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

Objective: To accurately verify the pathogenicity of variants of uncertain significance (VUS) in and genes through mass spectrometry and analysis.

Methods: 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.

Results: 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.

Conclusion: 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.