Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase.

5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.

Induced pluripotent stem cell-derived hepatocytes reveal TCA cycle disruption and the potential basis for triheptanoin treatment for malate dehydrogenase 2 deficiency.

Mitochondrial malate dehydrogenase 2 (MDH2) is crucial to cellular energy generation through direct participation in the tricarboxylic acid (TCA) cycle and the malate aspartate shuttle (MAS). Inherited MDH2 deficiency is an ultra-rare metabolic disease caused by bi-allelic pathogenic variants in the MDH2 gene, resulting in early-onset encephalopathy, psychomotor delay, muscular hypotonia and frequent seizures. Currently, there is no cure for this devastating disease. We recently reported symptomatic improvement of a three-year-old girl with MDH2 deficiency following treatment with the triglyceride triheptanoin. Here, we aimed to better characterize this disease and improve our understanding of the potential utility of triheptanoin treatment. Using fibroblasts derived from this patient, we generated induced pluripotent stem cells (hiPSCs) and differentiated them into hepatocytes (hiPSC-Heps). Characterization of patient-derived hiPSCs and hiPSC-Heps revealed significantly reduced MDH2 protein expression. Untargeted proteotyping of hiPSC-Heps revealed global dysregulation of mitochondrial proteins, including upregulation of TCA cycle and fatty acid oxidation enzymes. Metabolomic profiling confirmed TCA cycle and MAS dysregulation, and demonstrated normalization of malate, fumarate and aspartate following treatment with the triheptanoin components glycerol and heptanoate. Taken together, our results provide the first patient-derived hiPSC-Hep-based model of MDH2 deficiency, confirm altered TCA cycle function, and provide further evidence for the implementation of triheptanoin therapy for this ultra-rare disease. Synopsis: This study reveals altered expression of mitochondrial pathways including the tricarboxylic acid cycle and changes in metabolite profiles in malate dehydrogenase 2 deficiency and provides the molecular basis for triheptanoin treatment in this ultra-rare disease.

Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria.

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria, present unique challenges to energetic homeostasis by disrupting energy-producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut)-type methylmalonic aciduria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared with littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these shed light on the mechanisms and adaptations behind energy imbalance in methylmalonic aciduria and provide insight into metabolic responses to chronic energy shortage, which may have important implications for disease understanding and patient management.

The complex machinery of human cobalamin metabolism.

Vitamin B12 (cobalamin, Cbl) is required as a cofactor by two human enzymes, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylmalonyl-CoA mutase (MMUT). Within the body, a vast array of transporters, enzymes and chaperones are required for the generation and delivery of these cofactor forms. How they perform these functions is dictated by the structure and interactions of the proteins involved, the molecular bases of which are only now being elucidated. In this review, we highlight recent insights into human Cbl metabolism and address open questions in the field by employing a protein structure and interactome based perspective. We discuss how three very similar proteins-haptocorrin, intrinsic factor and transcobalamin-exploit slight structural differences and unique ligand receptor interactions to effect selective Cbl absorption and internalisation. We describe recent advances in the understanding of how endocytosed Cbl is transported across the lysosomal membrane and the implications of the recently solved ABCD4 structure. We detail how MMACHC and MMADHC cooperate to modify and target cytosolic Cbl to the client enzymes MTR and MMUT using ingenious modifications to an ancient nitroreductase fold, and how MTR and MMUT link with their accessory enzymes to sustainably harness the supernucleophilic potential of Cbl. Finally, we provide an outlook on how future studies may combine structural and interactome based approaches and incorporate knowledge of post-translational modifications to bring further insights.

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency.

Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.

Cellular and computational models reveal environmental and metabolic interactions in MMUT-type methylmalonic aciduria.

Methylmalonyl-coenzyme A (CoA) mutase (MMUT)-type methylmalonic aciduria is a rare inherited metabolic disease caused by the loss of function of the MMUT enzyme. Patients develop symptoms resembling those of primary mitochondrial disorders, but the underlying causes of mitochondrial dysfunction remain unclear. Here, we examined environmental and genetic interactions in MMUT deficiency using a combination of computational modeling and cellular models to decipher pathways interacting with MMUT. Immortalized fibroblast (hTERT BJ5ta) MMUT-KO (MUTKO) clones displayed a mild mitochondrial impairment in standard glucose-based medium, but they did not to show increased reliance on respiratory metabolism nor reduced growth or viability. Consistently, our modeling predicted MUTKO specific growth phenotypes only for lower extracellular glutamine concentrations. Indeed, two of three MMUT-deficient BJ5ta cell lines showed a reduced viability in glutamine-free medium. Further, growth on 183 different carbon and nitrogen substrates identified increased NADH (nicotinamide adenine dinucleotide) metabolism of BJ5ta and HEK293 MUTKO cells compared with controls on purine- and glutamine-based substrates. With this knowledge, our modeling predicted 13 reactions interacting with MMUT that potentiate an effect on growth, primarily those of secondary oxidation of propionyl-CoA, oxidative phosphorylation and oxygen diffusion. Of these, we validated 3-hydroxyisobutytyl-CoA hydrolase (HIBCH) in the secondary propionyl-CoA oxidation pathway. Altogether, these results suggest compensation for the loss of MMUT function by increasing anaplerosis through glutamine or by diverting flux away from MMUT through the secondary propionyl-CoA oxidation pathway, which may have therapeutic relevance.

Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate.

Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0-4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.

HIF1 and DROSHA are involved in MMACHC repression in hypoxia.

The MMACHC gene encodes for an enzyme involved in intracellular vitamin B12 metabolism, and autosomal recessive defects in MMACHC represent the most common disorder of intracellular vitamin B12 metabolism. Recent studies have identified increased levels of reactive oxygen species in cells and tissues with MMACHC dysfunction, suggesting a role for oxidative stress in disease. To investigate the link between oxidative stress and MMACHC, we exposed mice as well as human and mouse cells to hypoxia, and found significant repression of MMACHC in all investigated tissues (retina, eyecup, liver, kidney) and cell lines (HeLa, ARPE-19, human and mouse fibroblasts, 661W). Furthermore, in HeLa cells, we found transcriptional repression already at 5% oxygen, which was stable during prolonged hypoxia up to 5 days, and a return of MMACHC transcripts to normal levels only 24 h after reoxygenation. This hypoxia-induced downregulation of MMACHC was not due to altered function of the known MMACHC controlling transcription factor complex HCFC1/THAP11/ZNF143. Using in vitro RNA interference against hypoxia-induced transcription factors (HIF1A, HIF2A and REST) as well as the microRNA transcription machinery (DROSHA), we observed release of hypoxia-dependent downregulation of MMACHC expression by HIF1A and DROSHA knockdowns, whose combined effect was additive. Together, these results strongly indicate that MMACHC is a hypoxia-regulated gene whose downregulation appears to be partially mediated through both hypoxia-induced transcription factor and microRNA machinery. These findings suggest that oxidative stress could impair vitamin B12 metabolism by repression of MMACHC in healthy as well as in diseased individuals.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12.

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.

Spectrum and characterization of bi-allelic variants in MMAB causing cblB-type methylmalonic aciduria.

Pathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5'-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 µM; Kd2 = 8.4 µM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.

Mitochondrial disease, mitophagy, and cellular distress in methylmalonic acidemia.

Mitochondria-the intracellular powerhouse in which nutrients are converted into energy in the form of ATP or heat-are highly dynamic, double-membraned organelles that harness a plethora of cellular functions that sustain energy metabolism and homeostasis. Exciting new discoveries now indicate that the maintenance of this ever changing and functionally pleiotropic organelle is particularly relevant in terminally differentiated cells that are highly dependent on aerobic metabolism. Given the central role in maintaining metabolic and physiological homeostasis, dysregulation of the mitochondrial network might therefore confer a potentially devastating vulnerability to high-energy requiring cell types, contributing to a broad variety of hereditary and acquired diseases. In this Review, we highlight the biological functions of mitochondria-localized enzymes from the perspective of understanding-and potentially reversing-the pathophysiology of inherited disorders affecting the homeostasis of the mitochondrial network and cellular metabolism. Using methylmalonic acidemia as a paradigm of complex mitochondrial dysfunction, we discuss how mitochondrial directed-signaling circuitries govern the homeostasis and physiology of specialized cell types and how these may be disturbed in disease. This Review also provides a critical analysis of affected tissues, potential molecular mechanisms, and novel cellular and animal models of methylmalonic acidemia which are being used to develop new therapeutic options for this disease. These insights might ultimately lead to new therapeutics, not only for methylmalonic acidemia, but also for other currently intractable mitochondrial diseases, potentially transforming our ability to regulate homeostasis and health.

Shifting landscapes of human MTHFR missense-variant effects.

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice.

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B12 metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.

Decrease of disease-related metabolites upon fasting in a hemizygous knock-in mouse model (Mut-ko/ki) of methylmalonic aciduria.

