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Human Molecular Genetics Advance Access originally published online on October 15, 2004
Human Molecular Genetics 2004 13(23):2991-2995; doi:10.1093/hmg/ddh322
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

Case–control study and transmission disequilibrium test provide consistent evidence for association between schizophrenia and genetic variation in the 22q11 gene ZDHHC8

Wu-Yan Chen1,3,{dagger}, Yong-Yong Shi1,3,{dagger}, Yong-Lan Zheng2,3,{dagger}, Xin-Zhi Zhao1,3, Guang-Ji Zhang4, Sheng-Qi Chen5, Pei-Di Yang6 and Lin He2,3,*

1Bio-X Life Science Research Center and 2NHGG, Bio-X Center, Shanghai Jiao Tong University, Shanghai, China, 3Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai, China, 4Shanghai Zhabei Institute of Mental Health, Shanghai, China, 5Shanghai Yangpu Institute of Mental Health, Shanghai, China and 6Shanghai Changning Institute of Mental Health, Shanghai, China

Received August 14, 2004; Revised September 19, 2004; Accepted September 29, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic variants in the 22q11 gene ZDHHC8, which encodes a putative transmembrane palmitoyltransferase, has been associated to schizophrenia in family-based linkage disequilibrium (LD) studies. The single nucleotide polymorphism (SNP) rs175174 (A/G), which had the strongest association, has been shown recently to regulate the level of the fully functional transcript by modulating the retention of intron 4 of ZDHHC8. In this work, we genotyped three genetic variants within the ZDHHC8 locus and conducted association studies in both population- and family-based samples of the Han Chinese population. The three polymorphisms spanning ~5.5 Kb were detected to be in significant LD. Our results provided compelling supportive evidence for association of the variants within the ZDHHC8 locus with schizophrenia but revealed different risk allele at SNP rs175174. The G allele was significantly more common in cases than in controls (69.47 : 59.96%; P=0.000018) and excess transmission of the same allele was confirmed in the family-based transmission disequilibrium test (transmitted/non-transmitted=87 : 54; P=0.0055). Both sample sets even shared the same risk haplotype with similar frequency. Our current data presents consistent association results obtained from both case–control and family-based samples in a same laboratory under the same experimental condition. Despite the potential genetic heterogeneity, our independent findings further support that the 22q11 region is likely to harbor candidate schizophrenia susceptibility genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Schizophrenia (MIM 181500) is a complex disorder affecting ~1% of the population worldwide. The disorder is characterized by psychotic symptoms and by cognitive, affective and psychosocial impairment. Although epidemiologic evidence, together with recent linkage and association studies, clearly demonstrates high heritability of schizophrenia (~80%), the genetic mechanism underlying the disorder is still obscure (1,2).

An increased prevalence of microdeletions at chromosome 22q11 has been reported in patients with schizophrenia and 22q11 microdeletions represent the highest known genetic risk factor for the development of the disorder (3). Chromosome 22q11 is therefore believed to be a region that harbors susceptibility genes to schizophrenia, which is also supported by the results of independent linkage studies (4,5). Whereas deletions of chromosome 22q11 may account for only a small proportion of schizophrenia cases in the general population (up to ~2%), non-deletion common variants of specific gene within this region may make a larger contribution to susceptibility to schizophrenia in the wider population (6). On the basis of this assumption, Liu et al. (6) conducted detailed linkage disequilibrium (LD) studies in family samples to capture association signal of specific genes with schizophrenia by studying a dense collection of 72 SNPs across the 1.5 Mb deletion locus on 22q11. SNP rs175174 within the gene ZDHHC8, which encodes a putative transmembrane palmitoyltransferase, had the strongest association of all 72 SNPs. This SNP was recently indicated to regulate the level of the fully functional transcript by modulating the retention of intron 4 of ZDHHC8 (7). The results from Mukai et al. (7) suggest that ZDHHC8 affects behavior at least partly by interfering with glutamateric transmission, an important system involved in the development of schizophrenia.

