Cobalamin Malabsorption Research Articles

Severe pancytopenia at the presentation of Imerslund-Gräsbeck syndrome in a 23-month-old Italian boy

BackgroundImerslund-Gräsbeck syndrome (IGS) is a rare autosomal recessive disorder characterized by megaloblastic anemia due to selective cobalamin malabsorption and benign proteinuria. IGS is caused by a disfunction of the cubam receptor, which mediates the reabsorption of cobalamin in the ileum and the reuptake of albumin in renal proximal tubules.Case presentationWe describe the case of a 23-month-old-italian infant presenting with severe pancytopenia and failure to thrive in whom the diagnosis of IGS was made and vitamin B12 replacement therapy was resolutive. Genetic analysis (NGS with CNV analysis including 214 genes involved in bone marrow failure and anemia), showed the presence of two pathogenetic variants in the AMN gene (c-208–2 A > G and c.1006 + 34_1007-31del). These variants have been previously described in the literature, but their combination has never been reported.ConclusionsImerslund-Gräsbeck syndrome should be considered in the differential diagnosis of children with severe pancytopenia even in those without neurological involvement. This case emphasizes the importance of an early diagnosis and prompt treatment, to prevent irreversible neurological injury.

Update on Serum Biomarkers in Autoimmune Atrophic Gastritis.

Autoimmune atrophic gastritis (AAG) is a persistent, corpus-restricted immune-mediated destruction of the gastric corpus oxyntic mucosa with reduced gastric acid and intrinsic factor secretion, leading to iron deficiency and pernicious anemia as a consequence of iron and cobalamin malabsorption. Positivity toward parietal cell (PCA) and intrinsic factor (IFA) autoantibodies is very common. AAG may remain asymptomatic for many years, thus making its diagnosis complex and often delayed. Due to the increased risk of gastric neoplasms, a timely diagnosis of AAG is clinically important. The gold standard for AAG diagnosis is histopathological assessment of gastric biopsies obtained during gastroscopy, but noninvasive, preendoscopic serological screening may be useful in some clinical scenarios. Serum biomarkers for AAG may be divided into 2 groups: gastric autoimmunity-related biomarkers, such as PCA and IFA, and gastric corpus atrophy/reduced gastric acid secretion-related biomarkers, such as serum gastrin and pepsinogens. The present review focuses on the clinical significance and pitfalls of serum biomarkers related to gastric autoimmunity and gastric corpus atrophy, including some discussion of analytical methods. Serum assays for PCA, IFA, gastrin, and pepsinogen I show good diagnostic accuracy for noninvasive diagnostic work-up of AAG. Diagnostic performance may increase by combining >1 of these tests, overcoming the problem of seronegative AAG. However, appropriately designed, comparative studies with well-characterized patient cohorts are needed to better define the reliability of these biomarkers in the diagnosis of patients with AAG. Currently, positive serum tests should always be followed by the state-of-art diagnostic test, that is, histopathological assessment of gastric biopsies obtained during gastroscopy to definitively confirm or rule out AAG and eventually neoplastic complications.

Chronic Atrophic Gastritis Presenting as Hemolytic Anemia due to Severe Vitamin B12 Deficiency

Vitamin B12 is an essential nutrient which plays an important role in neurological function, hematopoiesis, and DNA synthesis. Low levels usually stem from either poor intake or a malabsorptive process. Presently, the most common cause of vitamin B12 deficiency is food-bound cobalamin malabsorption, which occurs when there is impaired release of vitamin B12 from ingested food due to an outstanding factor preventing the release of the nutrient from its transport protein. Such causes include achlorhydria, gastritis, gastrectomy, or the use of PPIs or antacids. A rarer cause is autoimmune chronic atrophic gastritis, resulting in pernicious anemia. In this disease process, there is destruction of parietal cells and thus a reduction in intrinsic factor, which is essential to the absorption of vitamin B12. Deficiency will result in a variety of abnormalities including but not limited to pancytopenia, paresthesias, and neuropsychiatric symptoms. A rare manifestation of vitamin B12 deficiency is hemolytic anemia, which occurs due to intramedullary and extramedullary dysfunction. This case describes a 46-year-old male with no past medical history who presented with chest pain, fatigue, and progressive weakness, found to have hemolytic anemia, ultimately attributed to vitamin B12 deficiency. Antiparietal cell antibodies and intrinsic factor antibodies (IFA) were both negative. Still, the patient underwent an endoscopy with biopsies of the stomach; pathology was consistent with chronic metaplastic atrophic gastritis. The patient improved with intramuscular vitamin B12 supplementation. This case highlights both a rare cause and presentation of vitamin B12 deficiency. Patients with autoimmune chronic atrophic gastritis should have antiparietal cell or intrinsic factor antibodies. Still, seronegative patients have been reported, like this patient. Additionally, hemolytic anemia secondary to vitamin B12 deficiency is uncommon. The presentation will usually mirror that of a thrombotic microangiopathy (TMA), including hemolytic anemia with schistocytes on peripheral blood smear and thrombocytopenia, as it did in this patient. This clinical entity is described as pseudothrombotic microangiopathy and is crucial to identify in order to prevent the initiation of invasive treatment strategies such as plasmapheresis.

