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

Abstract

Objective: To accurately verify the pathogenicity of variants of uncertain significance (VUS) in MUT and MMACHC genes through mass spectrometry and silico 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 MMUT and MMACHC variants were analyzed using silico analysis.

Results: The percentage of those carrying VUS cases was 68.15% (659/967). In the MMUT 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 (p < 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 (p < 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 MUT 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 silico analysis the VUS may be elevated to a likely pathogenic variant. The results also suggest that mass spectrometry and silico analysis may be feasible screening methods for verifying the pathogenicity of VUS in other inherited metabolic diseases.

Keywords: mass spectrometry; methylmalonic acidemia; neonatal period; silico analysis; variants of uncertain significant.

FIGURE 1

Flow diagram of VUS variant gene research strategy.

FIGURE 2

Distribution characteristics and metabolic levels of newborns carrying related variants site. (A) Study the distribution of variants (carrier rate≧1/11,000) in MMUT and MMACHC genes. (B) Percentage of variant sites (carrier rate≧1/11,000) in MMUT and MMACHC genes. (C) Changes in C3 levels when carrying 11 VUS in MMUT. Data were expressed as medians (P₂₅, P₇₅). (D) Change of C3/C0 ratio when carrying 11 VUS variants in MMUT. Data were expressed as medians (P₂₅, P₇₅). (E) Change of C3/C2 ratio when carrying 11 VUS in MMUT. Data were expressed as medians (P₂₅, P₇₅). (F) Comparison of C3 levels in newborns carrying the (C) 1159A>C or (C) 1286A>G variant with other carrying VUS newborns. All data were analyzed using independent sample nonparametric test. Data of Panel C,D,E compared with non-variant group. Data of Panel F compared with other carrying VUS variants except for (C) 1159A>C and (C) 1286A>G.

FIGURE 3

Metabolic level of abovementioned three patients with methylmalonic acidemia. (A–D) Changes in MMA, C3, C3/C0, C3/C2 levels in newborns carrying c.1159A>C variation with methylmalonic acidemia (n = 3/group). All the data were tested using the independent sample nonparametric test.