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.