Daily oral cyanocobalamin supplementation in Beagles with hereditary cobalamin malabsorption (Imerslund-Gräsbeck syndrome) maintains normal clinical and cellular cobalamin status.

BackgroundEfficacy of PO cobalamin (Cbl) supplementation in dogs with hereditary Cbl malabsorption (Imerslund‐Gräsbeck syndrome, IGS) is unknown.ObjectivesTo evaluate PO Cbl supplementation in Beagles with IGS previously treated parenterally. We hypothesized that 1 mg cyano‐Cbl daily PO would maintain clinical and metabolic remission.AnimalsThree client‐owned Beagles with IGS and 48 healthy control dogs.MethodsProspective study. Daily PO cyanocobalamin (cyano‐Cbl; 1 mg) supplementation was monitored for 13 (2 dogs) and 8 months (1 dog). Health status was assessed by owner observations. Methylmalonic acid (MMA)‐to‐creatinine concentrations were measured using an ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐TMS) method on urine samples collected monthly. Concurrent measurements of serum MMA concentration (n = 7; UPLC‐TMS) were available for 1 dog.ResultsAll dogs remained in excellent health during PO supplementation. Urine MMA remained consistently low in 2 dogs (median, 2.5 mmol/mol creatinine; range, 1.2‐9; healthy dogs [n = 30], median, 2.9 mmol/mol creatinine; range, 1.3‐76.5). Urine MMA ranged from 38.9‐84.9 mmol/mol creatinine during the first 6 months in 1 dog already known to excrete comparable amounts when supplemented parenterally. Brief antibiotic treatment for an unrelated condition after 6 months resulted in low urine MMA (median, 2.8 mmol/mol creatinine; range, 1.9‐4.8) for the next 7 months. All concurrent serum MMA concentrations (median, 651 nmol/L; range, 399‐919) before and after month 6 were within the established reference interval (393‐1476 nmol/L; n = 48).Conclusions and Clinical ImportanceOne milligram of cyano‐Cbl daily PO appears efficacious for maintaining normal clinical status and normal cellular markers of Cbl metabolism in Beagles with IGS.

Inherited selective cobalamin malabsorption in Komondor dogs associated with a CUBN splice site variant

BackgroundThree Komondor dogs in a small family and 3 sporadic cases exhibited a constellation of signs that included juvenile-onset of failure-to-thrive, inappetence, vomiting and/or diarrhea, and weakness. In each we documented dyshematopoiesis, increased anion gap, methylmalonic acidemia/-uria, and serum cobalamin deficiency. Urine protein electrophoresis demonstrated excretion of cubam ligands. All clinical signs and metabolic abnormalities, except proteinuria, were reversed by regular parenteral cobalamin administration. The pattern of occurrence and findings in the disorder suggested an autosomal recessive inheritance of cobalamin malabsorption with proteinuria, a condition in humans called Imerslund-Gräsbeck syndrome. The purpose of this study was to determine the molecular cause of this disorder in Komondors.ResultsWhole genome sequencing of two affected Komondor dogs of unknown relatedness and one parent and a clinically-normal littermate of an affected dog revealed a pathogenic single-base change in the CUBN intron 55 splice donor consensus sequence (NM_001003148.1: c.8746 + 1G > A) that was homozygous in affected dogs and heterozygous in the unaffected parents. Alleles of the variant co-segregated with alleles of the disease locus in the entire family and all more distantly-related sporadic cases. A population study using a simple allele-specific DNA test indicated mutant allele frequencies of 8.3 and 4.5% among North American and Hungarian Komondors, respectively.ConclusionsDNA testing can be used diagnostically in Komondors when clinical signs are suggestive of cobalamin deficiency or to inform Komondor breeders prospectively and prevent occurrence of future affected dogs. This represents the third cubilin variant causing inherited selective cobalamin malabsorption in a large animal ortholog of human Imerslund-Gräsbeck syndrome.

