The scary world of variants of uncertain significance (VUS): A hitchhiker’s guide to interpretation

This short review will focus on how to interpret variants of uncertain significance (VUS) in the setting newborn screening (NBS) for severe combined immunodeficiency (SCID). Many tenets this can be extrapolated other settings; however, NBS SCID represents a best-case scenario variant interpretation because nearly all genes are known and phenotype is easily defined, usually as T-cell count. The gene rapidly evolving, with individual being reclassified regularly new data become available.1Weck K.E. Interpretation genomic sequencing: should considered until proven guilty.Genet Med. 2018; 20: 291-293Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 2Andreasen C. Nielsen J.B. Refsgaard L. Holst A.G. Christensen A.H. Andreasen et al.New population-based exome questioning pathogenicity previously cardiomyopathy-associated genetic variants.Eur J Hum Genet. 2013; 21: 918-928Crossref (167) 3Manrai A.K. Funke B.H. Rehm H.L. Olesen M.S. Maron B.A. Szolovits P. al.Genetic misdiagnoses potential health disparities.N Engl 2016; 375: 655-665Crossref (358) Scholar Therefore, start current nomenclature schema, recognizing that classification not fixed. A fairly unique aspect immunology gain-of-function (GOF) increasingly associated disease they portrayed figure although loss-of-function (LOF) more often encountered (Fig 1, A).4Tangye S.G. Al-Herz W. Bousfiha A. Chatila T. Cunningham-Rundles Etzioni al.Human inborn errors immunity: 2019 update from International Union Immunological Societies Expert Committee.J Clin Immunol. 2020; 40: 24-64Crossref (485) GOF refers protein increased function, even if function deleterious cell. In contrast, LOF compromised function. Protein participates cell biology; sequence results do directly report effect most cases it unknown. Sequencing reports categorize into likelihood altered American College Medical Genetics has recommended 5-tier system classifying variants,5Richards S. Aziz N. Bale Bick D. Das Gastier-Foster J. al.Standards guidelines variants: joint consensus recommendation Genomics Association Molecular Pathology.Genet 2015; 17: 405-424Abstract (11640) terminology used sequencing methods differ B). Reporting VUS happen Sanger single gene, panel, whole-exome sequencing, or whole-genome sequencing.6Heimall Genetic testing diagnose primary disorders identify targeted therapy.Immunol Allergy North Am. 2019; 39: 129-140Abstract (4) Variants reported same way regardless technique used. Fig 2 an approach understanding next steps when arrives infant presumed SCID. some where narrow range was assayed, algorithm suggest expanding approach. moving smaller number larger considering copy variation. may also matching SCID+ patients, low count CD4/CD45RA along any syndromic features. Finally, determining whether segregates pedigree refer question found affected children. references key step autosomal-recessive (AR) conditions determination each separate chromosomes, referred alleles. Generally, one allele chromosome inherited father second mother. There identified but 1 parent, then there potentially still classified normal parent. hidden cannot presumed. situation quite different X-linked recessive such due mutations IL2RG. Here, cause must assumed disease. Similarly, autosomal-dominant condition RAC2 SCID, pathogenic sufficient disease; causal. about 10% performed no reported. Awaiting diagnosis necessary move toward therapy. 3 main approaches very frustrating circumstance: (1) escalate expansive (2) perform variation analysis, (3) consider necessarily basis. These workflows use Clinical Laboratory Improvement Amendments (CLIA)-approved studies invoke research approaches. If pathogenic, could useful try garner additional support its assignment possibly Rarely, identified. helpful these circumstances. Several easy:1.Matchmaker see similar patients by othersClinical Immunology Society listservMatchmakerexchange.org2.Check Gnomad at amino acid. Check tolerant variants. Two scores reported: observed/expected. Low observed/expected (<0.5) indicate tolerance LOF. pLI score. High (>0.9) LOF.Gnomad.broadinstitute.org3.Look up Catalogue Somatic Mutations Cancer (COSMIC). Highly enriched cancer imply function.Cancer.sanger.ac.uk/cosmic Of much greater difficulty defining functional consequences specific vitro. laboratory interest, have willing make expression vector variant. significant effort part laboratory, important recognize assay fully reflect cellular role protein. degree higher production animal model. require expertise, dedicated funding. Moving era sequence-based diagnosis7Smith L.D. Willig L.K. Kingsmore S.F. Whole-exome critically ill neonates suspected single-gene disorders.Cold Spring Harb Perspect 6a023168PubMed requires skills technology8Chinn I.K. Chan A.Y. Chen K. Chou Dorsey M.J. Hajjar al.Diagnostic diseases: working group Primary Immunodeficiency Diseases Committee Academy Allergy, Asthma & Immunology.J 145: 46-69Abstract (27) lead improved management.9Stray-Pedersen Sorte H.S. Samarakoon Gambin Chinn Coban Akdemir Z.H. al.Primary delineate heterogeneous Mendelian disorders.J 2017; 139: 232-245Abstract (156) Scholar,10Mahlaoui Picard Bach Costes Courteille V. Ranohavimparany immunodeficiencies: survey French national registry.J 143: 1646-1649.e10Abstract (12) few cautions bear mind assessing pathogenicity. •Common isolated populations cataloged large databases.•Variant classifications change over time, both less pathogenic.•Do accept causal just makes sense.•For AR conditions, ensure alleles.•Be wary heterozygous inheritance unless allele.•Copy accounts 5% effects. provides overview clinicians faced T cells. Similar complex needed broader, consistent phenotype, variable penetrance. ongoing efforts improve foundational information allow analysis conundrum fortunately frequent time. grateful acknowledgment many lessons colleagues.