Inborn errors of cobalamin absorption, transport and 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.

CblX (MIM309541) is an X-linked recessive disorder characterized by defects in cobalamin (vitamin B12) metabolism and other developmental defects. Mutations in HCFC1, a transcriptional co-regulator which interacts with multiple transcription factors, have been associated with cblX. HCFC1 regulates cobalamin metabolism via the regulation of MMACHC expression through its interaction with THAP11, a THAP domain-containing transcription factor. The HCFC1/THAP11 complex potentially regulates genes involved in diverse cellular functions including cell cycle, proliferation, and transcription. Thus, it is likely that mutation of THAP11 also results in biochemical and other phenotypes similar to those observed in patients with cblX. We report a patient who presented with clinical and biochemical phenotypic features that overlap cblX, but who does not have any mutations in either MMACHC or HCFC1. We sequenced THAP11 by Sanger sequencing and discovered a potentially pathogenic, homozygous variant, c.240C > G (p.Phe80Leu). Functional analysis in the developing zebrafish embryo demonstrated that both THAP11 and HCFC1 regulate the proliferation and differentiation of neural precursors, suggesting important roles in normal brain development. The loss of THAP11 in zebrafish embryos results in craniofacial abnormalities including the complete loss of Meckel's cartilage, the ceratohyal, and all of the ceratobranchial cartilages. These data are consistent with our previous work that demonstrated a role for HCFC1 in vertebrate craniofacial development. High throughput RNA-sequencing analysis reveals several overlapping gene targets of HCFC1 and THAP11. Thus, both HCFC1 and THAP11 play important roles in the regulation of cobalamin metabolism as well as other pathways involved in early vertebrate development.

BackgroundThe most common disorder of the intracellular cobalamin metabolism pathway is the combined methylmalonic acidemia and homocysteinemia, cblC type (cblC). There is a variation in its clinical spectrum ranging from severe neonatal-onset forms that are highly fatal to later-onset forms which are milder. In this study, the first case of an asymptomatic Chinese woman with a defect in congenital cobalamin (cblC type) metabolism at prenatal diagnosis due to elevated homocysteine level is identified.Case presentationThe proband, a male child born to a 29-year-old G1P0 mother, admitted to local hospital with feeding disorder, intellectual disability, seizures, microcephaly, as well as heterophthalmos. The level of the urine methylmalonic was elevated. Equally found were increased blood propionylcarnitine (C3) and propionylcarnitine/free carnitine ratio (C3/C0) and decreased methionine levels. The plasma total homocysteine level was elevated at 101.04 μmol/L (normal < 15 μmol/L). The clinical diagnosis of combined methylmalonic acidemia and homocysteinemia was supported. Four years later, the mother of the boy married again and came to us for prenatal diagnosis exactly 15 weeks after her last menstrual period. Subsequently, there is an increase in the amniotic fluid methylmalonate. The level of the amniotic fluid total homocysteine was marginally high. A considerably elevated amniotic fluid C3 was equally observed. In addition, there is a respective significant increase in the plasma and urine total homocysteine at 31.96 and 39.35 μmol/L. After the sequencing of MMACHC genes, it is found that the boy, a proband carried a homozygous mutation of the MMACHC at c.658_660delAAG. While the boy's mother, she carries two mutations in MMACHC: c.658_660delAAG and c.617G>A. The fetus is a carrier of the MMACHC gene. Following the administration of routine treatment, the mother remained symptom-free in the course of pregnancy, and she gave birth to a healthy boy.ConclusionVariable and nonspecific symptoms characterized the cblC type of methylmalonic acidemia combined with homocysteinemia. Both biochemical assays and mutation analysis are recommended as crucial complementary techniques.

SIRT1 activation rescues the mislocalization of RNA-binding proteins and cognitive defects induced by inherited cobalamin disorders

Derivatives of cobalamin (vitamin B(12)) are required for activity of two enzymes in humans. Adenosylcobalamin is required for activity of mitochondrial methylmalonylCoA mutase and methylcobalamin is required for activity of cytoplasmic methionine synthase. Deficiency in cobalamin, or inability to absorb cobalamin normally, can result in accumulation of methylmalonic acid and homocysteine in blood and urine. Methylmalonic acidemia can result in metabolic acidosis which in severe cases may be fatal. Hyperhomocysteinemia along with hypomethioninemia can result in hematologic (megaloblastic anemia, neutropenia, thrombocytopenia) and neurologic (subacute combined degeneration of the cord, dementia, psychosis) defects. Inborn errors affecting cobalamin absorption (inherited intrinsic factor deficiency, Imerslund–Gra¨ sbeck syndrome) and transport (transcobalamin deficiency) have been described. A series of inborn errors of intracellular cobalamin metabolism, designated cblA-cblG, have been differentiated by complementation analysis. These can give rise to isolated methylmalonic acidemia (cblA, cblB, cblD variant 2), isolated hyperhomocysteinemia (cblD variant 1, cblE, cblG) or combined methylmalonic acidemia and hyperhomocysteinemia (cblC, classic cblD, cblF). All these disorders are inherited as autosomal recessive traits. The genes underlying each of these disorders have been identified. Two other disorders, haptocorrin deficiency and transcobalamin receptor deficiency, have been described, but it is not clear that they have any consistent clinical phenotype.