Given the complexity of complex disorders, it is believed that supportive evidence in other independent samples would enhance the significance of the original association considerably. In light of the recent findings of association between schizophrenia and ZDHHC8, we decided to conduct an association study using both population- and family-based samples of the Han Chinese population. We investigated three genetic variants [rs175174 in intron 4; rs175179 and a C ins/del polymorphism in the 3'-untranslated region (3'-UTR)] within ZDHHC8 locus in attempt to confirm the initial study and to discover potential susceptible variants or haplotypes in the Han Chinese.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To confirm the association of ZDHHC8 with schizophrenia, we used a two-stage association study design in this work. First we performed a case–control study as this generally has greater power to detect association than the family-based transmission disequilibrium test (TDT) design. Allele and genotype frequencies of SNP rs175179 and C ins/del polymorphism are described in Table 1. Because SNP rs175174 (A/G) was in complete LD with rs175179 (A/G) (r2=1), the data of rs175179 here were used to surrogate that of rs175174. In fact, SNP rs175179 and C ins/del polymorphism were also in significant LD (r2=0.61). Genotype distributions of each polymorphism had no deviation from Hardy–Weinberg equilibrium. Both allele and genotype distributions of SNP rs175179 and C ins/del polymorphism differed significantly between cases and controls (Table 1).


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  		Table 1. Allele and genotype frequencies of SNP rs175179 and C ins/del polymorphisms in cases (n=465) and controls (n=467)
 
In the second stage, to rule out the potential risk of false positives arising from population stratification in case–control studies, a family-based TDT was subsequently conducted in the same population. The TDT results (Table 2) indicated that the G allele of SNP rs175179 (or rs175174) was transmitted from parent to offspring more frequently (transmitted/non-transmitted=87 : 54; P=0.0055; OR= 1.61). With regard to C ins/del polymorphism, the deletion allele was overtransmitted to affected offspring (transmitted/non-transmitted=92 : 66; P=0.039; OR=1.40).


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  		Table 2. TDT for family-based sample of SNP rs175179 and C ins/del polymorphisms
 
The allele G of SNP rs175179 was significantly more common in the patients than in the controls (69.47 : 59.96%; P=0.000018; OR=1.52, 95% CI 1.25–1.83) and excess transmission of the same allele was confirmed in the family-based analysis. The current data indicate that allele G of rs175174 could be the susceptibility allele for schizophrenia or allele A may be a protective factor. This observation is in contrast to the results reported by Mukai et al. (7) in which A was shown to be the risk allele.

We also performed a haplotype analysis based on all the three polymorphisms (rs175174–rs175179–C ins/del). One risk 1–1–1 (G–G–del) and one protective haplotype 2–2–2 (A–A–ins) were identified (Table 3), which is consistent with the results of the single markers, since the three polymorphisms were in significant LD. The estimated haplotype frequencies obtained from the family-based sample and the cases of population-based sample were very similar. The global P-value (both df=2) from case–control study and TDT were 0.000065 and 0.02 (using UNPHASED), respectively. To verify whether the disease association to C ins/del is caused by the LD to rs175174–rs175179 haplotype, we conducted a case–control analysis in the individuals who carried G–G at rs175174–rs175179. No significance (P=0.93) was found in this analysis (Table 4), which revealed that the disease association to C ins/del can be explained by LD to the rs175174–rs175179 haplotype.


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  		Table 3. Haplotype analysis (rs175174–rs175179–C ins/del) for case–control and family-based TDT studies
 

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  		Table 4. The disease association to C ins/del can be explained by LD to the rs175174–rs175179 haplotype
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using both case–control and family-based samples of the Han Chinese population, we attempted to confirm the original and the subsequent association studies implicating ZDHHC8 at 22q11 as a susceptibility gene for schizophrenia. We investigated genetic variants within the ZDHHC8 locus, including the most significant SNPs rs175174 and rs175179 which were revealed to be in complete LD in our sample. In addition, we genotyped a C ins/del polymorphism within 13 bp downstream to rs175179 in 3'-UTR. All these polymorphisms showed significant association with schizophrenia in both case–control study and the family-based analysis.

