The enigma of VUS

With the increasing availability and affordability of next-generation sequencing, more patients are undergoing genetic testing to resolve diagnostic uncertainties or improve disease management. Although genetic testing has advanced our understanding of many diseases and improved family counselling, it can also generate additional questions. A common challenge arises when a test result identifies a variant of uncertain significance (VUS) rather than a clear pathogenic or benign outcome. This issue is especially true in syndromes with multiple genetic causes, where VUS results may be more common than interpretable findings. To support consistent variant classification, the American College of Medical Genetics and Genomics (ACMG) issued 2015 guidelines allowing variants to be classified as pathogenic, likely pathogenic, likely benign, benign, or VUS based on genetic, computational, population, and functional data.1 Many variants remain classified as VUS due to limited evidence, leaving clinicians and families in a state of uncertainty that can contribute to confusion. The ACMG criteria face several limitations: variable interpretation of the guidelines, disease- and gene-specific nuances that are sometimes overlooked, and the potential for new evidence to alter classifications. Many clinicians are not fully aware of these subtleties. In the current issue of Annals of Movement Disorders, two reports present interesting cases of dystonia in which novel VUS were identified: biallelic compound-heterozygous variants in the COL6A3 gene in one report and a heterozygous variant in ANO3 in another. These cases illustrate the complexity of variant interpretation and the importance of reviewing the published literature. To better understand the terminology, readers are advised to refer to Tables 3, 4, and 5 of the ACMG guideline paper.1 Novel variants of uncertain significance in the COL6A3 gene in a case of parkinsonism and dystonia. In this report describing a patient with a parkinsonism and dystonia phenotype, the authors identified two compound heterozygous variants in the COL6A3 gene: c.8839G>A;p.Ala2947Thr and c.8726C>T;p.Ala2909Val. Each parent carried one variant, confirming that the variants were inherited in trans, that is, one from each parent, resulting in biallelic involvement. Both variants, c.8839G>A;p.Ala2947Thr and c.8726C>T;p.Ala2909Val, are nonsynonymous missense variants that are rare in the general population (PM2), with an allele frequency of A;p.Ala2947Thr is reported in the ClinVar database as benign (https://www.ncbi.nlm.nih.gov/clinvar/variation/3620750/). However, the phenotype in that case is Bethlem myopathy rather than dystonia, so it does not meet BP6 of the ACMG criteria (a reputable source reports the variant as benign). This distinction is particularly important for genes associated with multiple phenotypes. Although the variants are in trans, the PM3 rule for recessive disorders— applicable when a variant is found in trans with a pathogenic variant—cannot be applied here, as neither variant is classified as pathogenic or likely pathogenic. Thus, the variants are best classified as VUS (PM2BP4), and the currently available evidence is insufficient to attribute the phenotype to the identified variants. Additional evidence, such as nonsegregation in family members (BS4) confirmed by Sanger sequencing, functional studies (PS3 or BS3), or demonstration that the variant is more common in dystonia patients than in healthy individuals (PS4), might support reclassification. Major clinical decisions, including treatment and family planning, should not be based solely on these findings. First report of DYT-COL6A3 COL6A3 gene abnormalities can cause Bethlem myopathy 1C and Ullrich muscular dystrophy 1C, with both dominant and recessive inheritance. In 2015, Zech et al.2 identified compound heterozygous COL6A3 variants in trans in a family with two siblings Table 1, Family-I affected by isolated segmental dystonia, with these variants segregating with the disease (PP1). They also identified two additional families Table 1, Family-II and III with compound heterozygous variants in trans while screening 367 patients with isolated dystonia. In all three families, at least one variant was located in exon 41, and in a zebrafish model, exon 41 skipping caused abnormal development (PS4). The variant c.8966-1G>A in Families II and III was a splice variant causing exon 41 skipping (PS4), is rare (PM2), present in two affected families (PS3), and segregates with the disease (PP1), classifying it as pathogenic. The other two variants were rare (PM2), in trans to a pathogenic variant (PM3), segregated with the disease (PP1), and predicted deleterious (PP3), with c.7502G>A considered likely pathogenic and c.7660G>A of uncertain significance Table 1. Variants in Family I were likely benign due to their frequency and benign prediction Table 1.Table 1: Interpretations of reported COL6A3 variants in patients with movement disordersHowever, Zech et al. argued that: (i) In a particular population, the variant carrier frequency of a recessive gene may be high and does not necessarily suggest a benign nature of the variant. (ii) The occurrence of these variants in a homozygous state in an apparently normal population may be due to the population being screened only for severe paediatric abnormality and not for isolated dystonia, and affected individuals may develop dystonia at a later age. They