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Hereditary spherocytosis (HS) is often misdiagnosed due to lack of specific diagnostic methods. Our study summarized clinical characteristics and described the diagnostic workflow for mild and moderate HS in Chinese individuals, using data from 20 adults, 8 of whom presented a familial history for HS. We used scanning electron microscopy (SEM) to diagnose HS. We observed reduced eosin maleimide fluorescence activity (5.50 mean channel fluorescence (MCF) units) in the 10 cases of HS, which differed significantly when compared with 10 normal adults (15.50 units), iron deficiency anemia (15.50 MCF units), and megaloblastic anemia (12.00 MCF units) values (P

Thiamine is a crucial cofactor involved in the maintenance of carbohydrate metabolism and participates in multiple cellular metabolic processes within the cytosol, mitochondria, and peroxisomes. Currently, four genetic defects have been described causing impairment of thiamine transport and metabolism: SLC19A2 dysfunction leads to diabetes mellitus, megaloblastic anemia and sensory-neural hearing loss, whereas SLC19A3, SLC25A19, and TPK1-related disorders result in recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability, and early death. In order to achieve early diagnosis and treatment, biomarkers play an important role. SLC19A3 patients present a profound decrease of free-thiamine in cerebrospinal fluid (CSF) and fibroblasts. TPK1 patients show decreased concentrations of thiamine pyrophosphate in blood and muscle. Thiamine supplementation has been shown to improve diabetes and anemia control in Rogers' syndrome patients due to SLC19A2 deficiency. In a significant number of patients with SLC19A3, thiamine improves clinical outcome and survival, and prevents further metabolic crisis. In SLC25A19 and TPK1 defects, thiamine has also led to clinical stabilization in single cases. Moreover, thiamine supplementation leads to normal concentrations of free-thiamine in the CSF of SLC19A3 patients. Herein, we present a literature review of the current knowledge of the disease including related clinical phenotypes, treatment approaches, update of pathogenic variants, as well as in vitro and in vivo functional models that provide pathogenic evidence and propose mechanisms for thiamine deficiency in humans.

Background The Solute Carrier Family 19 Member 2 (SLC19A2, OMIM *603941) encodes the thiamine transporter 1 (THTR‐1) that brings thiamine (Vitamin B1) into cells. THTR‐1 is the only thiamine transporter expressed in bone marrow, cochlear, and pancreatic beta cells. THTR‐1 loss‐of‐function leads to the rare recessive genetic disease Thiamine‐Responsive Megaloblastic Anemia (TRMA, OMIM #249270). Methods In vitro stimulated blood lymphocytes were used for cytogenetics and the isolation of genomic DNA used to perform whole exome sequencing (WES). To validate identified mutations, direct Sanger sequencing was performed following PCR amplification. Results A 6‐year‐old male born from a consanguineous couple presenting bone marrow failure and microcephaly was referred to our clinic for disease diagnosis. The patient presented a normal karyotype and no chromosomal fragility in response to DNA damage. WES analysis led to the identification of a new pathogenic variant in the SLC19A2 gene (c.596C>G, pSer199Ter) allowing to identify the young boy as a TRMA patient. Conclusion Our analysis extend the number of inactivating mutations in SLC19A2 leading to TRMA that could guide future prenatal diagnosis for the family and follow‐up for patients.