The consistent polymorphisms (rs175174 and rs175179) within ZDHHC8 were successfully confirmed to be associated to schizophrenia in our current study. In contrast to the original results reported by Mukai et al. (7), however, our findings indicated that allele G instead of A was the susceptibility allele at SNP rs175174. In the report of Mukai et al. (7), SNP rs175174 was shown to affect the relative abundance of the unspliced form of ZDHHC8, thereby regulating the level of the gene activity. The effect of rs175174 was reported to be modest and the extent that the unspliced product bearing a premature termination codon efficiently escapes nonsense-mediated decay in types of cells is still unknown (7). On the basis of the inconsistent findings with regard to the risk allele at rs175174, the exact functional effect of rs175174 requires to be further clarified.

The presence of complex patterns of association findings in complex disorders such as schizophrenia has been illustrated (8–10). Since our association study was based on a distinct ethnic population, genetic heterogeneity could account for the discrepancy between the different studies. The difference between the initial report and our findings regarding the risk allele of SNP rs175174 implicates that genetic variants conferring susceptibility for schizophrenia may be inherited in the Han Chinese on a different haplotype. In the previous associations of neuregulin 1 gene with schizophrenia, different risk haplotypes have been reported in Icelandic/Scottish population and the Han Chinese (11). These findings may reflect differences in allele frequency or in LD structure across different populations. It is possible that the same disease causative variants to be identified exist in both populations but they are associated with different haplotypes. Therefore, identification of the genuine functional variants sending risk to the disorder will clarify this point.

The significant LD among the three polymorphisms spanning ~5.5 kb implicates the presence of strong LD around the ZDHHC8 locus and even in the larger region embracing the gene. Therefore, our observation suggests that existence of additional functional variants in LD with SNP rs175174 or even an interaction effect on the risk haplotype may not be excluded (12). The functional variants could locate within ZDHHC8 or other genes around this locus at 22q11 and may independently or synergistically exert increased risk for the manifestation of schizophrenia. Further inquiry is needed for a detailed insight into the connection between genetic variations in this region and the disorder. Nevertheless, so far, our current data has introduced further support for the notion that the 22q11 region is likely to harbor candidate schizophrenia susceptibility genes.

In summary, this study provided supportive evidence for the potential association of ZDHHC8 gene with schizophrenia. Our current data represents consistent results obtained from both case–control and family-based samples in a same laboratory under the same experimental condition. The family-based TDT following the case–control study indicates well that the positive association is not likely attributable to population stratification. Since our findings implicate potential genetic heterogeneity in different populations, further work from different populations will still be required to investigate the exact role of ZDHHC8 in the pathogenesis of schizophrenia. A gene-based replication strategy, which aims to capture specific risk-conferring variations in different populations, will be an efficient approach to clarify the complexity of association findings (13). In addition, the region surrounding ZDHHC8 at 22q11 should be the next goal in further exploration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
For the case–control study, 465 unrelated individuals with schizophrenia (245 males and 220 females with a mean age of 46.39 years, SD=12.87) and 467 control subjects (216 males and 251 females with a mean age of 29.10 years, SD=9.98) were recruited. The average onset age of disease is 26.71 years (SD=8.22). For the family-based analysis, 158 unrelated schizophrenia probands (100 males and 58 females with a mean age of 23.86 years, SD=6.60) and their biological parents were recruited. Those with schizophrenia were diagnosed strictly according to the criteria of DSM-III-R (American Psychiatric Association, 1987). Written informed consent was obtained from either the participants or the participants' relatives, after the procedure had been fully explained. All subjects were Han Chinese in origin from Shanghai.

Variants identification and genotyping
All the genetic variants tested in this study were genotyped by direct DNA sequencing. The genomic sequence involved in this work was on the basis of NM_013373 (GenBank accession no.). At the beginning, we selected four SNPs [the three positive SNPs reported (6) and a non-synonymous SNP rs5748539] within the ZDHHC8 locus and examined their allele frequencies in 32 healthy Han Chinese individuals. The SNPs rs175164 in the 5' upstream region and rs5748539 in exon 10 were found to be not polymorphic and then were excluded in the later study. When we genotyped rs175179 in the 3'-UTR, a C ins/del polymorphism was detected to be within 13 bp downstream to rs175179. Upon individual genotyping of 600 persons (300 cases and 300 controls) for rs175174 and rs175179, we found the two SNPs to be in complete LD (r2=1). SNP rs175174 (intron 4) is ~5.4 Kb apart from rs175179 (3'-UTR).

