Search

Hemoglobinopathies are common genetic disorders of the hemoglobin (Hb) molecule. Globally, 7.0% of the population are carriers of thalassemia with 300,000-400,000 affected births each year. There are >40 million carriers of β-thalassemia (β-thal) in India with 10,000-12,000 affected births every year. This makes control programs crucial in this vast and diverse country. The present study was undertaken to find out the burden of hemoglobinopathies, and in particular, the prevalence of β-thal carriers in the population of Saurashtra region of Gujarat in Western India. A total of 16,780 individuals, including school and college students, were screened. Complete blood counts (CBCs) and high performance liquid chromatography (HPLC) analysis were performed. We detected 1891 (11.26%) individuals with different hemoglobinopathies, of whom 758 (4.52%) were diagnosed to carry β-thal trait, 104 (0.62%) carried Hb D-Punjab ( HBB : c.364G>C) trait, 61 (0.36%) carried sickle cell trait, 32 (0.19%) carried δβ-thal trait/HPFH (hereditary persistence of fetal Hb) trait, and other hemoglobinopathies were identified in smaller numbers (0.15%). We encountered 27 individuals with mean corpuscular Hb (MCH) <27.0 pg and mean corpuscular volume (MCV) <80.0 fL levels, who had borderline Hb A 2 levels (3.2-3.5%). Twenty castes showed the presence of β-thal or other hemoglobinopathies. A high prevalence of β-thal was found in the Sindhis (11.67%), Lohanas (9.71%), Brahmins (6.31%), Bharvads (6.94%), Harijans (7.57%) and Vankars (7.77%). All the heterozygotes were given appropriate counseling. A multi pronged approach, including screening of high school and college students, needs to be considered for this vast and ethnically diverse country to reduce the burden of hemoglobinopathies.

Management and control of hemoglobinopathies are a challenge in India where 67.0% of people reside in rural regions. The GDP spent on health is one of the lowest (1.3%) resulting in high out-of-pocket expenses. The β-thalassemias are prevalent with an estimated 7500-12000 new births each year. Hb S ( HBB : c.20A>T) and Hb E ( HBB : c.79G>A) are also common regionally. Over 80 β-thalassemia (β-thal) mutations have been characterized in Indians. The δ gene mutations are increasingly being described and their coinheritance in β-thal carriers leads to a reduction in Hb A 2 levels and a misdiagnosis of carriers. Around 15-20 centers offer prenatal diagnosis (PND) mainly in urban regions. The projected annual cost of care of β-thal patients over a decade (2016-2026) will increase from INR30,000 (US$448) million to INR55,000 (US$820) million if all patients are adequately treated. Cost comparisons are difficult to make with other international studies as the standard of care, cost of medicines and other services vary in different countries. Several centers provide hematopoietic stem cell transplants (HSCTs) for thalassemias, however, only around 250 HSCTs are done annually. Although the cost is high, financial assistance is available for a few patients. There are disparities in the quality of care and to address this a National Policy has been proposed for the management and prevention of hemoglobinopathies that will embark on a comprehensive program, providing adequate care and augmenting the existing public health care services. It will also include training, genetic counseling and easier access to preventive options and a National Registry.

The β-thalassemias and sickle cell disorders pose a considerable health burden in India. Of the more than 10,000 annual births of children with a severe hemoglobinopathy, only around 10.0% are managed optimally. Thus, genetic counseling and prenatal diagnosis (PND) is a valid option for a large and diverse country. Our center was one of the first to initiate PND and we present our experience over 30 years to evaluate the impact of awareness in changing the trends of PND of hemoglobinopathies. Both second and first-trimester diagnoses were undertaken by fetoscopy/cordocentesis and globin biosynthesis/high-performance liquid chromatography (HPLC) analysis of fetal blood and chorionic villus sampling (CVS) and DNA analysis. Over 30 years, 3478 couples (first trimester: 2475; second trimester: 1003) from all over India were offered PND. The number of couples coming in the first trimester increased significantly over each decade and couples coming prospectively increased from 2.5 to 18.4%. A cost-effective stepwise approach was used for molecular analysis. Eight hundred and one fetuses (23.0%) were affected and all except three couples opted for termination of these pregnancies. Genetic counseling and PND is the only way to reduce the burden of disease. With awareness, there was a shift from second trimester to first trimester PND over each decade, with an increasing number of couples coming during the first pregnancy. There are only 15 to 20 centers in India offering PND. We have compared our study with other reports on PND from different regions in India.

