Inborn Error Of Cobalamin Metabolism Research Articles

Multiomic analysis in fibroblasts of patients with inborn errors of cobalamin metabolism reveals concordance with clinical and metabolic variability

The high variability in clinical and metabolic presentations of inborn errors of cobalamin (cbl) metabolism (IECM), such as the cblC/epicblC types with combined deficits in methylmalonyl-coA mutase (MUT) and methionine synthase (MS), are not well understood. They could be explained by the impaired expression/activity of enzymes from other metabolic pathways. We performed metabolomic, genomic, proteomic, and post-translational modification (PTM) analyses in fibroblasts from three cblC cases and one epi-cblC case compared with three cblG cases with specific MS deficits and control fibroblasts. CblC patients had metabolic profilings consistent with altered urea cycle, glycine, and energy mitochondrial metabolism. Metabolomic analysis showed partial disruption and increased glutamate/ketoglutarate anaplerotic pathway of the tricarboxylic acid cycle (TCA), in patient fibroblasts. RNA-seq analysis showed decreased expression of MT-TT (mitochondrial tRNA threonine), MT-TP (mitochondrial tRNA proline), OXCT1 (succinyl CoA:3-oxoacid CoA transferase deficiency), and MT-CO1 (cytochrome C oxidase subunit 1). Proteomic changes were observed for key mitochondrial enzymes, including NADH:ubiquinone oxidoreductase subunit A8 (NDUFA8), carnitine palmitoyltransferase 2 (CPT2), and ubiquinol-cytochrome C reductase, complex III subunit X (UQCR10). Propionaldehyde addition in ornithine aminotransferase was the predominant PTM in cblC cells and could be related with the dramatic cellular increase in propionate and methylglyoxalate. It is consistent with the decreased concentration of ornithine reported in 3 cblC cases. Whether the changes detected after multi-omic analyses underlies clinical features in cblC and cblG types of IECM, such as peripheral and central neuropathy, cardiomyopathy, pulmonary hypertension, development delay, remains to be investigated. The omics-related effects of IECM on other enzymes and metabolic pathways are consistent with the diversity and variability of their age-related metabolic and clinical manifestations. PTMs are expected to produce cumulative effects, which could explain the influence of age on neurological manifestations. French Agence Nationale de la Recherche (Projects PREDICTS and EpiGONE) and Inserm.

Overexpression of MMACHC Prevents Craniofacial Phenotypes Caused by Knockdown of znf143b.

ZNF143 is a sequence-specific DNA binding protein that regulates the expression of protein-coding genes and small RNA molecules. In humans, ZNF143 interacts with HCFC1, a transcriptional cofactor, to regulate the expression of downstream target genes, including MMACHC, which encodes an enzyme involved in cobalamin (cbl) metabolism. Mutations in HCFC1 or ZNF143 cause an inborn error of cobalamin metabolism characterized by abnormal cbl metabolism, intellectual disability, seizures, and mild to moderate craniofacial abnormalities. However, the mechanisms by which ZNF143 mutations cause individual phenotypes are not completely understood. Defects in metabolism and craniofacial development are hypothesized to occur because of decreased expression of MMACHC. But recent results have called into question this mechanism as the cause for craniofacial development. Therefore, in the present study, we implemented a loss of function analysis to begin to uncover the function of ZNF143 in craniofacial development using the developing zebrafish. The knockdown of znf143b, one zebrafish ortholog of ZNF143, caused craniofacial phenotypes of varied severity, which included a shortened and cleaved Meckel's cartilage, partial loss of ceratobranchial arches, and a distorted ceratohyal. These phenotypes did not result from a defect in the number of total chondrocytes but were associated with a mild to moderate decrease in mmachc expression. Interestingly, expression of human MMACHC via endogenous transgene prevented the onset of craniofacial phenotypes associated with znf143b knockdown. Collectively, our data establishes that knockdown of znf143b causes craniofacial phenotypes that can be alleviated by increased expression of MMACHC.

