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Premacular retrohyaloid hemorrhage is a rare complication of acute severe anemia. The authors report two cases of premacular hemorrhage in which no treatment other than clinical and spectral-domain optical coherence tomography observation was performed. The natural history of this condition reveals that complete clinical resolution is not accompanied by full anatomical restoration. [ Ophthalmic Surg Lasers Imaging Retina . 2014;45:E5–E7.]

A 49-year-old man presented with neurosis, hyperpigmentation of the skin, and depigmentation of the hair. On examination, hyperpigmentation was observed on the oral mucosa and the skin of the forearms, elbows, palmar creases and periunguinal area, knees, and feet. He had megaloblastic anemia with a low serum level of vitamin B12 due to malabsorption resulting from a gastrectomy 10 years previously. His hyperpigmentation was resolved with vitamin B12 supplementation. Histology showed an increase of melanin in the basal layer. In electron microscopic study, many melanosomes were observed in melanocytes and surrounding keratinocytes. We consider that the dominant mechanism of hyperpigmentation due to vitamin B12 deficiency is not a defect in melanin transport but is rather an increase in melanin synthesis.

Thiamine-responsive megaloblastic anemia (TRMA) syndrome (OMIM No. 249270) comprises a distinctive triad of clinical features: megaloblastic anemia with ringed sideroblasts, diabetes mellitus, and progressive sensorineural deafness. The TRMA gene has been mapped and cloned. Designated "SLC19A2" as a member of the solute carrier gene superfamily, this gene is mutated in all TRMA kindreds studied to date. The product of the SLC19A2 gene is a membrane protein which transports thiamine (vitamin B1) with sub-micromolar affinity. Cells from TRMA patients are uniquely sensitive to thiamine depletion to the nanomolar range, while pharmacologic doses of vitamin B1 ameliorate the anemia and diabetes. Here we review the current status of studies aimed at understanding the pathophysiology of this unique transport defect.

A case of transient splenic accumulation of the bone-seeking agent Tc-99m HMDP is reported. This effect was caused by transient megaloblastic anemia induced by 5-fluorouracil chemotherapy. The extent of splenic uptake reflected the development and severity of megaloblastic anemia. The mechanism of splenic accumulation is thought to be similar to transient iron deposition in the spleen by megaloblastic anemia.

Classically, deficiency of folic acid (folates) or vitamin B 12 (cobalamins) was recognized by the presence of a macrocytic anemia which was the result of megaloblastic changes in the bone marrow. A markedly changing paradigm has identified both new mechanisms for altered folate and cobalamin status and new sequelae and clinical interrelationships that include altered mechanisms of absorption, a changing pattern of neurologic deficits, an increased risk of vascular occlusive lesions, and an important relationship with the mechanisms of neoplastic transformation. Several of these newer characterizations relate to issues of neoplasia in the nonpregnant woman and to issues in pregnancy, such as the potential for developmental abnormalities of the fetal nervous system. In pregnancy the predominant expression of folate or cobalamin deficiency is in the form of a megaloblastic anemia. 48 Historically the existence of megaloblastic anemia in pregnancy was considered rare, because the anemia was presumed to produce infertility; in 1919, however, Osler 81 described such cases. In the 1960s, the presence of macrocytic anemias, marrow megaloblastosis, and altered serum levels of folate and cobalamins in pregnancy refocused interest in these metabolic changes. The recognition of the critical role of folate in the first trimester of pregnancy in the development of the fetal neurologic system indicated a new pattern of deficiency unrelated to the presence or absence of a megaloblastic-macrocytic anemia.

Background: Elevation of mean cell volume (MCV) is a common clinical problem, but the etiologic spectrum and optimal diagnostic evaluation of macrocytosis are not well defined. Methods: We studied 300 consecutive hospitalized adult patients with MCV values ≥ 100 fL. Assessment included complete blood counts, morphologic review, liver function tests, and levels of serum cobalamin (Cbl), methyl malonic acid, and total homocysteine. Results: The most common cause of macrocytosis was drug therapy, followed by alcohol, liver disease, and reticulocytosis. Megaloblastic hematopoiesis accounted for less than 10% of cases. MCV values > 120 fL were usually caused by Cbl deficiency. Anisocytosis, macro-ovalocytosis, and teardrop erythrocytes were most prominent in megaloblastic hematopoiesis. Elevated levels of serum methyl malonic acid and total homocysteine were useful in the diagnosis of Cbl deficiency. Conclusions: Drugs and alcohol are the most common causes of macrocytosis in hospitalized patients in a New York City teaching hospital. We have formulated tentative guidelines for the evaluation of high MCV values in this setting.

