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 successful twin pregnancy in a patient with HbE-ß-thalassemia in western India.

Improvements in medical facilities have helped a large number of clinically severe hemoglobin E (HbE)-ß-thalassemia patients reach adulthood. Consequently, there is a new challenge, that of managing women with HbE-ß-thalassemia during pregnancy. In particular, they have a high risk of abortion, preterm delivery, intrauterine growth restriction, and thromboembolism. A 27-year-old HbE-ß-thalassemia patient on regular transfusion, who was splenectomized and heptatitis C (HCV)-positive, conceived for the first time without any infertility treatment. However, there was incomplete abortion with heavy bleeding at 3 months of gestation, which required bilateral uterine artery angiography. The angiogram showed the left uterine artery to be moderately hypertrophied. This was embolized with 300-500 micron polyvinyl alcohol (PVA) to stop the bleeding. Soon after, she conceived again with a twin pregnancy, and at 33.3 weeks of gestation, there was a normal delivery of twin girls without any postpartum hemorrhage or perineal tear. Both babies were given prematurity care. The mother and children were both normal up till the last follow-up 18 months after delivery, and both the girls are HbE heterozygous. Thorough monitoring of endocrine functions along with proper management of transfusions and iron overload can help in reducing the complications related to pregnancy in these patients.

Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma.

BACKGROUND: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk. AIM: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. SETTINGS AND DESIGN: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. MATERIALS AND METHODS: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation. RESULTS AND CONCLUSIONS: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.

Invasive & non-invasive approaches for prenatal diagnosis of haemoglobinopathies: experiences from India.

The thalassaemias and sickle cell disease are the commonest monogenic disorders in India. There are an estimated 7500 - 12,000 babies with ß-thalassaemia major born every year in the country. While the overall prevalence of carriers in different States varies from 1.5 to 4 per cent, recent work has shown considerable variations in frequencies even within States. Thus, micromapping would help to determine the true burden of the disease. Although screening in antenatal clinics is being done at many centres, only 15-20 per cent of pregnant women register in antenatal clinics in public hospitals in the first trimester of pregnancy. There are only a handful of centres in major cities in this vast country where prenatal diagnosis is done. There is considerable molecular heterogeneity with 64 mutations identified, of which 6 to 7 common mutations account for 80-90 per cent of mutant alleles. First trimester foetal diagnosis is done by chorionic villus sampling (CVS) and DNA analysis using reverse dot blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Second trimester diagnosis is done by cordocentesis and foetal blood analysis on HPLC at a few centres. Our experience on prenatal diagnosis of haemoglobinopathies in 2221 pregnancies has shown that >90 per cent of couples were referred for prenatal diagnosis of ß-thalassaemia after having one or more affected children while about 35 per cent of couples were referred for prenatal diagnosis of sickle cell disorders prospectively. There is a clear need for more data from India on non-invasive approaches for prenatal diagnosis.

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.

Microsatellite diversity among the primitive tribes of India.

The present study was undertaken to determine the extent of diversity at 12 microsatellite short tandem repeat (STR) loci in seven primitive tribal populations of India with diverse linguistic and geographic backgrounds. DNA samples of 160 unrelated individuals were analyzed for 12 STR loci by multiplex polymerase chain reaction (PCR). Gene diversity analysis suggested that the average heterozygosity was uniformly high ( >0.7) in these groups and varied from 0.705 to 0.794. The Hardy-Weinberg equilibrium analysis revealed that these populations were in genetic equilibrium at almost all the loci. The overall G(ST) value was high (G(ST) = 0.051; range between 0.026 and 0.098 among the loci), reflecting the degree of differentiation/heterogeneity of seven populations studied for these loci. The cluster analysis and multidimensional scaling of genetic distances reveal two broad clusters of populations, besides Moolu Kurumba maintaining their distinct genetic identity vis-à-vis other populations. The genetic affinity for the three tribes of the Indo-European family could be explained based on geography and Language but not for the four Dravidian tribes as reflected by the NJT and MDS plots. For the overall data, the insignificant MANTEL correlations between genetic, linguistic and geographic distances suggest that the genetic variation among these tribes is not patterned along geographic and/or linguistic lines.

Frequency of partial D in Western India.

