrenia (frequency ¼ 15%, Z ¼ 3.08, uncorrected P ¼ 0.0021). We searched transcription factor binding sites of the promoter region through MatInspector of Genomatix (http:==www. genomatix.de=) and found that all the four SNPs were within some putative transcription factor (TF) binding. For V1 (rs737923), the change from A to G caused the loss of STAT.01 and BCL6.02 TF binding sites, while created GRE.01 TF binding sites. For V2 (rs1936950), the change from A to T created four new TF binding sites (ARNT.01, HIF1.02, EGR3.01 and XBP1.01). For V3 (rs1936951), the change from G to A caused the loss of ROAZ.01 TF binding sites, while created MZF1.01 TF binding sites. For V4 (rs3810598), the change from T to C caused the loss of COMP1.01 TF binding sites, while created PAX6.01 TF binding sites. Furthermore, it is intriguing that Hoogendoorn and his colleagues (2004) reported that either V2 or V3 or both combined might affect transcription of the DGCR14 gene by a factor of two. An alternative possibility is that these SNPs are in LD with other polymorphisms that affect transcription and=or splicing. Our family-based association analysis revealed that the V2-A and V3-G were preferentially transmitted to schizophrenic males. In our previous study using the same panel of samples, we also found differences between genders in respect of the 18q12.3 gene PIK3C3, which was proved to be a susceptible gene in three independent research studies including our own. Although our evidence for the distorted transmission of DGCR14 alleles to male individuals with schizophrenia could be a chance finding, it

fits well with the meta analysis evidence of gender differences in the risk of schizophrenia which shows that males are more often affected than females, with a mean risk ratio of 1.32 (Aleman et al., 2003). Power analysis showed that the male group (147 trios) had powers of 59.4% and 85.7% (alpha<0.05) in an additive model with a genotype relative risk of 1.5 and allele frequencies of 0.15 and 0.3, respectively. By contrast, the female group (88 trios) only had powers of 39.7% and 64.9% under the same situation. Although our results suggest that the V2-A and V3-G alleles are risk factors only for males, it must be viewed as hypothesis generating rather than conclusive, given the smaller size of the female trios group compared to the male trios group and the consequent possibility of a type II error. To our knowledge, this is the first study to examine the association between the DGCR14 locus and schizophrenia. What we have established is an association between schizophrenia and a possible susceptible haplotype V2–V3 (A–G) which possesses a genetic tendency for increased DGCR14 expression. Our work provides evidence that the DGCR14 gene is positively associated with schizophrenia in the Chinese Han population, although the association observed in the present study between the DGCR14 promoter variant and schizophrenia in males only might reflect differences between genders. Our findings need independent replication with different ethnic populations. Further studies are necessary to investigate the genetic mechanism of how the DGCR14 gene may be involved in the pathogenesis of schizophrenia. Acknowledgements We sincerely thank all the subjects for their participation in this study and all the medical staff involved in specimen collecting. This work was supported by grants from the Shanghai Municipal Commission of Science and Technology, and the Ministry of Education, China, the National 863 and 973 Programs of China, the National Natural Science Foundation of China.

DGCR14 and schizophrenia

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Ye L, Sun Z, Xie L, Liu S, Ju G, Shi J, Yu Y, Zhang X, Wei J, Xu Q, Shen Y (2005) Further study of a genetic association between the CLDN5 locus and schizophrenia. Schizophr Res 75: 139–141 Authors’ address: Dr. L. He, Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China, e-mail: [email protected]