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A common cause of hereditary nonspherocytic hemolytic anemia is pyruvate kinase deficiency, which is associated with lifelong chronic hemolysis. Pyruvate kinase deficiency has a worldwide distribution with a higher prevalence in the Caucasian population, and especially in Europe and North America. It is inherited in an autosomal fashion and over 180 different mutations have been described. Investigation of hemolytic anemia in Northern Ireland has uncovered 4 new cases of pyruvate kinase deficiency. Molecular investigation revealed a total of six different mutations. One mutation (p.Arg495Val) is reported here for the first time in a homozygous patient. Another mutant PKLR allele harbored a nonsense and frameshift mutation in cis: c.[721G>T; 826delG]. Considering the three previously described Irish cases of pyruvate kinase deficiency, this study raises the total number of pyruvate kinase-deficient Irish patients to seven in which a total of nine mutant PKLR alleles were identified. This indicates the absence of a founder pyruvate kinase mutation in the Northern Ireland population. Although pyruvate kinase deficiency is prevalent in the Caucasian population it is not reflected in the number of individuals diagnosed in Northern Ireland. Hence, many cases of pyruvate kinase deficiency may remain undetected possibly due to the resultant anemia being mild.

beta thalassemia is one of the most common genetic diseases worldwide resulting from aberrant beta-globin chain production. It is highly prevalent in regions with endemic malaria, but it is also present at low frequency in the indigenous populations of non-tropical areas such as Britain. Screening beta thalassemia trait individuals from Northern Ireland has detected 2 Mediterranean mutations, 39 (C --> T) and IVS-I-110 (G --> A); the previously reported IVS-II-850 (G --> A) mutation originally described in individuals of Scottish/English ancestry; and 2 novel mutations, initiation codon A --> C and 109 delG. Haplotype analysis indicates that the Mediterranean mutations are present on previously described haplotypes, suggesting that they have arisen due to migration. It remains to be established whether the novel mutations have arisen de novo in Northern Ireland.

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

A baby centrally cyanosed from birth was investigated for a congenital cardiac defect. Echocardiography and angiography revealed patent foramen ovale without any other cardiac abnormality. Congenital methaemoglobinaemia was considered as the methaemoglobin level was 27%, suggesting either Hb M or a deficiency of the NADH-cytochrome b5 reductase (cytb5r) enzyme. Measurement of the cytb5r enzyme activity of this patient indicated a reduced level of 7.3 IU/g Hb (normal range 11.5–26.9 IU/g Hb). Sequencing the DIA 1 gene that encodes cytb5r revealed a novel C403T base change, predicting a proline to serine change at codon 144. This amino-acid change is not located in the enzyme’s active site and does not cause loss of function. Instead it results in reduced stability of the enzyme and development of the less severe or Type I form of recessive congenital methaemoglobinaemia. The infant was started on daily ascorbic acid treatment. She has very mild cyanosis and normal growth and developmental parameters on follow-up at 10 months of age. The Hematology Journal (2004) 5, 367–370. doi:10.1038/sj.thj.6200380

Deficiency of folate during pregnancy is associated with megaloblastic anaemia. Lower levels of folate and vitamin B12 have been reported in mothers whose offspring had neural tube defects compared to unaffected controls. Increased methylmalonic acid levels are a sensitive indicator of mild vitamin B12 deficiency and elevated homocysteine levels denote vitamin B12 or folate deficiency. We have investigated the relationship between serum concentration of total homocysteine, methylmalonic acid, vitamin B12 and folate in pregnancy. A significant inverse correlation was found between homocysteine and red cell folate and, to a lesser extent, serum folate. In addition, a significant inverse correlation was found between methylmalonic acid and vitamin B12. No significant relationship was found between homocysteine and vitamin B12. The relationship between red cell folate and serum folate and homocysteine may be useful for detecting borderline folate deficiency in pregnancy and indicate pregnancies at risk of neural tube defect. These sensitive assays are useful tools for the further investigation of folate vitamin B12 and metabolism in normal and abnormal pregnancy.

Aims—To determine eVects of maternal iron depletion and smoking on iron status of term babies using serum transferrin receptors (STfR) and their ratio to ferritin (TfR-F index) in cord blood. Methods—Iron, ferritin, STfR, and haemoglobin (Hb) concentration were measured and TfR-F index calculated in 67 cord /maternal blood pairs. Twenty six mothers were iron depleted (ferritin

The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O2 and enhances Epo production at 1% O2, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist.