Genomic DNA was prepared from venous blood using the standard phenol chloroform extraction. Genotyping was conducted according to our standard protocol (14) apart from the annealing temperature of individual PCR condition (55°C for rs175179 and C ins/del; 58°C for rs175174). The PCR primers were 5'-CCCCTGGGTCAACAACTGC-3' (forward) and 5'-CCAATGACAGGGATGAAGAAGAG-3' (reverse) for rs175174; 5'-CTCTGTCTTGCTGCCTCCTC-3' (forward) and 5'-CCAGTGAGCCGACTTCCC-3' (reverse) for rs175179 and C ins/del. Both forward primers were used as sequencing primers.

Statistical analysis
For case–control study, CLUMP (version 2.2) (15) implementing a Monte Carlo simulation strategy was used to compare the differences of allele, genotype and haplotype frequencies between cases and controls. The multiple markers' haplotype frequencies were estimated using the program EHPLUS (16,17). For transmission disequilibrium test, ETDT (18) was used to test the number of transmitted and non-transmitted target alleles in single marker analysis. Haplotype analysis was carried out by TRANSMIT program (version 2.5.4) (19) and TDTPHASE program of UNPHASED set (version 2.403) (20). The measure of LD for each pair of markers, denoted as r2, was estimated with program EMLD. All test were two tailed and significance was accepted at P<0.05.


   ACKNOWLEDGEMENTS
 
We would 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 national 973 and 863 programs, the National Natural Science Foundation of China, Shanghai Municipal Commission for Science and Technology, and the Ministry of Education.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Bio-X Center, Shanghai Jiao Tong University, Hao Ran Building, 1954 Hua Shan Road, Shanghai 200030, China. Tel/Fax: +86 2162822491; Email: helin{at}nhgg.org

{dagger} The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Baron, M. (2001) Genetics of schizophrenia and the new millennium: progress and pitfalls. Am. J. Hum. Genet., 68, 299–312.[CrossRef][Web of Science][Medline]

  2. O'Donovan, M.C., Williams, N.M. and Owen, M.J. (2003) Recent advances in the genetics of schizophrenia. Hum. Mol. Genet., 12, R125–133.[Abstract/Free Full Text]

  3. Karayiorgou, M., Morris, M.A., Morrow, B., Shprintzen, R.J., Goldberg, R., Borrow, J., Gos, A., Nestadt, G., Wolyniec, P.S., Lasseter, V.K. et al. (1995) Schizophrenia susceptibility associated with interstitial deletions of chromosome 22q11. Proc. Natl Acad. Sci. USA, 92, 7612–7616.[Abstract/Free Full Text]

  4. Blouin, J.L., Dombroski, B.A., Nath, S.K., Lasseter, V.K., Wolyniec, P.S., Nestadt, G., Thornquist, M., Ullrich, G., McGrath, J., Kasch, L. et al. (1998) Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21. Nat. Genet., 20, 70–73.[CrossRef][Web of Science][Medline]

  5. Shaw, S.H., Kelly, M., Smith, A.B., Shields, G., Hopkins, P.J., Loftus, J., Laval, S.H., Vita, A., De Hert, M., Cardon, L.R. et al. (1998) A genome-wide search for schizophrenia susceptibility genes. Am. J. Med. Genet., 81, 364–376.[CrossRef][Web of Science][Medline]

  6. Liu, H., Abecasis, G.R., Heath, S.C., Knowles, A., Demars, S., Chen, Y.J., Roos, J.L., Rapoport, J.L., Gogos, J.A. and Karayiorgou, M. (2002) Genetic variation in the 22q11 locus and susceptibility to schizophrenia. Proc. Natl Acad. Sci. USA, 99, 16859–16864.[Abstract/Free Full Text]

