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.

Significance of borderline HbA2 levels in ß thalassemia carrier screening.

Increased HbA2 levels are the characteristic feature of ß-thalassemia carriers. A subset of carriers however do not show HbA2 levels in the typical carrier range (≥ 4.0%) but show borderline HbA2 levels. As a result, these carriers escape diagnosis and carry the risk of having ß-thalassemia major offspring. Borderline HbA2 values may occur as a consequence of mild ß-thalassemia mutations, co-inherited ß-thalassemia and α- or δ- thalassemia or iron deficiency anemia. However, there is insufficient knowledge regarding the cause of borderline HbA2 levels in specific populations. This study aimed to identify the determinants of borderline HbA2 levels (which we have considered as HbA2 3.0-3.9%) in 205 individuals. Primary screening involved detecting the presence of iron deficiency anemia followed by molecular analysis of α, ß and δ globin genes. Remarkably, 168 of 205 individuals were positive for a defect. 87% (149/168) of positive individuals were heterozygous for ß thalassemia with (59/149) or without (90/149) the presence of co-existing IDA, α or δ gene defects. Notably, 20 of 149 ß thalassemia carriers showed HbA2 < 3.5% and MCV > 80fL. 7 of these 20 carriers were married to carriers of hemoglobinopathies. Our findings describe the genetic basis of borderline HbA2 levels and emphasize the necessity of a molecular diagnosis in these individuals in the routine clinical setting.

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.

Genotypic-phenotypic heterogeneity of 뫧-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) in India.

Large deletions in the ß-globin gene cluster lead to increased HbF levels by delaying the γ- to ß-globin switch process. However, these deletions when inherited as a homozygous condition or when co-inherited with ß-thalassemia result in variable clinical phenotypes. Individuals or families with a clinically presenting child, where the parents had HbF levels ≥ 10%, were further screened for the presence of large ß-globin cluster deletions. Six deletions in the ß-globin gene cluster were screened by GAP-PCR, and the uncharacterized deletions were further analyzed by gene dosage or by multiplex ligation-dependent probe amplification (MLPA). Among 192 individuals suspected for the inheritance of large deletions, 138 were heterozygous for large deletions, 45 were compound heterozygous of a large ß-globin cluster deletion and ß-thalassemia, and 9 were found to be homozygous for deletions. Among the heterozygotes, the Asian Indian inversion-deletion was found to be the most common deletion (39.9%), followed by the HPFH-3 deletion (30.0%). Other deletions 49.3 kb, δß-thalassemia (21.2%), and 32.6 kb deletion (4.4%) were also found to be prevalent in our population. Patients compound heterozygous or homozygous for HPFH-3 and 32.6 kb deletions showed a milder clinical presentation, as compared with the patients compound heterozygous or homozygous for the Asian Indian inversion-deletion and 49.3 kb δß-thalassemia. This comprehensive study highlights the mutation spectrum of large ß-globin cluster deletions and the clinical heterogeneity in the patients homozygous or compound heterozygous with ß-thalassemia, thus asserting the need for molecular characterization of these deletions.

Prevalence of globin gene modifiers encountered in fetuses during antenatal diagnosis of hemoglobinopathies.

INTRODUCTION: The hemoglobinopathies are the commonest group of single gene disorders in the Indian subcontinent. Although genetic modifiers are known to have a remarkable effect on phenotypic expression, the effects of the possible co-inheritance of different modifiers are not taken into account during prenatal diagnosis. The present study was undertaken to look for the frequency of globin gene modifiers like the types of ß-globin gene mutations, α thalassemia, α gene triplication, and the Xmn1 polymorphism in fetuses during antenatal diagnosis of hemoglobinopathies. MATERIALS AND METHODS: A total of 580 fetuses with different diagnoses were screened for the presence of genetic modifiers. RESULTS: Twenty-two different ß-globin gene mutations were identified of which 3.5% were milder mutations. Among the affected fetuses, 29.6% of the ß-thalassemia major and 52.9% of the sickle cell anemia (SCA) fetuses had one genetic modifier while 3.7% of the ß-thalassemia major and 41.1% of the SCA fetuses had co-inherited two modifiers. α-gene triplication was detected in 16 (3.5%) ß-thalassemia/sickle cell heterozygous and normal fetuses of which 5 babies (2 ß-thalassemia heterozygous and 3 normal) could be followed up. Of the 2 ß-thalassemia heterozygous babies, one had a severe clinical presentation. CONCLUSION: Many fetuses had one or two gene modifiers. However, the impact of these on ameliorating the severity of the disease could not be evaluated as all the fetuses with ß thalassemia major or sickle cell disease were terminated. Parents having heterozygous fetuses with α gene triplication should be followed up periodically after birth for better management of these babies.

