Insights into Severe 5,10-Methylenetetrahydrofolate Reductase Deficiency: Molecular Genetic and Enzymatic Characterization of 76 Patients

Abstract

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.

Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency

“…Therefore it must be kept in mind that these in vitro studies are not to be used as prognostic markers in individual patients or families. Correlations between type and location of mutation and enzyme level and kinetic characteristics had been observed in a study in fibroblasts of 72 patients, including the 33 patients presented here (Burda et al 2015). However, at least for the 33 patients reported here, no correlation between genotype and clinical phenotype could be delineated.…”

“…DNA was extracted and sequenced from primary fibroblasts derived from skin biopsies obtained for diagnostic purposes. MTHFR activity was measured using the physiological forward assay as described earlier, which allows reliable detection of residual activities and measurement of kinetic parameters (Suormala et al 2002) with minor modifications (Burda et al 2015). In cell lines with very low MTHFR activity (<1.5 % mean control value) only enzymatic activity and FAD-responsiveness were measured.…”

“…In cell lines with very low MTHFR activity (<1.5 % mean control value) only enzymatic activity and FAD-responsiveness were measured. For those with higher residual activity (>1.5 % mean control value), further enzymatic characterization (FAD responsiveness, km for NADPH) was performed (Burda et al 2015).…”

“…Since most of the mutations causing severe MTHFR deficiency are private (Tonetti et al 2003;Burda et al 2015) and significant intrafamiliar variation of the clinical phenotype has been described (Haworth et al 1993), no clear genotypephenotype correlations have been outlined. A relation between in vitro enzyme activity and clinical course has occasionally been postulated (Goyette et al 1995;Birnbaum et al 2008;Forges et al 2010) but has not yet been systematically investigated.…”

“…The enzyme MTHFR catalyses the reduction of 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the most common form of folate in blood, cerebrospinal fluid and tissues and serves as a methyl donor for the methylation of homocysteine (Hcy) to methionine (Met). In MTHFR deficiency, methylation of Hcy to Met is decreased resulting in highly elevated plasma total Hcy (tHcy) and low plasma Met concentrations (Fattal-Valevski et al 2000; Thomas and Rosenblatt 2005;Burda et al 2015). Low Met results in a depletion of Sadenosylmethionine, which is the main donor for many methylation reactions including the synthesis of creatine, and RNA and DNA methylation (Thomas and Rosenblatt 2005).…”