Analysis of fibroblasts from patients with cblC and cblG genetic defects of cobalamin metabolism reveals global dysregulation of alternative splicing.

Vitamin B12 or cobalamin (Cbl) metabolism can be affected by genetic defects leading to defective activity of either methylmalonyl-CoA mutase or methionine synthase or both enzymes. Patients usually present with a wide spectrum of pathologies suggesting that various cellular processes could be affected by modifications in gene expression. We have previously demonstrated that these genetic defects are associated with subcellular mislocalization of RNA-binding proteins (RBP) and subsequent altered nucleo-cytoplasmic shuttling of mRNAs. In order to characterize the possible changes of gene expression in these diseases, we have investigated global gene expression in fibroblasts from patients with cblC and cblG inherited disorders by RNA-seq. The most differentially expressed genes are strongly associated with developmental processes, neurological, ophthalmologic and cardiovascular diseases. These associations are consistent with the clinical presentation of cblC and cblG disorders. Multivariate analysis of transcript processing revaled splicing alterations that led to dramatic changes in cytoskeleton organization, response to stress, methylation of macromolecules and RNA binding. The RNA motifs associated with this differential splicing reflected a potential role of RBP such as HuR and HNRNPL. Proteomic analysis confirmed that mRNA processing was significantly disturbed. This study reports a dramatic alteration of gene expression in fibroblasts of patients with cblC and cblG disorders, which resulted partly from disturbed function of RBP. These data suggest to evaluate the rescue of the mislocalization of RBP as a potential strategy in the treatment of severe cases who are resistant to classical treatments with co-enzyme supplements.

The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology.

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.

Mouse models to study the pathophysiology of combined methylmalonic acidemia and homocystinuria, cblC type.

Combined methylmalonic acidemia and homocystinuria, cblC type, is the most common inherited disorder of cobalamin metabolism and is characterized by severe fetal developmental defects primarily impacting the central nervous system, hematopoietic system, and heart. CblC was previously shown to be due to mutations in the MMACHC gene, which encodes a protein thought to function in intracellular cobalamin trafficking and biosynthesis of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). These coenzymes are required for the production of succinyl-CoA and methionine, respectively. However, it is currently unclear whether additional roles for MMACHC exist outside of cobalamin metabolism. Furthermore, due to a lack of sufficient animal models, the exact pathophysiology of cblC remains unknown. Here, we report the generation and characterization of two new mouse models to study the role of MMACHC in vivo. CRISPR/Cas9 genome editing was used to develop a Mmachc floxed allele (Mmachcflox/flox), which we validated as a conditional null. For a gain-of-function approach, we generated a transgenic mouse line that over-expresses functional Mmachc (Mmachc-OE+/tg) capable of rescuing Mmachc homozygous mutant lethality. Surprisingly, our data also suggest that these mice may exhibit a partially penetrant maternal-effect rescue, which might have implications for in utero therapeutic interventions to treat cblC. Both the Mmachcflox/flox and Mmachc-OE+/tg mouse models will be valuable resources for understanding the biological roles of MMACHC in a variety of tissue contexts and allow for deeper understanding of the pathophysiology of cblC.

Cobalamin J disease detected on newborn screening: Novel variant and normal neurodevelopmental course.

Cobalamin J disease (CblJ) is an ultra-rare autosomal recessive disorder of intracellular cobalamin metabolism associated with combined methylmalonic acidemia and homocystinuria. It is caused by pathogenic variants in ABCD4, which encodes an ATP-binding cassette (ABC) transporter that affects the lysosomal release of cobalamin (Cbl) into the cytoplasm. Only six cases of CblJ have been reported in the literature. Described clinical features include feeding difficulties, failure to thrive, hypotonia, seizures, developmental delay, and hematological abnormalities. Information on clinical outcomes is extremely limited, and no cases of presymptomatic diagnosis have been reported. We describe a now 17-month-old male with CblJ detected by newborn screening and confirmed by biochemical, molecular, and complementation studies. With early detection and initiation of treatment, this patient has remained asymptomatic with normal growth parameters and neurodevelopmental function. To the best of our knowledge, this report represents the first asymptomatic and neurotypical patient with CblJ.

Subcortical lesions due to cobalamin deficiency: an unusual MRI lesion pattern.