Homozygous sickle cell disease in Central India & Jamaica: A comparison of newborn cohorts.

Background & objectives: Homozygous sickle cell (SS) disease in Central India runs a more severe clinical course than reports from other areas of India. The current study was undertaken to compare the disease in Central India (Nagpur) with that in Jamaica, both populations defined by newborn screening. Methods: The Nagpur cohort included infants born to sickling-positive mothers from May 2008 to 2012, examined by high-pressure liquid chromatography and DNA analysis. The Jamaican cohort screened 100,000 consecutive non-operative deliveries between June 1973 and December 1981, analyzed by haemoglobin (Hb) electrophoresis and confirmed by family studies and compatible HbA2levels. Results: In Nagpur, 103 SS patients were detected, but only 78 (76%) were followed up. In Jamaica, 311 cases were followed from birth and compliance with follow up remained 100 per cent up to 45 years. In the Nagpur cohort all had the Asian haplotype, and 82 per cent of Jamaicans had at least one Benin chromosome; none had the Asian haplotype. Compared to Jamaica, Nagpur patients had higher foetal Hb, less alpha-thalassaemia, later development of splenomegaly and less dactylitis. There were also high admission rates for febrile illness and marked anaemia. Invasive pneumococcal disease occurred in 10 per cent of Jamaicans but was not seen in Nagpur. Interpretation & conclusions: There were many differences between the disease in Nagpur, Central India and the African form observed in Jamaica. The causes of severe anaemia in Nagpur require further study, and reticulocyte counts may be recommended as a routine parameter in the management of SS disease. The role of pneumococcal prophylaxis needs to be determined in Nagpur patients. Future studies in India must avoid high default rates.

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.

Sickle Cell Disease and Pregnancy.

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy and is associated with increased risk of complications and early mortality. Nowadays, with improved health care facilities, antibiotic prophylaxis, vaccination, and availability of drugs like hydroxyurea, the life expectancy of SCD patients has improved. More women are reaching reproductive age group and are expressing their desire to reproduce. Though SCD adversely affects pregnancy, leading to increased incidence of maternal and perinatal complications like pre-eclampsia, preterm labor, IUGR, abortions etc., adequate care throughout pregnancy ensures a better outcome. Also, recent advancements in the fields of prenatal diagnosis and preimplantation genetic diagnosis, help couples suffering from SCD to have a healthy baby. This paper focuses on the effects of SCD on pregnancy outcomes and effective management of complications during pregnancy, also comparing maternal and perinatal outcomes in studies conducted in different countries. The second part of the paper summarizes pregnancy management in SCD for better maternal and fetal outcomes.

Red Cell Indices and Hemoglobin Profile of Newborn Babies with Both the Sickle Gene and Alpha Thalassaemia in Central India.

This study evaluated the effect of alpha thalassemia on the red cell indices and hemoglobin profiles of normal, sickle heterozygous and sickle homozygous newborn babies in central India where the sickle gene is linked to the Arab-Indian haplotype. 265 newborn babies were analysed with complete blood count and hemoglobin analysis on high performance liquid chromatography (Variant Hb Testing System, BioRad Laboratories, Hercules, CA, USA) using the ß-thal short program. The sickle genotypes was confirmed by DNA analysis. The two common alpha gene deletions (- α3.7 and - α4.2) were detected by multiplex PCR. Among the 102 normal, 106 sickle heterozygous and 57 sickle homozygous newborns, the prevalence of a single alpha gene deletion (- α/αα) was 28.3% and that of deletion of 2 alpha genes (- α/- α) was 21.5%. In all, 57 normal (55.9%), 35 (33.0%) sickle heterozygous and 41 (71.9%) sickle homozygous newborns had a normal α genotype while - α/- α was seen in 23 (22.5%) normal, 30 (28.3%) sickle heterozygous and 4 (7.0%) sickle homozygous newborns respectively. The presence of associated alpha thalassemia resulted in a reduction in the hemoglobin levels and red cell indices in normal, sickle heterozygous and sickle homozygous newborn babies, MCV and MCH being strong discriminators of alpha thalassemia with two alpha gene deletions in all the three groups. This study also helped us to know the variations in hematological parameters in normal, sickle heterozygous and sickle homozygous newborns with and without associated α thalassemia.

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.

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.

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.