Using metabolic abnormalities of carriers in the neonatal period to evaluate the pathogenicity of variants of uncertain significance in methylmalonic acidemia

To accurately verify the pathogenicity of variants of uncertain significance (VUS) in and genes through mass spectrometry and analysis. This multicenter retrospective study included 35 participating units (ClinicalTrials.gov ID: NCT06183138). A total of 3,071 newborns (within 7 days of birth) were sorted into carrying pathogenic/likely pathogenic (P/LP) variants and carrying VUS, non-variant groups. Differences in metabolites among the groups were calculated using statistical analyses. Changes in conservatism, free energy, and interaction force of and variants were analyzed using analysis. The percentage of those carrying VUS cases was 68.15% (659/967). In the gene variant, we found that C3, C3/C2, and C3/C0 levels in those carrying the P/LP variant group were higher than those in the non-variant group ( < 0.000). The conservative scores of those carrying the P/LP variant group were >7. C3, C3/C0, and C3/C2 values of newborns carrying VUS (c.1159A>C and c.1286A>G) were significantly higher than those of the non-variant group and the remaining VUS newborns ( < 0.005). The conservative scores of c.1159A>C and c.1286A>G calculated by ConSurf analysis were 9 and 7, respectively. Unfortunately, three MMA patients with c.1159A>C died during the neonatal period; their C3, C3/C0, C3/C2, and MMA levels were significantly higher than those of the controls. Common variants of methylmalonic acidemia in the study population were categorized as VUS. In the neonatal period, the metabolic biomarkers of those carrying the P/LP variant group of the gene were significantly higher than those in the non-variant group. If the metabolic biomarkers of those carrying VUS are also significantly increased, combined with analysis the VUS may be elevated to a likely pathogenic variant. The results also suggest that mass spectrometry and analysis may be feasible screening methods for verifying the pathogenicity of VUS in other inherited metabolic diseases.