Thiamine-responsive megaloblastic anemia (TRMA) is a rare autosomal recessive disorder characterized by megaloblastic anemia, diabetes mellitus and progressive sensorineural deafness. We report the cases of two infants, aged 4 and 5 months, hospitalized for diabetic ketoacidosis requiring insulin therapy. Laboratory tests revealed megaloblasic anemia, thrombocytopenia and normal thiamine level. Neurosensorial investigations showed bilateral deafness and ophthalmic involvement. Treatment with oral thiamine normalized hematological disorders and controlled diabetes; however, thiamine therapy had no impact on neurosensorial disorders.

Methionine synthase reductase (MSR) deficiency is an autosomal recessive disorder of folate/cobalamin metabolism leading to hyperhomocysteinemia, hypo- methioninemia and megaloblastic anemia. Deficiency in MSR activity occurs as the result of a defect in the MSR enzyme, which is required for the reductive activation of methionine synthase (MS). MS itself is responsible for the folate/cobalamin-dependent conversion of homo- cysteine to methionine. We have recently cloned the cDNA corresponding to the MSR protein, a novel member of the ferredoxin-NADP(+)reductase (FNR) family of electron transferases. We have used RT-PCR, heteroduplex, single-strand conformation poly- morphism (SSCP) and DNA sequence analyses to reveal 11 mutations in eight patients from seven families belonging to the cblE complementation group of patients of cobalamin metabolism that is defective in the MSR protein. The mutations include splicing defects leading to large insertions or deletions, as well as a number of smaller deletions and point mutations. Apart from an intronic substitution found in two unrelated patients, the mutations appear singular among individuals. Of the eleven, three are nonsense mutations, allowing for the identification of two patients for whom little if any MSR protein should be produced. The remaining eight involve point mutations or in-frame disruptions of the coding sequence and are distributed throughout the coding region, including proposed FMN, FAD and NADPH binding sites. These data demonstrate a unique requirement for MSR in the reductive activation of MS.

An 18-month-old girl presented with macrocytic megaloblastic anaemia followed by haemolytic uraemic syndrome. Metabolic investigations led to the identification of an inborn error of cobalamin metabolism consisting of defective methylcobalamin biosynthesis, probably cobalamin G, since methionine synthase activity was decreased under standard reducing conditions. Despite treatment, pulmonary hypertension progressively developed and responded to oxygen therapy. Renal involvement evolved to terminal failure and haemodialysis, while pulmonary hypertension was controlled by oxygen therapy. Such clinical manifestations have never been reported in association with a defect of methylcobalamin and thus of methionine biosynthesis. A congenital abnormality of cobalamin metabolism was suspected then confirmed in the presence of typical haematological features associated with unusual clinical manifestations such as progressive renal failure and pulmonary hypertension.

Thiamine-responsive megaloblastic anaemia (TRMA), also known as Rogers syndrome, is an early onset, autosomal recessive disorder defined by the occurrence of megaloblastic anaemia, diabetes mellitus and sensorineural deafness, responding in varying degrees to thiamine treatment1,2 (MIM 249270). We have previously narrowed the TRMA locus from a 16-cM to a 4-cM interval on chromosomal region 1q23.3 (Refs 3, 4) and this region has been further refined to a 1.4-cM interval5. Previous studies have suggested that deficiency in a high-affinity thiamine transporter may cause this disorder6,7. Here we identify the TRMA gene by positional cloning. We assembled a P1-derived artificial chromosome (PAC) contig spanning the TRMA candidate region. This clarified the order of genetic markers across the TRMA locus, provided 9 new polymorphic markers and narrowed the locus to an approximately 400-kb region. Mutations in a new gene, SLC19A2, encoding a putative transmembrane protein homologous to the reduced folate carrier proteins8,9, were found in all affected individuals in six TRMA families, suggesting that a defective thiamine transporter protein (THTR-1) may underlie the TRMA syndrome.

