In Canada, rare disorders are defined as conditions affecting less than one in 2000 individuals. Although individually rare, in aggregate, one in every 12 Canadians is severely impacted by one of these rare disorders, placing a significant burden on the affected individuals, their families, and the health care system. Gene discovery is the critical starting point in understanding the molecular mechanisms underlying these diseases, providing robust diagnoses, and developing targeted treatments. An illustrative example is isolated brachydactyly, which is an umbrella term describing disproportionately short fingers and toes, often part of an autosomal dominant complex malformation syndrome. To date, various forms of brachydactyly have been characterized and 11 causative genes have been found, but many forms remain genetically undefined. Here, we describe a fifteen-year old unsolved medical case where an Ontario family with a history of brachydactyly remained genetically undiagnosed after Sanger sequencing. We hypothesize that the brachydactyly phenotype of this family is caused by a single large-effect variant, or a combination of small-to-moderate effect variants acting synergistically.

To characterize the genetic basis of brachydactyly in these patients, DNA was extracted from whole blood of 18 family members (14 definitely affected) who were previously Sanger sequenced but not found to have mutations in known genes underlying the disorder. One patient was recently whole- exome sequenced at the London Regional Genomics Centre, and a custom bioinformatics pipeline and non-synonymous rare variant prioritization approach was used to identify variants of interest. In this patient, a novel candidate variant (exon 1, c. 285_287dupGAA, p.Glu95_Asn96insLys) was identified in the Indian hedgehog (IHH) gene, and was confirmed through Sanger sequencing to co-segregate with affected status in the pedigree.

IHH is a member of the hedgehog family of proteins essential in secreting signaling molecules that regulate processes such as growth, patterning, and morphogenesis. The novel, heterozygous in-frame insertion in the IHH gene (p.Glu95_Asn96insLys) is predicted to be disease-causing in this Ontario family. Mutations in this gene are known to cause autosomal dominant brachydactyly type A1, which features the shortening or absence of the middle phalanges. This genetic diagnosis correlates with the clinical phenotype of the family. Using statistical analyses, we find that in comparison to an unaffected control population, the IHH variant is associated with shortened middle phalange length by 21.1% (P<0.001), palm length by 13.8% (P<0.01), digit-palm ratio by 6.8% (P<0.03) and stature by 9.5% (P<0.001). Follow-up studies will include ex vivo and in vitro functional assessment to confirm the pathogenicity of the IHH variant. These data highlight the power of whole-exome sequencing as a tool in identifying the genetic basis of rare disorders. They also indicate the power of next-generation sequencing to solve or clarify the genetic basis of “cold cases”. Determining the molecular basis of these disorders will allow for the understanding of disease mechanisms, and will help other health care providers caring for families with the same condition caused by the same gene.