We present a 44-year-old male patient with new onset of right focal epilepsy and bilateral hand hypesthesia. Cerebral MRI showed bilateral T2w/DWI hyperintense subcortical lesions in the cingulate gyrus, insula, and amygdala, whereas spinal MRI revealed a cervical posterior column lesion, corresponding to subacute combined degeneration. Laboratory workup revealed a cobalamin deficiency due to type A gastritis, and no evidence of antibodies associated with limbic encephalitis. After sufficient cobalamin substitution, the cerebral and spinal lesions gradually regressed. Our case represents a unique cerebral subcortical MRI lesion pattern in a patient with epilepsy and cobalamin deficiency. Thus, the latter represents an important differential diagnosis for autoimmune encephalitis.

Hereditary intrinsic factor deficiency in China caused by a novel mutation in the intrinsic factor gene-a case report.

BACKGROUND: Hereditary intrinsic factor deficiency is a rare disease characterized by cobalamin deficiency with the lack of gastric intrinsic factor because of gastric intrinsic factor (GIF) mutations. Patients usually present with cobalamin deficiency without gastroscopy abnormality and intrinsic factor antibodies. CASE PRESENTATION: A Chinese patient presented with recurrent severe anemia since age 2 with low cobalamin level and a mild elevation of indirect bilirubin. The hemoglobin level normalized each time after intramuscular vitamin B12 injection. Gene test verified a c.776delA frame shift mutation in exon 6 combined with c.585C > A nonsense early termination mutation in exon 5 of GIF which result in the dysfunction of gastric intrinsic factor protein. The hereditary intrinsic factor deficiency in literature was further reviewed and the ancestry of different mutation sites were discussed. CONCLUSIONS: A novel compound heterozygous mutation of GIF in a Chinese patient of hereditary intrinsic factor deficiency was reported. It was the first identified mutation of GIF in East-Asia and may indicate a new ancestry.

Vitamin B12 deficiency from the perspective of a practicing hematologist.

B12 deficiency is the leading cause of megaloblastic anemia, and although more common in the elderly, can occur at any age. Clinical disease caused by B12 deficiency usually connotes severe deficiency, resulting from a failure of the gastric or ileal phase of physiological B12 absorption, best exemplified by the autoimmune disease pernicious anemia. There are many other causes of B12 deficiency, which range from severe to mild. Mild deficiency usually results from failure to render food B12 bioavailable or from dietary inadequacy. Although rarely resulting in megaloblastic anemia, mild deficiency may be associated with neurocognitive and other consequences. B12 deficiency is best diagnosed using a combination of tests because none alone is completely reliable. The features of B12 deficiency are variable and may be atypical. Timely diagnosis is important, and treatment is gratifying. Failure to diagnose B12 deficiency can have dire consequences, usually neurological. This review is written from the perspective of a practicing hematologist.

Helicobacter pylori and extragastric diseases.

Many studies have been performed in the last year concerning the potential role of Helicobacter pylori in different extragastric diseases, reinforcing the idea that specific microorganisms may cause diseases even far from the primary site of infection. While the role of H. pylori on idiopathic thrombocytopenic purpura, sideropenic anemia, and vitamin B12 deficiency has been well established, there is a growing interest in other conditions, such as cardiovascular, neurologic, dermatologic, obstetric, immunologic, and metabolic diseases. Concerning neurologic diseases, there is a great interest in cognitive impairment and neurodegeneration. The aim of this review was to summarize the results of the most relevant studies published over the last year on this fascinating topic.

Computer-assisted interventions in the clinical laboratory process improve the diagnosis and treatment of severe vitamin B12 deficiency.

BACKGROUND: Severe vitamin B12 deficiency can result in serious complications if undiagnosed or untreated. Our aim was to test the efficacy of interventions in the laboratory process to improve the detection and the treatment of severe vitamin B12 deficiency. METHODS: Quasi-experimental investigation with a retrospective 7-year pre-intervention period and 29-month post-intervention follow-up in a university hospital. Two interventions were designed to improve the detection and treatment of subjects with vitamin B12 deficiency: the laboratory information system (LIS) automatically added seru vitamin B12 (s-vitamin B12) based on certain conditions; and created a comment in the report and scheduled an appointment with the general practitioner (GP). We calculated the number of new diagnoses of severe vitamin deficiency (s-vitamin B12 <73.8 pmol/L) and the proportion of identified patients that were correctly treated in the pre- and post-intervention periods. We compared the number of tests needed to detect a new case when ordered by GPs vs. added by the strategy. Finally, we investigated the economic cost of each new case. RESULTS: The strategy added 699 s-vitamin B12 and detected 66 new cases of severe vitamin deficiency. The number of tests needed to identify a new case when s-vitamin B12 was ordered by GPs was 187, as opposed to 10 when added through the intervention (p<0.001). The intervention reagent cost was €26.7 per new case. In the post-intervention cohort, 88% of patients were correctly treated, as opposed to 52% in the pre-intervention (p<0.001). CONCLUSIONS: Interventions in the clinical laboratory process improved the diagnosis and treatment of severe vitamin B12 deficiency.

