Wide spectrum of novel and rare hemoglobin variants in the multi-ethnic Indian population: A review.

The hemoglobin (Hb) variants are qualitative abnormalities due to production of structurally abnormal globin proteins. They are categorized based on the type of mutation present in the α1, α2, ß, Gγ, Aγ and δ globin genes. So far, more than 1550 Hb variants are reported in the database. They could lead to Hb polymerization, Hb instability, altered oxygen affinity and decreased oxygen-carrying capacity of Hb or have no clinical manifestations. In India, ethnic diversity, consanguinity, regional variations and migration result in the presence of different Hb variants. We have compiled all the variants of α, ß and δ globin chains in heterozygous, homozygous and in compound heterozygous forms reported from India in the last 52 years. Of the 63 rare and novel hemoglobin variants reported from India, 22 were α-globin chain variants, 37 were ß-globin chain variants and 4 were δ-globin chain variants. Twelve novel Hb variants (Hb J Rajappan, Hb Koya Dora, Hb Rampa, Hb Godavari, Hb Chandigarh, Hb D Agri, Hb Lucknow, Hb Vellore, Hb Midnapore, Hb Bijnor, Hb A2Tianhe and Hb A2Saurashtra) were identified among persons of Indian origin. Majority of them were picked up on HPLC. Some of the variants like Hb Titusville, Hb Shimonoseki, Hb Chandigarh, Hb D Agri, Hb Yaizu and Hb Vellore eluted in the HbS window whereas variants like HbD Iran, Hb St. Louis, Hb G Coushata, HbM Saskatoon, Hb Lucknow, Hb Grange-Blanche and Hb Tianshui showed falsely elevated HbA2. Hence, careful and systematic investigations are required to identify them.

Spectrum of ß-Thalassemia and Other Hemoglobinopathies in the Saurashtra Region of Gujarat, India: Analysis of a Large Population Screening Program.

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 ß-thalassemia (ß-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 ß-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 ß-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 δß-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 A2 levels (3.2-3.5%). Twenty castes showed the presence of ß-thal or other hemoglobinopathies. A high prevalence of ß-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.

Role of Oxidative Stress and the Protective Effect of Fermented Papaya Preparation in Sickle Cell Disease.

Fermented papaya preparation (FPP) is the source of antioxidants that may help in reducing the complications associated with oxidative stress and may improve the quality of life in sickle cell disease patients. In this study, we assessed the in vitro effect of FPP on sickled red blood cells (RBCs) using oxidative stress markers and observed that FPP has the potential to reduce the oxidative stress. Scanning electron microscopy (SEM) and eosin 5' malaemide (E5'M) dye test showed that FPP protects red cell morphology against the oxidative stress. Liquid chromatography mass spectrometry (LCMS) analysis of FPP suggests the presence of essential amino acids, vitamin D3, and its derivatives. Fermented papaya preparation can be of benefit either in reducing oxidative stress parameters or in preventing pathophysiological events in the sickle cell disease patients.

Thalassemia in India.

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 ß-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 ß-thalassemia (ß-thal) mutations have been characterized in Indians. The δ gene mutations are increasingly being described and their coinheritance in ß-thal carriers leads to a reduction in Hb A2 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 ß-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 Changing Trends in Prenatal Diagnosis of Hemoglobinopathies in India: The Quest of a Single Center to Reduce the Burden of Disease over Three Decades.

The ß-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.

Newborn Screening for Sickle Cell Disease Among Tribal Populations in the States of Gujarat and Madhya Pradesh in India: Evaluation and Outcome Over 6 Years.

