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 β-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 β-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.

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 GST value was high (GST = 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.

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.

HPLC studies in hemoglobinopathies.

An accurate diagnosis of beta -thalassemia carriers, homozygous patients and identification of different structural hemoglobin variants is important for epidemiological studies as well as for management and prevention of the major hemoglobin disorders. There are many electrophoretic and chromatographic approaches for estimation of HbA2 and Hb F but cation exchange HPLC (CE-HPLC)using automated dedicated machines like the Variant Hb testing system have become the method of choice for these investigations. CE-HPLC also helps in the presumptive identification of many abnormal hemoglobin variants and has been useful for both neonatal screening of sickle cell disease as well as second trimester prenatal diagnosis of thalassemia by fetal blood analysis. Other applications of HPLC in hemoglobinopathies include separation of globin chains, measuring the ratio of gamma globin chains (Ggamma/Agamma) and the recently described denaturing HPLC for detecting mutations in both alpha and beta globin genes.

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.

Hemoglobin sickle D Punjab-a case report.

Compound heterozygosity for bS/bD results in a severe hemolytic anemia and a clinical syndrome similar to that of sickle cell disease. Here, we report a case of HbSD Punjab disease. A 10 year old female child residing at Nagpur, Maharashtra presented with severe hemolytic anemia, hepatosplenomegaly and occasional pains in bones and abdomen. Initially, she was thought to be a case of sickle cell anemia, however, with the help of HPLC and molecular analysis it was confirmed as HbSD Punjab disease.

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.

Is Fetal Hemoglobin Level A Prognostic Indicator For The Severity Of Sickle Cell Disease.

Genetic and environmental factors are believed to pay a role in the variation in clinical severity of sickle cell disease in different populating group. Among these, fetal Hb expression is one such epistatic factor which may ameliorate severity of the disease as it can reduced the polymerization sickle RBCs. The present work was undertaken to look for correlation between severity of the disease and the expression of HbF. A total of 110 sickle cell disease cases in the age group of 2-49 years were studied. The HbF levels varied from 2-24% with mean of 10.53+ 406%. Our findings did not shown any statistically significant correlation between HbF levels and clinical severity. Nevertheless the mean HbF levels were slightly higher in the group of patients not having any history of painful crisis, infections, need for hospitalisation or blood transfusions. Further the levels of HbS were significantly lower when the HbF levels were high (>10%). This suggests that variation in clinical severity in sickle cell disease may be more due to role of other genetic factors like associated alpha thalassaemia and the higher HbF may only offer an added advantage to these patients.