Prevalence of hemoglobinopathies among Malayali tribes of Jawadhu hills, Tiruvannamalai district, Tamil Nadu, India: a community-based cross-sectional study.

BACKGROUND: Hemoglobin (Hb), a red pigment of red blood cells (RBCs), carries oxygen from the lungs to different organs of the body and transports carbon dioxide back to the lungs. Any fault present in the Hb structure leads to undesirable functional effects of the RBCs, such as sickle cell anemia (SCA), thalassemia, etc. Hemoglobinopathies affect around 7% of people in both developed and developing countries globally. The aim of the present study was to determine the prevalence and carrier frequencies of hemoglobinopathies including SCA, thalassemia, and other abnormal Hb variants among Malayali tribes in the Jawadhu hills of Tiruvannamalai district, Tamil Nadu, India. METHODS: A community-based cross-sectional study was carried out among 443 Malayali tribes inhabiting the Jawadhu hills of Tiruvannamalai district from July 2022 to September 2022. The RBC indices were analyzed using an automated 5-part hematology analyzer (Mindray, BC-5150) and hemoglobin fractions were done using the HPLC system (Bio-Rad, D-10) following standard protocols. FINDINGS: A total of 443 participants were screened, out of whom 14.67% had an abnormal Hb fraction, 83.30% were identified as normal, and 2.03% were borderline. Notably, the study revealed a prevalence of 0.68% for the α-thalassemia trait and 13.99% for the ß-thalassemia trait. INTERPRETATION: Haemoglobinopathies, specifically the ß-thalassemia trait, were most prevalent among the Malayali tribal population of Tamil Nadu residing in the Jawadhu hills of Tiruvannamalai district. Hence, we need special attention for creating awareness, increasing hemoglobinopathies screening programs, and improving the importance of tribal health conditions by the government and non-governmental organizations (NGOs) for the betterment of the ethnic tribes.

Challenges in screening for sickle cell disease among newborns from the tribal region of Palghar, Maharashtra during the COVID-19 pandemic.

BACKGROUND OBJECTIVES: Despite several adversities imposed by the COVID-19 pandemic, it was crucial to sustain research having public health relevance such as investigations around sickle cell disease (SCD). Against this background, an ongoing ICMR-multicentric study for newborn screening of SCD in the tribal population at Model Rural Health Research Unit (MRHRU-Dahanu) in Palghar District, Maharashtra constituted the current study setting. This was a descriptive study wherein, certain measures were undertaken and strategies were developed in view of the challenges in newborn screening for SCD due to the COVID-19 pandemic during December 2019-September 2021 at Sub District Hospital, MRHRU-Dahanu. METHODS: During the onset of the pandemic, (December 2019-March 2020), the follow up was possible in 26.7 per cent (20/75) of the newborns. Subsequently, challenges such as travel restrictions, fear of COVID-19, shortage of staff were experienced with respect to enrolment and follow up visits. RESULTS: After implementing certain pragmatic strategies (ASHA involvement, usage of virtual platform and flexible visits), follow up rate increased to 47.5 per cent (66/139) between July 2020-April 2021 (post first lockdown) and to 66 per cent (65/98) during the second wave (May 2021-August 2021). INTERPRETATION CONCLUSIONS: The study emphasizes the importance of network building, use of virtual platform and engaging health workers in tribal settings. Such pragmatic approaches have the potential to pave a path for further implementation research involving specific interventions to improve health outcomes in tribal settings.

Effect of hydroxyurea on erythrocyte apoptosis in hemoglobinopathy patients.