Abnormal chondrocyte development in a zebrafish model of cblC syndrome restored by an MMACHC cobalamin binding mutant

Variants in the MMACHC gene cause combined methylmalonic acidemia and homocystinuria cblC type, the most common inborn error of intracellular cobalamin (vitamin B12) metabolism. cblC is associated with neurodevelopmental, hematological, ocular, and biochemical abnormalities. In a subset of patients, mild craniofacial dysmorphia has also been described. Mouse models of Mmachc deletion are embryonic lethal but cause severe craniofacial phenotypes such as facial clefts. MMACHC encodes an enzyme required for cobalamin processing and variants in this gene result in the accumulation of two metabolites: methylmalonic acid (MMA) and homocysteine (HC). Interestingly, other inborn errors of cobalamin metabolism, such as cblX syndrome, are associated with mild facial phenotypes. However, the presence and severity of MMA and HC accumulation in cblX syndrome is not consistent with the presence or absence of facial phenotypes. Thus, the mechanisms by which mutations in MMACHC cause craniofacial defects are yet to be completely elucidated. Here we have characterized the craniofacial phenotypes in a zebrafish model of cblC (hg13) and performed restoration experiments with either a wildtype or a cobalamin binding deficient MMACHC protein. Homozygous mutants did not display gross morphological defects in facial development but did have abnormal chondrocyte nuclear organization and an increase in the average number of neighboring cell contacts, both phenotypes were fully penetrant. Abnormal chondrocyte nuclear organization was not associated with defects in the localization of neural crest specific markers, sox10 (RFP transgene) or barx1. Both nuclear angles and the number of neighboring cell contacts were fully restored by wildtype MMACHC and a cobalamin binding deficient variant of the MMACHC protein. Collectively, these data suggest that mutation of MMACHC causes mild to moderate craniofacial phenotypes that are independent of cobalamin binding.

Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

PRDX1 gene-related epi-cblC disease is a common type of inborn error of cobalamin metabolism with mono- or bi-allelic MMACHC epimutations

BackgroundThe role of epigenetics in inborn errors of metabolism (IEMs) is poorly investigated. Epigenetic changes can contribute to clinical heterogeneity of affected patients but could also be underestimated determining factors in the occurrence of IEMs. An epigenetic cause of IEMs has been recently described for the autosomal recessive methylmalonic aciduria and homocystinuria, cblC type (cblC disease), and it has been named epi-cblC. Epi-cblC has been reported in association with compound heterozygosity for a genetic variant and an epimutation at the MMACHC locus, which is secondary to a splicing variant (c.515-1G > T or c.515-2A > T) at the adjacent PRDX1 gene. Both these variants cause aberrant antisense transcription and cis-hypermethylation of the MMACHC gene promotor with subsequent silencing. Until now, only nine epi-cblC patients have been reported.MethodsWe report clinical/biochemical assessment, MMACHC/PRDX1 gene sequencing and genome-wide DNA methylation profiling in 11 cblC patients who had an inconclusive MMACHC gene testing. We also compare clinical phenotype of epi-cblC patients with that of canonical cblC patients.ResultsAll patients turned out to have the epi-cblC disease. One patient had a bi-allelic MMACHC epimutation due to the homozygous PRDX1:c.515-1G > T variant transmitted by both parents. We found that the bi-allelic epimutation produces the complete silencing of MMACHC in the patient’s fibroblasts. The remaining ten patients had a mono-allelic MMACHC epimutation, due to the heterozygous PRDX1:c.515-1G > T, in association with a mono-allelic MMACHC genetic variant. Epi-cblC disease has accounted for about 13% of cblC cases diagnosed by newborn screening in the Tuscany and Umbria regions since November 2001. Comparative analysis showed that clinical phenotype of epi-cblC patients is similar to that of canonical cblC patients.ConclusionsWe provide evidence that epi-cblC is an underestimated cause of inborn errors of cobalamin metabolism and describe the first instance of epi-cblC due to a bi-allelic MMACHC epimutation. MMACHC epimutation/PRDX1 mutation analyses should be part of routine genetic testing for all patients presenting with a metabolic phenotype that combines methylmalonic aciduria and homocystinuria.

Redox-Linked Coordination Chemistry Directs Vitamin B12 Trafficking.