Genetic structure of the Indian population is influenced by waves of several immigrants from West Eurasia. Therefore, genetic information of various ethnic groups is valuable to understand their origins, the pattern of migration as well as the genetic relationship between them. No genetic data is available on Pathare Prabhu, which is a small indigenous Hindu community from Mumbai, Maharashtra State, India. The aim of this study was to screen the Pathare Prabhus for hemoglobinopathies, which is a major public health problem in India. Two hundred and fifty-seven unrelated Pathare Prabhus subjects were screened for various hemoglobinopathies. Complete blood counts (CBC) were done on an automated hematology counter. High performance liquid chromatography (HPLC) was used to identify β-thalassemia (β-thal) carriers. Molecular characterization of the β gene defects was done by reverse dot-blot hybridization, amplification refractory mutation system (ARMS) and DNA sequencing. Deletional α-thalassemia (α-thal) was detected by multiplex polymerase chain reaction (PCR). Hb A 2 -Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of β-thal was 3.89%, while deletional α-thal was 5.4%. The initiation codon (ATG>ACG) (HBB: c.2T>C) was seen in eight individuals (80.0%), Hb D-Punjab (HBB: c.364G>C) and Hb A 2 -Saurashtra, was found in two and one individual, respectively. A community-specific β-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.

Hemoglobinopathies are a group of inherited single gene disorders. There are reports on hemoglobin (Hb) variants identified in the tribal and non-tribal populations of Tripura State in northeastern India. This study aimed to determine the spectrum of hemoglobinopathies and enzymopathies by newborn screening in Tripura State and assess the extent of neonatal jaundice. A total of 2400 cord blood samples were collected and analyzed by high performance liquid chromatography (HPLC). Further confirmation of any abnormal HPLC was done by DNA analysis. The samples were also screened for deficiency of enzymopathies, glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase. Of 2400 cord blood samples screened, 225 (9.3%) were Hb E (HBB: c.79G>A) heterozygotes, 80 (3.3%) were Hb E homozygotes and one carried Hb E-β-thalassemia (β-thal). Other Hb abnormalities were also detected including 15 Hb S (HBB: c.20A>T) heterozygotes, two Hb D-Punjab (HBB: c.364G>C) heterozygotes and two compound heterozygotes for Hb D-Punjab and Hb E. Of the 80 homozygous Hb E babies, four were non-tribal and 76 babies were tribal, and 225 patients carried Hb E trait, 141 were tribal, while 84 were non-tribal. Of 40 G6PD deficient babies identified, 13 had coinherited Hb E and two babies had pyruvate kinase deficiency. α Genotyping was performed in 162 affected babies, 50 of them carried α gene deletions. Newborn screening programs for Hb E, other hemoglobinopathies and G6PD deficiency must be encouraged in the malaria-endemic northeastern region of India. Drug-induced hemolysis can also be avoided by screening for G6PD deficiency at birth.

Haemoglobinopathies particularly haemoglobin S and E (HbS, HbE) and β-thalassaemia are important challenges for tribal populations in India. The HbS, HbE and β-thalassaemia genes are variably distributed across various tribal populations of India. HbE is mainly restricted in tribals of North-East, West Bengal, Odisha and those in Andaman and Nicobar islands. HbS has more extensive distribution in the country (10-40% trait frequency) and the homozygotes and double heterozygotes present with a wide array of morbidities. The morbidity varies greatly in different areas of the country due to differential co-inheritance of α-thalassaemia gene and interaction of various epistatic and environmental factors. Though substantial data on prevalence of these disorders exist, there is an urgent need to develop integrated hierarchical core facilities to manage the disease. Such centres will generate more data and will also explore areas of management which need more local attention. Newborn screening, genetic counselling, carrier detection, prenatal diagnosis along with management of cases should form the basic infrastructure of haemoglobinopathy management. Research in this areas should continue focusing on various challenges in care delivery, prevention and basic sciences on interaction of haemoglobinopathies with various other infections.

