Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia

Abstract

Linkage studies have implicated 10q22-q23 as a schizophrenia (SZ) susceptibility locus in Ashkenazi Jewish (AJ) and Han Chinese from Taiwan populations. To further explore our previous linkage signal in the AJ population (NPL score: 4.27, empirical p = 2 x 10(-5)), we performed a peakwide association fine mapping study by using 1414 SNPs across approximately 12.5 Mb in 10q22-q23. We genotyped 1515 AJ individuals, including 285 parent-child trios, 173 unrelated cases, and 487 unrelated controls. We analyzed the binary diagnostic phenotype of SZ and 9 heritable quantitative traits derived from a principal components factor analysis of 73 items from our consensus diagnostic ratings and direct assessment interviews. Although no marker withstood multiple test correction for association with the binary SZ phenotype, we found strong evidence of association by using the "delusion" factor as the quantitative trait at three SNPs (rs10883866, rs10748842, and rs6584400) located in a 13 kb interval in intron 1 of Neuregulin 3 (NRG3). Our best p value from family-based association analysis was 7.26 x 10(-7). We replicated this association in the collection of 173 unrelated AJ cases (p = 1.55 x 10(-2)), with a combined p value of 2.30 x 10(-7). After performing 10,000 permutations of each of the phenotypes, we estimated the empirical study-wide significance across all 9 factors (90,000 permutations) to be p = 2.7 x 10(-3). NRG3 is primarily expressed in the central nervous system and is one of three paralogs of NRG1, a gene strongly implicated in SZ. These biological properties together with our linkage and association results strongly support NRG3 as a gene involved in SZ.

Figure 1

AJ Individuals Enrolled in This Study For analyses with SZ as a binary phenotype, the subjects for various scenarios are: “Trios,” (a)+(b); “CC-all” (case-control analysis with all SZ individuals), (b)+(c)+(d); “Combined” (all samples), (a)+(b)+(c)+(d). For analyses with factors as quantitative traits, the subjects for various scenarios are: “Trios,” (a)+(b); “Cases-all” (case-only analysis with all SZ individuals), (b)+(c); “Cases-independent” (case-only analysis with independent SZ individuals), (c); “Combined” (all SZ individuals and trios parents), (a)+(b)+(c).

Figure 2

Results of Association Analyses and Genes in the ∼12.5 Mb Region at Chromosome 10q22-q23 The horizontal axis is the sequence position (chr10:79,550,189-92,037,551) according to NCBI genome build 35.1. (A) Results with SZ as a binary phenotype. Values along the vertical axis are the negative logarithm (based 10) of the p values. CC-all, case-control study of all cases and all controls. Subjects analyzed in various scenarios are described in Figure 1. (B) Results with the nine factors as the quantitative traits. For clarity, we did not assign different colors to different factors. The upper horizontal pink line marks the study-wide significance cutoff value for Bonferroni correction of all 1414 SNPs and all 9 factors (p = 3.93 × 10⁻⁶). The lower horizontal pink line marks the cutoff value for Bonferroni correction of 532 independent SNPs and all 9 factors (p = 1.04 × 10⁻⁵). (C) Genes and segmental duplications in this region, according to UCSC genome browser with the NCBI genome build 35.1 data.

Figure 3

Representative Quantile-Quantile Plots Based on 10,000 Permutations The values plotted are the negative logarithm (based 10) of p values, either of the observed results (vertical axis) or the permutation results (horizontal axis) of the corresponding phenotypes. The three lines in each plot represent the 97.5, 50, and 2.5 percentile values of the permutation. The area between the upper line and lower line represents the 95% confidence interval of the corresponding permutation. The solid circles are the observed values plotted against the permutation values. (A) The quantile-quantile (QQ) plot with SZ as a binary phenotype. Our results show no obvious inflation of type I error. (B) The QQ plot with data from all nine factors analyzed together. There are three observations above the 97.5 percentile of the study-wide permutation data. When those three observations are removed (the result shown in open circles), the distribution shows no evidence of type I error inflation. (C) The QQ plot with the “delusion” factor as a quantitative trait. The three observations with highly significant values are the same as those of (B); removal of those three observations (open circles) yields a distribution that fits the permutation data quite well.

Figure 4

Results of Association Analyses and Selected Genomic Features in and around NRG3 (A) Association analysis results (combined samples) with SZ as the binary phenotype and three factors (scholastic, delusion, and disorganization) as quantitative traits. As in Figure 2, the two short upper horizontal pink lines represent the study-wide significance cut-off p values (either for all SNPs, higher, or for independent SNPs, lower) for all nine factors. The long horizontal pink line (p = 1.34 × 10⁻³) marks the 20^th smallest p value from all 1414 SNPs of the ∼12.5 Mb region with any of the nine factors as the phenotype. We also plot the two segregating deletions (solid rectangles) and the three SNPs (and two flanking SNPs) with the most significant associations. We also provide the NRG3 gene and the LD block structure (based on r² values) for the CEPH (CEU) from the HapMap project and the LD block structure for the AJ population from 487 controls of our current study to show the relative positions of these association results. (B) A closer look at the 5′ region of NRG3. The p values of the same three SNPs (and two flanking SNPs) are indicated.