Metals are partners for an estimated one-third of the proteome and vary in complexity from mononuclear centers to organometallic cofactors. Vitamin B12 or cobalamin represents the epitome of this complexity and is the product of an assembly line comprising some 30 enzymes. Unable to biosynthesize cobalamin, mammals rely on dietary provision of this essential cofactor, which is needed by just two enzymes, one each in the cytoplasm (methionine synthase) and the mitochondrion (methylmalonyl-CoA mutase). Brilliant clinical genetics studies on patients with inborn errors of cobalamin metabolism spanning several decades had identified at least seven genetic loci in addition to the two encoding B12 enzymes. While cells are known to house a cadre of chaperones dedicated to metal trafficking pathways that contain metal reactivity and confer targeting specificity, the seemingly supernumerary chaperones in the B12 pathway had raised obvious questions as to the rationale for their existence.With the discovery of the genes underlying cobalamin disorders, our laboratory has been at the forefront of ascribing functions to B12 chaperones and elucidating the intricate redox-linked coordination chemistry and protein-linked cofactor conformational dynamics that orchestrate the processing and translocation of cargo along the trafficking pathway. These studies have uncovered novel chemistry that exploits the innate chemical versatility of alkylcobalamins, i.e., the ability to form and dismantle the cobalt-carbon bond using homolytic or heterolytic chemistry. In addition, they have revealed the practical utility of the dimethylbenzimidazole tail, an appendage unique to cobalamins and absent in the structural cousins, porphyrin, chlorin, and corphin, as an instrument for facilitating cofactor transfer between active sites.In this Account, we navigate the chemistry of the B12 trafficking pathway from its point of entry into cells, through lysosomes, and into the cytoplasm, where incoming cobalamin derivatives with a diversity of upper ligands are denuded by the β-ligand transferase activity of CblC to the common cob(II)alamin intermediate. The broad reaction and lax substrate specificity of CblC also enables conversion of cyanocobalamin (technically, vitamin B12, i.e., the form of the cofactor in one-a-day supplements), to cob(II)alamin. CblD then hitches up with CblC via a unique Co-sulfur bond to cob(II)alamin at a bifurcation point, leading to the cytoplasmic methylcobalamin or mitochondrial 5'-deoxyadenosylcobalamin branch. Mutations at loci upstream of the junction point typically affect both branches, leading to homocystinuria and methylmalonic aciduria, whereas mutations in downstream loci lead to one or the other disease. Elucidation of the biochemical penalties associated with individual mutations is providing molecular insights into the clinical data and, in some instances, identifying which cobalamin derivative(s) might be therapeutically beneficial.Our studies on B12 trafficking are revealing strategies for cofactor sequestration and mobilization from low- to high-affinity and low- to high-coordination-number sites, which in turn are regulated by protein dynamics that constructs ergonomic cofactor binding pockets. While these B12 lessons might be broadly relevant to other metal trafficking pathways, much remains to be learned. This Account concludes by identifying some of the major gaps and challenges that are needed to complete our understanding of B12 trafficking.

Value of amniotic fluid homocysteine assay in prenatal diagnosis of combined methylmalonic acidemia and homocystinuria, cobalamin C type

