Fig. 1: Overview of the challenges encountered based on a cohort of 4577 molecularly characterized families.

In a cohort of 4577 molecularly characterized families, we encountered 5 main scenarios we found to be challenging in 1570 families. First, there are phenotype-related challenges that can be further sub-categorized into phenotypic heterogeneity, phenotypic expansion, novel allelic disorders, blended phenotype, and misleading diagnoses. Gene-related challenges comprise the discovery of novel disease genes, novel mutation mechanisms, and cases where the animal model did not corroborate the phenotype observed in human patients. Pedigree-related challenges comprise gonadal mosaicism, allelic and genetic heterogeneity, and pseudo-dominance. Variant-related challenges comprise interpretation and technical level challenges. Pitfalls of autozygosity include the lack of detectable ROH at the disease locus and apparent sharing of the candidate ROH with an unaffected member of the family. We then dissect the prevalence of these challenges in 314 families referred to us with negative clinical exome or genome sequencing. We observed that most of the challenges encountered are either because the causal gene was novel at the time of analysis or because of non-technical variant related challenges. Logos for Gene-related, phenotype-related, and variant-related challenges are created using BioRender.com.