Sickle cell disease (SCD) poses considerable public health problems in India. This study was undertaken to understand the clinical course of SCD among children identified during newborn screening programmes in Gujarat and Madhya Pradesh where the frequency of the HbS gene is high. A total of 8,916 newborn babies 8,411 from Gujarat and 505 from Madhya Pradesh were screened over 6 years (2010-2016) using HPLC and the diagnosis was confirmed by molecular analysis in a subset. A total of 128 babies (122 Gujarat, 6 Madhya Pradesh) were identified with sickle cell disease, of whom 87 (69 HbSS, 18 HbS-ß thalassemia) from Gujarat were followed for 0.5-6.6 years. Acute painful events, severe anemia and fever with infections were the major complications and 23 babies required hospitalization. Severe to moderate clinical presentation was found in 13.8% babies with SCD whereas, 86.2% babies had a milder presentation. Presence of ameliorating factors (α-thalassemia and Xmn 1 polymorphism) did not have a discernible effect on the clinical severity. Parents of babies with SCD were educated and counseled for home care. Distribution of mobile phones to 44 families having babies with SCD was beneficial as it allowed regular contact with patients and their families. Genetic counseling to the affected families has increased the awareness and acceptance for prenatal diagnosis and 18 couples opted for prenatal diagnosis in subsequent pregnancies. SCD is not always mild among tribal groups in India. Therefore, facilities for early diagnosis and prophylactic treatment in the tertiary care centers should be made available. The difficulties in regular follow up of the babies in remote rural areas have also been highlighted.

Haemoglobinopathies in India: estimates of blood requirements and treatment costs for the decade 2017-2026.

The Government of India is presently engaged in the implementation of a prevention and control programme for two major forms of haemoglobinopathies, thalassaemia major and sickle cell disease, with guidelines for their prevention and management formulated under the National Health Mission. Based on projections for the population up to the year 2026, the annual blood requirement for treatment will increase to 9.24 million units, together with an 86% increase in budgetary requirements which then would account for over 19% of the current National Health Budget. To avert a public health crisis there is an urgent need to fully implement the prevention programme for haemoglobinopathies.

Multicenter Evaluation of HemoTypeSC as a Point-of-Care Sickle Cell Disease Rapid Diagnostic Test for Newborns and Adults Across India.

OBJECTIVES: Sickle cell anemia is the commonest genetic disorder in India, and the frequency of the sickle cell gene is very high in the remote tribal areas where facilities are generally limited. Therefore, a rapid and affordable point-of-care test for sickle cell disease is needed. METHODS: The diagnostic accuracy of HemoTypeSC was evaluated against automated high-performance liquid chromatography (HPLC) as the gold standard for its efficacy in a newborn screening program. RESULTS: A total of 1,559 individuals (980 newborns and 579 adults) from four participating centers were analyzed by both methods. HemoTypeSC correctly identified 209 of 211 total hemoglobin (Hb) SS cases, for a 99.1%/99.9% total HbSS sensitivity/specificity. Overall, HemoTypeSC exhibited sensitivity and specificity of 98.1% and 99.1% for all possible phenotypes (HbAA, HbAS, and HbSS) detected. HPLC is relatively expensive and not available in most laboratories in remote tribal areas. CONCLUSIONS: We conclude that the rapid, point-of-care testing device HemoTypeSC test is suitable for population and newborn screening for the HbS phenotype.

Red cell distribution width and its association with mortality in neonatal sepsis.

OBJECTIVE: Neonatal sepsis is a major cause of mortality in the developing countries. However, with current severity scores and laboratory parameters, predicting outcomes of neonatal sepsis is a serious challenge. Red cell distribution width (RDW) is a readily available pragmatic means to predict outcomes of various comorbidities in adults and children, without causing any additional blood loss. However, its utility in neonates remains unexplored. Hence, the objective of the present study was to evaluate the association of RDW with neonatal sepsis and its role as a predictive marker for mortality. METHODS: This Prospective observational study was carried out in a Level IIIB NICU for a period of 3 years. It involved comparison of RDW values of septic neonates with those of controls (matched for gestational age and birth weight) with an equal allocation ratio. A total of 251 septic neonates along with 251 controls >28 weeks of gestational age were enrolled. The RDW was derived from complete blood count done within first 6 hours of life. After arranging the RDW (median; interquartile range (IQR)), the values were categorized as those above the 50th percentile i.e. ≥20% and those below the 50th percentile i.e. <20%. The cumulative survival rates of the above two groups were assessed using the Kaplan-Meier curve and the log rank test. RESULTS: RDW levels were significantly higher among the neonatal sepsis cases (19.90%) as compared to the controls (18.90%) with a p value of < .001. RDW was significantly higher amongst the nonsurvivors than survivors (p < .003). Kaplan-Meier curve showed that septic neonates having RDW values ≥20% had significantly increased mortality (p < .02) with a hazard ratio of 0.5. CONCLUSIONS: High RDW is associated with neonatal sepsis and is an independent outcome predictor for mortality associated with neonatal sepsis.

