Prabhakar Kedar

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.

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.

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.

A novel R198H mutation in the glucose-6-phosphate dehydrogenase gene in the tribal groups of the Nilgiris in Southern India

AbstractGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common red cell enzymopathy among humans. In India, G6PD Mediterranean, G6PD Orissa, and G6PD Kerala–Kalyan are the three common mutations which account almost 90% of G6PD deficiency. Here we describe G6PD Coimbra, an unreported variant from India, and a novel 593 G → A mutation in exon 6 with an amino acid change of Arg 198 His, among the tribal groups of the Nilgiris in Southern India. Further, this novel mutation was structurally characterized and it was found that the mutation is located at the end of the coenzyme domain, which may cause enzyme instability.

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.

A new simple approach for the determination of pyrimidine 5'-nucleotidase activity in human erythrocytes using an ELISA reader.

Introduction:  Pyrimidine 5′ nucleotidase type I (P5′N‐1) deficiency is the most frequent abnormality of cell nucleotide metabolism causing hereditary non spherocytic hemolytic anemia (HNSHA). The aim of this study was to develop a simple method of determination of P5′N‐1 activity in human erythrocytes using an ELISA reader

Glucose phosphate isomerase (GPI) Tadikonda: Characterization of a novel Pro340Ser mutation

ABSTRACT After a thirty-year lag, we serendipitously reestablished contact with a patient with glucose phosphate isomerase deficiency and hydrops fetalis first reported in 1987. We now provide a clinical update and provide results of mutation analysis in this patient, from Southern India. The patient now an adult female of 36 years of age has moderate anemia but requires no transfusions except with some intercurrent illnesses. Exome sequencing studies showed a homozygous c.1018C>T (Pro340Ser) mutation in exon 12 of the glucose phosphate isomerase gene and later confirmed by direct sequencing. This mutation has not been previously described. To our knowledge, this is also the first known homozygous mutation in the hydrophobic core of the protein and is a highly deleterious mutation by in silico analysis and by clinical history in the family. Flow cytometry studies of band 3 content with eosin maleimide showed a unique tail of red cells on histograms, reflecting the dense red cells (presumably ATP depleted) seen on blood smears; similar findings were seen in patients with pyruvate kinase and phosphoglycerate kinase deficiency.

A novel nine base deletion mutation in NADH-cytochrome b5 reductase gene in an Indian family with recessive congenital methemoglobinemia-type-II

Recessive hereditary methemoglobinemia (RCM) associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r) deficiency (Type II). We report a case of 11 month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175). To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173–p.V175del3) is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM — Type II.