Thiamine-responsive megaloblastic anaemia with diabetes and deafness1 (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in thiamine (vitamin B1) transport2,3. Pharmacological doses of thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible4. Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref. 5), and fine-mapping has recently narrowed that region further6,7. We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity thiamine transport8. Expression of a gene encoding a known yeast thiamine transporter, THI10 (refs 8,9,10), in TRMA mutant cells prevents apoptotic cell death in thiamine-depleted medium. On the basis of these studies, we hypothesized that a defective thiamine transporter causes TRMA. We undertook a candidate gene approach to identify putative thiamine transporters in the 1q23.3 critical region. Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (thiamine transporter), member 2) encodes the first known mammalian thiamine transporter, which we designate thiamine transporter-1 (THTR-1).

Megaloblastic anaemia 1 (MGA1, OMIM 261100) is a rare, autosomal recessive disorder characterized by juvenile megaloblastic anaemia, as well as neurological symptoms that may be the only manifestations. At the cellular level, MGA1 is characterized by selective intestinal vitamin B12 (B12, cobalamin) malabsorption. MGA1 occurs worldwide, but its prevalence is higher in several Middle Eastern countries and Norway, and highest in Finland (0.8/100,000). We previously mapped the MGA1 locus by linkage analysis in Finnish and Norwegian families to a 6-cM region on chromosome 10p12.1 (ref. 8). A functional candidate gene encoding the intrinsic factor (IF)-B12 receptor, cubilin, was recently cloned; the human homologue, CUBN, was mapped to the same region. We have now refined the MGA1 region by linkage disequilibrium (LD) mapping, fine-mapped CUBN and identified two independent disease-specific CUBN mutations in 17 Finnish MGA1 families. Our genetic and molecular data indicate that mutations in CUBN cause MGA1.

We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400–550 nM; Vmax 11 pmol/min/106 cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death. J. Clin. Invest. 103:723–729 (1999).

Involvement of the gastrointestinal tract by plasmocytoma is rare. In a 78-year-old man with IgA lambda multiple myeloma stage IIIB, the evaluation of a megaloblastic anaemia revealed a subnormal vitamin B 12 level. Urinary excretion of isotope-labelled vitamin B 1 2 was reduced. Tests for gastric parietal cell and intrinsic factor antibodies were negative. There were no clinical signs of an insufficient absorption in the ileum. Biopsy specimens of the stomach showed a dense, diffuse infiltrate of malignant plasma cells in the lamina propria of fundus and corpus. A urease test for Helicobacter pylori was positive. There was a minor haematological improvement when vitamin B 12 was given parenterally. Several combinations of cytostatic drugs had no effect on the manifestations of the multiple myeloma. In our patient the vitamin B 12 deficiency may be related to a displacement or destruction of parietal cells by malignant plasma cells.

Background There has been little systematic study of the clinical spectrum of pancytopenia, and the optimal diagnostic approach to pancytopenia remains undefined. Methods The authors studied 134 hospitalized pancytopenie patients in Zimbabwe in both consecutive and nonconsecutive fashion. Results The most common cause of pancytopenia was megaloblastic anemia, followed by aplastic anemia, acute leukemia, acquired immunodeficiency syndrome (AIDS), and hypersplenism. Severe pancytopenia was usually due to aplastic anemia. Patients with aplastic anemia and acute leukemia were usually children, whereas those with megaloblastic anemia were adults. Moderate to severe anemia was noted throughout the series, but was most striking in patients with megaloblastic anemia, aplastic anemia, and acute leukemia. The mean corpuscular volume (MCV) was elevated in most patients with megaloblastic hematopoiesis, aplastic anemia, and acute nonlymphocytic leukemia. Normal or low MCV values were noted in almost one third of patients with megaloblastic anemia. Anisocytosis, poikilocytosis, macroovalocytosis, microcytosis, fragmentation, and teardrop erythrocytes were more prominent on the blood films of patients with megaloblastic anemia. Conclusions Megaloblastic anemia, aplastic anemia, and AIDS are the most common causes of pancytopenia in Zimbabwe. Aplasia is the most frequent cause of severe pancytopenia. The authors have formulated tentative guidelines for the evaluation of pancytopenie patients in this setting.

Nutritional B12 deficiency in childhood is an uncommon disorder. Most cases are due to maternal insufficiency, resulting from deficient stores and intake, and is generally seen in exclusively breast fed infants. This report describes a breast-fed infant with megaloblastic anemia secondary to maternal vitamin B12 deficiency. We describe this patient to remind readers that B12 deficiency may cause severe pancytopenia and regression of motor functions. These patients can present with unexpected signs and symptoms, such as developmental delay and regression as in our patient. It is also important to take the nutritional history of both the child and the mother of early prevention and treatment. With early awareness and appropriate measures potentially irreversible neurologic damage can be prevented in the infant.