Partial D is of clinical importance as the partial D-positive individuals who lack some epitopes of D antigen can develop anti-D if exposed to normal D antigen. The frequency of partial D varies in different populations. The majority of molecular studies on D variants have been reported in European, African and some East Asian populations, but no study has been reported in the Indian population so far. The aim of the study was to screen Indian population for detection of partial D by serology and classify them by multiplex polymerase chain reaction (M-PCR). The study population, consisting of 10,000 RhD-positive individuals from West India, was screened for detection of partial D using the partial D kit. In addition to these, blood samples referred because of serological RhD discrepant results from blood banks of West India were also investigated. The samples identified as partial D from these two groups were further characterized by M-PCR. Fifteen partial D cases were identified by population screening and 45 were identified from referred samples. Population screening revealed that one third of partial D was DFR when tested by partial D kit. We were able to classify 63.4 and 76.6% of partial D by partial D kit and M-PCR, respectively. The incidence of partial D in West India was found to be at least 0.15% when tested with partial D kit. DFR partial D was found to be predominant in the present study.

Iron deficiency anaemia in sickle cell disorders in India.

BACKGROUND & OBJECTIVE: Iron deficiency anaemia (IDA) is uncommon in individuals with sickle cell disease (SCD) because of availability of an adequate iron source potentially from increased red cell turnover and from blood transfusions. Also, iron deficiency anaemia can often go unnoticed because the sickle cell disease patients are already anaemic. Iron deficiency in sickle cell patients may result in lowering the intracellular haemoglobin concentration and this may ameliorate sickling. The present study was undertaken to determine the prevalence of iron deficiency anaemia and the response of iron supplementation in sickle cell disorders in tribal population of the four States viz. Maharashtra, Gujarat, Orissa and Tamil Nadu. METHODS: A total of 8434 individuals (7105 AA, 1267 AS and 62 SS) were tested for zinc protoporphyrin/haem (ZPP/H) ratio and haemoglobin levels. Twenty two sickle cell anaemia (SS), 47 sickle cell trait (AS) and 150 normal control (AA) individuals who were iron deficient, were given iron therapy for a period of 12 wk and the laboratory investigations were repeated at the 13th wk. RESULTS: Sixty seven per cent of subjects with sickle cell anaemia and 26 per cent with sickle cell trait had elevated ZPP/H ratios (>80 micromol/mol) as against 22.8 per cent of normal individuals. The elevated ZPP/H ratios is an indicator of microcytic anaemia of iron deficiency. Following iron therapy, an improvement in the Hb levels and ZPP/H ratios was observed in both sickle cell disorders and normal individual cases. INTERPRETATION & CONCLUSION: This study suggests that iron deficiency anaemia is an important problem in Indian sickle cell anaemia patients and iron supplementation should be given only in proven cases of iron deficiency anaemia.

First-trimester prenatal diagnosis of pyruvate kinase deficiency in an Indian family with the pyruvate kinase-Amish mutation.

Pyruvate kinase (PK) deficiency is a rare red cell glycolytic enzymopathy. The purpose of the present investigation was to offer prenatal diagnosis for PK deficiency to a couple who had a previous child with severe enzyme deficiency and congenital non-spherocytic hemolytic anemia. PK deficiency was identified in the family by assaying the enzyme activity in red cells. Chorionic villus sampling was performed in an 11-week gestation and the mutation was located in exon 10 of the PKLR gene characterized by polymerase chain reaction and using restriction endonuclease digestion with the MspI enzyme, which was confirmed by DNA sequencing on the ABI 310 DNA sequencer. Both the parents were heterozygous for the 1436G-->A [479 Arg-->His] mutation in exon 10 and the proband was homozygous for this mutation. The fetus was also heterozygous for this mutation and the pregnancy was continued. Prenatal diagnosis allowed the parents with a severely affected child with PK deficiency to have the reproductive choice of having the fetus tested in a subsequent pregnancy.

Hb Q(India) and its interaction with beta-thalassaemia: a study of 64 cases from India.

Haemoglobin Q (Hb Q), a relatively uncommon alpha-chain structural Hb variant, has been reported either in the heterozygous state or interacting with beta-thalassaemia. Individuals inheriting Hb Q generally are asymptomatic and are diagnosed by chance during population screening or as a part of a family study. This paper represents the first large study from India of 64 cases of Hb Q, documenting the haematological and molecular findings on 36 cases of Hb Q trait, 22 of Hb Q beta-thalassaemia trait and three of Hb Q beta-thalassaemia major, as well as a family of Hb Q homozygous cases. Hb Q is detected by Hb electrophoresis and chromatography. Hb Q levels in homozygous cases ranged from 32% to 35%, while in Hb Q heterozygotes the level was 20%. When there was an interaction of beta-thalassaemia heterozygotes the level was 14%, and in interacting beta-thalassaemia homozygotes the levels ranged from 7% to 9%. beta-thalassaemia mutations were characterised in cases showing elevated Hb A2 levels, which were markedly reduced in the majority of cases in which beta-thalassaemia was absent. Hb Q is rare and not a single homozygous case has been reported. However, Hb Q disease showed wide variation in clinical and haematological presentation in the same family.