Red Cell Distribution Width (RDW): Normative Data in Indian Neonates.

Red cell distribution width (RDW) is altered because of prematurity and fetal growth restriction (FGR). We conducted a prospective observational study to determine normal RDW values in Indian neonates (N=964) with significant FGR. Mean RDW values in preterm neonates were higher than term neonates (P<0.0004). The RDW values in Indian neonates (with significant FGR) were higher than their western counterparts (P<0.0001). The mean RDW values for different gestational ages in Indian neonates are higher than those observed in other studies. This could be attributable to the FGR component among Indian neonates.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center.

INTRODUCTION: The hemoglobinopathies pose a significant health burden in India. Apart from the ß thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. MATERIALS AND METHODS: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. RESULTS: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 ß thalassemia mutations. 143 ß thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in ß thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδß)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of ß thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon ß chain variants were identified. CONCLUSION: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Genetic lesions in the UGT1A1 genes among Gilbert's syndrome patients from India.

The present study was undertaken to investigate genetic variations present in the coding regions of the UGT1A1 gene among the Gilbert's syndrome patients. Analysis of genetic variations was performed by direct DNA sequencing among the patients that do not have any polymorphic variations in the promoter regions of the UGT1A1 gene. We identified seven different sequence variations among Gilbert's Syndrome patients, of which four were novel. Out of seven variants, six missense and one silent single nucleotide substitutions were present in the UGT1A1 gene. In addition, molecular modeling of UGT1A1 (H55R, P152S and N212H) variants suggested a reduced activity of the enzyme. This study demonstrates that different variations present in the UGT1A1 gene and specifically, the H55R variation had a significant effect on bilirubin levels and could be genetic risk factors for hyperbilirubinemia.

Newborn Screening for Hemoglobinopathies and Red Cell Enzymopathies in Tripura State: A Malaria-Endemic State in Northeast India.

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-ß-thalassemia (ß-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.

Rare ß- and δ-Globin Gene Mutations in the Pathare Prabhus: Original Inhabitants of Mumbai, 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 ß-thalassemia (ß-thal) carriers. Molecular characterization of the ß 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 A2-Saurashtra (HBD: c.301C>T) was identified by DNA sequencing; its modeling was also done. The prevalence of ß-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 A2-Saurashtra, was found in two and one individual, respectively. A community-specific ß-thal mutation was found in Pathare Prabhus in significant proportions. This information is useful in developing an algorithm for a prenatal diagnosis (PND) program.

Newborn Screening for Sickle Cell Disease: Indian Experience.

Sickle cell disease (SCD) is a major public health problem in India with the highest prevalence amongst the tribal and some non-tribal ethnic groups. The clinical manifestations are extremely variable ranging from a severe to mild or asymptomatic condition. Early diagnosis and providing care is critical in SCD because of the possibility of lethal complications in early infancy in pre-symptomatic children. Since 2010, neonatal screening programs for SCD have been initiated in a few states of India. A total of 18,003 babies have been screened by automated HPLC using either cord blood samples or heel prick dried blood spots and 2944 and 300 babies were diagnosed as sickle cell carriers and SCD respectively. A follow up of the SCD babies showed considerable variation in the clinical presentation in different population groups, the disease being more severe among non-tribal babies. Around 30% of babies developed serious complications within the first 2 to 2.6 years of life. These pilot studies have demonstrated the feasibility of undertaking newborn screening programs for SCD even in rural areas. A longer follow up of these babies is required and it is important to establish a national newborn screening program for SCD in all of the states where the frequency of the sickle cell gene is very high followed by the development of comprehensive care centers along with counselling and treatment facilities. This comprehensive data will ultimately help us to understand the natural history of SCD in India and also help the Government to formulate strategies for the management and prevention of sickle cell disease in India.

Effect of the Hemochromatosis Mutations on Iron Overload among the Indian ß Thalassemia Carriers.

