Family-based and caseтАУcontrol study of catechol-O-methyltransferase in schizophrenia among Palestinian Arabs.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 119B:35 – 39 (2003) Family-Based and Case–Control Study of Catechol-O-Methyltransferase in Schizophrenia Among Palestinian Arabs I. Kremer,1 M. Pinto,2 I. Murad,3 M. Muhaheed,4 I. Bannoura,3 D.J. Muller,5 T.G. Schulze,5 A. Reshef,1 M. Blanaru,1 S. Gathas,1 R. Goichman,1 M. Rietschel,5 M. Dobrusin,6 R. Bachner-Melman,2 L. Nemanov,2 R.H. Belmaker,6 W. Maier,5 and R.P. Ebstein2* 1 Emek Hospital, Afula, Israel Herzog Hospital, Jerusalem, Israel 3 Dr. Kemal Psychiatric Hospital, Bethlehem, Palestine 4 The Palestinian Research Center for Genetics of Mental Disorders, Palestine 5 Department of Psychiatry, University of Bonn, Germany 6 Beersheva Mental Health Center, Beersheva, Israel 2 COMT is a ubiquitous enzyme crucial to catechol metabolism. The molecular basis of COMT thermolability, that leads to three to fourfold differences in enzyme activity, is due to a substitution of valine with methionine in the Val158/108Met polymorphism. Of special interest is the role of this gene in major psychoses especially since a microdeletion (22q11) containing the COMT gene (velo-cardio-facial syndrome) also carries with it several types of behavioral disorders, including an increased prevalence of schizophrenia. Almost 20 genetic studies have examined the role of COMT in schizophrenia with ambiguous results. Towards clarifying the role of this polymorphism in conferring risk for psychosis, we examined a large group of culturally and ethnically akin Palestinian Arab schizophrenic triads (N ¼ 276) using both a case–control and family-based study. In 194 informative triads with at least one heterozygote parent, no preferential transmission of either COMT allele was observed in this sample (TDT statistic chi-square ¼ 0.14 NS; 131 COMT valine alleles were transmitted and 125 alleles not transmitted). However, using a case–control design a Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ri 908/3-1. *Correspondence to: R.P. Ebstein, Research Laboratory, S. Herzog Memorial Hospital, Rehov Givat Shaul, P.O. Box 35300, Givat Shaul, Jerusalem 91351, Israel. E-mail: firstname.lastname@example.org Received 14 October 2002; Accepted 9 December 2002 DOI 10.1002/ajmg.b.20008 ß 2003 Wiley-Liss, Inc. significant increase (Likelihood ratio ¼ 3.935, P ¼ 0.047) in the valine allele was observed in the group of schizophrenic patients (N ¼ 276) compared to an ethnically matched control group (N ¼ 77). The association was stronger in female patients (P ¼ 0.012) similar to other studies showing that some COMT behavioral effects are gender sensitive. In summary, by case–control design but not by a familybased study, there is a weak effect in female patients of the high activity COMT allele in conferring risk for schizophrenia. ß 2003 Wiley-Liss, Inc. KEY WORDS: catechol-O-methyltransferase; schizophrenia; Arab; transmission disequilibrium test; association; linkage INTRODUCTION Catechol-O-methyltransferase (COMT) is a ubiquitous enzyme crucial to catechol metabolism [Mannisto and Kaakkola, 1999]. The physiological substrates of COMT include L-dopa, catecholamines (dopamine, norepinephrine, epinephrine), their hydroxylated metabolites, and catecholestrogens. The level of COMT enzyme activity is genetically polymorphic in human tissues with a trimodal distribution of low, intermediate, and high activities [Weinshilboum and Raymond, 1977]. This polymorphism, which according to segregation analysis of family studies is caused by autosomal codominant alleles, leads to three to fourfold differences in COMT activity in human erythrocytes and liver. Low COMT activity is associated with enzyme thermolability, even at 378C. The molecular basis of the thermolability is due to a substitution of valine by methionine, (Val158/108Met) caused by transition of guanine to 36 Kremer et al. adenine at codon 158 of the COMT gene [Lotta et al., 1995; Lachman et al., 1996b]. Many genetic studies have focused on the possible clinical relevancy of the COMT polymorphism in psychiatric disorders. Although some of these yielded uncertain results there are several disorders in which some relationship has been observed. First, obsessivecompulsive disorder (OCD) has been correlated in some [Karayiorgou et al., 1997, 1999; Alsobrook et al., 2002] but not all studies [Ohara et al., 1998] to low COMT activity alleles. Second, low COMT activity allele appears to be associated with aggressive and highly antisocial impulsive schizophrenia [Strous et al., 1997a; Lachman et al., 1998; Kotler et al., 1999]. The high enzyme activity COMT valine allele has also been shown to confer risk for polysubstance abuse and heroin addiction [Vandenbergh et al., 1997; Horowitz et al., 2000]. Of special interest is the role of this gene in major psychoses especially since a microdeletion (22q11) containing the COMT gene (velo-cardio-facial syndrome) also carries with it several types of behavioral disorders, including a higher prevalence of schizophrenia than in the general population [Pulver et al., 1994; Lachman et al., 1996a; Murphy et al., 1998, 1999]. Almost 20 genetic studies have examined the role of COMT in schizophrenia with ambiguous results [Chen et al., 1996; Li et al., 1996; Karayiorgou