Delta globin gene variations leading to reduction in HbA2 levels.

INTRODUCTION: Mutations in the δ-globin gene are not pathogenically relevant, but co-inheritance of δ-globin variants along with ß-globin gene defects can mask the diagnosis of ß-thalassaemia trait. METHODS: Routine haematological parameters were carried out. Molecular analysis of ß-globin gene mutations was carried out by CRDB, ARMS and DNA sequencing. δ- globin gene analysis was carried out by DNA sequencing. RESULTS: In this case study, we report a ß-thalassaemia trait (IVS 1-5G→C) (HBB:c.92 + 5G→C) with HbA2 of 1% showing the presence of δ-globin gene variant HbA2 St. George CD 81 (C→T) (HBD:c.244C→T). A similar observation was reported in another unrelated patient who showed near absence of HbA2 level in HPLC. He showed a presence of δ-globin gene mutation HbA2 Saurashtra CD 100(C→T) (HBD: c.301C→T) and a single 3.7 kb deletion in the α-globin gene. CONCLUSION: In the countries, where ß-thalassaemia is prevalent, an awareness and detection of different δ-globin gene mutations is important, as complex interactions between these haemoglobinopathies can lead to the misdiagnosis of ß-thalassaemia carriers.

Molecular understanding of Indian untransfused thalassemia intermedia.

BACKGROUND: The term thalassemia intermedia describe a form of thalassemia of intermediate severity, between the major transfusion-dependent forms of the disease and the symptomless carrier states. The phenotypic diversity of ß-thalassemia results from its underlying genetic diversity. The wide clinical variability of these conditions leads to major difficulties in their management. The molecular basis of thalassemia intermedia is very heterogeneous. The clinical and hematological course of ß-thalassemia intermedia is influenced by a number of genetic factors. METHODS AND RESULTS: The main aim of the study was to evaluate the effect of globin and nonglobin genetic modifiers on clinical severity of the disease. The study group consisted of 66 homozygous patients with ß-thalassemia [40 transfusion-dependent thalassemia (TDT), 26 nontransfusion-dependent thalassemia (NTDT)]. Hepatosplenomegaly was pronounced in the NTDT group. The presence of associated α-thalassemia was significantly higher in untransfused patients (P < 0.05). The milder ß-thalassemia mutations, such as Cap site +1 (A → C), -88 (C → T), and -87 (C → G), were observed mainly in the NTDT group (9.61%) as against patients with TDT (1.25%). The cis-DNA haplotypes, motifs, or polymorphisms around the gamma-globin genes [(AT)x (T)y motif (38.4%), XmnI (76.92%)and the Aγ-δ intergenic region haplotype T (73.07%) and Pre Gγ globin gene haplotype TAG (46.15%)] contributed significantly in amelioration of the disease severity. CONCLUSION: Our study emphasizes the complexity of genetic interactions that underlie the phenotype of ß-thalassemia and highlights the importance of epistatic factors and the regulation of HbF production in ß-thalassemia syndromes.

ICSH recommendations for assessing automated high-performance liquid chromatography and capillary electrophoresis equipment for the quantitation of HbA2.

Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of ß-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.

Comparison of FPLC with cellulose acetate electrophoresis for the diagnosis of beta-thalassaemia trait.

Different electrophoretic and chromatographic techniques are described in the literature for the estimation of HbA2 levels. We compared the fast protein liquid chromatography (FPLC) technique with the conventional cellulose acetate electrophoresis (CAE) used routinely in most laboratories. Ninety five individuals from high risk groups were screened for beta-thalassaemia trait by both the techniques. The cut-off value for the diagnosis of beta-thalassaemia trait by both the techniques was 3.8 per cent and 27 heterozygotes were identified. As both techniques gave comparable results, CAE could be the cost effective method of choice for routine screening for beta-thalassaemia trait.

Trimodal distribution of HbS levels in sickle heterozygotes--an useful predictor of the alpha-genotype for population screening.

