Sanger confirmation, family segregation and reporting strategy
Neither Sanger confirmation nor family segregation was required for all cases. Verification was deemed necessary for variant(s) with borderline quality parameters (e.g. coverage) to preclude a false positive result and short INDELS (over 5 bp) to define their exact size and position. Variants(s) with clear pathogenicity, such as nonsense, frameshift or canonical splice site variants and variant(s) previously reported as pathogenic in similar cases required no family segregation studies, except in cases where trans or cis configuration of variants was important for the final resolution of genotypes.
Further analysis was also necessary for assessing VUS and determinede novo or inherited nature of variants, parental origin and final classification to likely pathogenic or benign. In the presence of one pathogenic/likely pathogenic variant associated with recessive inheritance exonic deletions/duplications along the candidate gene were investigated using Multiplex Ligation-dependent Probe Amplification (MLPA) or other techniques used for Copy Number Variant (CNVs) detection (e.g. SNP arrays). Recommendations for re-evaluation of WES data and clinical findings after 1-2 years were made to cases where no variant could explain the patient’s phenotype and cases when parental samples were unavailable to resolve the VUS detected.