Prospective long-term evaluation of parenteral hydroxocobalamin supplementation in juvenile beagles with selective intestinal cobalamin malabsorption (Imerslund-Gräsbeck syndrome).

BackgroundProspective studies on maintenance treatment for Beagles with hereditary selective cobalamin (Cbl) malabsorption (Imerslund‐Gräsbeck syndrome, IGS) are lacking. In our experience, measurement of methylmalonic acid (MMA), a Cbl‐dependent metabolite, seems more helpful to monitor Cbl status as compared with serum Cbl concentrations.ObjectivesTo evaluate a standardized Cbl supplementation scheme in Beagles with IGS. We hypothesized that a single parenteral dose of 1 mg hydroxocobalamin (OH‐Cbl) would maintain clinical and metabolic remission for up to 2 months.AnimalsSix client‐owned juvenile Beagles with genetically confirmed IGS and 28 healthy control dogs.MethodsProspective study. Monthly IM OH‐Cbl (1 mg) supplementation was done over a median of 9 months (range, 6‐13) in 6 dogs, followed by bimonthly (every 2 months) injections in 5 dogs over a median of 6 months (range, 3‐10). Health status was assessed by routine clinical examinations at injection time points and owner observations. Voided urine samples were collected immediately before OH‐Cbl injections for measurement of MMA‐to‐creatinine concentrations using a gas‐liquid chromatography‐tandem mass spectrometry (GC‐MS) method.ResultsAll dogs were clinically healthy while receiving monthly and bimonthly OH‐Cbl supplementation. Urinary MMA results in healthy dogs ranged from 1.3 to 76.5 mmol/mol creatinine (median, 2.9). Median urinary MMA concentrations did not differ between dogs with IGS receiving monthly (n = 49; 5.3 mmol/mol creatinine; range, 2.3‐50.4) and bimonthly (n = 31; 5.3 mmol/mol creatinine; range, 1.6‐50) injections.Conclusions and Clinical ImportanceA maintenance parenteral dose of 1 mg OH‐Cbl monthly or bimonthly appears adequate in Beagles with IGS monitored by metabolic testing.

Amnionless-mediated glycosylation is crucial for cell surface targeting of cubilin in renal and intestinal cells

Mutations in either cubilin (CUBN) or amnionless (AMN) genes cause Imerslund–Gräsbeck syndrome (IGS), a hereditary disease characterised by anaemia attributed to selective intestinal malabsorption of cobalamin and low-molecular weight proteinuria. Although cubilin protein does not have a transmembrane segment, it functions as a multi-ligand receptor by binding to the transmembrane protein, amnionless. We established a system to quantitatively analyse membrane targeting of the protein complex in cultured renal and intestinal cells and analysed the pathogenic mechanisms of mutations found in IGS patients. A novel CUBN mutation, several previously reported CUBN missense mutations and all previously reported AMN missense mutations resulted in endoplasmic reticulum (ER) retention and completely inhibited amnionless-dependent plasma membrane expression of cubilin. The ER retention of cubilin and amnionless was confirmed in renal proximal tubular cells of a patient with IGS. Notably, the interaction between cubilin and amnionless was not sufficient, but amnionless-mediated glycosylation of cubilin was necessary for their surface expression. Quantitative mass spectrometry and mutagenesis demonstrated that N-linked glycosylation of at least 4 residues of cubilin protein was required for its surface targeting. These results delineated the molecular mechanisms of membrane trafficking of cubilin in renal and intestinal cells.

THE RISK OF COBALAMIN DEFICIENCY SYMPTOMS RELATED TO LONG-TERM METFORMIN USE IN T2DM PATIENTS.

