Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular, and coronary heart disease. 1-4 Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 1,5-7 5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery diseases. 6 We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.

Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation.

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient’s clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children’s Hospital Foundation and others.)

The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed “next-generation metabolic screening” (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the “various forms of chromatography mass spectrometry (XCMS)” software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term “open the metabolome” analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs.Electronic supplementary materialThe online version of this article (10.1007/s10545-017-0131-6) contains supplementary material, which is available to authorized users.

Cardiovascular diseases (CVD), especially coronary heart disease (CHD), are the most important causes of death in industrialized countries. Increased concentrations of total plasma homocysteine (tHcy) have been associated with an increased risk of CHD. Assuming that this relation is causal, a lower tHcy concentration will reduce the occurrence and recurrence of CHD. Therefore, it is important to know which factors determine the tHcy concentration. In the general population, the most important modifiable determinants of tHcy are folate intake and coffee consumption. Smoking and alcohol consumption are also associated with the tHcy concentration, but more research is necessary to elucidate whether these relations are not originating from residual confounding due to other lifestyle factors. The most important nonmodifiable determinant is the 677 C>T polymorphism in the gene that encodes methylenetetrahydrofolate reductase (MTHFR), a regulating enzyme in homocysteine metabolism. Especially subjects with the homozygous form of this polymorphism (i.e., 677TT genotype) and a low folate status have elevated tHcy concentrations. Specific clinical conditions like the use of antiepileptic drugs or methotrexate, renal failure, cancer, rheumatoid arthritis, and hypothyroidism may lead to elevated tHcy concentrations. The available epidemiological evidence indicates that an increased tHcy concentration is not an important risk factor for CHD in healthy subjects. However, prospective studies, which included subjects at high risk of CHD, and secondary prevention trials with intermediary endpoints consistently show that elevations in the tHcy concentration may be an important risk factor in these subjects for a (recurrent) CHD event. The induction of vascular endothelial dysfunction by homocysteine may underlie this increased risk. Ongoing intervention trials will indicate whether homocysteine-lowering through vitamin supplementation, prevents CHD in the treatment groups.

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Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.

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