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External quality assurance (EQA) is crucial to monitor and improve the quality of biochemical genetic testing. ERNDIM (www.erndim.org), established in 1994, aims at reliable and standardized procedures for diagnosis, treatment and monitoring of inherited metabolic disease (IMD) by providing EQA schemes and educational activities. Currently, ERNDIM provides 16 different EQA schemes including quantitative schemes for various metabolite groups, and interpretive schemes such as diagnostic proficiency testing (DPT). DPT schemes focus on the ability of laboratories to correctly identify and interpret abnormalities in authentic urine samples across a wide range of IMDs. In the DPT schemes, six samples each year are distributed together with clinical information. Laboratories choose and perform the tests needed to reach a diagnosis. Data were collected on 345 samples, distributed to up to 105 laboratories worldwide. Diagnostic proficiency (the % of total points possible for all participating laboratories within a scheme for analysis and interpretation) ranged widely: amino acid disorders (n = 20), range 33%-100%, mean 84%; organic acid disorders (n = 35), range 14%-100%, mean 84%; lysosomal storage disorders (n = 13), range 20%-97%, mean 73%; purine/pyrimidine disorders (n = 9), range 37%-100%, mean 70%; miscellaneous disorders (n = 8), range 17%-100%, mean 65%; no IMD, range 65%-95%, mean 85%. When a sample with the same disorder was distributed in a subsequent survey, performance improved in 75 cases with no improvement seen in 32, suggesting overall improvement of performance. ERNDIM diagnostic proficiency testing is a valuable activity which can help to assess laboratory performance, identify methodological/technical challenges, be informative during quality audits and contribute to a better clinical appreciation of diagnostic uncertainty., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Vitamin B 12 (cobalamin, Cbl) is a nutrient essential to human health. Due to its complex structure and dual cofactor forms, Cbl undergoes a complicated series of absorptive and processing steps before serving as cofactor for the enzymes methylmalonyl-CoA mutase and methionine synthase. Methylmalonyl-CoA mutase is required for the catabolism of certain (branched-chain) amino acids into an anaplerotic substrate in the mitochondrion, and dysfunction of the enzyme itself or in production of its cofactor adenosyl-Cbl result in an inability to successfully undergo protein catabolism with concomitant mitochondrial energy disruption. Methionine synthase catalyzes the methyl-Cbl dependent (re)methylation of homocysteine to methionine within the methionine cycle; a reaction required to produce this essential amino acid and generate S-adenosylmethionine, the most important cellular methyl-donor. Disruption of methionine synthase has wide-ranging implications for all methylation-dependent reactions, including epigenetic modification, but also for the intracellular folate pathway, since methionine synthase uses 5-methyltetrahydrofolate as a one-carbon donor. Folate-bound one-carbon units are also required for deoxythymidine monophosphate and de novo purine synthesis; therefore, the flow of single carbon units to each of these pathways must be regulated based on cellular needs. This review provides an overview on Cbl metabolism with a brief description of absorption and intracellular metabolic pathways. It also provides a description of folate-mediated one-carbon metabolism and its intersection with Cbl at the methionine cycle. Finally, a summary of recent advances in understanding of how both pathways are regulated is presented., (© 2018 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material.

Background: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients., Methods: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts., Results: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious., Discussion: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

Background: The cobalamin E (cblE) (MTRR, methionine synthase reductase) and cobalamin G (cblG) (MTR, methionine synthase) defects are rare inborn errors of cobalamin metabolism leading to impairment of the remethylation of homocysteine to methionine., Methods: Information on clinical and laboratory data at initial full assessment and during the course of the disease, treatment, outcome and quality of life was obtained in a survey-based, retrospective study from physicians caring for patients with the CblE or CblG defect. In addition, data on enzyme studies in cultured skin fibroblasts and mutations in the MTRR and MTR gene were analysed., Results: In 11 cblE and 13 cblG patients, failure to thrive, feeding problems, delayed milestones, muscular hypotonia, cognitive impairment and macrocytic anaemia were the most frequent symptoms. Delay in diagnosis depended on age at first symptom and clinical pattern at presentation and correlated significantly with impaired communication abilities at follow-up. Eighteen/22 patients presented with brain atrophy or white matter disease. Biochemical response to treatment with variable combinations of betaine, cobalamin, folate was significant. The overall course was considered improving (n = 8) or stable (n = 15) in 96% of patients, however the average number of CNS symptoms per patient increased significantly over time and 16 of 23 patients were classified as developmentally delayed or severely handicapped. In vitro enzyme analysis data showed no correlation with outcome. Predominantly private mutations were detected and no genotype- phenotype correlations evident., Conclusions: The majority of patients with the cblE and cblG defect show limited clinical response to treatment and have neurocognitive impairment.