BACKGROUND: Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron absorption.HFE gene mutations C282Y and H63D are responsible for the majority of hereditary hemochromatosis cases. METHODS: We tried to look at the effect of HFE mutations on the iron status. A total of 100 ß thalassemia traits (BTT) with 100 normal individuals were screened for the C282Y and H63D mutations using PCR-RFLP. The serum ferritin levels were determined using ELISA kit. RESULTS: We did not find the C282Y mutation in our study group. The allelic frequencies for H63D mutation did not differ significantly between ß-thalassemia traits (8.5%) and normal controls (9%). ΒΤΤ with H63D genotype of H/D (143.16 ± 80.3 ng/ml) and D/D (504 ng/ml) showed higher ferritin levels as against H/H genotype (88.64 ± 92.43 ng/ml). The statistically significant difference was observed in the mean serum ferritin levels among the individuals showing H/H and D/D genotypes (P < 0.002) and H/D and D/D genotype (P < 0.01) in both the groups. CONCLUSION: This suggests that iron load in BTT tends to aggravated with the co-inheritance of the H63D mutation. The mutant H63D gene showed the presence of haplotype 6 which is reported in the European population suggesting a common origin.

Genetic Variations in Bilirubin Metabolism Genes and Their Association with Unconjugated Hyperbilirubinemia in Adults.

OBJECTIVE: The present study was undertaken to investigate the genotype and allele frequencies of the variants in the four bilirubin metabolism genes (UGT1A1, OATP2, HMOX1, and BLVRA) and their association with hyperbilirubinemia. MATERIAL AND METHODS: Genotyping of 17 genetic variants was performed in 115 adults with hyperbilirubinemia and 150 controls by PCR-RFLP, GeneScan analysis, and direct DNA sequencing. RESULTS: Genetic polymorphisms of the UGT1A1 promoter, specifically the T-3279G phenobarbital-responsive enhancer module and (TA)7 dinucleotide repeat, as well as the intron and coding region variants of the OATP2, HMOX1, and BLVRA genes, were significantly higher among the cases than the controls. Further, nearly 82% of the cases showed the presence of significantly four or more variants as compared to 37% of the controls (P < 0.0001) and the mean total serum bilirubin levels also increased according to the number of variants co-expressed. CONCLUSIONS: This study demonstrates that polymorphisms in the bilirubin metabolism genes had a significant effect on bilirubin levels and could be genetic risk factors for hyperbilirubinemia.

Hb E-ß-Thalassemia in Five Indian States.

Hb E [ß26(B8)Glu→Lys; HBB: c.79G > A]-ß-thalassemia (ß-thal) has an extremely variable clinical presentation. We report the clinical features of these patients from five Indian states together with their hematological and molecular characteristics. Seventy-eight Hb E-ß-thal patients from different regions [West Bengal (30), Maharashtra (21), Uttar Pradesh (13), Bihar (11), Orissa (3)] were clinically evaluated along with hematological profiles and molecular characteristics (ß-thal mutations, XmnI polymorphisms, α genotypes). Twenty-nine of the 78 patients had a mild clinical presentation (clinical score 2.2 ± 1.1), while 15 had moderate severity (clinical score 6.1 ± 1.2) with occasional transfusion needs, and 34 patients were severely affected (clinical score 8.2 ± 0.5) requiring regular blood transfusions. The age at clinical presentation in the severely affected patients was lower (6 months-10 years) as compared to those with milder symptoms (2 years-34 years). Thirty-four patients showed splenomegaly (spleen ≥3 cm below the costal margin) and five patients were splenectomized. The severe ß+ IVS1-5 (G > C) (HBB: c.92 + 5G > C) was the most common ß-thal mutation, while seven other mutations were also seen. The XmnI [+/+] and [-/-] polymorphisms were seen in 24.1 and 10.3% of mildly affected patients and 14.7 and 17.6% of severely affected patients respectively. A single α gene deletion (-α3.7/αα) was found in 20.7% of mildly affected and 5.9% of severely affected patients, respectively. No specific differences in the clinical, hematological or molecular characteristics were observed in the Hb E-ß-thal patients from various geographic regions or different ethnic groups.