acknowledged these limitations and proposed further functional studies to clarify the role of missense variants in the COL6A3 gene in dystonia. This underscores the complexities of applying BS1 and BS2 criteria and the limitations in the reliability of in-silico tools (BP4). Subsequent reports of COL6A3 variants in dystonia and Parkinson disease Shortly after the Zech et al.2 paper, Lohmann et al.3 examined 955 unrelated patients with dystonia and/or Parkinson disease (PD) for COL6A3 variants and identified only one family with bi-allelic compound heterozygous missense variants in COL6A3 (Exon 42:c.9245C>G;p.Pro3082Arg and Exon 6:c.2195C>T;p.Thr732Met). In that family, the variants did not segregate with affected siblings (BP5), and an alternative cause—a homozygous pathogenic PINK1 variant consistent with PD—was identified (BS4), suggesting that the COL6A3 variants are likely benign. Lohmann et al.3 suggested that the role of COL6A3 in dystonia remains uncertain. The variants reported by Lohmann et al.3 were in exons 42 and 6, unlike the exon 41 involvement in Zech et al.‘s cases, suggesting that the specific location of a variant in exon 41 may influence the phenotype. Reports have documented biallelic COL6A3 variants in cases of dystonia and parkinsonism, but none have been classified as likely pathogenic, and most lack functional data.4-9 Only one additional report mentioned a variant in exon 41, but it was in a heterozygous state.9 Zech et al.’s work remains the sole robust functional evidence linking COL6A3 to dystonia, highlighting the significance of the exon 41 variant. This case report underscores the difficulties in interpreting ACMG criteria and understanding the nuances within the current literature. A unique presentation of buckling and falling due to a novel ANO3 variant In this report, the authors describe a patient with adult-onset paroxysmal dyskinesia and a family history of similar illness in multiple family members. Exome sequencing revealed a novel missense variant in the ANO3 gene (c.2459G>A;p.Gly820Asp), which was also present in the affected sibling. Other family members, including the parents, were not tested for the variant. The variant is rare (PM2), predicted to be deleterious (PP3), and segregated with the disease (PP1). Based on these findings, the variant has been classified as a VUS (PM2, PP1, PP3). Even with the application of the PP4 rule, based on the recent report of paroxysmal dystonia in DYT-ANO3, the variant remains classified as a VUS.10 Consequently, the current evidence is still insufficient to establish a definitive link between the identified variant and the phenotype described in the report. The paroxysmal dyskinesia phenotype is not well described in DYT-ANO3. Two siblings carry this variant, suggesting it is not de novo and may be present in one unaffected parent. This means that the variant occurs in an unaffected family member and does not segregate with the disease, invalidating the PP1 criteria. However, incomplete penetrance in dystonia and ANO3 could explain the presence of an unaffected carrier parent. This highlights a limitation of the ACMG criteria, which were designed mainly for Mendelian inheritance; applying them to diseases with incomplete penetrance or imprinting can be challenging. There is a 25% chance that two siblings share a heterozygous variant from one parent, making the segregation rule (PP1) less robust in dominant disorders. For recessive diseases, this chance is 1 in 16 6.25%, see Figure 1. Increasing segregation data through Sanger sequencing of family members can strengthen the rule. With six siblings, each having two possibilities, the probability that all affected siblings—but no unaffected ones—carry the variant is 1 in 64 (1.5%), making chance occurrence unlikely. Additionally, the parent who might carry the variant should be examined to detect any subtle signs of dystonia.Figure 1: Segregation rule scenarios in dominant and recessive disordersConclusions Both reports highlight the challenges of interpreting VUS under the ACMG framework and highlight the need for careful review and communication with patients. In both reports, the cases remain unsolved based on the currently available evidence. Further investigation into alternative causative genes or variants may be warranted, including assessment of copy number variations, intronic variants, or structural variants through chromosomal microarray, whole-genome sequencing, and/or long-read sequencing. It is not recommended to base major clinical decisions solely on VUS findings, as this may lead to misdiagnosis, inappropriate management and counselling, and limit consideration of alternative diagnoses. Tracking VUS helps to resolve their classification as more data emerge. Publishing uncertain variants with explicit mention of their status, or creating an Indian database of such variants, could connect patients with the same variants across the country, increasing the chance of solving these cases. Author contribution A. Research project: Conception: Vikram V. Holla Organization: Vikram V. Holla Execution: Vikram V. Holla B. Statistical analysis: Design: Vikram V. Holla Execution: Vikram V. Holla Review and Critique: Vikram V. Holla C. Manuscript preparation: Writing of the first draft: Vikram V. Holla Review and Critique: Vikram V. Holla Ethical compliance statement We confirm that we have read the journal’s position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.