Thiamine-responsive megaloblastic anaemia (TRMA) is a rare autosomal recessive condition, characterized by megaloblastic anaemia, non-autoimmune diabetes mellitus, and sensorineural hearing loss. We describe three infants with TRMA from two consanguineous Pakistani families, who were not known to be related but originated from the same area in Pakistan. All children were homozygous, and the parents were heterozygous for a c.196G>T mutation in the SLC19A2 gene on chromosome 1q23.3, which encodes a high-affinity thiamine transporter. The result is an abnormal thiamine transportation and vitamin deficiency in the cells. Thiamine in high doses (100-200 mg/d) reversed the anaemia in all our patients. Two patients discontinued insulin treatment successfully after a short period, while the third patient had to continue with insulin. The hearing loss persisted in all three children. The diagnosis of TRMA should be suspected in patients with syndromic diabetes including hearing loss and anaemia, even if the latter is only very mild and, particularly, in the case of consanguinity.

Thiamine-responsive megaloblastic anemia (TRMA, also known as Rogers syndrome, OMIM 249270) is a rare autosomal recessive disorder characterized by a triad of megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Patients respond, to varying degrees, to treatment with megadoses of thiamine. We have recently shown genetic linkage of the TRMA gene to a 16-centimorgan (cM) region on 1q23.2-1q23.3 based on the analysis of four large, inbred families of Alaskan, Italian, and Israeli-Arab origin. Here we narrow the TRMA interval down to 4 cM based on genetic recombination, homozygosity mapping, and linkage disequilibrium (highest LOD score of 12.5 at D1S2799, at a recombination fraction of 0). We provide further evidence that the TRMA gene is located in this region and confirm the homogeneity of the disease. In this analysis, we genotyped seven additional families of diverse ethnic origin (Pakistani, Indian, Italian, Brazilian, and Japanese), and analyzed additional markers in two previously reported families showing evidence of linkage disequilibrium in a large area of their haplotypes. The multi-system manifestations of TRMA suggest that thiamine has a pivotal role in a multiplicity of physiological processes. Mapping the TRMA gene and understanding the molecular basis of the disease might, thus, shed light on the role of thiamine in common disorders such as deafness, anemia, and diabetes.

The utility of 1H nuclear magnetic resonance (NMR) diffusion-diffraction of water as a tool for characterising red cell shape was investigated. Experiments were conducted on various cell suspensions which contained different shapes/forms of erythrocytes prepared by manipulating the conditions of the suspension medium, such as osmolality, and altering metabolism to affect the adenosine triphosphate concentration. Abnormal red cells from patients with hereditary stomatocytosis and megaloblastic anemia were also studied in order to assess the practical application of this "new" technique. The results clearly show that NMR diffusion-diffraction is sensitive to very small changes in mean cell dimensions and that a "characteristic" q-space plot/profile can be ascribed to each erythrocyte form. It was also found that the homogeneity of the cell shape and/or size is an important factor that affects the intensity of the diffusion-diffraction peaks. This study demonstrates the potential of the NMR diffusion-diffraction technique as a diagnostic tool in hematology.

Serum erythropoietin (sEpo) concentration is primarily related to the rate of renal production and, under the stimulus of hypoxia, increases exponentially as hemoglobin (Hb) decreases. Additional factors, however, appear to influence sEpo, and in this work, we performed studies to evaluate the role of the red blood cell precursor mass. We first compared the relationship of sEpo with Hb in patients with low versus high erythroid activity. The first group included 27 patients with erythroid aplasia or hypoplasia having serum transferrin receptor (sTfR) levels < 3 mg/L (erythroid activity < 0.6 times normal), while the second one included 28 patients with β-thalassemia intermedia having sTfR levels > 10 mg/L (erythroid activity > 2 times normal). There was no difference between the two groups with respect to Hb (8.3 ± 1.6 v 8.0 ± 1.3 g/dL, P > .05), but sEpo levels were notably higher in patients with low erythroid activity (1,601 ± 1,542 v 235 ± 143 mU/mL,P < .001). In fact, multivariate analysis of variance (ANOVA) showed that, at any given Hb level, sEpo was higher in patients with low erythroid activity (P < .0001). Twenty patients undergoing allogeneic or autologous bone marrow transplantation (BMT) were then investigated. A marked increase in sEpo was seen in all cases at the time of marrow aplasia, disproportionately high when compared with the small decrease in Hb level. Sequential studies were also performed in five patients with iron deficiency anemia undergoing intravenous (IV) iron therapy. Within 24 to 72 hours after starting iron treatment, marked decreases in sEpo (up to one log magnitude) were found before any change in Hb level. Similar observations were made in patients with megaloblastic anemia and in a case of pure red blood cell aplasia. These findings point to an inverse relationship between red blood cell precursor mass and sEpo: at any given Hb level, the higher the number of red blood cell precursors, the lower the sEpo concentration. The most likely explanation for this is that sEpo levels are regulated not only by the rate of renal production, but also by the rate of utilization by erythroid cells.

