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Presence of Haemoglobin E in Durg, Chhatisgarh

  • February 2014
  • Conference: Pune Public Health Conference 2014-IPHA-IAPSM Joint State CME and Conference
  • At: Yashada, Pune 411007 INDIA
Authors:
Devendra Lingojwar at ADEETECHGENE BIOTECH PVT LTD
Devendra Lingojwar
  • ADEETECHGENE BIOTECH PVT LTD
Pramod Gupta
Pramod Gupta
Pramod Gupta
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Ravikant Jadhav at Atg Lab
Ravikant Jadhav
  • Atg Lab
Saurabh Gawande at KTH Royal Institute of Technology
Saurabh Gawande
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Abstract

The origin of haemoglobinopathies and prevalence of endemic malaria are inter-linked. Natural protection from malaria pathogenesis after infection might be the most likely and highly accepted hypothesis of origin of different alleles of normal adult haemoglobin in different population groups throughout the world, more specifically in the tropical countries where malaria is endemic. Mutation in some of the of genes encoding haemoglobin, red cell enzymes and membrane proteins are being extensively studied with reference to protection from P.falciparum malaria. Haemoglobin (Hb) E is one of the world's most common and important mutation. It results in a heterogeneous group of disorders whose phenotype range from asymptomatic to severe. Hb E trait and Hb EE are mild disorders. 2 The Hb E β β β β 26 (Glu Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. 3 Pathogenesis study suggested that patients who co-inherit a mild β-thalassaemia allele with Hb E may have disease on the mild end of the spectrum, while those who co-inherited severe β + or β 0-thalassaemia alleles might be more severely affected. 4 INTRODUCTION METHODS OBJECTIVE To assess presence of Hemoglobin E during sickle cell anaemia screening in Durg, Chhattisgarh RESULTS Slow migrating haemoglobin pattern of sample no. 17 was observed near point of application. After repeated application on the cellulose acetate membrane along with successive samples during sickle cell anaemia prevalence studies, it is confirmed that the haemoglobin pattern near point of application is of haemolglobin E. Separation pattern of sample no. 17 is different from that of HbA and HbS. After comparison with these abnormal hemoglobins, known mobility of Hb E from review of literature and repeated application during cellulose acetate membrane electrophoresis, it is confirmed that this is Hb E. In brief, in the present study, a case of homozygous haemoglobinE from Kurmi caste of OBC community is reported from Durg, Chhattisgarh, Central India. Future studies will include molecular analysis of the coding gene as well as parents studies.
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RESEARCH POSTER PRESENTATION DESIGN ©2012
www.PosterPresentations.com
The origin of haemoglobinopathies and prevalence of
endemic malaria are inter-linked. Natural protection from malaria
pathogenesis after infection might be the most likely and highly accepted
hypothesis of origin of different alleles of normal adult haemoglobin in
different population groups throughout the world, more specifically in the
tropical countries where malaria is endemic. Mutation in some of the of
genes encoding haemoglobin, red cell enzymes and membrane proteins are
being extensively studied with reference to protection from P.falciparum
malaria. Haemoglobin (Hb) E is one of the world’s most common and
important mutation. It results in a heterogeneous group of disorders whose
phenotype range from asymptomatic to severe. Hb E trait and Hb EE are mild
disorders. 2
The Hb E β
ββ
β26 (Glu Lys) is concentrated in parts of Southeast
Asia w here malaria is endemic, and HbE carrier status has been shown to
confer some protection against Plasmodium falciparum malaria.3
Pathogenesis study suggested that patients who co-inherit a mild β-
thalassaemia allele with Hb E may have disease on the mild end of the
spectrum, while those who co-inherited severe β+or β0-thalassaemia alleles
might be more severely affected. 4
INTRODUCTION METHODS
OBJECTIVE
To assess presence of Hemoglobin E during sickle cell anaemia screening in
Durg, Chhattisgarh
RESULTS
Slow migrating haemoglobin pattern of sample no. 17 was observed near
point of application. After repeated application on the cellulose acetate
membrane along with successive samples during sickle cell anaemia
prevalence studies, it is confirmed that the haemoglobin pattern near point
of application is of haemolglobin E.
Separation pattern of sample no. 17 is different from that of HbA and HbS.
After comparison with these abnormal hemoglobins, known mobility of Hb E
from review of literature and repeated application during cellulose acetate
membrane electrophoresis, it is confirmed that this is Hb E. In brief, in the
present study, a case of homozygous haemoglobinE from Kurmi caste of
OBC community is reported from Durg, Chhattisgarh, Central India. Future
studies will include molecular analysis of the coding gene as well as parents
studies.
1. http://en.wikipedia.org/wiki/File:Red_Blood_Cell_abnormalities.png
2. Vichinsky E. “haemoglobinE Syndromes” Hematology 2007 1: 79-83
3. Ohashi J et. al. “Extended linkage disequilibrium surrounding the haemoglobinE variant due to
malarial selection. ” Am J Hum Genet. 2004;74(6):1198
4. Winichagoon P et. al. “Severity differences in beta-thalassaemia/haemoglobin E syndromes:
implication of genetic factors”. Br J Haematol 1993; 83 : 633-9.