KEY POINTS: Folate (folic acid) deficiency and mutations in folate-related genes in humans result in megaloblastic anaemia. Folate metabolism, which requires the enzyme methionine synthase reductase (MTRR), is necessary for DNA synthesis and the transmission of one-carbon methyl groups for cellular methylation. In this study, we show that the hypomorphic Mtrrgt/gt mutation in mice results in late-onset and sex-specific blood defects, including macrocytic anaemia, extramedullary haematopoiesis and lymphopenia. Notably, when either parent carries an Mtrrgt allele, blood phenotypes result in their genetically wildtype adult daughters, the effects of which are parent specific. Our data establish a new model for studying the mechanism of folate metabolism in macrocytic anaemia aetiology and suggest that assessing parental folate status might be important when diagnosing adult patients with unexplained anaemia. ABSTRACT: The importance of the vitamin folate (also known as folic acid) in erythrocyte formation, maturation and/or longevity is apparent since folate deficiency in humans causes megaloblastic anaemia. Megaloblastic anaemia is a type of macrocytic anaemia whereby erythrocytes are enlarged and fewer in number. Folate metabolism is required for thymidine synthesis and one-carbon metabolism, though its specific role in erythropoiesis is not well understood. Methionine synthase reductase (MTRR) is a key enzyme necessary for the progression of folate metabolism since knocking down the Mtrr gene in mice results in hyperhomocysteinaemia and global DNA hypomethylation. We demonstrate here that abnormal folate metabolism in mice caused by Mtrrgt/gt homozygosity leads to haematopoietic phenotypes that are sex and age dependent. Specifically, Mtrrgt/gt female mice displayed macrocytic anaemia, which might be due to defective erythroid differentiation at the exclusion of haemolysis. This was associated with increased renal Epo mRNA expression, hypercellular bone marrow, and splenic extramedullary haematopoiesis. In contrast, the male response differed since Mtrrgt/gt male mice were not anaemic but did display erythrocytic macrocytosis and lymphopenia. Regardless of sex, these phenotypes were late onset. Remarkably, we also show that when either parent carries an Mtrrgt allele, a haematological defect results in their adult wildtype daughters. However, the specific phenotype was dependent upon the sex of the parent. For instance, wildtype daughters of Mtrr+/gt females displayed normocytic anaemia. In contrast, wildtype daughters of Mtrr+/gt males exhibited erythrocytic microcytosis not associated with anaemia. Therefore, abnormal folate metabolism affects adult haematopoiesis in an age-, sex- and parent-specific manner., Lister Institute of Preventative Medicine, Centre for Trophoblast Research, Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge joint research grant, Newnham College (Cambridge) studentship, A.G. Leventis scholarship, Wellcome Trust PhD studentship in Developmental Mechanisms

Thiamine responsive megaloblastic anemia syndrome (TRMAS) is a rare autosomal recessive disorder especially in countries where consanguinity is uncommon. Three main features are characteristic of the disease – megaloblastic anemia, early onset deafness, and non-type I diabetes. TRMAS is a Mendelian disorder; a gene SLC19A2 coding high affinity thiamine transporter mediating vitamin B1 uptake through cell membrane has been identified. We present the first patient with TRMAS in Lithuania – a 3-year-old boy born to a non-consanguineous family with a novel homozygous SLC19A2 gene mutation. The patient had insulin dependent diabetes (onset 11 months), respiratory illness (onset 11 months), bilateral profound hearing loss (onset at 7 months, verified at 20 months), refractory anemia (onset 2 years), and decreased vision acuity and photophobia (onset 2.5 years). The psychomotor abilities developed according to age. Phenotypic evaluation did not reveal any dysmorphic features. The clinical diagnosis of TRMAS was suspected and daily supplementation with thiamine 100 mg was started. The condition of the patient markedly improved several days after the initiation of treatment. The results of SLC19A2 gene molecular testing confirmed the clinical diagnosis – novel homozygous c.[205G>T], p.[(Val69Phe)] mutation changing conserved amino acid residue or even interfering the mRNA splicing. Clinical heterogeneity, diverse dynamics, and wide spectrum of symptoms are aggravating factors in the diagnosis. The possibility of treatment demands early recognition of disorder to facilitate the improvement of the patient's condition. © 2015 Wiley Periodicals, Inc.

Cobalamin (vitamin B12) deficiency is a common cause of megaloblastic anemia in Western populations. Laboratory evaluation of megaloblastic anemia frequently includes the assessment of patient cobalamin and folate status. Current total serum cobalamin measurements are performed in the clinical laboratory with competitive binding luminescence assays, whose results may not always accurately reflect actual cobalamin stores. Surrogate markers of cobalamin deficiency such as methylmalonic acid and homocysteine have been utilized to improve diagnostic accuracy; however, the specificity of these tests by themselves is rather low. Measurement of the biologically active fraction of cobalamin, holotranscobalamin, has been proposed as a replacement for current total cobalamin assays. Although holotranscobalamin measurements appear to have slighter better sensitivity, the specificity of this assay remains to be determined. The relative merits and demerits of commonly available methods to assess cobalamin deficiency in patients with suspected megaloblastic anemia are discussed.

