Genome-wide copy number analysis uncovers a new HSCR gene: NRG3

Abstract

Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.

Figure 1. CNV burden for syndromic (Syn) and non-syndromic (Non-syn) HSCR patients relative to controls (Ctrl).

The burden was measured with reference to the (A) size, (B) rate and (C) gene count of CNVs. Red and blue bars denote the mean value of the corresponding test for deletion and duplication respectively. Summary statistics as well as conditional permutation p-value was shown in (D).

Figure 2. NRG3 deletions identified in HSCR patients.

(A) Intensity signals of 5 HSCR patients (CN = 1; red) with NRG3 deletions together with other samples of normal copy number (CN = 2; grey). Deleted regions are shown by the dark red bar and are highlighted in pink. (B) Consensus CNV segments of the 5 NRG3 deletions (red) and the overlapping DGV segments (blue; with DGV ID). (C, D and E) Box plot of NRG3 copy number estimates by real-time PCR. Samples were grouped according to the called copy number states (CN = 1, red; CN = 2, white; CN = 3, blue); (C) Validation of 5 deletions (CN = 1) and 24 copy-neutral (CN = 2) HSCR patients in the discovery phase; (D) Follow-up analysis on independent case-controls set and (E) Transmission analysis for probands with NRG3 deletions (child CN = 1) or duplications (child CN = 3). (F) Sequence of the NRG3 deletion boundary region showing the breakpoint (upstream boundary chr10: 84032610; downstream boundary chr10: 84052262).