BackgroundCombined methylmalonic acidemia and homocystinuria, cobalamin C type (cblC defect) is the most common inborn error of cobalamin metabolism, and different approaches have been applied to its prenatal diagnosis. To evaluate the reliability of biochemical method for the prenatal diagnosis of cblC defect, we conducted a retrospective study of our 10-year experience at a single center.Methods248 pregnancies whose probands were diagnosed as cblC defect were referred to our center for prenatal diagnosis from January 2010 to December 2019. Prenatal data of Hcy levels determined by enzymatic cycling assay, acylcarnitine analysis using liquid chromatography tandem mass spectrometry, organic acid analysis using gas chromatography mass spectrometry, and genetic analysis by direct sequencing of 248 at-risk fetuses were retrospectively reviewed.ResultsFor 2.0 and 16.0 μmol/L levels of Hcy AF samples, the relative errors were − 2.5% and 2.8%, respectively. The respective measurement uncertainties were 13.07% and 14.20%. For the 248 at-risk fetuses, 63 fetuses were affected and 185 fetuses were unaffected. Hcy level of 13.20 (6.62–43.30) μmol/L in 63 affected fetuses was significantly higher than that in 185 unaffected fetuses of 2.70 (0.00–5.80) μmol/L, and there was no overlap between the affected and unaffected groups. The diagnostic sensitivity and specificity of Hcy were 100% and 92.05%, respectively. The positive and negative predictive values of the combination of Hcy, propionylcarnitine (C3), ratio of C3 to acetylcarnitine (C2; C3/C2), methylmalonic acid (MMA), and methylcitric acid (MCA) were both 100%. Sixteen fetuses displayed inconclusive genetic results of MMACHC variants, in which seven fetuses were determined to be affected with elevated levels of Hcy, C3, C3/C2 and MMA, and their levels were 18.50 (6.70–43.30) μmol/L, 8.53(5.02–11.91) μmol/L, 0.77 (0.52–0.97), 8.96 (6.55–40.32) mmol/mol Cr, respectively. The remaining nine fetuses were considered unaffected based on a normal amniotic fluid metabolite profile.ConclusionsHcy appears to be another characteristic biomarker for the prenatal diagnosis of cblC defect. The combination of Hcy assay with acylcarnitine and organic acid analysis is a fast, sensitive, and reliable prenatal diagnostic biochemical approach. This approach could overcome the challenge of the lack of genetic analysis for families with at-risk cblC defect fetuses.

Brain Circuit Alterations and Cognitive Disability in Late-Onset Cobalamin D Disorder.

Neuroimaging studies describing brain circuits’ alterations in cobalamin (vitamin B12)-deficient patients are limited and have not been carried out in patients with inborn errors of cobalamin metabolism. The objective of this study was to assess brain functionality and brain circuit alterations in a patient with an ultra-rare inborn error of cobalamin metabolism, methylmalonic aciduria, and homocystinuria due to cobalamin D disease, as compared with his twin sister as a healthy control (HC). We acquired magnetic resonance imaging (including structural, functional, and diffusion images) to calculate brain circuit abnormalities and combined these results with the scores after a comprehensive neuropsychological evaluation. As compared with HC, the patient had severe patterns of damage, such as a 254% increment of ventricular volume, pronounced subcortical and cortical atrophies (mainly at striatum, cingulate cortex, and precuneus), and connectivity alterations at fronto-striato-thalamic circuit, cerebellum, and corpus callosum. In agreement with brain circuit alterations, cognitive deficits existed in attention, executive function, inhibitory control, and mental flexibility. This is the first study that provides the clinical, genetic, neuroanatomical, neuropsychological, and psychosocial characterization of a patient with the cobalamin D disorder, showing functional alterations in central nervous system motor tracts, thalamus, cerebellum, and basal ganglia, that, as far as we know, have not been reported yet in vitamin B12-related disorders.

Clinical feature and outcome of late-onset cobalamin C disease patients with neuropsychiatric presentations: a Chinese case series.

ObjectiveThe Cobalamin C (cblC) disease is an inborn error of cobalamin metabolism. Late-onset cblC disease was diagnosed in patients having overt symptoms after 4 years of age. The late-onset cblC disease patients were rare and easily misdiagnosed. This study analyzed the clinical presentations, gene mutations, and treatments of Chinese patients with late-onset cblC disease.MethodsThe clinical data of 26 Han Chinese patients diagnosed with late-onset cblC disease were retrospectively analyzed. All patients underwent serum homocysteine level exam, urine concentrations of organic acids measurement, neuroimaging scans, gene analysis, and treatments evaluations.ResultsThe mean age at disease onset and diagnosis was 17.8±7.0 years. The most frequent neuropsychiatric disturbances were lower limb weakness (50%), psychiatric disturbances (46.2%), and gait instability (42.3%). The mean methylmalonic acid level in urine was 107.4±56.6 μmol/L, and mean serum total homocysteine was 105.4±41.0 μmol/L. The most common abnormal radioimaging changes were observed in the spinal cord (88%) and brain (32%). Scoliosis was detected in 85.7% of patients. The methylmalonic aciduria and homocystinuria type C protein gene analysis showed that c.482G>A (57.7%) and c.609G>A (34.6%) mutations were the most frequent genotypes. After treatments with hydroxycobalamin, betaine, folic acid, L-carnitine, and compound vitamin B, the clinical features and biochemical parameters of patients with late-onset cblC disease were found to be alleviated.ConclusionIn our late-onset cblC disease cases, lower limb weakness, psychiatric disturbances, and gait instability were the most frequent manifestations. Patients responded well to the drug treatments with hydrocobalamin and betaine. When juvenile or adult patients with hyperhomocysteinemia present with neurological symptoms, cblC disease needs to be considered.