et al., 1997, 1999; Kunugi et al., 1997; Strous et al., 1997a,b; Ohmori et al., 1998; Wei and Hemmings, 1999; Nolan et al., 2000; de Chaldee et al., 2001; Egan et al., 2001; Liou et al., 2001; Semwal et al., 2001]. Six of these studies were based on a family-based design [Kunugi et al., 1997; de Chaldee et al., 1999, 2001; Wei and Hemmings, 1999; Egan et al., 2001; Semwal et al., 2001]. The most recent familybased study in a Chinese population examined the transmission of five SNP markers in the COMT gene from parents to schizophrenic offspring in 166 trios and found no evidence for preferential transmission of any allele or haplotype [Fan et al., 2002]. A recent case–control study of a large number of non-related Ashkenazi Jews showed a highly significant association between haplotypes at the COMT locus and schizophrenia [Shifman et al., 2002]. On the other hand, a British study also with a substantial number of patients failed to find an association using a case–control design when two polymorphisms (Val158/108Met and a promoter region variant) in the COMT gene were examined [Norton et al., 2002]. A recent article by Egan et al.  rekindled interest regarding the role of COMT in schizophrenia. These authors found that the COMT genotype was related in allele dosage fashion to performance on the Wisconsin Card Sorting Test of executive cognition. Consistent with other evidence that dopamine enhances prefrontal neuronal function the low-activity methionine allele predicted enhanced cognitive performance. The effect of COMT genotype on prefrontal physiology during a working memory task assayed with functional MRI was then examined. Subjects with the methionine allele showed a more efficient physiological response in prefrontal cortex. Finally, in a family-based association analysis of 104 trios, Egan et al.  found a significant increase in transmission of the valine allele to the schizophrenic offspring. The authors interpreted their results to suggest that the COMT valine allele, because it increases prefrontal dopamine catabolism, impairs prefrontal cognition and physiology, and by this mechanism slightly increases risk for schizophrenia. The intriguing finding of Egan et al.  prompted us to examine the COMT polymorphism and schizophrenia in the largest group of nuclear families (parents and proband) so far examined for this gene. The robust transmission disequilibrium test design [Spielman et al., 1993; Ewens and Spielman, 1995] that is not sensitive to population admixture, a common cause of type I errors in case–control studies, was employed in the analysis. Schizophrenic subjects were recruited from a culturally and ethnically akin Palestinian Arab population that we are studying in schizophrenia [Dobrusin et al., 2001; Murad et al., 2001] and bipolar disorder [Mujaheed et al., 2000]. In addition to the TDT design we also examined the association between COMT and schizophrenia using a case–control strategy. MATERIALS AND METHODS Clinical Sample All patients were interviewed by an experienced psychiatrist using the SCID interview. Diagnosis of schizophrenia was assigned on the basis of the interview and medical records according to DSM IV criteria [American Psychiatric Association, 1994]. The sample was constituted of 248 nuclear families (276 probands) from 3 different centers: 136 families from Bethlehem, 62 families from Afula, and 50 families from Beersheba. Twenty-four families contained more than one affected child. The mean age of the schizophrenic probands was 34.8 13.6 years and included 31% men and 69% women. The average age of onset was 21.0 4.8. The control sample consisted of Palestinian Arabs recruited from the Northern area of Israel. Their average age was 39.5 15.5 years and consisted of 31% men and 69% women. The control group was randomly recruited from subjects visiting a family medicine (not psychiatric) national health care clinic (Kupat Holim) or minor medical ailments. The protocol for recruiting both control and schizophrenic families was approved by the local IRB committees and all subjects or their legal representative gave informed consent. Genotyping Genomic DNA containing the COMT polymorphism was amplified as previously described in our laboratory [Eisenberg et al., 1999] using the following primers: F 50 ACT GTG GCT ACT CAG CTG TG 30 R 50 CCT TTT TCC AGG TCT GAC AA 30 . The PCR product was purified using exonuclease 1 and SAP (shrimp alkaline phosphatase) for 15 min at 808C. Following purification the Val158/108Met polymorphism was genotyped using a ABI PRISM1 SNaPshotTM Multiplex System for SNP Analysis (Applied COMT and Schizophrenia Biosystems, Foster City, CA). The primer used in the sequencing/extension reaction was 50 TGC ACA CCT TGT CCT TCA 30 . The polymorphism extension products were identified using an ABI Prism 310 Genetic Analyzer. TABLE II. COMT Allele Frequency Tabulated by Diagnosis Sex Male Val Met Statistical Procedures All statistical analyses were carried out using SPSS for Windows. Power calculations were carried out courtesy of a convenient web site (http://statgen.iop. kcl.ac.uk/gpc/) kindly maintained by S. Purcell and P. Sham at the Social, Genetic & Developmental Research Centre, London. The TDT test was carried out as described in the article by Daly and his colleagues [Daly et al., 1999]. All significance