BACKGROUND: Hydroxyurea (HU) therapy improves the clinical severity of patients with hemoglobinopathies. Few studies have documented some mechanisms of HU, but the exact mechanism of action is unknown. Phosphatidylserine on erythrocytes is responsible for apoptosis. In this study, we investigate the expression of phosphatidylserine on the erythrocytes surface of hemoglobinopathies before and after HU treatment. RESEARCH DESIGNS AND METHODS: Blood samples from 45 thalassemia intermedia and 40 SCA and 30 HbE-b-thalassemia patients were analyzed before and after 3 and 6 months of HU treatment. The profile of phosphatidylserine was determined by flow-cytometry using the Annexin V-RBC apoptosis kit. RESULTS: Hydroxyurea proved effective in improving clinical severity of hemoglobinopathies. After treatment with hydroxyurea, the percentage of phosphatidylserine-positive cells was significantly reduced in all 3 patient groups (p < 0.0001). Correlation analysis using different hematological parameters as independent variables and % phosphatidylserine  as dependent variable showed a negative relationship with HbF, RBC, and hemoglobin in all 3 patient groups. CONCLUSION: Hydroxyurea reduces the expression of phosphatidylserine on erythrocytes, contributing to the beneficial effects of this therapy. We suggest that the use of such a biological marker in conjunction with HbF levels may provide valuable insights into the biology and consequences of early RBC apoptosis.

The study investigated the role of hydroxyurea in reducing the externalization of phosphatidylserine on the surface of the erythrocyte membrane of patients with hemoglobinopathies. In patients treated with hydroxyurea for 3 and 6 months, the percentage of phosphatidylserine exposure on the erythrocyte surface was reduced compared with baseline. The decreased percentage of phosphatidylserine correlated negatively with hematologic parameters such as red blood cell (RBC), hemoglobin, and fetal hemoglobin (HbF) in patients at baseline and after HU therapy. Treatment with hydroxyurea decreases the percentage of PS exposure on the surface of RBCs, contributing to the beneficial effects of this therapy. We, therefore, suggest that the use of such a biological marker on the erythrocyte cell surface in conjunction with HbF levels may provide valuable insights into the biology and consequences of early erythrocyte apoptosis.

Impact of COVID-19 infection among indian sickle cell disease patients.

Background: Sickle cell disease (SCD) is one of the most common hemoglobinopathy disorders and is widely prevalent in India, especially in the tribal population. SCD patients are prone to develop recurrent respiratory infections and related complications owing to the microvascular occlusion and impaired immunological response. Objectives: We aimed to determine the prevalence and impact of COVID-19 in SCD patients from India. Methodology: We conducted a cross-sectional study in Chandrapur district of Maharashtra, India, between August and October 2021. After taking informed consent, details of 300 SCD patients' demographic data, history of COVID-19 testing, infection, symptoms related to COVID-19 in the past 1 year, hospitalization, complications, mortality, COVID-19 vaccination, and side effects were recorded. Results: We found that 93 (31%) of SCD patients had influenza-like symptoms during the COVID-19 pandemic with symptoms of fever (81.72%), cough (35.48%), sore throat (18.27%), headache (15.05%), and breathlessness (7.52%). A total of 13 (4.33%) SCD among 300 SCD were tested as COVID positive. Majority of them were mild cases and the 1st dose of COVID-19 vaccine was received by 47 (29.37%) of SCD patients and 10 (6.02%) of the patient had received second dose of vaccine. Conclusion: Low incidence of COVID-19 and milder disease spectrum in our study cohort suggests that there is no increased risk of COVID-19 mortality and morbidity in SCD patients compared to general population. However, the reason for low COVID vaccination in our study could be due to the fear of complications of COVID vaccine.

Role of microRNA in hydroxyurea mediated HbF induction in sickle cell anaemia patients.

Hydroxyurea (HU) is found to be beneficial in sickle cell anaemia (SCA) patients, due to its ability to increase foetal haemoglobin (HbF), however, patients show a variable response. Differences in HbF levels are attributed to many factors; but, the role of miRNA in HbF regulation is sparsely investigated. In this study, we evaluated the effect of miRNA expression on HbF induction in relation to hydroxyurea therapy in 30 normal controls, 30 SCA patients at baseline, 20 patients after 3 and 6 months of hydroxyurea (HU) therapy. HbF levels were measured by HPLC. Total RNA and miRNA were extracted from CD71+ erythroid cells and the expression was determined using Taqman probes. The mean HbF level increased 7.54 ± 2.44 fold, after 3 months of HU therapy. After the HU therapy 8 miRNAs were significantly up-regulated while 2 were down-regulated. The increase in miR-210, miR16-1, and miR-29a expression and decrease in miR-96 expression were strongly associated with the HU mediated HbF induction. Post HU therapy, decreased miR-96 expression negatively correlate with HbF and γ-globin gene while increased expression of miR-210, miR-16-1 and miR-29a post HU therapy positively corelate with HbF and γ-globin gene. Thus, suggest that miR-210, miR-16-1 and miR-29a are positive regulator of γ-globin gene and miR-96 is negative regulator of γ-globin gene. The study suggests the role of miR-210, miR16-1, miR-29a, and miR-96 in γ-globin gene regulation leading to HbF induction. Identification of the relevant protein targets might be useful for understanding the HU mediated HbF induction.

