Previous studies have provided evidence for an important role of CNVs in the pathogenesis of several developmental conditions, including congenital heart disease. These studies have predominantly relied on identification of de novo CNVs in sporadic cases. Here, we present the first family-based CNV study in LS-CHD, a disorder characterized by familial clustering, reduced penetrance and variable expressivity.
Based on a carefully phenotyped cohort recruited from the French-Canadian founder population and a large number of controls with cardiac evaluation, our findings provide several lines of evidence for a strong association of novel CNVs with LS-CHD. Four plausible syndromic regions and 25 candidate genes either known to be involved in congenital heart pathogenesis or highly likely to impact the risk for LS-CHD were identified.
The use of a family-based cohort allowed us to make use of segregation patterns to strengthen the association between rare CNVs and LS-CHD. In our cohort enriched for multiplex families, CNVs can occur both on an inherited and on a de novo basis, mostly with intrafamilial phenotypic variability of LS-CHD. This is compatible with a model in which structural genomic variation contributes to both heritability and variable expressivity of this trait. Interestingly, the vast majority of causative CNVs identified in our study qualify as private in nature, despite our intentional selection bias towards multiplex families within a founder population.
In our studies, we used a sequential filtering approach to increase the biological plausibility of identified LS-CHD candidate genes. Several lines of evidence support enrichment for genes involved in angiogenesis in this disease spectrum. We identified a significant enrichment for genes implicated in angiogenesis, pointing to a role of disturbances in endothelial development in disease pathogenesis. In silico
analyses, SAGE libraries and mining of public databases identified several known and novel cardiac-specific candidate genes. The in situ
expression patterns of CTHRC1
are striking examples for enrichment in developing valve structures and endothelium. Interestingly, both of these genes act in known pathways of valvulogenesis and are copy-number gains, suggesting that mechanisms other than haploinsufficiency may contribute to disease pathogenesis in these two examples. Moreover, CTHRC1
was found to be significantly overexpressed in calcific aortic stenosis, underscoring that hits to developmental genes may predispose to both early and adult onset valve disease 
This evidence is further corroborated by the identification of a novel role for known syndromic loci in LS-CHD. Overall, CNVs intersecting with four known syndromic loci were identified, and for all loci, cardiovascular phenotypes were reported. Our study widens the genotype-phenotype correlations in these syndromes; of note, none of the patients had been a priori
suspected to manifest the associated clinical phenotypes. We suspect that for these loci, the gene dosage – phenotype correlations are not perfect, and that they represent predisposing loci which require further hits for full penetrance of specific clinical features. Taking family 54 as an example, the most severely affected individual showed three unique CNVs, two inherited (one gain, one loss) from the affected father, plus a de novo
gain overlapping the previously described 1q21 locus (). One of the inherited CNVs intersected with LIMS1
, which plays an essential role in outflow tract development through TGF-β signalling. Interestingly, the clinical phenotypes within this family partially overlapped, strengthening the idea that multiple hits explain reduced penetrance or variable expressivity. Based on this observation, we speculate that other CNVs may also buffer phenotypes; i.e., two antagonistic hits within a single cascade may render cardiac development tolerant against perturbations in an epistatic fashion. Such a model would also be consistent with insight from animal studies in which modifier genes can govern normal or abnormal cardiac development on certain backgrounds 
. As another example, endothelial-specific knockout of GATA5
in mice leads to BAV in only 20% of the offspring, compatible with the reduced penetrance even of strong alterations of gene dosage 
. Other mouse models - examples include mice haploinsufficient for eNOS
- also display reduced penetrance of CHD traits, with complex gene-dosage effects of interacting alleles 
. Of note, our study was designed to identify CNVs which would not be detectable by linkage analysis, using an algorithm that prevented the discovery of incompletely penetrant alleles since CNVs seen in unaffected family members or the well-phenotyped control cohort were excluded. Two limitations of our study need to be kept in mind: first, our results do not exclude the possibility that additional, incompletely penetrant CNVs play a role in LS-CHD; second, our design could have missed CNVs containing important non-coding sequences such as regulatory elements since we required further validation through expression studies. Further studies with much larger cohorts are warranted to dispose of sufficient power for the detection of incompletely penetrant alleles, rare double hits and gene deserts 
Strengths of our study comprise the stringent, uniform CNV analysis workflow for both the LS-CHD as well as the control cohort, which yielded similar results in respect to reported de novo
CNV transmission rates 
. Importantly, we used a rigorous approach limited to CNVs which were unique or statistically enriched in our cases. All controls had adequate cardiac screening to account for mild phenotypes not detectable by conventional clinical examination. Furthermore, the founder character of our cohort theoretically facilitates detection of recurrent hits; nevertheless, this was not the case with our current sample size. On the other hand, several limitations of our study should be noted. At this point, it is unknown whether a cohort enriched for multiplex families with LS-CHD is in itself genetically distinct from a normal population sample. Due to the high stringency of our filtering mechanism, our design precludes the discovery of CNVs with incomplete penetrance and may underestimate the true impact of CNVs on LS-CHD. Furthermore, we recognize that adequate CNV boundary calling remains an issue which will best be resolved using NextGeneration sequencing in future studies.
Taken together, our study suggests that unique CNVs contribute significantly to LS-CHD, and that the majority of genetic events are of private nature. CNVs were found to contribute to 10% of our LS-CHD cases after statistical, biological and genetic validation. Combinatorial interactions between several different genetic factors disturbing key developmental events in left ventricular outflow tract development - such as angiogenesis – may modify the risk for LS-CHD, with important implications for an oligogenic origin for the entire spectrum of LS-CHD.
Future work should aim at more precisely defining gene inventories in larger cohorts and at replication of combinatorial hits in animal models. Insight gained from these studies will assist in identifying the underlying pathophysiological mechanisms of LS-CHD and help clarify the diversity of outcomes in individual patients despite similar morphologies.