Background: Grfisbeck-lmerslund disease (congenital familial selective vitamin B12-malabsorption with proteinuria, MGAI, MIM No. 261100) is a rare disorder displaying autosomal recessive inheritance. This study was designed to investigate the usefulness of measuring the activity of the urinary receptor for the intrinsic factor-cobalamin complex as a tool to diagnose this disease. Methods: The receptor activity was measured by a radioisotope-binding assay, using phenyl-Sepharose gel as the adsorbant solid phase of the receptor. Results: In 10 Finnish patients, urinary receptor activity was on the average 640 times (15-1400 times) lower than that in 13 healthy control subjects: mean values of 0.1 nmol/mol (range. 0.01-0.32 nmol/mol) and 6.4 nmol/mol (range, 3.8-12.4 nmol/ mol) creatinine, respectively. The mean value of urinary receptor activity in 11 first-degree, healthy relatives of the patients was 4.6 nmol/mol (range, 1.1-10.4 nmol/mol) creatinine, a difference from levels in control subjects that is not statistically significant. When the first-degree relatives were divided into heterozygotes (parents and siblings heterozygous for the haplotype of genetic markers associated with the disease gene) and wild-type homozygotes (siblings not displaying the disease haplotype), no difference was seen. Conclusion: Determination of receptor activity in the urine is a highly accurate method for diagnosis of Grhsbeck-lmerslund disease at an early stage, but it does not detect carriers of the disorder.

OBJECTIVE To describe a 15-year follow-up of diabetes and to present data regarding pancreatic β-cell function in two adolescents affected by the thiamine-responsive megaloblastic anemia (TRMA) syndrome. CASE REPORTS The first patient (PMR) is a 17.5-year-old Italian girl who presented megaloblastic anemia at 7.5 months of age. At age 2.5 years, because of the presence of diabetes and sensorineural deafness, she was diagnosed with TRMA syndrome and started treatment with thiamine-HCl, followed very early by benzoyloxymethyl-thiamine (BOM-T). The second patient (PF) is a 16.8-year-old Italian boy born to consanguineous parents. Sensorineural deafness was diagnosed at age 1.5 years, while diabetes with ketoacidosis and megaloblastic anemia were diagnosed at age 3 years. Treatment with thiamine HCl was started immediately after diagnosis and changed to BOM-T 2 months later. Subsequent to the initiation of the vitamin, the two patients did not require insulin for ∼ 7 and 10 years, respectively. Puberty was determinant in deteriorating the metabolic control in these patients, leading to treatment with an oral hypoglycemic agent and finally to a reinstitution of insulin therapy. CONCLUSIONS The hormonal assessment in our patients (normal insulin response to oral glucose in childhood, preserved C-peptide secretion in case 2) and the good response to an oral hypoglycemic agent would indicate that the pancreatic disease may initiate as type 2 diabetes and may progress after several years to an insulin-requiring diabetes, as indicated by the exhaustion of the insulin secretory capacity.

We investigated the haematological effects of a massive dose of oral cobalamin (vitamin B12) on patients with cobalamin deficiency anaemia who had severe anaemia with a few neurological impairments and found that oral treatment was almost as effective as conventional injection therapy. The recovery of haematological indices with oral cobalamin preparations was slightly slower than with parenteral preparations, although the subjective symptoms disappeared soon after the start of therapy. The results of this study indicate that oral treatment keeps the cobalamin body stores satisfactorily filled and might be useful for older patients in whom injecting might be difficult.

We report here a case of megaloblastic anaemia in late pregnancy, which leads us to question whether folate supplements should be recommended in the UK routinely throughout pregnancy and not just in the preconception period and first trimester.