Metformin is the first-line oral antidiabetic agent used in the treatment of diabetes mellitus. One of the adverse reactions of the long term use of metformin is cobalamin malabsorption. Clinical and laboratory findings are important in the diagnosis of cobalamin deficiency. This study aimed to evaluate the risk of cobalamin deficiency symptoms related to long-term use of metformin in type 2 diabetes mellitus patients at Pasar Rebo General Hospital in Jakarta. This quantitative, observational study with retrospective cohort design was conducted in outpatient department Pasar Rebo General Hospital November 2015 until January 2016. 200 subjects were recruited and divided into two groups, patients who had been taking metformin for 1-3 years and patients who had been taking metformin for more than 3 years. Each patient was assessed for the presence of cobalamin deficiency symptoms. Cobalamin deficiency symptoms evaluated were symptoms of neuropathy (measured by DN4 questionnaire) and hematologic abnormalities associated to cobalamin deficiency, i.e. macrocytic erythrocyte, hypersegmented neutrophils, and giant bands. There are significant differences in the proportions of neuropathy symptoms (RR 2.36, 95%, p=0.000) and hematologic abnormalities (RR 1.5, 95%, p=0.007) between the two groups. Long-term use of metformin (≥3 years) may increase the risk of cobalamin deficiency symptoms in type 2 diabetes mellitus patients.

Vitamine B12 Deficiency Presenting As Severe Pancytopenia in Adult Patients. a Monocentric Study of 104 Cases

▪Vitamin B12 deficiency should be suspected in all patients with unexplained anemia and/or neuropsychiatric symptoms, and special attention should be paid to patients at risk (the elderly, vegetarians, patients with autoimmune thyroiditis or vitiligo, patients receiving proton pump inhibitors or biguanides for prolonged periods). Documented symptomatic pancytopenia related to cobalamin deficiency is very rare representing less than 5% cases in different series. We aim to report an important series of pancytopenia related to cobalamin deficiency to analyze the clinical, biological and outcomes of those patients.Methods :Consecutive patients presented with B12 deficiency and pancytopenia were included. Charts were retrospectively reviewed from an internal medicine department during a period of 15 years (2000-2015). Diagnosis of cobalamine deficiency used the vitamin B12 dosage bellow 200 pg/ml. Assessment included clinical features, blood count and morphological review and a statistical analysis of those parameters comparing pancytopenic and non-pancytopenic patients.ResultsThe medical records of 268 consecutive patients hospitalized for cobalamin deficiency from January 2000 to December 2015 were identified and retrospectively analyzed. Among them, 104 patients had pancytopenia (38.8%). The median age was 55+17 years and the sex ratio : 1. Pancytopenia revealed vitamin B12 deficiency in all cases. Neurological manifestations were associated in 13 cases and digestive involvement in 16 cases. Twenty five patients presented with febrile neutropenia. Fourteen patients (13.4%) presented with "pseudo" thrombotic microangiopathy.The mean hemoglobin level was 58+19 g/L, the neurtophils count was 1188+579/µl and median platelets count was 69.616+34.379/µl. Macrocytosis was present in 80.7% of patient with mean MCV was 106+11 fl, but was normal (<95 fl) in 20 patients. The mean serum vitamine B12 levels was 80+11 pg/ml. The median serum lactate dehydrogenase level was high (3.204 IU/L).The causes of B12 vitamin deficiency were mainly food cobalamin malabsorption (63.4%) and pernicious anemia (28.8%). Correction of the hematological abnormalities was achieved in all patients treated with either intramuscular, subcutaneous or oral cyanocobalamin.Significant differences appears comparing patients with or without pancytopenia. Pancytopenic patients had significantly lower MCV mean (p=0.001), lower polynuclear neutrophils count and lower platelets count. Atrophic gastritis was also significantly associated with pancytopenia (p=0.005). There was no significant difference in mean haemoglobin level (p=0.3), serum cobalamin level (p=0.17). Looking at other variables that could explain the high prevalence of pancytopenia in our series, there were no difference in ferritinemia level or number of patients with of iron deficiency .ConclusionIn this study, based a single institution with a large number of consecutive patients with well-documented cobalamin deficiency, we found a high prevalence (38,8%) of severe pancytopenia. We don't find yet an explanation for those findings. Those results can also remind us that severe vitamin B12 deficiency may present with findings mimicking malign hematologic disorder and timely recognition and supplementation lead to resolution of symptoms and blood abnormalities. DisclosuresNo relevant conflicts of interest to declare.

