Prashant Warang

Flow cytometric osmotic fragility—An effective screening approach for red cell membranopathies

Among the red cell membrane disorders, hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. The aim of this study was to compare the flow‐cytometric approach for screening of red cell membrane disorders based on osmotic fragility with the eosin‐5‐maleimide (E5′M) dye test. A group of β‐thalassemia heterozygotes were also studied.

Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D

Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India.

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.

Severe mental retardation and recessive congenital methemoglobinemia in three Indian patients: Compound heterozygous for NADH‐cytochrome b5 reductase gene mutations

The diagnosis of Type II congenital methemoglobinemia has been established in three Indian patients in a single family based on persistent cyanosis and neurological manifestations with severe enzyme deficiency in erythrocytes. Clinical evaluation showed very severe encephalopathy, microcephaly, generalized dystonia, and mild cyanosis. Molecular studies demonstrated compound heterozygosity for two mutations in the reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) gene. One was a novel mutation 705G→A, which leads to the substitution of a Trp by a stop codon at residue 235 within exon 8 and the other was a previously reported missense mutation 608G→A leading to a replacement of Cys-203 (TGC) by Try (TAC) in exon 7. Although both amino acid substitutions are located in the NADH-binding domain, the whole protein structure, especially the region between the flavin adenine dinucleotide and NADH-binding domains, is disturbed. The presence of a premature stop codon results in the production of truncated b5R and should explain the severe enzyme deficiency seen in these cases.

Molecular and clinical heterogeneity in pyruvate kinase deficiency in India.

We studied the PK-LR gene in 10 unrelated Indian patients with congenital haemolytic anemia associated with erythrocyte pyruvate kinase deficiency. The patients had a variable presentation ranging from a very mild compensated hemolysis to severe anemia. Nine different mutations were detected among the 20 mutated alleles identified: one deletion (c.1042-1044del) p.Lys348del and eight single-nucleotide (nt) substitutions resulting in amino acid exchanges c.397A>G (p.Asn133Asp), c.992A>G (p.Asp331Gly), c.1072G>A (p.Gly358Arg), c.1076G>A (p.Arg359His), c.1219G>A (p.Glu407Lys), c.1241C>T (p.Pro414Leu), c.1436G>A (p.Arg479His) and c.1529G>A (p.Arg510Gln) were identified. Although all the exons, the flanking regions and the promoter region were sequenced in all cases, we failed to detect the second expected mutation in two subjects. Two mutations [c.397A>G; c.1241C>T] were novel. These novel missense mutations involved highly conserved amino acids. Two mutations were identified for the first time in the homozygous state globally (c1042-1044del; c.1072G>A) and two other mutations were identified for the first time in our population (c.1076G>A; c.1529G>A). This study along with our earlier report suggests that the most frequent mutations in India would appear to be c.1436G>A (18.33%), followed by c.992A>G (11.66%) and c.1456C>T (11.66%). Structural implications of amino acid substitutions were correlated with the clinical phenotypes seen.

Molecular Diversity of Hemoglobin H Disease in India

This study was undertaken to evaluate the variable clinical expression of hemoglobin (Hb) H disease in India. For the study, alpha genotyping was done in 8 patients with Hb H disease using multiplex polymerase chain reaction and DNA sequencing. The study revealed that 4 genotypes (- -(SEA)/ -alpha(3.7), - -(SA)/-alpha(3.7), - -(SEA)/-alpha(3.7 Sallanches), - -alpha(3.7)/-alpha(3.7 Sallanches)) were responsible for Hb H disease, the alpha+ thalassemia mutation (-alpha(3.7) deletion) being the most common defect. The nondeletional mutation Hb Sallanches (alpha 2 codon 104 G --> A) was seen in 3 cases. Two unique and novel genotypes leading to Hb H disease were characterized (- -(SEA)/-alpha(3.7 Sallanches) and -alpha(3.7)/-alpha(3.7 Sallanches)). Because a majority of patients with Hb H disease do not have severe manifestations, prenatal diagnosis is usually unwarranted in India.

Spectrum of novel mutations in the human PKLR gene in pyruvate kinase-deficient Indian patients with heterogeneous clinical phenotypes

Eighteen unrelated pyruvate kinase (PK)‐deficient Indian patients were identified in the past 4 years with varied clinical phenotypes ranging from a mild chronic haemolytic anaemia to a severe transfusion‐dependent disorder. We identified 17 different mutations in the PKLR gene among the 36 mutated alleles. Ten novel mutations were identified: 427G>A, 499C>A, 1072G>A, 1180G>T, 1216G>A, 1220A>G, 644delG, IVS5 (+20) C>A, IVS9 (+44) C>T, and IVS9 (+93) A>C. A severe syndrome was commonly associated with some mutations, 992A>G, 1436G>A, 1220A>G, 644delG and IVS9 (+93) A>C, in the PKLR gene. Molecular graphics analysis of human red blood cell PK (RPK), based on the crystal structure of human PK, shows that mutations located near the substrate or fructose 1,6‐diphosphate binding site may change the conformation of the active site, resulting in very low PK activity and severe clinical symptoms. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. In particular, the 1216G>A and 1219G>A mutations significantly affect the interdomain interaction because they are located near the catalytic site in the A/B interface domains. The most frequent mutations in the Indian population appear to be 1436G>A (19.44%), followed by 1456C>T (16.66%) and 992A>G (16.66%). This is the first study to correlate the clinical profile with the molecular defects causing PK deficiency from India where 10 novel mutations that produce non‐spherocytic haemolytic anaemia were identified.