  7. Mukai, J., Liu, H., Burt, R.A., Swor, D.E., Lai, W.S., Karayiorgou, M. and Gogos, J.A. (2004) Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia. Nat. Genet., 36, 725–731.[CrossRef][Web of Science][Medline]

  8. Straub, R.E., Jiang, Y., MacLean, C.J., Ma, Y., Webb, B.T., Myakishev, M.V., Harris-Kerr, C., Wormley, B., Sadek, H., Kadambi, B. et al. (2002) Genetic variation in the 6p22.3 gene DTNBP1, the human ortholog of the mouse dysbindin gene, is associated with schizophrenia. Am. J. Hum. Genet., 71, 337–348.[CrossRef][Web of Science][Medline]

  9. Schwab, S.G., Knapp, M., Mondabon, S., Hallmayer, J., Borrmann-Hassenbach, M., Albus, M., Lerer, B., Rietschel, M., Trixler, M., Maier, W. et al. (2003) Support for association of schizophrenia with genetic variation in the 6p22.3 gene, dysbindin, in sib-pair families with linkage and in an additional sample of triad families. Am. J. Hum. Genet., 72, 185–190.[CrossRef][Web of Science][Medline]

  10. Williams, N.M., Preece, A., Morris, D.W., Spurlock, G., Bray, N.J., Stephens, M., Norton, N., Williams, H., Clement, M., Dwyer, S. et al. (2004) Identification in 2 independent samples of a novel schizophrenia risk haplotype of the dystrobrevin binding protein gene (DTNBP1). Arch. Gen. Psychiatry, 61, 336–344.[Abstract/Free Full Text]

  11. Li, T., Stefansson, H., Gudfinnsson, E., Cai, G., Liu, X., Murray, R.M., Steinthorsdottir, V., Januel, D., Gudnadottir, V.G., Petursson, H. et al. (2004) Identification of a novel neuregulin 1 at-risk haplotype in Han schizophrenia Chinese patients, but no association with the Icelandic/Scottish risk haplotype. Mol. Psychiatry, 9, 698–704.[Web of Science][Medline]

  12. Peltekova, V.D., Wintle, R.F., Rubin, L.A., Amos, C.I., Huang, Q., Gu, X., Newman, B., Van Oene, M., Cescon, D., Greenberg, G. et al. (2004) Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat. Genet., 36, 471–475.[CrossRef][Web of Science][Medline]

  13. Neale, B.M. and Sham, P.C. (2004) The future of association studies: gene-based analysis and replication. Am. J. Hum. Genet., 75, 353–362.[CrossRef][Web of Science][Medline]

  14. Chen, W., Duan, S., Zhou, J., Sun, Y., Zheng, Y., Gu, N., Feng, G. and He, L. (2004) A case–control study provides evidence of association for a functional polymorphism –197C/G in XBP1 to schizophrenia and suggests a sex-dependent effect. Biochem. Biophys. Res. Commun., 319, 866–870.[CrossRef][Web of Science][Medline]

  15. Sham, P.C. and Curtis, D. (1995) Monte Carlo tests for associations between disease and alleles at highly polymorphic loci. Ann. Hum. Genet., 59(1), 97–105.[Web of Science][Medline]

  16. Xie, X. and Ott, J. (1993) Testing linkage disequilibrium between a disease gene and marker loci. Am. J. Hum. Genet., 53, 1107.[Web of Science][Medline]

  17. Zhao, J.H., Curtis, D. and Sham, P.C. (2000) Model-free analysis and permutation tests for allelic associations. Hum. Hered., 50, 133–139.[Web of Science][Medline]

  18. Sham, P.C. and Curtis, D. (1995) An extended transmission/disequilibrium test (TDT) for multi-allele marker loci. Ann. Hum. Genet., 59, 323–336.[Web of Science][Medline]

  19. Clayton, D. (1999) A generalization of the transmission/disequilibrium test for uncertain-haplotype transmission. Am. J. Hum. Genet., 65, 1170–1177.[CrossRef][Web of Science][Medline]

  20. Dudbridge, F. (2003) Pedigree disequilibrium tests for multilocus haplotypes. Genet. Epidemiol., 25, 115–121.[CrossRef][Web of Science][Medline]


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