levels were two-sided. No correction was made for Type I error rate. 37 Female Val Met Control Patients Total 22 91.67 2 8.33 24 31 58.49 22 41.51 53 124 77.02 37 22.98 161 73 77.66 21 22.34 94 146 Count % Count % Total count Count % Count % Total count 39 185 104 43 147 statistic ¼ 1.64, P ¼ 0.2; female probands TDT statistic ¼ 0.576, P ¼ 0.45). RESULTS Genotype and allele frequency of the COMT Val158/ 108Met polymorphism in schizophrenic patients and an ethnically matched control group is shown in Table I. There is an insignificant excess of the valine/valine genotype (Likelihood ratio ¼ 4.02, P ¼ 0.134, 2df) in the schizophrenic subjects. However, when allele frequencies are compared excess of the valine allele in schizophrenia attains significance (Likelihood ratio ¼ 3.935, P ¼ 0.047, 1df). The odds ratio indicated a minor effect of the valine allele in this population (OR ¼ 1.19; 95% CI 1.01–1.402) as observed in some [Egan et al., 2001] but not all previous studies of schizophrenia. Interestingly, when subjects are examined by gender diagnosis (Table II) the association between the valine allele and schizophrenia is significant in female patients (Likelihood ratio ¼ 5.89, P ¼ 0.015) but not in male subjects (Likelihood ratio ¼ 3.21, P ¼ 0.073). Indeed in male subjects, the trend is in the opposite direction for decreased prevalence of the valine allele in the schizophrenic group. Possible preferential transmission of alleles was also examined in informative families using the TDT design that examines transmission of either the valine or methionine allele from a heterozygote parent to the proband. As shown in Table III, no preferential transmission of either allele was observed (TDT statistic ¼ 0.14, NS) in the entire sample and when each geographical recruitment center was separately examined. Nor was any preferential transmission observed when probands were sorted by gender (male probands TDT DISCUSSION By case–control analysis the current study offers weak support to some findings showing an excess of the COMT high enzyme valine allele in schizophrenia. Interestingly, these results are only significant in female subjects. This tendency may be related to consistent gender effects in schizophrenia; male patients having an earlier age of onset, poorer functional outcome, greater negative symptoms and cognitive impairment, and less severe positive symptoms [Leung and Chue, 2000; Moriarty et al., 2001; Roy et al., 2001]. Regarding cognitive function the direction of the gender difference is unclear and two recent articles suggest that female patients show more cognitive impairment [Faraone et al., 2000; Weiser et al., 2000]. In a recent case–control study of a large number of Ashkenazi Jews the association with schizophrenia was also stronger for some haploytpes in female patients [Shifman et al., 2002]. When we attempted to validate our case–control findings using a family based design, no evidence for preferential transmission of the valine allele from heterozygous parent to proband was observed overall as well as when families with male and female probands were examined separately. Failure to observe a significant association using the TDT design suggests that the results obtained using the case–control approach is a false positive, perhaps related to undetected population stratification in our sample of Palestinian Arabs [Hamer and Sirota, 2000]. However, an alternative TABLE I. COMT Genotype and Allele Frequency of Schizophrenic Patients and Ethnically Matched Control Subjects Genotype Control SCZ Count % Count % Total Allele Val/val Val/met Met/met Total Val Met Total 14 18.18 77 27.90 91 39 50.65 136 49.28 175 24 31.17 63 22.83 87 77 100 276 100 353 67 43.51 290 52.54 357 87 56.49 262 47.46 349 154 100 552 100 706 38 Kremer et al. TABLE III. TDT Analysis of Val108/Met108 Allele Transmission From a Heterozygote Parent to Schizophrenic Proband All locations Val Met Bethlehem Val Met Afula Val Met Beersheva Val108 Met108 Transmitted Not transmitted de Chaldee M, Laurent C, Thibaut F, Martinez M, Samolyk D, Petit M, Campion D, Mallet J. 1999. Linkage disequilibrium on the COMT gene in French schizophrenics and controls. Am J Med Genet 88(5):452– 457. 131 125 125 131 de Chaldee M, Corbex M, Campion D, Jay M, Samolyk D, Petit M, Thibaut F, Laurent C, Mallet J. 2001. No evidence for linkage between COMT and schizophrenia in a French population. Psychiatry Res 102(1):87–90. 77 72 72 77 32 33 33 32 22 20 20 22 explanation related to power considerations also needs to be addressed. For example, the minor risk conferred by the valine allele for schizophrenia calculated from the current case–control design suggests that our sample of 276 triads is unlikely to provide sufficient power to detect preferential transmission of the valine allele using the TDT design. Indeed, a sample size of several thousand (3,792 triads, 80% power) would be required for detecting preferential transmission of the valine allele assuming a genotype relative risk (valine/valine) of 1.2 for the valine/valine homozygotes. The COMT Val158/108Met allele in schizophrenia should be evaluated in light of a recent article by Ioannidis et al.  who evaluated by meta-analysis 370 studies addressing 36 genetic associations for various outcomes of disease. 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