We evaluated the spectrum of hemoglobinopathies among the primitive tribal groups from 4 states in India. A total of 15,200 individuals from 14 primitive tribal groups were studied by automated high-performance liquid chromatography. The hemoglobin S (HbS) allele frequency varied from 0.011 to 0.120 and the β-thalassemia allele frequency from 0.005 to 0.024. It is interesting to note that a very high HbS allele frequency was observed among the Dravidian (0.060-0.120) and Indo-European (0.060-0.076) as compared with Austro-Asiatic (0.011-0.022) speaking tribal groups. Although statistical analysis of the data did not show any ethnic differences within the states, regional differences were observed between the states for both HbS and β-thalassemia traits. HbS was found to be the most common hemoglobinopathy followed by β-thalassemia. A health plan for identifying sickle-cell homozygotes in the neonatal period with proper medical intervention is desirable., (© 2013 APJPH.)

Background: Sickle cell disease is a major health burden in India. The aim of the study was to compare the diagnostic utility of two different approaches on automated high performance liquid chromatography (HPLC) for newborn screening for sickle cell disorders and other haemoglobinopathies in India., Methods: Newborn babies of sickle heterozygous mothers were tested by HPLC using two different kits, the β-thal short kit, which is routinely used for screening for haemoglobinopathies in most laboratories, and the sickle cell short kit which is specific only for neonatal samples. Confirmation of the sickle and α genotypes was done by molecular analysis., Results: Of the 601 babies tested, 276 were normal, 284 were sickle heterozygous and 41 were sickle homozygous using the β-thal short kit. Using the sickle cell short kit, a discrepancy was seen in one newborn sample where a normal baby was identified as a sickle heterozygous baby. α-Genotyping was done in 42 babies and 16 of them had α gene deletions. The presence of α thalassaemia could be suspected in 15 of these 16 babies based on a spike at the start of the chromatogram using the β-thal short kit. In comparison, using the sickle cell short kit the diagnosis of α thalassaemia was difficult based on the percentage of the FAST peak. Further, other rare α chain Hb variants were also missed., Conclusions: The β-thal short kit was more versatile than the sickle cell short kit for screening for haemoglobinopathies in newborns in our population.

Although iron deficiency anemia is very common in India, systematic large studies on the prevalence and hematological consequences of iron deficiency among carriers of β-thalassemia (β-thal) and other hemoglobinopathies are lacking. A multi center project was undertaken to screen college/university students and pregnant women for iron deficiency anemia and various hemoglobinopathies. Fifty-six thousand, seven hundred and seventy-two subjects from six states, Maharashtra, Gujarat, Karnataka, West Bengal, Assam and Punjab, were studied. Iron deficiency anemia was evaluated by measuring zinc protoporphyrin (ZPP) and hemoglobin (Hb) levels, while β-thal and other hemoglobinopathies were detected by measuring the red cell indices and by Hb analysis using high performance liquid chromatography (HPLC). College boys (2.2%), college girls (14.3%) and antenatal women (27.0%) without any hemoglobinopathies had iron deficiency anemia. Among the β-thal carriers, the prevalence of iron deficiency anemia was 17.3% in college boys, 38.1% in college girls and 55.9% in pregnant women, while in the Hb E [β26(B8)Glu→Lys; HBB: c.79G>A] carriers, it was 7.3% in college boys, 25.4% in college girls and 78.0% in antenatal women. In individuals with Hb E disease, the prevalence of iron deficiency anemia varied from 31.2-77.3% in the three groups. A significant reduction in Hb levels was seen when iron deficiency anemia was associated with hemoglobinopathies. However, the Hb A2 levels in β-thal carriers were not greatly reduced in the presence of iron deficiency anemia.

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.