Red cell pyruvate kinase deficiency in neonatal jaundice cases in India

ObjectivePyruvate Kinase (PK) deficiency is the most common enzymopathy of the glycolytic pathway in erythrocytes. It constitutes one of the common causes of hereditary non-spherocytic hemolytic anemia. The aim of this study was to screen newborns in India for pyruvate kinase (PK) deficiency in relation to unconjugated hyperbilirubinemia.MethodsLaboratory investigations done included complete blood counts, reticulocyte counts, direct and indirect bilirubin, assay of G6PD and PK activity, ATP and 2,3 DPG levels. All variables were studied in 50-cord blood samples from normal deliveries and 218 neonates with hyperbilirubinemia.Results7 of the 218 cases of neonatal jaundice were PK deficient with 30–40% reduction in PK activity. These cases also had a 3–4-fold increase in 2,3 DPG:ATP ratios, which is one of the additional indicators for PK deficiency. Six of the 7 infants had a severe clinical course.ConclusionThis study shows that the prevalence of PK deficiency in Indian neonatal jaundice cases is 3.21%, which is relatively high. This emphasizes the need for screening neonatal hyperbilirubinemia cases in India for PK deficiency.

Erythrocytosis, methemoglobinemia, and the saturation gap

Case A24-year-old software engineer was found to have high hematocrit at a local blood bank where he had volunteered for a blood donation. He was subsequently evaluated at another hospital where his clinical examination was reportedly normal; his Hb was 19.1 g/dL, hematocrit 57.6 %, leukocytes 5.2×10/L, and platelets 523×10/L. Arterial blood gas analysis while breathing room air showed pH 7.4, pCO2 34 mm Hg, pO2 95 mm Hg, and oxygen saturation (SaO2) 98%. Results of routine biochemical tests, serology for HIV 1 and 2, computed tomographic scan of thorax and abdomen, and echocardiography were normal or negative. Assay for JAK2-V617F and exon 12 mutations was negative. Serum erythropoietin level was 7.3 mU/mL (reference range 4– 27 mU/mL). Capillary zone electrophoresis showed no evidence of hemoglobin variant. Bone marrow aspiration and trephine biopsy showed normal findings. He was diagnosed with polycythemia vera and underwent two units of therapeutic phlebotomy. Following the phlebotomy, he was seen in consultation seeking a second opinion. The patient was born of non-consanguineous marriage. He belonged to the Brahmin community from West Godavari district in South India. There was no one else in his family with a history of cyanosis or polycythemia. On examination, his nail beds showed bluish discoloration. The lower lip and the conjunctiva looked a dusky hue. His Oxygen saturation using a pulse oximeter (SpO2) was 60 %. His blood methemoglobin level was 30 % and the erythrocyte NADH-cytochrome b5 reductase (Cb5R) activity was 21.0 IU/g Hb (reference range 35.0±5.00 IU/g Hb). He was found to be homozygous for Cb5R p.Arg58Gly mutation on DNA analysis. Both his parents were found to be heterozygotes for the mutation, and their erythrocyte Cb5R activity was 24.5 and 26.8 IU/g Hb, respectively. Hereditary methemoglobinemia due to NADH-Cb5R deficiency is sometimes associated with erythrocytosis mediated by a left-shift in the oxygen dissociation curve [2]; erythrocytosis is inconstant and is usually mild although cases with hemoglobin values of 18.6 and 19.5 g/dL have been reported [3, 4]. The condition appears to be not widely recognized [5, 6]. Dual-wavelength pulse oximeters show spuriously low SpO2 readings in the presence of methemoglobin due to optical interference. The difference between the SaO2 and SpO2 values exceeding five percentage points is called the saturation gap and points to the presence of methemoglobinemia or hemoglobin M variant [7]. Pulse oximetry is a widely available, simple test that has been considered the fifth vital sign [8]. The inclusion of SpO2measurement in algorithms for the diagnosis of erythrocytosis can eliminate the problem of “missed cyanosis” and help avoid unnecessary investigations in the rare patient with erythrocytosis due to inherited methemoglobinemia. P. R. Koduri (*) The Division of Hematology-Oncology, Mediciti Hospital, Secretariat Road, Hyderabad 500063, India e-mail: prkoduri@yahoo.com

Hemoglobinopathy screening by osmotic fragility test based on flow cytometer or naked eye

BACKGROUND Diagnosis of hemoglobin disorders is based mostly on abnormal red blood cell indices, elevated levels of HbA2, HbF or any other Hb on the Variant HPLC system and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate and cost effective technique. We have tried to compare the sensitivity of the widely used NESTROFT and the osmotic fragility described as percentage residual RBCs through flow cytometry for population screening. METHODS The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal hemoglobins were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis. RESULTS The normal group showed a significantly lower percentage of residual RBCs (48.08±11.87) as compared to cases (β thalassemia trait- 82.97±12.20, α thalassemia trait-72.58±8.34 and HbS trait- 85.00±4.05). The sensitivity and specificity of NESTROFT was high for both β thalassemia traits (98.33% and 96.72% respectively) and α thalassemia traits (100% and 96.72 % respectively) but very low sensitivity for HbS traits (54.84%). CONCLUSION Flow cytometric osmotic fragility was a more sensitive method to discriminate normals from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of Hb S carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. This article is protected by copyright. All rights reserved.