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A single nucleotide polymorphism (rs7341475) in RELN has recently been shown to be associated with schizophrenia (SZ) in an Ashkenazi Jewish (AJ) case-control study specifically in women by Shifmen et al. We have replicated this association in women in another large independent AJ collection (721 cases, 259 female; 1455 controls, 834 female) and confirmed that it applies to both SZ and schizoaffective disorder. Further, we explore the effects of this polymorphism through quantitative trait loci (QTL) analysis of 9 SZ related factors providing information on sex-specific genotype-phenotype correlations.
Schizophrenia (SZ) is a common, disabling, neuropsychiatric disorder with onset typically in young adults and characterized by multiple sex-specific phenotypic differences: females make up only ~30% of the cases, have a later age of onset, higher familiality and a somewhat different presentation (more affective symptoms). Consistent with these sex-specific phenotypic differences, a recent study by Shifman et al. (Shifman et al. 2008) reported that a common DNA variant (rs7341475) in RELN was associated with an increased risk for SZ in Ashkenazi Jewish (AJ) females but not males. RELN is expressed at peak levels in the fetal brain during the 2nd trimester and encodes a ~388 kDa protein whose function is important for corticogenesis. Consistent with the hypothesis that SZ is a neurodevelopmental disorder (Sawa, Snyder 2002), certain regions of brains from SZ patients show a significant reduction in RELN expression (~50%) (Impagnatiello et al. 1998).
We have a large, independent collection of AJ SZ patients and controls and therefore sought to replicate the association results and observed sex effect of Shifman et al. We also explored the possible relationship between phenotype and rs7341475 genotype in this sample by performing quantitative trait loci (QTL) analysis with 9 phenotypic factors, derived from our recently published principal components factor analysis of 73 items from our consensus diagnostic ratings and direct assessment interviews (McGrath et al. 2009).
Our analysis sample of 721 AJ SZ probands and 720 AJ controls was collected by the Johns Hopkins Epidemiology-Genetics Program in Psychiatry (http://www.hopkinsmedicine.org/epigen/), and has been described in detail elsewhere (Fallin et al. 2003). Of the 721 AJ SZ probands, 124 (~17%) are schizoaffective patients. We also included an additional 735 control individuals of AJ descent from the New York Cancer Project (NYCP) Biorepository (Foulkes et al. 2002).
We genotyped SNP rs7341475 using the Taqman 5′-exonuclease allelic discrimination assay according to the manufacturer’s recommendations in 721 independent AJ SZ cases and 1455 AJ controls. We performed a one-tailed Chi-Square test, considering only results consistent with the original study of Shifman et al. as statistically significant, i.e, AJ female SZ cases would carry more G alleles than controls. In our sample 17% of the affected individuals have the diagnosis of schizoaffective disorder (SZA). Patients with this diagnosis were specifically eliminated from the analysis performed by Shifman et al. (Shifman et al. 2002), but explored in a later study and found to show the same association (Pisante et al. 2009). For this reason we also explored the effect of including or removing SZA individuals.
To further investigate the possibility of complexity in the genotype-phenotype relationship, we performed a QTL analysis (UNPHASED v3.0.9) using the 9 factor scores derived from 73 dichotomous signs and symptoms from each of the 721 AJ SZ cases (McGrath et al. 2009; *Dudbridge 2006). These factors were labeled: affective (AFF), child/adolescent sociability (CHA), disorganization (DIS), hallucination (HAL), disability/impairment (IMP), negative (NEG), delusion (DEL), prodromal (PRO), and scholastic (SCH).
The SNP rs7341475 was successfully genotyped in totaling 2176 individuals and the genotype frequencies were in Hardy-Weinberg equilibrium. The distribution of genotypes and alleles of rs7341475 is shown in the Table 1. As predicted, we observed a consistent and significant (one tail p=0.0475) difference in case versus control allele frequencies only in AJ women with an Odds Ratio of 1.23.
When we subtracted the SZA individuals from our cases and repeated the analysis, we observed the same trend but with reduced statistical significance (one tail p=0.065, data not shown) again in AJ females only. We conclude that the SZA patients contribute to the association and excluding them reduces the sample size and, consequently, the statistical power. This result is consistent with the result published later by the same group (Pisante et al. 2009).
For our QTL analysis with the 9 factors from the McGrath et al factor analysis (McGrath et al. 2009) , as shown in the Figure 1, two factors (CHA, HAL) show nominal association with rs7341475 with the best signal (p=0.018) in male-allelic analysis for factor CHA and the second best signal (p=0.035) in female-genotypic analysis for factor HAL. In both cases, the G allele is nominally associated with higher scores (i.e., more symptoms). However, these results do not withstand multiple test correction and we include them only to suggest approaches that could improve the power of future replication attempts and provide information on the complex nature of the observed associations.
In summary, we have replicated the genetic association between a common variant in RELN and SZ in AJ women reported by Shifman et al. in a population of the same origin. Our results strengthen the evidence for RELN’s involvement in SZ and provide sex specific connections of the disease-associated variant with phenotypic features of SZ. It is of interest that RELN expression has also been shown to be sex-related in SZ patients (Eastwood, Harrison 2003). Our data add to the accumulating evidence that implicate RELN in SZ through sex specific mechanism(s).
We thank all the patients and controls for their participation in this research study. This study was supported by a grant from the Johns Hopkins Brain Science institute (DV and AP). The collection of the Ashkenazi Jewish patients and controls was funded by National Institutes of Mental Health grant RO1MH057314 and RO1MH068406 (AP). None of the authors have any conflict of interest concerning the manuscript.
Grant from the Johns Hopkins Brain Science Institute (DV, AP)
Grant from National Institutes of Mental Health grant RO1MH057314 and RO1MH068406 (AP)
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