Methylmalonyl-CoA mutase (MMUT) is part of the propionyl-CoA catabolic pathway, responsible for the breakdown of branched-chain amino acids, odd-chain fatty acids and the side-chain of cholesterol. Patients with deficient activity of MMUT suffer from isolated methylmalonic aciduria (MMAuria), frequently presenting in the newborn period with failure to thrive and metabolic crisis. Even well managed patients remain at risk for metabolic crises, of which one known trigger is acute illness, which may lead to poor feeding and vomiting, putting the patient in a catabolic state. This situation is believed to result in increased breakdown of propionyl-CoA catabolic pathway precursors, producing massively elevated levels of disease related metabolites, including methylmalonic acid and propionylcarnitine. Here, we used fasting of a hemizygous mouse model (Mut-ko/ki) of MMUT deficiency to study the role of induced catabolism on metabolite production. Although mice lost weight and displayed markers consistent with a catabolic state, contrary to expectation, we found strongly reduced levels of methylmalonic acid and propionylcarnitine in fasted conditions. Switching Mut-ko/ki mice from a high-protein diet to fasted conditions, or from a standard diet to a no-protein diet, resulted in similar reductions of methylmalonic acid and propionylcarnitine levels. These results suggest, in our mouse model at least, induction of a catabolic state on its own may not be sufficient to trigger elevated metabolite levels.

Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain.

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.

Naturally occurring cobalamin (B12) analogs can function as cofactors for human methylmalonyl-CoA mutase.

Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds - cobamides - that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.

Clinical and structural insights into potential dominant negative triggers of proximal urea cycle disorders.

Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.

Biochemical analysis of patients with mutations in MTHFD1 and a diagnosis of methylenetetrahydrofolate dehydrogenase 1 deficiency.

MTHFD1 is a trifunctional protein containing 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase activities. It is encoded by MTHFD1 and functions in the cytoplasmic folate cycle where it is involved in de novo purine synthesis, synthesis of thymidylate and remethylation of homocysteine to methionine. Since the first reported case of severe combined immunodeficiency resulting from MTHFD1 mutations, seven additional patients ascertained through molecular analysis have been reported with variable phenotypes, including megaloblastic anemia, atypical hemolytic uremic syndrome, hyperhomocysteinemia, microangiopathy, infections and autoimmune diseases. We determined the level of MTHFD1 expression and dehydrogenase specific activity in cell extracts from cultured fibroblasts of three previously reported patients, as well as a patient with megaloblastic anemia and recurrent infections with compound heterozygous MTHFD1 variants that were predicted to be deleterious. MTHFD1 protein expression determined by Western blotting in fibroblast extracts from three of the patients was markedly decreased compared to expression in wild type cells (between 4.8 and 14.3% of mean control values). MTHFD1 expression in the fourth patient was approximately 44% of mean control values. There was no detectable methylenetetrahydrofolate dehydrogenase specific activity in extracts from any of the four patients. This is the first measurement of MTHFD1 function in MTHFD1 deficient patients and confirms the previous molecular diagnoses.

In-depth phenotyping reveals common and novel disease symptoms in a hemizygous knock-in mouse model (Mut-ko/ki) of mut-type methylmalonic aciduria.

Isolated methylmalonic aciduria (MMAuria) is primarily caused by deficiency of methylmalonyl-CoA mutase (MMUT or MUT). Biochemically, MUT deficiency results in the accumulation of methylmalonic acid (MMA), propionyl-carnitine (C3) and other metabolites. Patients often exhibit lethargy, failure to thrive and metabolic decompensation leading to coma or even death, with kidney and neurological impairment frequently identified in the long-term. Here, we report a hemizygous mouse model which combines a knock-in (ki) missense allele of Mut with a knock-out (ko) allele (Mut-ko/ki mice) that was fed a 51%-protein diet from day 12 of life, constituting a bespoke model of MMAuria. Under this diet, mutant mice developed a pronounced metabolic phenotype characterized by drastically increased blood levels of MMA and C3 compared to their littermate controls (Mut-ki/wt). With this bespoke mouse model, we performed a standardized phenotypic screen to assess the whole-body impairments associated with this strong metabolic condition. We found that Mut-ko/ki mice show common clinical manifestations of MMAuria, including pronounced failure to thrive, indications of mild neurological and kidney dysfunction, and degenerative morphological changes in the liver, along with less well described symptoms such as cardiovascular and hematological abnormalities. The analyses also reveal so far unknown disease characteristics, including low bone mineral density, anxiety-related behaviour and ovarian atrophy. This first phenotypic screening of a MMAuria mouse model confirms its relevance to human disease, reveals new alterations associated with MUT deficiency, and suggests a series of quantifiable readouts that can be used to evaluate potential treatment strategies.

Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry.

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.