Methylmalonic Aciduria Secondary to Selective Cobalamin Malabsorption in a Yorkshire Terrier

An 8 wk old male Yorkshire terrier was presented with a 2 wk history of recurrent hypoglycemia, lethargy, and seizures. Investigations revealed a marked increase in blood ammonia, low serum cobalamin, and increased levels of urinary methylmalonic acid (MMA) excretion. No liver vascular abnormality was detected. The patient was diagnosed with methylmalonic aciduria due to cobalamin malabsorption. The patient responded well to parenteral cobalamin administration, and the urinary MMA levels normalized rapidly following instigation of treatment. Due to the suspected hereditary nature of selective cobalamin deficiency, one sibling of this dog was screened and found to be normal. This is the first reported case of MMA secondary to hypocobalaminemia in Yorkshire terriers, and the second report of this disease in a dog in the United Kingdom. Given the fact that clinical signs of MMA are similar to those seen in dogs with portosystemic shunts and that Yorkshire terriers are predisposed to liver vascular abnormalities, this case report adds important clinical information to the current available literature.

Congenital cobalamin malabsorption (Imerslund-Gräsbeck syndrome) in two Beagles in the UK

Two immature Beagles, one nine months old and one six months old, presented with a variety of clinical signs, including pyrexia, lethargy, weight loss and intermittent vomiting and diarrhoea in...

Degenerative Liver Disease in Young Beagles with Hereditary Cobalamin Malabsorption Because of a Mutation in the Cubilin Gene

Degenerative Liver Disease in Young Beagles with Hereditary Cobalamin Malabsorption Because of a Mutation in the Cubilin Gene

Selective Intestinal Cobalamin Malabsorption with Proteinuria (Imerslund‐Gräsbeck Syndrome) in Juvenile Beagles

BackgroundSelective intestinal cobalamin malabsorption with mild proteinuria (Imerslund‐Gräsbeck syndrome; I‐GS), is an autosomal recessive disorder of dogs caused by mutations in AMN or CUBN that disrupt cubam function and which can present as a medical emergency.ObjectivesTo describe the clinical, metabolic, and genetic bases of I‐GS in Beagles.AnimalsFour cobalamin‐deficient and 43 clinically normal Beagles and 5 dogs of other breeds.MethodsClinical description and candidate gene genetic study. Urinary organic acid and protein excretion were determined by gas‐chromatography and SDS‐PAGE, respectively. Renal cubilin protein expression was assessed on immunoblots. Mutation discovery was carried out by PCR amplification and DNA sequencing of exons with flanking splice sites and cDNA of CUBN and AMN. Genotyping was performed by restriction enzyme digestion of PCR amplicons.ResultsJuvenile‐affected Beagles exhibited failure to thrive, dyshematopoiesis with neutropenia, serum cobalamin deficiency, methylmalonic aciduria, hyperammonemia, and proteinuria. Affected dogs' kidneys lacked detectable cubilin protein. All affected dogs were homozygous for a single‐base deletion in CUBN exon 8 (CUBN c.786delC), predicting a translational frameshift, and the 2 parents tested were heterozygous.ConclusionsThe CUBN mutation in juvenile I‐GS Beagles causes a more severe cobalamin malabsorption than in Border Collies with a different CUBN defect, but is similar to I‐GS caused by AMN mutations in Giant Schnauzers and Australian Shepherds. Awareness of the disorder and breed predispositions to I‐GS is crucial to precisely diagnose and promptly treat hereditary cobalamin malabsorption and to prevent disease in those dogs at risk in future generations.

A frameshift mutation in the cubilin gene (CUBN) in Beagles with Imerslund-Gräsbeck syndrome (selective cobalamin malabsorption)

Mammals are unable to synthesize cobalamin or vitamin B12 and rely on the uptake of dietary cobalamin. The cubam receptor expressed on the intestinal endothelium is required for the uptake of cobalamin from the gut. Cubam is composed of two protein subunits, amnionless and cubilin, which are encoded by the AMN and CUBN genes respectively. Loss-of-function mutations in either the AMN or the CUBN gene lead to hereditary selective cobalamin malabsorption or Imerslund-Gräsbeck syndrome (IGS). We investigated Beagles with IGS and resequenced the whole genome of one affected Beagle at 15× coverage. The analysis of the AMN and CUBN candidate genes revealed a homozygous deletion of a single cytosine in exon 8 of the CUBN gene (c.786delC). This deletion leads to a frameshift and early premature stop codon (p.Asp262Glufs*47) and is, thus, predicted to represent a complete loss-of-function allele. We tested three IGS-affected and 89 control Beagles and found perfect association between the IGS phenotype and the CUBN:c.786delC variant. Given the known role of cubilin in cobalamin transport, which has been firmly established in humans and dogs, our data strongly suggest that the CUBN:c.786delC variant is causing IGS in the investigated Beagles.