Hypoxia induces a number of physiological responses including changes in blood gases, changes in acid-base status, and an increase in circulating erythropoietin (Epo) (1). Epo is a glycoprotein hormone that controls the red cell mass and is produced in the kidney. Chronic renal failure (CRF) is almost invariably accompanied by anemia caused by underproduction of Epo (2). However, the diseased kidney retains the ability to respond to short-term hypoxia; Blumberg et al. (3) have shown that an elevation in erythroid stimulating factor (ESF) occurred in patients with dialysis-dependent CRF who were transferred to altitude for a day. Metabolic acidosis and an increase in red cell 2,3-DPG levels, leading to a shift in the oxygen dissociation curve, are also usually found in CRF. We have investigated the physiological changes and increases in circulating Epo caused by short-term hypobaric hypoxia using a 5 6 -nephrectomized rabbit model of CRF.

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)–cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

OBJECTIVE—To study iron status at different gestational ages using cord blood serum transferrin receptors (STfRs). METHODS—STfRs, iron, ferritin, total iron binding capacity, haemoglobin, and reticulocytes were measured in 144 cord blood samples. The babies were divided into three groups according to gestation (26 very preterm (24-29 weeks); 50 preterm (30-36 weeks); 68 term (37-41 weeks)). RESULTS—Serum iron, ferritin, and total iron binding capacity were highest at term, whereas reticulocytes were highest in the very preterm. STfR levels were not influenced by gestation. Haemoglobin (r = 0.46; p < 0.0001) and reticulocytes (r = 0.42; p < 0.0001) were the only indices that independently correlated with STfR levels. CONCLUSIONS—STfR levels in cord blood are not directly influenced by gestation and probably reflect the iron requirements of the fetus for erythropoiesis.

Effect of Maternal Iron Status and Smoking on Cord Blood Transferrin Receptor Concentration

The Travenol Hem-O-Scan and the Instrumentation Laboratory (IL) system for determination of the oxygen dissociation curve and P50 values have been evaluated. Using the Hem-O-Scan, an oxygen dissociation curve and P50 value may be obtained on 2 microliter of blood in 40 min, though the pH is not measured. In the IL system, approximately 3 ml of blood is required to establish four points on the oxygen dissociation curve in 80 min, and pH data are available. Both systems give reproducible results for P50, giving standard deviations of 0.786 mm Hg for the Hem-O-Scan and 0.949 mm Hg for the IL system, compared with 1.740 mm Hg for the manual system. Good agreement was found between the manual method and Hem-O-Scan (t = 0.363, degrees of freedom = 19, 0.8 greater than P greater than 0.7; r = 0.892, P less than 0.001). There was also satisfactory agreement between the IL system and the Hem-O-Scan (t = 0.370, degrees of freedom = 20, 0.8 greater than P greater than 0.7; r = 0.760, P less than 0.001). P50 values obtained by the manual and by both automated technics agree well with published values.

This study was undertaken to establish whether there are differences or peculiarities in handwritings produced by the deaf that would uniquely distinguish them from writings of the normal hearing population.

In pyruvate kinase (PK)-deficient blood with high levels of reticulocytes, the degree of haemolysis after incubation at 37 degrees C for 48 h was halved by the inclusion of adenine in the medium. the decrease in haemolysis was associated with a higher ATP level but was not related to the pH of the incubation mixture. The incorporation of adenine into nucleotides studied over a 3 h period was similar in normal and PK-deficient blood. The observed increase in cellular ATP was equivalent to that shown by radioactive measurements to have been synthesized from added adenine. Inhibition of mitochondrial oxidative phosphorylation in the reticulocytes of the PK-deficient blood by KCN reduced the amount of adenine taken up by the cells by a factor of 3 and altered the pattern of incorporation into the nucleotides. Only 25% of the adenine which entered the blood cells was converted into ATP compared with 85% in the absence of cyanide. Despite the synthesis of small amounts of ATP from labelled adenine, the overall ATP content fell to less than 50% of its original level. It is suggested that incubation with adenine increased the ATP level in the reticulocytes by virtue of mitochondrial oxidative phosphorylation thereby reducing the haemolysis of these cells.