Subacute Combined Degeneration: A Retrospective Study of 68 Cases with Short-Term Follow-Up.

PURPOSE: The study aimed to analyze the clinical characteristics, laboratory test results, neuroimaging findings, and outcomes in patients diagnosed with subacute combined degeneration (SCD). MATERIALS AND METHODS: A total of 68 patients with SCD who had been appropriately treated for no less than 6 months were included in our study. Histories, results of routine blood tests, biochemical indices, serum vitamin B12 levels, and spinal magnetic resonance imaging (MRI) findings from the patients were studied and analyzed. Clinical signs and symptoms, graded using a functional disability rating scale, were scored at the time of admission and 3 and 6 months after admission. RESULTS: Limb numbness, limb weakness, and gait disturbances were the most common symptoms in patients with SCD. All patients showed clinical improvement to different degrees at the follow-up visits after vitamin B12 treatment. No differences in rating score were found in patients grouped by sex, hemoglobin level, serum vitamin B12, or MRI manifestations at the time of admission or at the follow-up visits. Younger patients and those with shorter disease courses had better rating scores at the short-term follow-up visits. CONCLUSION: Anemia, low levels of serum vitamin B12, and MRI abnormalities in the spinal cord are not expected to be associated with worse clinical manifestations. The age of onset and course of disease are important in evaluating the short-term prognosis of patients with SCD.

Low vitamin B12 increases risk of gastric cancer: A prospective study of one-carbon metabolism nutrients and risk of upper gastrointestinal tract cancer.

Previous studies have found associations between one-carbon metabolism nutrients and risk of several cancers, but little is known regarding upper gastrointestinal tract (UGI) cancer. We analyzed prediagnostic serum concentrations of several one-carbon metabolism nutrients (vitamin B12, folate, vitamin B6, riboflavin and homocysteine) in a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of male smokers, which was undertaken in Finland between 1985 and 1988. We conducted a nested case-control study including 127 noncardia gastric adenocarcinoma (NCGA), 41 esophagogastric junctional adenocarcinoma and 60 esophageal squamous cell carcinoma incident cases identified within ATBC. Controls were matched to cases on age, date of serum collection and follow-up time. One-carbon nutrient concentrations were measured in fasting serum samples collected at baseline (up to 17 years prior to cancer diagnosis). Odds ratios and 95% confidence intervals (CI) were calculated using conditional logistic regression. Lower prediagnostic vitamin B12 concentrations at baseline were associated with a 5.8-fold increased risk of NCGA (95% CI = 2.7-12.6 for lowest compared to highest quartile, p-trend <0.001). This association remained in participants who developed cancer more than 10 years after blood collection, and after restricting the analysis to participants with clinically normal serum vitamin B12 (>300 pmol/L). In contrast, pepsinogen I, a known serologic marker of gastric atrophy, was not associated with NCGA in this population. As vitamin B12 absorption requires intact gastric mucosa to produce acid and intrinsic factor, our findings suggest vitamin B12 as a possible serologic marker for the atrophic gastritis that precedes NCGA, one more strongly associated with subsequent NCGA than pepsinogen.

The changing of serum vitamin B12 and homocysteine levels after gastrectomy in patients with gastric cancer: do they associate with clinicopathological factors?