The foundation of laboratory hematologic diagnosis is the complete blood count and review of the peripheral smear. In patients with anemia, the peripheral smear permits interpretation of diagnostically significant red blood cell (RBC) findings. These include assessment of RBC shape, size, color, inclusions, and arrangement. Abnormalities of RBC shape and other RBC features can provide key information in establishing a differential diagnosis. In patients with microcytic anemia, RBC morphology can increase or decrease the diagnostic likelihood of thalassemia. In normocytic anemias, morphology can assist in differentiating among blood loss, marrow failure, and hemolysis-and in hemolysis, RBC findings can suggest specific etiologies. In macrocytic anemias, RBC morphology can help guide the diagnostic considerations to either megaloblastic or nonmegaloblastic causes. Like all laboratory tests, RBC morphologies must be interpreted with caution, particularly in infants and children. When used properly, RBC morphology can be a key tool for laboratory hematology professionals to recommend appropriate clinical and laboratory follow-up and to select the best tests for definitive diagnosis.

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

International audience; Transcobalamin (transcobalamin II, TC) transports plasma vitamin B(12) (cobalamin, Cbl) into cells. TC deficiency is a rare autosomal recessive disorder causing intracellular Cbl depletion, which in turn causes megaloblastic bone marrow failure, accumulation of homocysteine and methylmalonic acid, and methionine depletion. The clinical presentation reflects intracellular Cbl defects, with early-onset failure to thrive with gastrointestinal symptoms, pancytopenia, and megaloblastic anemia, sometimes followed by neurological complications. We report the clinical, biological, and molecular findings and the outcome in five TC-deficient patients. The three treated early had an initial favorable outcome, whereas the two treated inadequately had late-onset severe neuro-ophthalmological impairment. Even if the natural course of the disease over time might also result in late-onset symptoms in the aggressively treated patients, these data emphasize that TC deficiency is a severe disorder requiring early detection and probably long-term aggressive therapy. Mutation analysis revealed six unreported mutations in the TCN2 gene. In silico structural analysis showed that these mutations disrupt the Cbl-TC interaction domain and/or the putative transcobalamin-transcobalamin receptor interaction domain.

Urine methylmalonic acid (MMA) concentrations were detected in 79 Chinese patients by gas chromatography mass spectrometry (GC/MS), using a selected ion monitoring program. 10 of the 79 patients were found to have cobalamin deficiency. Their urine MMA amounts were all elevated with a mean value 1376 ng/microliter (11.66 mmol/l), ranging from 40.46 to 3900 ng/microliter (0.34-33.05 mmol/l). The remaining 69 cases were found to be unrelated to cobalamin deficiency. Their mean urine MMA was 3.62 ng/microliter (30.0 mumol/l), ranging from 0-17.47 ng/microliter (0-148.0 mumol/l). In this study, we found that urine MMA detected by GC/MS was a simple, rapid, convenient, specific and sensitive method for the diagnosis of cobalamin deficiency. The urine MMA concentrations in cases not due to cobalamin deficiency would not exceed 20 ng/microliter (169.5 mumol/l), whereas in cobalamin deficiency the urine MMA levels always exceeded 20 ng/microliter, or were even much higher. No overlapping of the results of urine MMA between these 2 groups of patients could be seen in our study. Detection of urine MMA is useful in the demonstration or exclusion of cobalamin deficiency in any suspect patients.

Lactic dehydrogenase activity (LDH) was examined in patients with megaloblastic anaemia and other types of anaemia. Only in untreated megaloblastic anaemia was LDH consistently increased. The increase was not related to the concentration of vitamin B12 and bilirubin in serum. Nor was there any definite relation to the degree of anaemia, but the number of patients examined was too small to permit any final conclusion on this point. The mean LDH value in megaloblastic anaemia was 3,800 units, while the mean value among controls was 257 units. There was a rapid fall in LDH activity during treatment with vitamin B12, corresponding to the increase in reticulocyte counts. In the other types of anaemia studied – acute haemorrhage, chronic haemorrhage and iron deficiency, myelomatosis, aplastic anaemia and anaemia due to renal failure – LDH activity was usually normal or only slightly increased, except in renal failure. In the latter condition the highest LDH value was 830 units, whereas the lowest value in patients with megaloblastic anaemia was 1,510 units. It is therefore concluded that LDH determinations are of diagnostic aid in differentiating megaloblastic anaemia from other types of anaemia.

A patient with hepatocellular carcinoma, megaloblastic anaemia and increased concentration of serum cobalamin is described. Plasma TC I was increased to 10,000 times the normal concentration, thus explaining the increased concentration of serum cobalamin and a false Schilling test. The increase in plasma TC I in concurrence with undetectable amounts of plasma TC II was a likely explanation for the anaemia. The electron microscopic picture of the hepatocellular carcinoma was in accordance with TC I being produced by the tumour cells.