Late onset of symptoms in an atypical patient with the cblJ inborn error of vitamin B12 metabolism: Diagnosis and novel mutation revealed by exome sequencing

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

Inherited methylmalonicacidemia due to deficiency of methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase; EC 5.4.99.2) activity results from at least three classes of biochemically distinct defects affecting cobalamin (Cbl: vitamin B12) metabolism (cbl A, cbl B, and cbl C mutants) and a fourth class producing a defective mutase apoenzyme. We have obtained genetic evidence in support of this biochemical heterogeneity, using heterokaryons prepared by Sendai-virus-mediated cell fusion. Nine fibroblast lines from patients with defective Cbl metabolism (4 cbl A, 3 cbl B, and 2 cbl C), two from patients with defective mutase apoenzyme, and two from controls were fused in pairwise combinations and tested for functional mutase holoenzyme using a radioautographic procedure which detects [14C]propionate incorporation into trichloroacetic-acid-precipitable material in fibroblast monolayers in situ. Each of the mutants incorporates negligible radioactivity compared to control cells. Activity is also negligible when different mutants are mixed without virus or when homokaryons are produced by self-fusion. Heterokaryons produced by fusing members of each of the four mutant classes with representatives of any other class recover the ability to incorporate [14C]propionate to levels comparable to those of control cells. However, heterokaryons produced between members of the same class fail to complement in all cases. We conclude that the mutants with defective Cbl metabolism (cbl A, cbl B, cbl C) comprise three complementation groups, that a fourth group corresponds to mutase apoenzyme deficiency, and that all four classes of mutations are recessively inherited.

We describe an inborn error of vitamin B12 metabolism in an infant who had severe developmental delay, megaloblastic anemia, and homocystinuria. There was no evidence of methylmalonic aciduria or deficiency of folate or vitamin B12. Treatment with hydroxocobalamin, but not with cyanocobalamin and folic acid, resulted in rapid clinical and biochemical improvement. Cultured fibroblasts showed an absolute growth requirement for methionine, defective incorporation of radioactivity from [14C]5-methyltetrahydrofolate into protein, and normal incorporation of radioactivity from [14C]propionate, thus assigning the intracellular defect to methionine synthesis. The proportion of intracellular methylcobalamin in the fibroblasts was decreased, but that of 5'-deoxyadenosylcobalamin was normal. Methionine synthetase activity in cell extracts was normal, as was cobalamin incorporation into cultured cells. This defect differs from those described previously in being limited to methylcobalamin accumulation and defective use of 5-methyltetrahydrofolate by intact cells with normal activity of methylmalonyl CoA mutase.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

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.

Based on Victor Herbert’s model for sequential stages in the development of vitamin B12 deficiency, the holotranscobalamin (HoloTC) immunoassay has controversially been promoted as a more specific and sensitive replacement for the total vitamin B12 test, for the diagnosis of deficiency. There have been no longitudinal studies, by means of experimental cobalamin deficiency, because ethical considerations prevent such risky studies on patients or healthy human volunteers. The objective was to provide a detailed record of the response of HoloTC, compared to total vitamin B12 and metabolites, to the development of experimental vitamin B12 deficiency in an initially replete human subject. This 54 year old male, with a vitamin B12 deficiency possibly caused by a defect in the intracellular cobalamin metabolism, ensured an initially replete condition by means of oral doses of cyanocobalamin supplements at 1000 μg/day for 12 weeks. The subject then depleted himself of vitamin B12, by withholding treatment and using a low-cobalamin diet, until significant metabolic disturbances were observed. The responses of serum total vitamin B12 and HoloTC and the two metabolites, plasma methylmalonic acid and homocysteine, were monitored by weekly blood tests. HoloTC was not significantly more sensitive than either total serum vitamin B12 or total homocysteine, and was much less sensitive than methylmalonic acid. HoloTC decreased from an initial concentration of >128 pmol/L to a minimum of 33 pmol/L on day 742, the only day on which it fell below the lower limit of the reference interval. Total vitamin B12 decreased from an initial concentration of 606 pmol/L to a minimum of 171 pmol/L on day 728. Total homocysteine increased from an initial concentration of 8.4 μmol/L to a maximum of 14.2 μmol/L on day 609. Methylmalonic acid unexpectedly contained four distinct peaks; initially at 0.17 μmol/L, it first exceeded the upper limit of the reference interval on day 386, finally reaching a maximum peak of 0.90 μmol/L on day 658. The results of this experiment are inconsistent with Herbert’s hypothesis that HoloTC is the earliest marker of vitamin B12 deficiency, and therefore do not support his model for the staged development of vitamin B12 deficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1740-5) contains supplementary material, which is available to authorized users.

Methylmalonic aciduria and homocystinuria, cobalamin C type (cblC), constitute the most common inborn error of intracellular cobalamin metabolism. Here, we report the case of a 6-month-old child, presenting severe subacute neurological decline associated with failure to thrive. Biochemical tests indicated a disorder of intracellular cobalamin metabolism, with elevated urinary and plasma methylmalonic acid levels associated with high plasma homocysteine concentrations, with normal plasma vitamin B12 concentrations. Diagnosis was later confirmed by genetic analysis which identified two pathogenic variants on the MMACHC gene: c.271dupA (p.Arg91lysfs∗14) paternal allele and c.388T>C (p.Tyr130His) maternal allele. The patient responded well to hydroxocobalamin treatment, with a rapid recovery of symptoms and a normal growth at 2.8 years of follow-up. This case illustrates the importance of early diagnosis of cobalamin metabolism disorders by prescribing adequate biochemical tests.

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.

Disorders of cobalamin (vitamin B12) metabolism are increasingly recognized in small animal medicine and have a variety of causes ranging from chronic gastrointestinal disease to hereditary defects in cobalamin metabolism. Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in clinical practice. While the detection of hypocobalaminemia has therapeutic implications, interpretation of cobalamin status in dogs can be challenging. The aim of this review is to define hypocobalaminemia and cobalamin deficiency, normocobalaminemia, and hypercobalaminemia in dogs, describe known cobalamin deficiency states, breed predispositions in dogs, discuss the different biomarkers of importance for evaluating cobalamin status in dogs, and discuss the management of dogs with hypocobalaminemia.