After total (TG) or distal subtotal gastrectomy (DG), patients are at high risk of vitamin B12 (vit-B12) deficiency, which results in elevation of homocysteine levels. The changing of serum vit-B12 and homocysteine levels in patients with gastric cancer is not well known. Seventy-two patients with gastric cancer who had undergone currative gastrectomy and 50 healthy controls were included. Serum vit-B12 and homocysteine levels were analyzed in gastric cancer patients. In addition, these parameters were compared with those of healthy control subjects. While serum vit-B12 levels in gastrectomized patients were significantly lower than that of healthy controls (221.8 ± 125.6 pg/mL vs. 309.9 ± 174.3 pg/mL, p = 0.002), homocysteine levels were significantly higher in patients with gastric cancer (14.2 ± 6.7 µmol/L vs. 12.5 ± 6.1 µmol/L, p = 0.016). Mean serum folate level was found to be high in healthy controls (7.3 ng/mL) compared to patients (9.2 ng/mL, p = 0.027). Out of 72 patients, 40 patients (55.6 %) with gastric cancer developed vit-B12 deficiency after gastrectomy. Vit-B12 deficiency was found to be related with gastrectomy type (p = 0.02) and homocysteine levels (p = 0.014). In patients who underwent TG, the incidence of vit-B12 deficiency was significantly higher compared with those with DG (67.5 vs. 32.5 %). In addition, serum vit-B12 level in patients with DG was significantly higher than that of patients with TG (248.3 ± 122.0 pg/mL vs. 200.8 ± 126.7 pg/mL, p = 0.041), whereas homocysteine levels were significantly lower in DG group compared with TG group (12.1 ± 6.1 µmol/L vs. 15.8 ± 6.9 µmol/L, p = 0.014). A logistic regression analysis showed that the extent of gastrectomy was found to be an independent factor for predicting the occurrence of vit-B12 deficiency (p < 0.001, odds ratio 1.38). Our results showed that cumulative vit-B12 deficiency rate was significantly higher after TG compared with that after DG, while homocysteine levels were significantly higher in TG group compared with DG group. The extent of gastrectomy was found to be an independent factor for predicting the occurrence of vit-B12 deficiency. Vit-B12 deficiency and hyperhomocysteinemia are imperious clinical situation for patients with gastric cancer after surgery. Hence, both preoperative and regular postoperative monitoring of vit-B12 and homocysteine levels for all gastrectomized patients with gastric cancer are important and necessary for early detection and prevention of vit-B12 deficiency and hyperhomocysteinemia as a risk factor for cardiovascular diseases.

Nephrotic syndrome and thrombotic microangiopathy caused by cobalamin C deficiency.

BACKGROUND: Cobalamin C (CblC) defects are inherited autosomal recessive disorders of vitamin B12 metabolism due to mutations in the MMACHC gene. Renal manifestations include thrombotic microangiopathy (TMA), acute or chronic renal failure, tubulointerstitial nephritis, and proximal renal tubular acidosis. However, reports about glomerular pathologies are scarce. CASE REPORT: A 4-year-old boy presented with nephrotic syndrome, arterial hypertension, and chronic anemia but no signs of hemolysis. Renal biopsy showed TMA with ischemic glomerular collapse, foot process effacement, and tubulointerstitial fibrosis. Elevated serum levels of homocysteine suggested a cobalamin C disorder. This was confirmed by the identification of compound heterozygous mutations in the MMACHC gene. Initial therapy consisted of antihypertensive treatment including angiotensin converting enzyme inhibitor (ACEi) leading to blood pressure control and a significant reduction of proteinuria. After a definite diagnosis of CblC deficiency, hydroxocobalamin was introduced. Thereafter, homocysteine levels decreased, anemia resolved, and a further decline of proteinuria with normalization of serum protein levels was noted. Renal function remained stable. CONCLUSIONS: Although uncommon, the clinical picture of CblC defects may be ruled by nephrotic syndrome mimicking glomerulonephritis, minimal change disease, or primary focal and segmental glomerulosclerosis. Key to a correct diagnosis is elevated serum levels of homocysteine, and a definite diagnosis can be confirmed by genetic testing.

REVERSIBLE CLINICAL AND MAGNETIC RESONANCE IMAGING FINDINGS IN LATE-ONSET COBALAMIN C DEFECT.

Cobalamin C (Cbl C) disease is an inborn error of intracellular cobalamin metabolism. Two distinct clinical types are defined according to the age of onset. We describe an 8 year old girl with late-onset Cbl C disease presenting with neuropsychiatric symptoms. Mutation analysis revealed homozygous c.394C>T (p.R132X) mutation in the MMACHC gene. Serial magnetic resonance imaging (MRI) before and after the treatment are provided. MRI of the brain before treatment showed bilateral patchy focal hyperintensities in the white matter and cortical atrophy. After treatment with intramuscular hydroxycobalamin, oral folinic acid, oral betaine, normalization of MRI findings can be achieved in addition to clinical improvement. We present this case to draw attention to the reversibility of clinical and MRI findings in the late onset Cbl C disease after treatment.

Failure to thrive and life-threatening complications due to inherited selective cobalamin malabsorption effectively managed in a juvenile Australian shepherd dog.

A juvenile Australian shepherd dog exhibited failure to grow, inappetence, weakness, nonregenerative anemia, neutropenia, and cobalamin deficiency. DNA testing confirmed homozygosity of an amnionless mutation (AMN c.3G > A). Clinical signs resolved with supportive care and parenteral cobalamin supplementation. Inherited selective intestinal cobalamin malabsorption requiring lifelong parenteral supplementation should be considered in Australian shepherds, giant schnauzers, border collies, and beagles that fail to thrive.