Myasthenia gravis (MG) is an autoimmune disease often associated with other autoimmune disorders. A case history of MG with a coexisting atypical megaloblastic anaemia with vitamin B12 deficiency and anti Intrinsic Factor (IF) antibodies, led to a study of humoral and cellular immunity to IF in 81 MG patients. Within this series, 3 other patients had a disturbed humoral and cellular immunity to IF. These 3 patients presented no other features of pernicious anaemia. The possible origins and significance of the anti IF antibodies in MG patients are discussed.

Imerslund-Gräsbeck syndrome, an autosomal recessive trait of defective uptake of intrinsic factor-vitamin B12 complex by terminal ileum, is described in a Saudi family (two siblings and their first cousin). This rare disease has previously been reported only in Northern Europe and North African Jewish ethnic groups.

Peripheral neuropathy is frequently observed in B(12) deficiency. In spite of this, there is little knowledge about peripheral neuropathy in B(12) deficiency because the severity of clinical involvement of the central nervous system clearly outweighs signs and symptoms due to peripheral nervous system involvement. We primarily investigated peripheral neuropathy with dorsal sural conduction study, which is a new method for detection of early peripheral neuropathy, in B(12) deficiency with megaloblastic anemia. Conventional nerve conduction studies and tibial sensory-evoked potential (SEP) recording were also performed. Twenty-eight B(12)-deficient patients (15 male, 13 female, mean age 65.8 years) with megaloblastic anemia and 18 age- and sex-matched controls were included in the study. Although dorsal sural sensory nerve action potentials (SNAPs) were not recorded in 15 (54%) of 28 patients, only 9 (32%) of them were found to have polyneuropathy by conventional conduction studies. Furthermore, patients with dorsal sural SNAP had mean lower amplitude, mean longer latency, and slower velocity response when compared with controls. Twenty patients (71%) were diagnosed as having myelopathy by the combination of tibial SEP and neurological findings. Two patients whose dorsal sural SNAPs were not recorded had normal tibial SEP responses; therefore, these patients were considered to have isolated peripheral neuropathy. As a result, we conclude that dorsal sural nerve conduction study is a reliable method for detection of early peripheral neuropathy in B(12) deficiency.

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.

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.

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.

This study introduces a patient who has thiamine and thiamine pyrophosphokinase (TPKase) enzyme deficiency associated with diabetes mellitus, sensorineural deafness and thiamine-responsive megaloblastic anemia. Diabetes mellitus was diagnosed when she was 20 months old. After 1 year, macrocytic anemia developed and the thiamine therapy was started at 75 mg/day. During the follow-up, the insulin requirement decreased and even ceased, and macrocytic anemia improved with thiamine treatment. After thiamine therapy was ceased an increase in insulin requirement was observed and macrocytic anemia developed again.

A 9-year study of thiamine metabolism and cellular transport was performed in two patients with thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of thiamine and thiamine compounds were reduced by about 40% as compared to controls. Thiamine pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of thiamine and thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, less than 2 mumol/L, physiological concentrations of thiamine) and the non-saturable component of thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from thiamine-responsive megaloblastic anaemia patients contain low levels of thiamine compounds, probably due to their inability to take up and retain physiological concentrations of thiamine, as a result of the lack of the saturable, specific component of transport and reduced thiamine pyrophosphokinase.

Megaloblastic anaemia is uncommon in Hong Kong. Eighty-four consecutive Chinese patients with megaloblastic anaemia were studied. There were 48 males and 36 females, with a median age at presentation of 67 years. Vitamin B12 deficiency was found in all cases, with none of the patients showing folate deficiency. The frequency of pernicious anaemia in our patients was higher than in other south-east Asian series but comparable with western ones. When compared with patients in the West, our cases showed the following main differences: virtual absence of folate deficiency, even in alcoholics; absence of associated gastric malignancies; and a high frequency of tuberculosis.

We reviewed six cases of infantile megaloblastosis secondary to maternal vitamin B12 deficiency, the most common cause of infantile megaloblastosis in our institution. Two patients had long-term neurological sequelae, with a further patient remaining abnormal but at short follow-up. In 50% of cases the mother was asymptomatic, with subtle or no peripheral blood abnormalities, having early pernicious anaemia. Any infant which fails to thrive, with progressive neurological deterioration and haematological cytopenias should have their vitamin B12 and folate status rapidly assessed. This is one of the few potentially reversible causes of failure to thrive and neurological deterioration. Early diagnosis and treatment may prevent significant long-term sequelae.