An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs

Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome.

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.

Production and characterization of cyanocobalamin-enriched lettuce (Lactuca sativa L.) grown using hydroponics.

When lettuces (Lactuca sativa L.) grown for 30 days in hydroponic culture were treated with various concentrations of cyanocobalamin for 24 h, its content in their leaves increased significantly from nondetectable to 164.6 ± 74.7 ng/g fresh weight. This finding indicated that consumption of only two or three of these fresh leaves is sufficient to meet the Recommended Dietary Allowance for adults of 2.4 μg/day. Analyses using a cobalamin-dependent Escherichia coli 215 bioautogram and LC/ESI-MS/MS demonstrated that the cyanocobalamin absorbed from the nutrient solutions by the leaves did not alter any other compounds such as coenzymes and inactive corrinoids. Gel filtration indicated that most (86%) of the cyanocobalamin in the leaves was recovered in the free cyanocobalamin fractions. These results indicated that cyanocobalamin-enriched lettuce leaves would be an excellent source of free cyanocobalamin, particularly for strict vegetarians or elderly people with food-bound cobalamin malabsorption.

Beware of the Ailments of Vitamin B12 Deficiency

Vitamin B12 or cobalamin deficiency is a common problem in adult patients, which is however frequently missed. The most common cause of cobalamin deficiency is the food cobalamin malabsorption syndrome (> 60% of all cases). Neuropsychiatric manifestations can be the presenting and only early sign of cobalamin deficiency even in the absence of hematologic abnormalities. The deficiency can occur despite serum cobalamin levels at low normal values; thus, normal vitamin B12 levels have been revised upward to >350 pg/ml. Early detection and treatment are important to prevent structural and irreversible damage. The causes and ailments of vitamin B12 deficiency are herein overviewed and a diagnostic and therapeutic strategy is outlined.

Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

BackgroundInherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult.MethodsWe present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients.ResultsOur results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved.ConclusionsCbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.

Ancient founder mutation is responsible for Imerslund-Gräsbeck Syndrome among diverse ethnicities

BackgroundImerslund-Gräsbeck syndrome (IGS) was described just over 50 years ago by Olga Imerslund and Ralph Gräsbeck and colleagues. IGS is caused by specific malabsorption of cobalamin (Cbl) due to bi-allelic mutations in either the cubilin gene (CUBN) or the human amnionless homolog (AMN). Mutations in the two genes are commonly seen in founder populations or in societies with a high degree of consanguineous marriages. One particular mutation in AMN, c.208-2A>G, causing an out-of-frame loss of exon 4 in the mRNA, is responsible for some 15% of IGS cases globally. We present evidence that this founder mutation causes a substantial percentage of cases among diverse ethnicities and that the mutation is as old as human civilization.MethodsPartial genotyping indicated a founder event but its presence in diverse peoples of Arabic, Turkish, Jewish, and Hispanic ancestry suggested that the mutation might be recurrent. We therefore studied the flanking sequence spanning 3.5 Mb to elucidate the origin of the haplotype and estimate the age of the mutation using a Bayesian inference method based on observed linkage disequilibrium.ResultsThe mutation's distribution, the size of the shared haplotype, and estimates of growth rate and carrier frequency indicated that the mutation was a single prehistoric event. Dating back to the ancient Middle East around 11,600 BC, the mutation predates the advent of writing, farming, and the monotheistic religions of the region.ConclusionsThis mutation causes over 50% of the IGS cases among Arabic, Turkish, and Sephardic Jewish families, making it a primary target for genetic screening among diverse IGS cases originating from the Middle East. Thus, rare founder mutations may cause a substantial number of cases, even among diverse ethnicities not usually thought to be related.