Summary Thiamine-responsive megaloblastic anemia, also known as "TRMA" or "Rogers syndrome," is an early-onset autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, and sensorineural deafness, responding in varying degrees to thiamine treatment. On the basis of a linkage analysis of affected families of Alaskan and of Italian origin, we found, using homozygosity mapping, that the TRMA-syndrome gene maps to a region on chromosome 1q23.2-23.3 (maximum LOD score of 3.7 for D1S1679). By use of additional consanguineous kindreds of Israeli-Arab origin, the putative disease-gene interval also has been confirmed and narrowed, suggesting genetic homogeneity. Linkage analysis generated the highest combined LOD-score value, 8.1 at a recombination fraction of 0, with marker D1S2799. Haplotype analysis and recombination events narrowed the TRMA locus to a 16-cM region between markers D1S194 and D1S2786. Several heterozygote parents had diabetes mellitus, deafness, or megaloblastic anemia, which raised the possibility that mutations at this locus predispose carriers in general to these manifestations. Characterization of the metabolic defect of TRMA may shed light on the role of thiamine deficiency in such common diseases.

This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G 2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic 'ladder pattern' which might have been expected in classic apoptosis. In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTag TM in situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.

It is proven that folic acid supplied in the periconceptional period can lower the recurrence and occurrence rate of neural tube defects (NTDs). Our research team on prevention of birth defects could demonstrate that folic acid preventable NTDs are partly based on hyperhomocystinemia and a genetic predisposition (mutation of the methylenetetrahydrofolate-reductase gene (MTHFR)). Reduced activity of the folate methylation cycle seems to be an attractive working hypothesis in the aetiology of NTDs. This genetic metabolic defect can be overcome by treatment with folic acid and/or vitamin B 12 .

International audience; The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalamin (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as 'cblG-variant'. In four 'cblG-variant' cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

A 46-year-old man was referred to us by his physician with a chief complaint of red patches on the tongue associated with a burning sensation that had occurred for approximately one year. His medical and dental histories were unremarkable. The patient had not encountered anything, including any type of food, that made the lesions improve or worsen. During a routine examination of the head and neck region, we observed no extraoral alterations, such as changes in the color of the face, swelling, facial asymmetry and palpable lymph nodes. When we conducted an intraoral examination, we observed multiple extensive erythematous areas on the inferior labial mucosa, buccal mucosa, tongue (dorsal, ventral and lateral aspects) and hard palate (Figures 1-3). Because the patient had diffuse mucosal erythema with a generalized burning sensation, we prescribed 20 milligrams of prednisone daily and topical benzocaine three times per day. After we noted no improvement following one week of treatment, we discontinued the prednisone and topical benzocaine and performed incisional biopsies on the tongue and buccal mucosa. Microscopic examination of the biopsied tissues revealed an atrophic surface of stratified squamous epithelium with a mild chronic inflammatory infiltrate within the lamina propria. One month after undergoing the biopsy, the patient complained of gastric distress, cramps, bloating, constipation, hair loss, and lack of energy and appetite. We requested testing for complete blood cell count, vitamin B12 level, and anti-endomysial, antitransglutaminase and anti-immunoglobulin A antibodies. The results of these tests showed a vitamin B12 deficiency with a level of serum cobalamin less than 150 picograms per milliliter (normal range [male], 193-982 pg/mL), a red blood cell (RBC) count of 3.96 million per cubic millimeter (normal range [male], 4.60-6.20 million/mm), a mean corpuscular volume (MCV) of 105.5 femtoliters (normal range [male], 76-96 fL), a mean corpuscular hemoglobin (MCH) weight of 35.1 pg (normal range [male], 27-32 pg) and an RBC distribution width of 17.3 percent (normal range [male], 11.5-14.5 percent). In addition, we found that the patient had negative levels of antiendomysial antibodies (normal titer, 35 units) and anti-immunoglobulin A antibody levels of 259 mg per deciliter (normal range [adults], 40-350 mg/dL).

Despite advances in device closure for atrial septal defect, post-closure heart failure remains a clinical problem in adult patients but is seen only rarely in children. An eight-year-old boy, who had been followed by a local pediatrician with the diagnosis of diabetes mellitus and congenital heart disease, was consulted to us for cardiac re-evaluation. Electrocardiography demonstrated absent P waves, and echocardiography revealed enlargement of the right ventricle and both atria and secundum atrial septal defect. With the diagnosis of atrial standstill, secundum atrial septal defect and thiamine-responsive megaloblastic anemia, acute heart failure developed after transvenous closure of the atrial septal defect, which improved dramatically with thiamine and supportive treatment.

Background Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. Methods Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. Results Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. Conclusions In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.