The case of a 59-year-old Japanese woman with post-gastrectomy megaloblastic anaemia having urinary and faecal incontinence and paraesthesia in four extremities is described. While the haematological abnormalities were improved by administration of a total dose of 17 mg of intramuscular mecobalamin, the neurological abnormalities remained unchanged. Five months later, a total dose of 7.5 mg of mecobalamin was injected intravenously over a period of 5 weeks, although the serum level of vitamin B12 was greater than 1180 pmol l-1. Immediately after initiation of the therapy, the urinary and faecal incontinence were gradually improved, and were completely cured within 2 months. The peripheral neuropathy was also ameliorated. The effectiveness of intravenous vitamin B12 injection for the neurological abnormalities due to vitamin B12 deficiency is emphasized.

Approximately 20% of women show a physiologic drop in serum vitamin B12 level during pregnancy. Establishment of the latter requires direct measurement of metabolites related to the vitamin-B12-dependent pathways. Although the fetal compartment contains the products of fetal and maternal metabolism and provides a window into fetal metabolism the dynamics and physiology of the transplacental transport of these metabolites has been hardly studied. We sought to assess the relationship between the maternal and fetal compartments with regard to the metabolism of folate cobalamin and homocysteine (Hcy) and their correlation to hematological indices. (excerpt)

A case of megaloblastic anaemia and pancytopenia associated with prophylactic cotrimoxazole therapy is reported and discussed.

Summary. Cytoplasmic and intranuclear annulate lamellae in the erythroblasts from patients with dyserythropoietic anaemia (megaloblastic anaemia, dysplastic anaemia and erythroleukaemia) are described. Annulate lamellae have mainly been observed in oocytes, in embryonic tissues and in malignant cells. Their occurrence in dyserythropoietic anaemia may be related to the reappearance of fetal characteristics in the erythroblasts and erythrocytes.

Summary. Thymidine kinase has been measured in phytohaemagglutinin (PHA)-stimulated lymphocytes from 13 normal subjects and eight patients with megaloblastic anaemia. The levels in normal subjects ranged from 0.20 to 2.10 units/mg protein (mean 0.903 units/mg protein) and in megaloblastic anaemia from 2.99 to 9.97 units/mg protein (mean 5.76 units/mg protein). All the patients showed raised levels of the enzyme which were partly but not completely reduced to normal by addition of folic acid in vitro. Vitamin B12in vitro had a lowering effect in the five vitamin-B12-deficient patients and two patients with combined deficiencies but not in one‘pure’folate-deficient patient. Thymidine kinase activity was highest in the cells of the least anaemic patients, suggesting that the degree of anaemia in megaloblastic anaemia may be determined in part by the ability of the cells to utilize thymidine by the 'salvage’pathway when the de novo pathway of thymidylate synthesis is failing. The rise in thymidine kinase activity in megaloblastic anaemia is presumably due to induction of the enzyme. Addition of methotrexate or 5-fluorouracil, drugs known to inhibit de novo thymidylate synthesis, caused an increase in thymidine kinase activity in normal PHA-stimulated lymphocytes after 24 h (but not after 1 h) which could be completely blocked by addition of puromycin. Thymidine mono- and di-phosphate kinases were also measured in normal PHA-stimulated lymphocytes. The activities were substantially higher than that of thymidine kinase and their activities were unaffected by methotrexate addition.

Periodontal therapy should involve an assessment of the patient's nutritional status. Anticonvulsant drugs play an important role in folic acid deficiency. Individuals with gingival hyperplasia are most commonly seen by the periodontist and correction of their nutrient deficiency should be an integral aspect of treatment.

We report a case of methionine synthase deficiency associated with cellular immune deficiency discovered in a 14‐year‐old boy. Principal findings were: developmental delay, recurrent upper and lower respiratory tract infections, megaloblastic anemia, discovered at 3 months of age, unresponsive to cyanocobalamin and poorly responsive to folinic acid. Biochemical studies showed: an abnormal deoxyuridine suppression test despite normal serum folate, cobalamin and transcobalamin levels; a normal intracellular uptake of these two coenzymes; and an absolute requirement of methionine for fibroblast growth, suggestive of defective methionine synthesis. An absence of methionine synthase activity in the patient's bone marrow and a profound depression of this activity in lymphocytes and liver were found. Hypergammaglobulinemia with variable lymphopenia, depressed lymphocyte transformation after lectin or recall‐antigen stimulation, defective delayed‐type hypersensitivity and decreased natural killer activity were noted as well. The patient died at the age of 14.