Dynamic interplay of microRNA in diseases and therapeutic.

MicroRNAs are the major class of small non-coding RNAs, evolutionary conserved post-transcriptional regulators of gene expression. Since their discovery in 1993, they have been implicated as master regulators in numerous cellular processes. MicroRNA (miRNA)s regulate gene expression by attenuation and/or mRNA degradation and are commonly associated with cell development, differentiation, and homeostasis. Extensive research in past two decades has provided new insights into the potential implications of miRNA in the onset, progression, and therapeutic nature of miRNAs in disease manifestation. Owing to the novel discoveries, "miRNAs" would probably pave a new direction in therapeutic research. However, "micro" in length miRNAs have attracted considerable attention in numerous other fields. Understanding the functionality of miRNAs, in this review article, we discussed the mechanistic role of miRNAs in human diseases and have outlined most of the recent published work in clinical therapeutics. We have constructed different network models for miRNA and its targets which made us understand their interrelationship and association with diseases. Future research would surely overcome challenges and would introduce new strategies for the utility of miRNAs in a broader setting.

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.

Borderline HbA2 levels: Dilemma in diagnosis of beta-thalassemia carriers.

There is inconsistency in the exact definition of diagnostic levels of HbA2 for ß thalassemia trait. While many laboratories consider HbA2 ≥4.0 % diagnostic, still others consider HbA2 ≥3.3 % or HbA2 ≥3.5 % as the cut-off for establishing ß thalassemia carrier diagnosis. This is because, over the years, studies have described ß thalassemia carriers showing HbA2 levels that lie above the normal range of HbA2 but below the typical carrier range of ß thalassemia. These, "borderline HbA2 levels", though not detrimental to health, are significant in ß thalassemia carrier diagnosis because they can lead to misinterpretation of results. In this review, we have evaluated the prevalence of borderline HbA2 levels and discussed the causes of borderline HbA2 values. We have also compiled an extensive catalogue of ß globin gene defects associated with borderline HbA2 levels and have discussed strategies to avoid misdiagnosing borderline HbA2 ß thalassemia carriers. Our analysis of studies that have delineated the cause of borderline HbA2 levels in different populations shows that 35.4 % [626/1766] of all individuals with borderline HbA2 levels carry a molecular defect. Among the positive samples, 17 % [299/1766] show ß globin gene defects, 7.7 % [137/1766] show α thalassemia defects, 2.7 % [49/1766] show KLF1 gene mutations, 2.3 % [41/1766] show the co-inheritance of ß and α thalassemia, 2.0 % [37/1766] show the co-inheritance of ß and δ thalassemia and 1.8 % [32/1766] show α globin gene triplication. It appears that a comprehensive molecular work up of the ß globin gene is the only definite method to detect borderline HbA2 ß thalassemia carriers, especially in populations with a high prevalence of the disease. The presence of associated genetic or acquired determinants may subsequently be assessed to identify the cause of borderline HbA2.

Significance of genetic modifiers of hemoglobinopathies leading towards precision medicine.

