title: High resolution melting analysis of the MMAA gene in patients with cblA and in those with undiagnosed methylmalonic aciduria.
abstract: The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA gene for variants. DNA was scanned by HRMA from 96 unaffected reference individuals, 72 cblA patients confirmed by complementation, and 181 patients with isolated elevated methylmalonic acid, who could not be diagnosed using complementation analysis. Suspected variants were confirmed by Sanger sequencing. In the cblA cohort, HRMA correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (c.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).
authors.affiliation: Department of Human Genetics, McGill University, Montreal, QC, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Dempsey-Nunez
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Illson
authors.forename: Jana
authors.initials: J
authors.lastname: Kent
authors.forename: Qiuying
authors.initials: Q
authors.lastname: Huang
authors.forename: Alison
authors.initials: A
authors.lastname: Brebner
authors.forename: David
authors.initials: D
authors.lastname: Watkins
authors.forename: Brian M
authors.initials: BM
authors.lastname: Gilfix
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: David S
authors.initials: DS
authors.lastname: Rosenblatt
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000097:blood / Q000175:diagnosis* / Q000235:genetics* / Q000652:urine; D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D006801:Humans; D007438:Introns; D008764:Methylmalonic Acid / Q000097:blood / Q000652:urine; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D014805:Vitamin B 12 / Q000378:metabolism
chemical_list: C497512:MMAA protein, human; D033681:Mitochondrial Membrane Transport Proteins; D008764:Methylmalonic Acid; D014805:Vitamin B 12