Objectives: Hydroxyurea is known to reduce ineffective erythropoiesis and thereby hemolysis leading to a reduction in bilirubin levels in patients with hemoglobinopathies. However, the effect of hydroxyurea on hyperbilirubinemia in relation to the UGT1A1 gene promoter polymorphism is not known in Indian patients with different hemoglobinopathies., Design and Methods: We studied 112 patients (77 sickle cell anemia, 22 β-thalassemia intermedia and 13 HbE-β-thalassemia) who were on hydroxyurea therapy for 2 years for their response towards hyperbilirubinemia associated with UGT1A1 promoter polymorphism., Results: The patients with (TA)(7)/(TA)(7) repeats had significantly higher serum bilirubin levels than those with (TA)(6)/(TA)(6) repeats in all the groups and the reduction in serum bilirubin after hydroxyurea therapy was still higher among patients with (TA)(7)/(TA)(7) repeats when compared with (TA)(6)/(TA)(6) repeats., Conclusions: Higher bilirubin levels were associated with the (TA)(7)/(TA)(7) sequence however they did not come down to normal levels after hydroxyurea therapy., (Copyright © 2010. Published by Elsevier Inc.)

Accurate estimation of hemoglobin (Hbs) A, Hb A(2), Hb F and abnormal Hb is required for diagnosis of hemoglobinopathies and genetic counseling. High pressure liquid chromatography (HPLC) is the most suitable approach available. But for 70% of the rural Indian population, HPLC analysis facilities are not available and screening would require transportation of samples to laboratories in bigger cities. We thus evaluated the feasibility of using a kit designed for measuring Hb A(1c) using capillary blood for collection and preservation of samples over a period of 15 days at different temperatures for screening for hemoglobinopathies. Capillary blood (5 microl) of 90 individuals was collected in the capillary collection system and run on the Variant Hemoglobin Testing System on days 1, 3, 5, 8, 12 and 15 after incubation at 4, 22, 37, 42 and 50 degrees C. The stability of different Hbs varied at different temperatures. The stability was maintained for 12 to 15 days by most of the samples up to 37 degrees C. Hb E was stable for 3 days up to at 37 degrees C and Hb D and Hb Q for 3 days up to 42 degrees C. This capillary blood collection system would have tremendous potential for sample collection and transportation under adverse climatic conditions for screening of hemoglobinopathies in remote areas in different countries.

Our purpose was to develop and evaluate isolation and enrichment of fetal erythroblasts and a nested polymerase chain reaction (PCR) approach using fetal erythroblasts for detecting the beta-globin gene mutations for a noninvasive prenatal diagnosis of hemoglobinopathies. Maternal blood at different periods of gestation was layered on a Percoll density gradient for enrichment of fetal nucleated RBCs (NRBCs). A combination of 3 monoclonal antibodies (CD45-peridinin chlorophyll protein, glycophorin A-phycoerythrin, and anti-hemoglobin F-fluorescein isothiocyanate) was used for flow cytometric sorting of fetal NRBCs from enriched cells. Different nested PCR-based approaches were used for identification of fetal mutations. Owing to heterogeneity of beta-thalassemia mutations in the population of India, we had to screen for 12 mutations and were able to give an accurate diagnosis in 84 (84.0%) of 100 cases when compared with chorionic villus sampling or cordocentesis and DNA analysis.This nested PCR approach enabled amplification of small quantities of DNA from fetal erythroblasts, providing a cost-effective method for noninvasive diagnosis.

Prenatal diagnosis is an option for couples at risk of having a child affected with hemoglobinopathies. Chorionic villus sampling (CVS) and cordocentesis are accurate but a finite risk of fetal loss exists. A non invasive, risk free strategy that has emerged is isolation of fetal erythroblasts from maternal blood. Enrichment of nucleated red blood cells (nRBCs) from 7.0 mL maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies: CD45 per CP, glycophorin A-phycoerythrin (PE) and Hb F-fluorescein isothiocyanate (FITC) in 43 cases between 7 and 21 weeks' gestation. The percentage of nRBCs ranged from 0.0001-2.03%. The presence of dual fluorescence (glycophorin A-PE and Hb F-FITC) was confirmed by confocal microscopy. A sufficient number of nRBCs could be isolated in the first and second trimester of pregnancy to provide a simple flow cytometric approach as a potential for non invasive diagnosis of beta-globin defects.