Functional methionine synthase deficiency is generally characterized by homocystinuria and hypomethioninemia in the absence of methylmalonic aciduria. Patients are divided into two classes, cblE and cblG, on the basis of complementation analysis. Presentation has usually been in the first 2 years of life, but one patient came to medical attention at age 21 years with symptoms initially diagnosed as multiple sclerosis. Common findings among 11 patients (4 with cblE and 7 with cblG) have included megaloblastic anemia (all patients) and various neurological deficits including developmental retardation (10 patients), cerebral atrophy (8 patients), hypotonia (7 patients), EEG abnormalities (6 patients), and nystagmus (5 patients). Hypertonia, seizures, blindness, and ataxia were less frequent. All patients have responded to therapy with cobalamin with resolution of anemia and biochemical abnormalities; neurological deficits resolved more slowly and in some cases incompletely. Hydroxycobalamin has been more effective than cyanocobalamin. Fibroblasts from patients with cblE (5 patients) and cblG (6 patients) all showed decreased intracellular levels of methylcobalamin (MeCbl) and decreased incorporation of label from 5-methyltetrahydrofolate into macromolecules, suggesting decreased activity of the MeCbl-dependent enzyme methionine synthase. Methionine synthase specific activity in extracts of all cblE fibroblasts was normal or near-normal under standard reducing conditions; synthase specific activity in extracts of 5 cblG patients was low but was high in a 6th patient measured in another laboratory. Thus, there is heterogeneity among patients with functional methionine synthase deficiency both in clinical presentation and in the results of biochemical studies of cultured cells.

Three different inherited disorders are known in which thiamine may exert a beneficial effect: maple syrup urine disease (MSUD), lactic acidaemia and the syndrome of megaloblastic anaemia with sensorineural deafness and diabetes mellitus. The amounts of thiamine which were used for long-term treatment varied from 20 to 2400 mg day-1. Additional treatment, such as the reduction of dietary branched chain amino acids in MSUD, could not be omitted in some cases. It has been shown that the vitamin improves the stability of the branched chain ketoacid decarboxylase, although some weeks may be needed to observe the in vivo effect of treatment. A prolonged trial with high doses of thiamine should always be given.

The expression of Fc(IgG) and C3b membrane receptors by granulocytes and their precursors was examined in 23 cases of megaloblastic anaemia which were graded I-III according to morphological severity. Fractionated bone marrow and peripheral blood granulocyte receptors were assessed by rosette formation with optimally sensitized ox erythrocytes and the results compared with those found in 14 normal marrows. Promyelocyte Fc and C3b receptor activities in megaloblastic anaemia did not differ from normal whilst the number of Fc receptor positive myelocytes and later cells showed a significant increase (P less than 0.05 in Grade I, P less than 0.01 in Grades II and III) proportional to the severity of megaloblastosis. An increase in the number of C3b receptor positive granulocytes was seen in early megaloblastic anaemias and, in contrast to Fc receptor expression, showed the highest receptor activities in the Grade II cases. The most significant changes in receptor expression were seen at the metamyelocyte stage and appear to be related to the numbers of these cells found in the megaloblastic marrows. It is suggested that these alterations are primarily related to asynchronous granulocyte maturation and the application of these findings to the study of granulocytic disorders is discussed.

SUMMARY. A direct method for measuring red cell lifespan in vivo using the radioiron occupancy method (Dagg et al, 1972) has been applied to eight normal subjects. The mean red cell lifespan was 116 d with a range of 104-124 d. To establish the method clinically in the presence of haemolysis and ineffective erythropoiesis, 22 patients with haemolytic disease and six patients with megaloblastic and sideroblastic anaemia were studied. All 22 patients with haemolytic anaemia had shortening of the red cell lifespan, with a range from 62 to 10 d; the results were compared with red cell lifespan derived from simultaneous radiochromium studies (Bentley et al, 1974). A close agreement between the two methods was obtained (r = 0.87; P