Challenging Atypical Findings in Two Patients Diagnosed as mut by Somatic Cell Complementation Analysis and Studied with a Novel NGS Panel
1. Department of Human Genetics, McGill University, Montreal, Quebec, Canada; 2. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States
Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Patients with classical methylmalonic aciduria, denoted as the mut complementation class, are characterized by biallelic mutations in the MUT gene coding for methylmalonyl-CoA mutase. Presented here are two patients diagnosed as mut by somatic cell complementation analysis that have been studied using a novel next-generation sequencing panel developed at the Baylor College of Medicine comprising MUT and 23 other genes that have known associations with methylmalonic aciduria. This panel has an average coverage depth of 500-1000X and is capable of detecting protein coding and splicing mutations, in addition to intragenic deletions.
In the first patient no mutations in MUT or any other gene on the NGS panel were detected. The second patient had heterozygous mutations in both the MUT (c.682C>T) and MCEE (c.428G>A) genes. MCEE codes for methylmalonyl-CoA epimerase, a protein responsible for catalyzing the conversion of D- to L-methylmalonyl-CoA. These findings were surprising considering that fibroblasts from both patients exhibited a clear inability to complement with mut fibroblasts, while complementing with cblA and cblB fibroblasts. Incorporation of label from [14C] propionate into TCA-precipitable cellular macromolecules is a measure of methylmalonyl-CoA mutase function and was considerably low for both patients at 0.4 and 0.6nmol/mg protein/18h (reference [mean ± SD]: 13.1 ± 4.1, n=217; mut patients: 0.9 ± 0.7 n=187).
Considering the high reliability of somatic cell complementation analysis in diagnosing inborn errors of cobalamin metabolism, it is suspected that regulatory or deep intronic mutations could be causative of the mut phenotype in some patients in whom no mutations are found on the NGS panel. Transfection of the wild-type MUT gene into cultured cells from these patients and sequence analysis of the entire genomic region of MUT will be used to look for this possibility. The patient with heterozygous mutations in MUT and MCEE presents a potential model of digenic inheritance, a phenomenon that has recently been suggested in a patient with cobalamin deficiency and heterozygous mutations in MTR and LMBRD1. Alternatively, the patient with no coding mutations in MUT might have mutations in unexpected genes controlling the regulation, modification, or localization of MUT as has recently been shown for HCFC1 and MMACHC.