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.

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 METHODS: Determination of P5'N-1 activity is based on the liberation of inorganic phosphorus (Pi) after incubation with uridine monophosphate/cytidine monophosphate. Inorganic phosphorus (Pi), a product of the enzymatic reaction is directly quantitated from its ultraviolet absorbance. Purine/Pyrimidine nucleotides ratio (OD 260: OD 280) was also measured RESULTS: P5'N-1 deficient patients showed reduction in P5'N-1 activity (Mean ± SD; 4.06 ± 0.66 using an ELISA reader & 6.25 ± 1.37 using a spectrophotometer) as compared to the normal control group (ELISA reader: 13.24 ± 3.42 & Spectrophotometer: 18.25 ± 3.20). Heterozygotes showed intermediate activity (ELISA reader: 6.06 ± 0.48 & Spectrophotometer: 8.06 ± 1.28), however they would have been missed on screening using the Purine/Pyrimidine nucleotides ratio CONCLUSION: Determination of P5'N-1 activity by using an ELISA reader is a new, simple, less time consuming and reliable method. It also avoids the use of radioactive material or HPLC which is a significant advantage.

Five alpha globin chain variants identified during screening for haemoglobinopathies.

BACKGROUND: This study was undertaken to analyse cases of microcytosis, and/or haemolytic anaemia where an unusual peak on HPLC or an abnormal electrophoretic mobility in isolation or along with common beta-globin gene defects was found, and to identify the molecular abnormality in them. PATIENTS AND METHODS: Investigations included a complete blood count, HPLC analysis, cellulose acetate electrophoresis (pH 8.9), heat stability test and DNA sequencing. RESULTS: Five alpha chain variants were identified. This is the first report of Hb Jackson and Hb O Indonesia in the Indian population. The presence of Hb J Meerut along with Hb E and Hb J Paris I with heterozygous beta-thalassaemia are uncommon associations. Hb Sun Prairie would have remained undetected in the heterozygous state. The presence of a homozygous child in the family helped to identify this variant. CONCLUSIONS: This study emphasizes the need to undertake systematic investigations while screening for the beta haemoglobinopathies to identify rare alpha chain variants in a population.

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.