Skin changes associated with vitamin B₁₂ deficiency are common. In most cases, patients present primarily with systemic involvement (eg, megaloblastic anemia and/or neurologic effects), and additional cutaneous changes related to the diagnosis are noted as incidental findings. The role of vitamin B₁₂ deficiency in the etiology of Addisonian pigmentation has not been well studied. We discuss the importance of testing vitamin B₁₂ levels in patients who present for evaluation of generalized hyperpigmentation. Various cutaneous changes associated with vitamin B₁₂ deficiency also are reviewed.

Background Selective intestinal cobalamin malabsorption with mild proteinuria (Imerslund-Grasbeck 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. Objectives To describe the clinical, metabolic, and genetic bases of I-GS in Beagles. Animals Four cobalamin-deficient and 43 clinically normal Beagles and 5 dogs of other breeds. Methods Clinical 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. Results Juvenile-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. Conclusions The 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.

Imerslund-Gräsbeck syndrome is a rare autosomal recessive disorder, characterized by vitamin B12 deficiency due to selective malabsorption of the vitamin and usually results in megaloblastic anemia appearing in childhood. It is responsive to parenteral vitamin B12 therapy. The estimated prevalence (calculated based on Scandinavian data) is less than 6:1,000,000. However, many cases may be misdiagnosed. When there is reasonable evidence to suspect that a patient suffers from IGS, a new and straightforward approach to diagnosis is mutational analysis of the appropriate genes. We report for the first time the case of a girl of Italian ancestry with IGS genetically confirmed by the detection of a homozygous missense mutation in the AMN gene (c.208-2 A > G).

This study was aimed to explore the change characteristics of cell differentiation antigen (CD) on bone marrow (BM) granulocytes in patients,with megaloblastic anemia (MA). In combination with BM cell morphology, hemogram, level of blood serum folic acid, level of Vit B(12), cell genetics and biological examination data, the BM granulocytes differentiation antigens in 13 patients with MA were detected by flow cyto metry and analyzed retrospectively, in order to summarize the variation characteristics of CD13, CD33 and CD15 expressed on myeloid cells in patient with MA, including forward scatter light (FSC) and side scatter light (SSC) signal intensity, then these findings were compared with that in normal healthy persons. The results showed that the expression rates of CD13, CD15 and CD33 on granulocytic in patients with MA and normal healthy persons were (44.53 ± 16)%, (96.16 ± 2.67)%, (80.81 ± 14.71)% and (62.33 ± 11.02)%, (99.53 ± 0.46)%, (70.00 ± 7.81)% respectively, in which the expression rate of CD13 and CD15 in patients with MA decreased (P0.01), while the expression rate of CD33 increased (P0.01). The mean fluorescence intensity (MFI) of CD13, CD15, CD33, SSC and FSC in MA patients and normal healthy persons were 3.39 ± 1.41, 14.29 ± 6.59, 1.95 ± 0.94, 478.78 ± 70.43, 633.46 ± 75.53 and 5.12 ± 1.15, 20.67 ± 5.13, 1.04 ± 0.17, 332.00 ± 38.16, 537.00 ± 16.70 respectively, in which the MFI of CD13 and CD15 on granulocytes in MA patients decreased (P0.01),while the MFI of FSC,SSC and CD33 increased (P0.01 and P0.05). It is concluded that not only the morphology of BM granulocytes in patents with MA shows dysmaturity, but the expressing feature of differentiation antigens on BM granulocytes in MA patients also displays dysmaturity.These findings will contribute to the clinical diagnosis of MA patients.

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.