The trimodal distribution of HbS levels in sickle heterozygotes has been used as an indirect approach to determine the prevalence of alpha-thalassaemia in different population groups. We used this approach to predict the alpha-genotypes of 124 sickle cell heterozygotes where the HbS concentration varied from 20 to 46 per cent with antimodes at 28.0 and 33.0. The alpha-genotypes in these individuals were also determined by Southern blot hybridization. We predicted homozygous (-alpha/-alpha) or heterozygous (-alpha/alpha alpha) alpha-thalassaemia-2 in 78 subjects by the trimodal distribution of HbS. However, actual genotyping showed that 75 patients had alpha-thalassaemia. Forty six of the 47 subjects with a normal alpha-globin genotype (alpha alpha/alpha alpha) could be predicted indirectly. The overall sensitivity was 100 per cent and specificity was 94.2 per cent with a positive predictive value of 96.2 per cent and negative predictive value of 100 per cent. As alpha-genotyping is very expensive and not feasible in most laboratories in India, we conclude that the trimodal distribution of HbS levels is a suitable method for screening for alpha-thalassaemia in population studies.

Characterization of G6PD Rohini--a new class III Indian variant.

A new Indian G6PD variant was detected in a 15 yr old Maratha male during a population screening programme in high school children in Bombay (India). The propositus and two family members having the same variant were apparently healthy. This enzyme variant has a mild erythrocyte G6PD deficiency and a slow electrophoretic mobility. It is characterized by a high Michaelis-Menton constant for G6P, a bimodal curve for pH optima and a slight decrease in the thermostability. The rate of utilization of substrate analogue is similar to that of normal. These observations suggest identification of a new class III variant, designated as G6PD Rohini.

The influence of alpha-thalassaemia on the haematological & clinical expression of sickle cell disease in western India.

We evaluated the clinical and haematological features of 29 sickle cell anaemia patients with associated alpha-thalassaemia and 22 sickle cell homozygotes with a normal alpha-globin genotype from western India. The presence of alpha-thalassaemia resulted in significantly higher haemoglobin (Hb), haematocrit (HCT), red blood cells counts (RBC) and haemoglobin A2 (HbA2) levels but lower mean cell haemoglobin (MCH) and mean cell volume (MCV). The clinical presentation in these patients was also milder with fewer episodes of painful crisis, chest syndromes, infections, requirement of hospitalization and blood transfusions. However, splenomegaly was more common as compared to the patients with a normal alpha-globin genotype. It is evident from the present study that alpha-thalassaemia could be an important genetic factor modulating the clinical expression and haematological severity of sickle cell anaemia in this region.

Detection of the beta s gene: an evaluation of the solubility test against automated chromatography and haemoglobin electrophoresis.

The solubility test is evaluated against automated high-performance liquid chromatography (HPLC) and haemoglobin (Hb) electrophoresis for its efficacy in screening for the beta s gene in population groups in remote areas. Blood samples taken from 3246 individuals from the tribal populations of the Dhule and Gadchiroli districts of Maharashtra state were analysed by all three methods. The solubility test detected 871 out of 932 individuals positive for the beta s gene by HPLC and Hb electrophoresis, and showed an overall sensitivity of 93.8% and specificity of 100%, with a positive predictive value of 100% and negative predictive value of 97.4%. Both HPLC and Hb electrophoresis are relatively expensive and not available in most laboratories in remote tribal areas, where the frequency of the beta s gene is very high. We conclude that the solubility test could be used for preliminary screening to determine the prevalence of the beta s gene in different population groups, particularly in remote areas where other facilities are not available. Individuals who test positive for the beta s gene by the solubility test require further investigation by either HPLC or Hb electrophoresis.

Evaluation of the single tube osmotic fragility test in detection of ß-thalassaemia trait.

NESTROFT (the Naked Eye Single Tube Red Cell Osmotic Fragility Test) is a simpleand inexpensivemethod for detecting the ß-thalassaemiatrait in the general population. Its results, however, depend on visual interpretation. We assessed interobserver variability in the recording of results by three individuals on 380 samples of blood collected intravenously. We also tested 745 samples by finger prick using a drop of blood or a uniform (20 µl) volume and found that when a uniform volume was tested the proportion of false positive results was7% (28/380)as compared to 22% (79/365) when a drop was used. We found that although there-was little variation in recording negative results, there was more variation in recording positive or doubtful results.We conclude that when the NESTROFT in field tests is positive or doubtful, then the blood should be further tested in a laboratory for Hb A2 and a uniform volume of blood rather than an arbitrary drop should be used in the test. NESTROFT is suitable for large scale preliminary screening for the ß-thalassaemiatrait in India.