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Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (NOTCH1, ARHGAP31, MAML1, SMARCA4, JARID2, JAG1) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in NOTCH1 highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in NOTCH1 associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO., Author Summary Left-ventricular outflow tract obstructions comprise a group of developmental heart disorders that are genetically and phenotypically heterogeneous, with no single gene accounting for the majority of cases. In order to identify mutations contributing to disease, we selected patients from 51 families with a history of congenital cardiac malformations. We interrogated the entire coding sequences of 106 patients and identified a small but highly pathogenic fraction of mutations that are likely to contribute to disease in 12 families (23.5%). Furthermore, we present a strategy for identifying candidate mutations based on familial segregation in a genetically heterogeneous disorder.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC., Author Summary The underlying genetic mutations involved in 60% of inherited gastric cancer cases remain unknown. Here we present a large, extended pedigree with familial gastric cancer and an association in part of the family with a mutation in MAP3K6. The conservation, predicted pathogenicity of the variant, tissue distribution, and known function of MAP3K6 made this a strong candidate that warranted further investigation. Examination of an additional 115 unrelated probands identified additional mutations in MAP3K6, including a truncating mutation.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730–129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred., Author Summary Sensory motor neuropathies are diseases of the peripheral nervous system, involving primarily the nerves which control our muscles. These can result from either genetic or non-genetic causes, with genetic causes usually referred to as Charcot-Marie-Tooth (CMT) disease after the three clinicians who first described the key diagnostic markers. CMT patients lose muscle function, mainly in their arms and legs, with increasing severity during their lives. There are almost two dozen known genes that can mutate to cause CMT, and these fall into a wide variety of biochemical cellular pathways. We identified a group of patients with CMT from a small rural community, with good reason to suspect a genetic basis for their disease. Using high-throughput mapping and DNA sequencing technologies developed as part of the Human Genome Project, we were able to find the likely mutated gene, which was not any of the previously known CMT genes. Based on its sequence, the gene, called LRSAM1, probably plays a role in the correct metabolism of other proteins in the cell. Among the known CMT genes, some are also involved in protein metabolism, suggesting that this is a generally important pathway in the neurons that control muscle activity.