Reversible encephalopathy caused by an inborn error of cobalamin metabolism

Reversible encephalopathy caused by an inborn error of cobalamin metabolism

CblC Type Methylmalonic Aciduria with a Novel Homozygous Mutation: A Case Report

Background CombinedMethylmalonic Aciduria (MMA), and homocystinuria CblC type is the most common inborn error of cobalamin metabolism with 77 mutations identified till date in the MMACHC gene. The disease has early and late presentations with varied clinical features. Case report A pair of preterm monochorionic twins was born to non-consanguineous parents with history of 2 previous infant deaths with suspected MMA. The twins became symptomatic in second week with severe neutropenia, thrombocytopenia, progressive encephalopathy and eczema without anemia, metabolic acidosis or hyperammonemia. Investigation revealed elevated plasma Complement 3 (C3) acyl carnitine and very high MMA levels in urine. Genetic testing revealed a novel large deletion mutation in chromosome 1 which comprised of the whole MMACHC gene confirming the diagnosis of CblC type of MMA. The mutation was homozygous despite parents being unrelated and was identified in both parents later. Both twins responded transiently to treatment with hydroxycobalamin. Conclusion Large deletion in the MMACHC genewhich was identified in a homozygous state in our twins is previously unreported. The phenotypic features are described.

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR.

The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.

Late-onset cobalamin C deficiency Chinese sibling patients with neuropsychiatric presentations.

The Cobalamin C deficiency (cblC), characterized with elevated methylmalonic acidemia and homocystinuria in plasma, is an inborn error of cobalamin metabolism. The late-onset cblC siblings patients were rarely reported. In this study, we analyzed the clinical presentations and treatment outcomes of late-onset cblC in Chinese sibling patients with neuropsychiatric presentations. The clinical data of four pairs of Chinese patients were retrospectively analyzed. Serum homocysteine, urine organic acids measurements, neuroimaging exams and gene analysis were carried out in all patents. Patients were reevaluated after treatments with cobalamin, folate, betaine, L-carnitine and compound vitamin B. The mean age at disease onset was 13.7 (range 2-19) years. The neuropsychiatric disturbances including cognitive decline (3/8), psychiatric disturbances (4/8), gait instability (2/8), lower extremity weakness and numbness (3/8) and thromboembolic events (1/8). Two patients suffered nephropathy. The mean serum homocysteine when patients were diagnosed was 109.4 (range 69.5-138) μM/L. The abnormal radioimaging included scoliosis by X-ray (5/6), cerebral atrophy (4/6) and spinal cord atrophy (3/6) by MRI scan. Three pairs of siblings showed heterozygous mutations of MMACHC gene including c.482G > A (4/6), c.354G > C (2/6), c.570insT (2/6), c.445_446del (2/6) and c.656_4658del (2/6). The other two siblings showed homozygous mutation with c.452A > G in MMACHC gene. After treatments, the psychiatric symptoms were obviously relieved in all the patients. In Chinese siblings with late-onset cblC, the main clinic manifestation and abnormal radioimaging were cognitive decline and cerebral atrophy respectively. The most common gene mutation was c.482G > A of MMACHC gene. The patients responded well to the treatments.

