Genome-wide copy number scan identifies IRF6 involvement in Van der Woude syndrome in an Indian family

Abstract

Summary Van der Woude syndrome (VWS) is an autosomal dominant developmental malformation presenting with bilateral lower lip pits related to cleft lip, cleft palate and other malformations. We performed a whole-genome copy number variations (CNVs) scan in an Indian family with members suffering from VWS using 2·6 million combined SNP and CNV markers. We found CNVs affecting IRF6, a known candidate gene for VWS, in all three cases, while none of the non-VWS members showed any CNVs in the IRF6 region. The duplications and deletions of the chromosomal critical region in 1q32-q41 confirm the involvement of CNVs in IRF6 in South Indian VWS patients. Molecular network analysis of these and other cleft lip/palate related module genes suggests that they are associated with cytokine-mediated signalling pathways and response to interferon-gamma mediated signalling pathways. This is a maiden study indicating the involvement of CNVs in IRF6 in causing VWS in the Indian population.

Fig. 1.

Illustration of the Van der Woude pedigrees under study. All the seven affected members were found to have lower lip anomalies, among these, two individuals (5S and 6S) also had cleft lip, while only one affected member (4S) was also found to have cleft palate. (a) The pedigree under study consisted of 15 individuals, of which, seven were affected with cleft lip/palate across all the three generations. The proband of this study was a 10 year old boy diagnosed with cleft lip and lower lip anomalies. (b) Shows subject 4S who is an affected female in the second generation of the pedigree with lower lip and cleft palate. (c) Shows subject 5S who is one of the affected daughters of case 4S having lower lip and cleft palate and (d) shows case 6S, who is the affected daughter of 5S with lower lip and cleft lip.

Fig. 2.

Schematic representation of copy number state of Van der Woude cases. An image of the copy number state (5S, 6S and 4S) indicates the quantitative assessments of genotyping used to determine copy number variation in the Van der Woude (VWS) cases. Blue bars indicate duplications and red indicates deletions. Case 4S shows a homozygous deletion copy number variation (CNV) of 3 kb from 209974442 bp to 209977378 bp at the 1q32 region encompassing IRF6. Case 5S shows a contiguous 53 kb heterozygous duplication CNV followed by a 18 kb deletion CNV from 209970522 bp to 209988149 bp in the 1q32 region, but in the homozygous state with the intermediate distance of ~16 kb between the duplication and deletion CNV. The end breakpoint of the duplication CNV and the start point of the deletion CNV partially disrupted IRF6. These two CNVs amounted to ~71 kb in variation. Case 6S shows a deletion CNV of 13 kb from 209974651 bp to 209988149 bp in the 1q32 region in the homozygous state. Though the deletion CNV in both the 5S and 6S cases have varied start points, they both have the same end point, which encompass IRF6.

Fig. 3.

IRF6 protein interaction network establishing the associated gene modules and their key hubs. The figure shows the network of genes involved in cleft lip/palate with hub genes coloured according to their respective Gene Ontology (GO). Network analysis of candidate genes of lip/palate, including the genes identified in this study, has established ~24 genes with protein and genetic interactions that have more than >70 links. Out of these, interactions involving some genes that participate in the facial determination region could be accounted for by the analysis in the present study. These interactions are more specific for interferon signalling as they directly participate in cytokine-mediated signalling pathways, response to interferon-gamma and interferon-gamma mediated signalling pathways. The geometrical establishment of the IRF6 network shows already identified candidate genes interacting with facial morphology genes in the present study. The smaller and larger circles depict network weights that are chosen based on the size of the input gene list. Based on the input gene list, a ‘GO-based weighting, biological process based’ weighting method is chosen. This weighting method assumes the input gene list is related in terms of biological processes (as defined by GO) and assigns weights based on maximizing connectivity between all input genes using the ‘assigned based on query gene’ strategy.

Fig. 4.

Ingenuity Pathway Analysis of the IRF6 gene and its interactions with target proteins indicating involvement in several biological functions and pathway.