Hemoglobinopathies though a monogenic disorder, show phenotypic variability. Hence, understanding the genetics underlying the heritable sub-phenotypes of hemoglobinopathies, specific to each population, would be prognostically useful and could inform personalized therapeutics. This study aimed to evaluate the role of genetic modifiers leading to higher HbF production with cumulative impact of the modifiers on disease severity. 200 patients (100 ß-thalassemia homozygotes, 100 Sickle Cell Anemia), and 50 healthy controls were recruited. Primary screening followed with molecular analysis for confirming the ß-hemoglobinopathy was performed. Co-existing α-thalassemia and the polymorphisms located in 3 genetic loci linked to HbF regulation were screened. The most remarkable result was the association of SNPs with clinically relevant phenotypic groups. The γ-globin gene promoter polymorphisms [- 158 C → T, + 25 G → A],BCL11A rs1427407 G → T, - 3 bp HBS1L-MYB rs66650371 and rs9399137 T → C polymorphisms were correlated with higher HbF, in group that has lower disease severity score (P < 0.00001), milder clinical presentation, and a significant delay in the age of the first transfusion. Our study emphasizes the complex genetic interactions underlying the disease phenotype that may be a prognostic marker for predicting the clinical severity and assist in disease management.

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.

Insight of fetal to adult hemoglobin switch: Genetic modulators and therapeutic targets.

The clinical heterogeneity of ß-hemoglobinopathies is so variable that it prompted the researchers to identify the genetic modulators of these diseases. Though the primary modulator is the type of ß-globin mutation which affects the degree of ß-globin chain synthesis, the co-inheritance of α-thalassemia and the fetal hemoglobin (HbF) levels also act as potent secondary genetic modifiers. As elevated HbF levels ameliorate the severity of hemoglobinopathies, in this review, the genetic modulators lying within and outside the ß-globin gene cluster with their plausible role in governing the HbF levels have been summarised, which in future may act as potential therapeutic targets.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients.

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR-RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

Role of MMP-2 and its inhibitor TIMP-2 as biomarkers for susceptibility to systemic lupus erythematosus.

Aim: To investigate the possible association between MMP-2 (-1575 G/A, -1306 C/T) and its inhibitor TIMP-2 (-418 G/C) functional polymorphisms with development of severity in systemic lupus erythematosus (SLE) patients. Materials & methods: 150 SLE patients and matched healthy controls were recruited. Polymorphisms were detected by PCR-RFLP and serum levels by ELISA. Results: Mean MMP-2 and TIMP-2 serum level and mRNA expression were significantly increased in SLE cases as compared with controls (p < 0.0001). The concomitant presence of both MMP-2 1575A and its inhibitor TIMP-2 418C alleles synergistically increased the risk of SLE by 3.25-fold (CI: 1.44-7.34, p = 0.003). Conclusion: MMP-2, TIMP-2 and MMP-2/TIMP-2 ratios may act as biomarkers for susceptibility to SLE.

Cytokine genes multi-locus analysis reveals synergistic influence on genetic susceptibility in Indian SLE - A multifactor-dimensionality reduction approach.

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with unclear etiology. Several loci associated with genetic susceptibility for lupus have been described. However, it lacks reports on cytokine gene-gene interactions among SLE patients from Asian population. Epistasis interaction among single nucleotide polymorphisms (SNPs) of cytokine genes in Indian SLE patients was tested using multifactor-dimensionality reduction (MDR) analysis. A total of fourteen SNPs lacking linkage disequilibrium among different cytokines genes were genotyped in a cohort of 200 SLE patients and 201 healthy individuals as controls of Indian origin. A high degree of synergism among Lymphotoxin-α (LT-α), Interleukin-1ß (IL-1ß) and Interleukin-10 (IL-10) gene polymorphisms was detected in our SLE patients. Furthermore, by virtue of biological inter-relations among different cytokines, a high strength of interactions was observed among pro-inflammatory (IL-1ß, IL-6) and anti-inflammatory (IL-10) cytokine gene SNPs. Also, among studied pro-inflammatory cytokines and chemokines, a strong synergistic effect among Tumor Necrosis Factor-α (TNF-α), LT-α and Monocyte Chemo-attractant Protein-1 (MCP-1) SNPs was occurred. A nature of strong interaction among the candidate cytokine genes may speculate a proactive role in causing genetic susceptibility to the disease in SLE patients with Indian origin.

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.