Neuropsychological implications of Cobalamin C (CblC) disease in Hispanic children detected through newborn screening

ABSTRACTCobalamin C (CblC) disease is the most common inborn error of cobalamin metabolism and recent data has indicated a higher prevalence among children of Hispanic heritage in particular. The purpose of this study was to (a) describe the neuropsychological characteristics of a pilot sample of Hispanic children with CblC disease and (b) explore potential differences in outcome based on underlying genetic mutation(s) and biochemical levels. Six Hispanic children (ages 2–10) diagnosed with CblC disease through newborn screening (NBS) underwent neuropsychological evaluation with a bilingual examiner. Biochemical levels and underlying mutation(s) were obtained through medical records. The overall sample performed below normative expectations across neuropsychological domains, including general cognition, adaptive functioning, language ability, and visual-motor integration. Underlying mutations and associative clinical phenotypes were found to significantly predict general cognitive abilities, while plasma methionine and Hcy at the time of diagnosis were significantly correlated with language outcomes. Despite limited sample size, results indicate that Hispanic children with CblC disease detected through NBS and treated early experience neuropsychological deficits even when treated with current standard treatments. However, consistent with prior research in non-Hispanic children with CblC disease, underlying mutations and early biochemical levels may predict better outcomes in this population as well.

Lessons in biology from patients with inherited disorders of vitamin B12 and folate metabolism

Over the last forty years, our laboratory has accumulated a collection of over 1000 cultured fibroblast lines derived from patients from around the world referred with signs of inborn errors of cobalamin or folate metabolism, including several hundred with complementation-confirmed diagnoses. By accurately classifying patient disorders into classes representing blocks affecting specific reactions, we have provide the basis for rational assessment of phenotypic heterogeneity, and development of methods for diagnosis, treatment and prognosis. These resources have been valuable in identification of causal genes for known inborn errors. Since 2000, we and our collaborators identified the genes for the cblA (MMAA), cblB (MMAB), cblC (MMACHC), cblD (MMADHC), and cblF (LMBRD1) disorders. Whole exome sequencing of DNA from a patient with severe combined immunodeficiency (SCID), megaloblastic anemia and hemolytic uremic syndrome identified mutations in the MTHFD1 gene, which encodes a trifunctional enzyme involved in interconversion of folate coenzyme derivatives. This disorder demonstrates the importance de novo pyrimidine synthesis in the etiology of SCID. Mutations in the ABCD4 gene have been identified in four patients with accumulation of unbound cobalamin in lysosomes; this gene encodes a lysosomal membrane protein that plays a role in the transport of cobalamin across this membrane. Mutations in the HCFC1 gene on the X chromosome have been identified in several male patients that had received a diagnosis of cblC on the basis of complementation studies in cultured fibroblasts. HCFC1 encodes a transcription factor that regulates expression of a number of genes, including MMACHC, the gene that is mutated in patients with the cblC disorder. These studies demonstrate that with the advent of affordable whole exome sequencing, it has been possible to identify genes for novel inborn errors of cobalamin metabolism, often working from a small number of affected patients.

46. The First Viable Mouse Model of cblC Type Combined Methylmalonic Acidemia and Homocysteinemia: AAV Gene Therapy Rescues Neonatal Lethality and Provides Insight into Disease-Associated Retinal Degeneration

