Effect of inherited red cell defects on growth of Plasmodium falciparum: An in vitro study

Background & objectives: High prevalence of certain polymorphic alleles of erythrocytes in malaria endemic area has been linked to the resistance provided by these alleles against parasitic infestations. Numerous studies undertaken to demonstrate this correlation have generated conflicting results. This study was undertaken to investigate the abilities of various polymorphic erythrocytes to support in vitro growth of Plasmodium falciparum parasites. Methods: In this study under in vitro condition the ability of P. falciparum parasites to grow was assessed in the erythrocytes obtained from a total of 40 patients with various haemoglobinopathies, such as ?-thalassaemia (?-Thal), sickle cell anaemia, erythroenzymopathy-like glucose-6-phosphate dehydrogenase deficiency and membranopathy-like hereditary spherocytosis. Results: Significantly reduced in vitro invasion and growth of parasites was seen in the cultures containing abnormal erythrocytes than in control cultures containing normal erythrocytes (P< 0.05). The mean per cent parasitaemia comparison was also carried out among the three polymorphic erythrocyte groups, i.e. ?-Thal, sickle cell anaemia and enzyme-membranopathies. Interpretation & conclusions: Erythroenzymopathies and membranopathies were found to provide a more hostile environment for parasites, as the least parasitaemia was observed in these erythrocytes. The present in vitro study showed that P. falciparum did not grow well and did not invade well in erythrocytes obtained from common inherited red cell disorders.

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.

Detection of two rare β -thalassemia mutations [-90 (C → T) and CD 26 (C → T)] among Indians.

BACKGROUND : β -Thalassemia (β -thal) is present in practically every caste group in Indians. Molecular characterization of β -thal in these groups has revealed an extremely heterogeneous picture. AIM : To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS : Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β -globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS : Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β -thal mutations [CD 26 (C→T) and -90 (C→T)]. CONCLUSION : DGGE is a useful mutation detection technique to identify β -thal mutations among the heterogeneous Indian population.

Variable clinical severity of Hb E beta-thalassemia among Indians.

OBJECTIVE: The aim of this preliminary report was to look at the effect of genetic variations in the alpha, beta and gamma globin genes in 7 cases of hemoglobin E/beta-thalassemia with diverse clinical expression of the disease. METHODS: beta-thalassemia mutations were characterized by PCR and dot blot hybridization. G gamma gene polymorphism (Xmnl) was determined by PCR followed by restriction enzyme digestion and polyacrylamide gel electrophoresis. alpha genotyping was done by Southern blot hybridization. RESULTS: Six cases had a severe beta+ mutation [IVS 1 nt 5 (G-->C)] and one case had a beta zero mutation [F/S 41/42 (-CTTT)]. Hence, the beta-thalassemia mutation does not seem to contribute towards the clinical diversity. alpha-genotyping showed a single alpha-gene deletion of the rightward type in three of the five milder cases. The -158 G gamma (C-->T) substitution was present at least in heterozygous state (+/-) in all the milder cases. CONCLUSIONS: Deletional alpha thalassemia and presence of the -158 G gamma (C->T) substitution are the two factors which appear to be more important in decreasing the severity of the disease rather than the nature of the beta thalassemia mutation.

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.

Beta-thalassemias: expression, molecular mechanisms and mutations in Indians.

The beta-thalassemias are a heterogenous group of inherited disorders of hemoglobin (Hb) synthesis characterized by a reduction (beta+) or absence (beta zero) of synthesis of the beta globin chains of Hb, resulting in an imbalanced chain synthesis. To understand their expression and molecular basis in Indians, it is essential to review briefly the genetic control of normal Hb production and the structure, organization and regulation of different globin genes. The Indian beta-thalassemia mutations and strategies for prevention are described.

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.

Prenatal diagnosis of haemophilia: a preliminary report.

BACKGROUND: Haemophilia A is the most common congenital bleeding disorder seen in man, affecting one in 5000 to 10,000 males. Because of the large size and heterogeneity of mutations in the factor VIII gene, direct detection of mutations is not practically feasible, except the recently detected intron 22 inversions. Hence, the indirect method of gene tracking using various polymorphic markers is the method of choice. Using this approach, we have performed antenatal diagnosis in four haemophilia A families. METHODS: The four families included 21 subjects who were used for gene-tracking analysis. Two families had a positive history with more than one member affected, while the remaining two families had a negative history with only one affected son. In all four families, the propositi and their affected relatives had severe haemophilia A with factor VIII:C less than 1%. All were negative for inhibitors. The polymorphic markers used were IVS 18 Bcl I, IVS 19 Hind III and the extragenic DXS 52 St 14 of the factor VIII gene. Prior to polymorphism analysis, the sex of the foetus was determined using Y chromosome-specific primers. All the analyses were carried out by polymerase chain reaction. RESULTS: Antenatal diagnosis in the four families showed three normal male foetuses and one normal female foetus. Two families provided evidence with only IVS 18 Bcl I and St 14 markers. One family provided information with only intron 19 Hind III marker. The fourth family provided information with all three markers. The coagulation parameters were almost in agreement with the results of DNA analysis. CONCLUSION: All three polymorphic markers yielded information. This suggests that these three markers can be effectively used in the antenatal diagnosis of haemophilia A in Indian families.

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.

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.

Assessment of transcervical chorion villus aspiration biopsy in early pregnancy.

BACKGROUND. Chorionic villus sampling for prenatal diagnosis is a relatively new technique and variable success rates have been reported by different authors depending on the methods and instruments used. We describe our experience with chorionic villus sampling in Bombay. METHODS. The procedure was attempted on 62 women before termination of their pregnancy via the transcervical route, under constant real-time ultrasound guidance. A metallic cannula was negotiated through the cervix into the uterine cavity to reach the chorionic frondosum and chorionic villi were aspirated by creating a negative pressure in the syringe attached to the cannula. The villus tissue was checked under a dissecting microscope. We calculated the success rate for obtaining a sample depending on the site of the chorionic frondosum, the physique of the mother, the position of the uterus and the size of the cannula. RESULTS. Villus tissue was aspirated in 47 of the 62 cases. The success rates of sampling at the first and second attempts were 48% and 27% respectively. The factors which were associated with a higher success rate were when the chorionic frondosum was situated posteriorly rather than anteriorly (61% v. 48%; p < 0.01), when the patient was thin rather than fat (58% v. 25%; p < 0.001), when the uterus was anteverted rather than retroverted (53% v. 41%). The commonest complication was bleeding which occurred in 15% of patients. CONCLUSION. Transcervical chorionic villus sampling is associated with a high success rate except in fat women with a retroverted uterus and in those with the chorionic frondosum situated in the fundus.

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.