Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia

Abstract

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.

Figure 1. Detection and validation of microduplications and triplications of 7q36.3

a) Map of CNVs detected in the primary and secondary cohorts from the UCSC genome browser. (b) Plots of probe intensity ratios for 16 CNVs detected in the primary and MGS datasets. All are cases, with the exception of two controls, who are indicated with an asterisk*. Regions with estimated copy numbers of 2, 3 and 4 are highlighted in gray, blue and green, respectively. Locations of four Sequenom validation assays are shown (dashed lines). (c-f) CNV genotypes were confirmed by MeZOD cluster plots of probe intensity ratios of the proximal and distal regions and in the primary dataset (c and d, respectively) and secondary dataset (e and f, respectively). Absolute copy numbers were confirmed for duplications and triplications of the proximal (g) and distal (h) regions by Sequenom MASSarray genotyping.

Figure 2. Patterns of CNV inheritance in families

Pedigree diagrams are shown for families (a) LW102, (b) 02-016 and (c) 02-135, along with the Sequenom validation for families (d) LW102, (e) 02-016 and (f) 02-135. Sequenom validation was performed on (a) mother and one of the affected sons, and (b) all three family members, along with 10 CEU HapMap controls. Sequenom assays confirmed that duplications were present in the patients and maternally inherited from LW102-2 and 02-0016-4.

Figure 3. Duplications and triplications of 7q36.3 result in increased VIPR2 transcription and cyclic-AMP signaling

(a) Quantitative PCR results of VIPR2 mRNA from lymphoblastoid cell lines. Two to four subjects were tested for each of four genotypes (subtelomeric duplication, VIPR2 duplication, exon 3/4 triplication, and normal diploid copy number as control). Results are expressed as the mean fold-change of CNV carriers relative to the mean of control samples. (b-c) Cyclic AMP accumulation was measured in the same cell lines in response to VIP (100nM) and the VPAC2 agonist BAY 55-9837 (100nM). Results are expressed as fold-change over forskolin/IBMX alone. (d) No significant differences were observed in cAMP response to another GPCR agonist, Prostaglandin E2 (PGE2, 1 μM), demonstrating that the effects are specific to VPAC2. For subjects, error bars represent standard error of the mean computed across replicates. Differences between the groups of 9 duplication carriers and 4 controls were tested using unpaired two sample t-tests.