Background: Diabetic peripheral neuropathy is now well recognized as the most common cause of Charcot arthropathy of the foot and ankle, but it may be associated with other peripheral neuropathies. While not well known, it is well documented that rheumatoid arthritis is correlated with peripheral neuropathy. However, despite rheumatoid neuropathy, Charcot arthropathy has never been associated with rheumatoid arthritis. We report a series of Charcot arthropathy patients with concomitant rheumatoid arthritis. Methods: The medical records of patients treated between 1986 and 2009 with Charcot arthropathy and rheumatoid arthritis were reviewed. Recorded data included neuropathy risk factors, medications, history of ulcerations, ambulatory status, shoe wear, and treatment course. Radiographs of Charcot joints were categorized according to the Brodsky anatomic classification. Patient care was based on published treatment algorithms, emphasizing accommodative, nonoperative treatment with selective surgical interventions. Surgery was indicated for recalcitrant, nonhealing lesions of the soft tissue and/or unbraceable, nonplantigrade feet. A successful outcome was considered an ambulatory patient without amputation and a closed skin envelope at last follow-up. Results: Four men and 16 women met the diagnostic criteria, resulting in 33 feet in the series. Average age was 61 years, and average follow-up was 4.3 years. In addition to rheumatoid arthritis, 4 patients (7 feet) had hypothyroidism, 4 patients (6 feet) had diabetes, 1 patient (2 feet) had megaloblastic anemia and diabetes, and 1 patient (1 foot) had hypothyroidism and diabetes; however, 17 feet (52%) had no known sources for neuropathy. Charcot involvement was type 1–midfoot in 21 feet (64%), type 2–hindfoot in 7 (21%), type 3a–ankle in 4 (12%), and type 3b–calcaneus in 1 (3%). Twenty-three feet (70%) were treated with conservative modalities. Ten feet (30%) required 15 surgeries, of which an exostectomy was the most common procedure. Of the 33 feet, 3 had persistent ulcerations and 1 underwent major amputation, representing 4 failures. Conclusions: Raising awareness within the orthopaedic community, we report a Charcot arthropathy population with a concomitant rheumatoid arthritis diagnosis, emphasizing a relationship between the 2 diseases. Through a conservative treatment regimen combined with selective surgical interventions, satisfactory outcomes were achieved in 88% of the rheumatoid Charcot feet. While several patients had additional neuropathy sources which could cause Charcot arthropathy (eg, diabetes), the majority of feet had no etiologies accounting for neuropathy or neuroarthropathy except rheumatoid arthritis. Further study is required to expand on this relationship between the 2 diseases. Level of Evidence: Level IV, retrospective case series.

Thiamine responsive megaloblastic anemia (TRMA) is characterized by a triad of megaloblastic anemia, non-type 1 diabetes mellitus and sensorineural deafness. Other clinical findings have been described in few cases. The SLC19A2 gene on chromosome 1q 23.3 is implicated in all cases with TRMA. Our aim is to discuss the clinical manifestations of all Omani children diagnosed with TRMA and determine genotype-phenotype relationship.Clinical and laboratory data of all patients diagnosed in Oman were retrospectively collected. Mutation analysis of affected families was conducted using two Microsatellite markers. Genotyping was performed with fluorescent-labeled PCR primers. To define the deletion breakpoint region, PCR reactions were carried out using different primer pairs located at the introns 3 and 3'-untranslated region with Expand Long Template PCR kit.A total of six children have been diagnosed with this syndrome. They were five females and one male. They all presented with sensorineural deafness at birth while the age of anemia presentation ranged between 6 weeks to 19 months. They all belong to same family with complex interfamilial marriages and presented with the typical triad. Of interest is the very rare presentation of one patient with Uhl cardiac anomaly (total absence of right ventricular myocardium with apposition of endocardium and pericardium) that has never been described before in patients with TRMA. All patients have a novel large deletion of 5,224 bp involving exons 4, 5, and 6 of SLC19A2.TRMA is a disease of expanding phenotypic spectrum with poor genotype-phenotype correlation.

India has the highest prevalence of severe acute malnutrition (SAM). Severe anemia is one of the comorbidities responsible for increased mortality in severely malnourished children, yet it has not received the attention it should. The aim of the present study was to determine the prevalence and type of anemia and to evaluate the possible etiologies for severe anemia, in these children.A cross-sectional study of patients with SAM in a tertiary care hospital in northern India over a period of 12 mo from Sept. 1, 2010 to Aug. 31, 2011 was conducted. We observed the prevalence of severe anemia (hemoglobin7 g/dL), morphologic type of anemia, number of patients requiring blood transfusion, hematologic profile of mothers, nature of feeding, duration of exclusive breastfeeding, and the demographic profile of these patients.Included in the study were 131 cases of SAM. The age group varied between 6 and to 59 mo. Of patients with SAM, 67.3% had severe anemia; 13.8% had moderate anemia. Of these patients, 25% required packed red blood cell transfusion. The most common type of anemia was microcytic (38.6%) followed by megaloblastic (30.5%).A high incidence of severe anemia in SAM with a large proportion (25%) requiring blood transfusion is a pointer toward nutritional anemia being a very common comorbidity of SAM requiring hospital admission. Because megaloblastic anemia closely followed microcytic anemia, supplementation with vitamin B12 in addition to iron and folic acid would be recommended.