Gestion efficace d'un retard de croissance et de complications potentiellement mortelles attribuables à une malabsorption de cobalamine sélective héréditaire chez un jeune chien Berger australien. Un jeune chien Berger australien a manifesté une absence de croissance, de l'inappétence, de la faiblesse, une anémie non régénérative, de la neutropénie et une déficience de cobalamine. Des tests d'ADN ont confirmé l'homozygotisme d'une mutation des récepteurs amnionless (AMN c.3G > A). Les signes cliniques se sont résorbés avec des soins de soutien et des suppléments de cobalamine parentéraux. Une malabsorption intestinale sélective héréditaire de cobalamine exigeant des suppléments parentéraux à vie devrait être considérée chez les Bergers australiens, les Schnauzers géants, les Border-Collies et les Beagles qui manifestent des problèmes de croissance.(Traduit par Isabelle Vallières).

Molecular mechanisms leading to three different phenotypes in the cblD defect of intracellular cobalamin metabolism.

The cblD defect of intracellular vitamin B(12) metabolism can lead to isolated methylmalonic aciduria (cblD-MMA) or homocystinuria (cblD-HC), or combined methylmalonic aciduria and homocystinuria (cblD-MMA/HC). We studied the mechanism whereby MMADHC mutations can lead to three phenotypes. The effect of various expression vectors containing MMADHC modified to contain an enhanced mitochondrial leader sequence or mutations changing possible downstream sites of reinitiation of translation or mutations introducing stop codons on rescue of adenosyl- and methylcobalamin (MeCbl) formation was studied. The constructs were transfected into cell lines derived from various cblD patient's fibroblasts. Expression of 10 mutant alleles from 15 cblD patients confirmed that the nature and location of the mutations correlate with the biochemical phenotype. In cblD-MMA/HC cells, improving mitochondrial targeting of MMADHC clearly increased the formation of adenosylcobalamin (AdoCbl) with a concomitant decrease in MeCbl formation. In cblD-MMA cells, this effect was dependent on the mutation and showed a negative correlation with endogenous MMADHC mRNA levels. These findings support the hypothesis that a single protein exists with two different functional domains that interact with either cytosolic or mitochondrial targets. Also a delicate balance exists between cytosolic MeCbl and mitochondrial AdoCbl synthesis, supporting the role of cblD protein as a branch point in intracellular cobalamin trafficking. Furthermore, our data indicate that the sequence after Met116 is sufficient for MeCbl synthesis, whereas the additional sequence between Met62 and Met116 is required for AdoCbl synthesis. Accordingly, western blot studies reveal proteins of the size expected from the stop codon position with subsequent reinitiation of translation.

Vitamin B12 status does not influence central motor conduction time in asymptomatic elderly people: a transcranial magnetic stimulation study.

INTRODUCTION: Vitamin B12 deficiency causes neurologic and psychiatric disease, especially in older adults. Subacute combined degeneration is characterized by damage to the posterior and lateral spinal cord affecting the corticospinal tract. OBJECTIVE: To test corticospinal tract projections using motor evoked potentials (MEPs) by transcranial magnetic stimulation (TMS) in asymptomatic older adults with low vitamin B12 (B12) levels. METHODS: Cross-sectional study of 53 healthy older adults (>70 years). MEPs were recorded in the abductor pollicis brevis and tibialis anterior muscles, at rest and during slight tonic contraction. Central motor conduction time (CMCT) was derived from the latency of MEPs and peripheral motor conduction time (PMCT). Neurophysiological variables were analyzed statistically according to B12 status. RESULTS: Median age was 74.3 ± 3.6 years (58.5% women). Twenty-six out of the 53 subjects had low vitamin B12 levels (B12 < 221 pmol/l). MEPs were recorded for all subjects in upper and lower extremities. There were no significant differences in either latency or amplitude of MEPs and CMCT between low and normal B12 groups. There was a significant PMCT delay in the lower extremities in the low B12 group (p = 0.014). CONCLUSIONS: No subclinical abnormality of the corticospinal tract is detected in asymptomatic B12-deficient older adults. The peripheral nervous system appears to be more vulnerable to damage attributable to this vitamin deficit. The neurophysiological evaluation of asymptomatic older adults with lower B12 levels should be focused mainly in peripheral nervous system evaluation.