VUS in WES

VUS, sometimes changed into (VOUS), stays for Variant Of Uncertain Significance. A VUS is a variant that, a priori, cannot be defined either as neutral/benign or as pathogenic. This term is pretty much used in clinical diagnostics, i.e. when performing a genetic test on an affected patient.

A VUS is usually considered a variant of possible pathogenic relevance because (1) it has a very low allele frequency across populations, (2) it’s falling in a gene where other mutations have been reported as pathogenic for a disorder similar to the one of the patient, (3) its biological features may indicate it’s pathogenic (e.g. in silico predictions say that it might affect the protein structure in a damaging way, or it sits nearby the exon/intron junctions – hence possibly impacting the splicing process – or it falls at the same codon, or the same nucleotide position, where another variant has been confirmed to be pathogenic).

VUS are a consistent proportion of the final results in genetic testing. Especially when doing large multigene panels, whole exome sequencing or whole-genome sequencing, the quantity of VUS is absolutely impressive. In affected patients, in whom a clearly pathogenic variant cannot be detected, several VUS may come into play as possible, candidate pathogenic variants.
Each VUS is then evaluated for its possible relevance to the phenotype of the patient based on (1) genotype (is this VUS heterozygous or homozygous?) (2) specific gene mutational spectrum (is the VUS biological type described as pathogenic in this gene) and, most of all, (3) the relevance of gene to the clinical presentation of the patient.

Well, there aren’t many ways, but something can be done and, sometimes, we can really shift the final interpretation to something certain. Especially in rare disorders, there are basically two ways:
(1) familial segregation studies: the VUS must be tested (usually by simple carrier testing with capillary electrophoresis – Sanger sequencing), to see if the variant has arisen de novo in the affected person (this is usually a very strong indicator for pathogenicity in isolated disorders) or to see if the variant appears only in affected family members and not in healthy members of the same family (this is also a very good indicator of pathogenicity);
(2) functional studies in-vitro: this is certainly the most complicated way, especially because a research assay must be designed from scratch and tested specifically for that variant. However, this may be the only accessible way, especially if other affected family members are not available (de novo disorder) or when it comes to verifying the real impact on the splicing of deep intronic variants.