Combined methylmalonic acidemia and homocysteinemia, cblC type (cblC), is the most common inborn error of cobalamin metabolism and is caused by mutations in the MMACHC gene. MMACHC transports and processes intracellular cobalamin (vitamin B12) into its two active cofactors, 5’-adenosylcobalamin and methylcobalamin, necessary for the enzymatic reactions of methylmalonyl-CoA mutase and methionine synthase, respectively. Mutations in MMACHC result in methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia. Disease manifestations include growth failure, anemia, heart defects, developmental delay and a progressive maculopathy and pigmentary retinopathy that causes blindness, usually by the end of the first decade. Despite the use of conventional therapies including cobalamin injections and other cofactors, the manifestions of cblC, in particular the eye disease, remain unresolved with treatment. In order to explore disease pathophysiology and develop AAV gene therapy for cblC deficiency, we first created a viable mouse model using TALENs to edit the murine Mmachc gene, near the location of the most common mutation in humans, c. 271dupA. Two alleles were investigated: c. 165_166delAC p. P56CfsX4 (Δ2) and c. 162_164delCAC p. S54_T55delinsR (Δ3). MmachcΔ2/Δ3 and MmachcΔ3/Δ3 homozygous mutant mice displayed reduced survival, severe growth retardation, and massive metabolic perturbations. The median survival was less than 7 days with 90% of the mutant mice perishing before 3 weeks (Δ2 n=13; Δ3 n=42). The weights of MmachcΔ3/Δ3 mice were reduced relative to heterozygote and wild type littermates (n=15, p<0.0001). MmachcΔ2/Δ2 and MmachcΔ3/Δ3 mice (n=4, n=6) recapitulated the biochemical features of cblC, with significantly elevated plasma methylmalonic acid, homocysteine, cystathionine and decreased methionine compared to littermate controls (n=7) (p<0.05 for all metabolites). To assess the potential for gene therapy as a treatment for cblC, we generated two AAV vectors: rAAVrh10-CBA-mMmachc and rAAV9-CBA-hMMACHC and compared AAV with biweekly injections of OH-cobalamin, the standard therapy. MmachcΔ3/Δ3 mice were then treated with a single vector dose (1 × 1011 GC) delivered via intrahepatic injection in the neonatal period. MmachcΔ3/Δ3 mice treated with AAV vectors (AAVrh10 n=11, AAV9 n=5) displayed dramatically improved clinical appearance with improved growth (p= 0.0568), and increased survival (p<0.0001 for both vectors), with the oldest treated mutants currently living beyond 9 months. Successful gene therapy in the MmachcΔ3/Δ3 mice also enabled us to model, for the first time, cblC associated ocular pathology: surviving MmachcΔ3/Δ3 mice displayed thinning of the outer nuclear layer and shortening of photoreceptor outer segments, consistent with the pathology described in patients. Our results demonstrate that AAV gene delivery of MMACHC represents a new therapy for cblC which can treat the systemic, and possibly ocular, manifestations of this relatively common and devastating inborn error of metabolism.

The proteome of cblC defect: in vivo elucidation of altered cellular pathways in humans.

Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.

Clinical onset and course, response to treatment and outcome in 24 patients with the cblE or cblG remethylation defect complemented by genetic and in vitro enzyme study data.

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. 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. 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. The majority of patients with the cblE and cblG defect show limited clinical response to treatment and have neurocognitive impairment.

Clinical, Biochemical, and Molecular Presentation in a Patient with the cblD-Homocystinuria Inborn Error of Cobalamin Metabolism.

Disorders of intracellular cobalamin (vitamin B12) metabolism result from deficient synthesis of the coenzymes derived from vitamin B12: adenosylcobalamin and methylcobalamin. Disturbances of cobalamin-cofactor synthesis result in elevated levels of homocysteine and/or methylmalonic acid. Nine defects of intracellular cobalamin metabolism have been defined. The most common of these disorders is cblC (combined methylmalonic aciduria and homocystinuria). The cblD disorder is rare with fewer than twenty cases reported in the literature. Some cblD patients have combined methylmalonic aciduria and homocystinuria (referred to as "cblD original," "cblD-combined," or herein "cblD-MMA/HC"); some have isolated homocystinuria (referred to as "cblD-variant 1" or herein "cblD-HC"); and others have isolated methylmalonic aciduria (called "cblD-variant 2" or herein "cblD-MMA"). Only six cases of cblD-HC have been defined thus far. We report the 7th case of cblD-HC. The clinical manifestations, biochemical profile, genetic mutation, and plausible ancestry are discussed.

Streptococcus Pneumonia -Associated Hemolytic Uremic Syndrome: A Case Report

Hemolytic uremic syndrome (HUS), a disease that destroys red blood cells, is the most common cause of sudden, short term acute kidney failure in children. By far, the commonest subgroup (>90% of childhood HUS) is induced by shiga toxin producing bacteria, usually enterohemorrhagic Escherichia coli (EHEC). These patients typically have a prodrome of diarrhea. Another important subgroup that is readily identified on clinical grounds follows invasive Streptococcus pneumonia infection. These infants tend to be younger than those with D + HUS, and the syndrome is very rare after the age of 4 years. They present with pneumonia, empyema, meningitis, and less often, isolated septicemia. Another group of patients may have mutations in complement regulators, and less often, inherited deficiency of von Willebrand protease or an inborn error of cobalamin metabolism. We report a 6-month-old child who developed hemolytic uremic syndrome following S. pneumonia meningitis.Keywords: Hemolytic-Uremic Syndrome; Meningitis; Streptococcus pneumonia; Child