Protocol for reevaluation and reclassification of variants of uncertain significance (VUS).

In the event that family members are asymptomatic, the detection of a de novo variant (with biochemical confirmation of parentage to exclude the possibility of falsely attributed paternity) in an autosomal dominant or X-linked gene, which has been previously implicated in a similar phenotype, reinforces its pathogenicity. However, it is necessary to remember that all individuals carry around 80-100 de novo mutations in their genome, of which 1-2 can potentially be found in the exome.

In some circumstances, demonstrating that the variant has been inherited from an asymptomatic parent can reinforce its benign nature. However, this information must be interpreted with caution, especially in autosomal dominant inheritance genes that exhibit variable penetrance, in which case, this information would not modify the pathogenicity classification.

In practice, it is difficult to demonstrate that the finding has not occurred by chance if only parental studies are available, unless a large pedigree is available and we have been able to study multiple family members across several generations, both affected and unaffected.

In the event that the gene involved is of autosomal recessive inheritance, and we have found two genetic variants, but only one of them can be classified as pathogenic or likely pathogenic, it may be useful to perform a family study, yielding 2 possible outcomes:

• Each variant has been inherited from one parent (and therefore, they are in trans in the index case), which is compatible with autosomal recessive inheritance.
• Both variants have been inherited from the same parent (and are in cis, i.e., on the same allele, in the index case), in which case the variants found would not be responsible for an autosomal recessive inheritance pattern.