5. http://www.mapsofindia.com/maps/chhattisgarh/tehsil/durg-tehsil-map.jpg
6. MB Agarwal "The Burden of Haemoglobinopathies in India - Time to Wake Up?"JAPI2005; 53; 1017-
1018
7. Panigrahi S "Neonatal screening of sickle cell anaemia: a preliminary report. "Indian J Pediatr. 2012
Jun;79(6):747-50
8. Balgir RS. "Community expansion and gene geography of sickle cell trait and G6PD deficiency, and
natural selection against malaria: experience from tribal land of India" CardiovascHematol Agents
Med Chem. 2012 Mar 1;10(1):3-13
9. Bhagat S. et. al. "Fetal Haemoglobin and β-globin Gene Cluster Haplotypes among Sickle Cell
Patients in Chhattisgarh" J Clin Diagn Res. 2013 Feb;7(2):269-72
Separation of haemoglobin composition was carried out by cellulose acetate
membrane electrophoresis method. Briefly, peripheral Blood samples (N=44)
after cell washing and solubility testing for screening haemoglobin S (Hb S)
were processed for hemolysate preparation by osmotic shock method. Pure
haemoglobin from hemolysate were subjected to cellulose acetate
membrane electrophoresis at pH 8.6 in Tris Glycine Buffer. After 45 minutes,
resultant haemoglobin pattern was stained by Ponseu S red dye followed by
destaining by 5% acetic acid. Appropriate known controls i.e. sickle cell
carrier samples were applied every time new samples loaded on cellulose
acetate membrane for electrophoresis.
1. ATG LAB, Biotechnology Research Laboratory, Ganesh Nagar, Pimple Nilakh, Pune 411027,
2. RESEARCH (Regional Society for Education and Research in Community Health), Pune 411027,
3. Dept. of Biotechnology, Govt. V.Y.T. PG. Autonomous College, G.E. Road, Durg Chhattisgarh 491001
Devendra Lingojwar 1,2, Pramod Gupta 2, Ravikant Jadhav 1, Saurabh Gawande 1,
Vaishnavi Digaskar1, Nikhil Mishra 3, Anil kumar 3and Sarita Lingojwar 1,2
Presence of haemoglobin E in Durg, Chhattisgarh
ACKNOWLEDGEMENT
www.biotechtrainingproject.com
www.sicklecellmaharashtra.com
CONCLUSION
REFERENCES
Fig. 2 Map of India, Chhattisgarh and Durg (Adapted from Maps of India) 5
First reported in Assam with 23% carrier prevalence, Hb E is widely distributed
in north-eastern states of India with high prevalence amongst 46.4 % in Ahoms
of Assam i.e. one of the highest for any abnormal haemoglobin reported from
any population in the world. Interestingly, only 1% in Mizoram, 3-33% in West
Bengal, while it is almost non-existing in South India.6
Chhattisgarh is a tribal state and well known for the high prevalence of P.
falciparum malaria. Presence of abnormal haemoglobin i.e. sickle cell
haemoglobin (Hb S) which is other that normal adult haemoglobin (Hb A), is
earlier reported and its prevalence pattern is being studied by different
groups. 7,8,9
Its presence in Central India is not completely understood. Prevalence of this
abnormal haemoglobin in Central India specifically in Durg, Chhattisgarh is
incompletely understood. Attempts were made to analyse urban samples
during sickle cell camp at Durg for finding haemoglobin E (Hb E) in the same
population group.
Fig. 1 Map of World showing all major abnormal haemoglobins (Adapted from Wikipedia)1
Fig. 3 Electrophoresis pattern of all the tested samples: Top and / or bottom samples (Sickle Cell
Anaemia Carrier Control samples (i.e. Hb A+S pattern); Sickle Cell Anaemia carriers samples (Hb A+S) : 1, 8,
11, 21 and 25. Sample No. 17: Hb E; All remaining samples belongs to normal individuals (HbA+A pattern)
Authors are thankful to faculti es, staff and students of academic institutions / post graduate
colleges who actively participated and contributed during study
+’ ve -’ ve
Poster presentation at Pune Public Health Conference 2014, Organized by Department of Health Sciences, University of Pune February 25-26, 2014 Pune and
Joint State Conference of IPHA-IAPSM (Maharashtra Chapter) 25th and 26th February,2014 NT 12
ResearchGate has not been able to resolve any citations for this publication.
Fetal Haemoglobin and β-globin Gene Cluster Haplotypes among Sickle Cell Patients in Chhattisgarh
Article
Full-text available
  • Feb 2013
Background: Foetal Haemoglobin (HbF) is the best-known genetic modulator of sickle cell anaemia, which varies dramatically in concentration in the blood of these patients. The patients with SCA display a remarkable variability in the disease severity. High HbF levels and the β-globin gene cluster haplotypes influence the clinical presentation of sickle cell disease. To identify the genetic modifiers which influence the disease severity, we conducted a β-globin haplotype analysis in the sickle cell disease patients of Chhattisgarh. Aim: The foetal haemoglobin and the β-globin gene haplotypes of the sickle cell trait and the sickle cell disease patients from Chhattisgarh were investigated. Materials and Method: A total of 100 sickle cell patients (SS), 50 sickle cell trait patients (AS) and 50 healthy control individuals were included in the present study. The distribution of the β-globin gene haplotype was done by the PCR-RFLP method. Result: PCR-RFLP showed that the homozygous Arab-Indian haplotype (65%) was the most frequent one, followed by the heterozygous Arab-Indian haplotype (11%) in the sickle cell patients (SS), while the AS patients had a higher frequency of the heterozygous Arab-Indian haplotype (38%) in comparison to homozygous one (32%). Four atypical haplotypes, 3 Benin and 1 Cameroon were also observed, although they were in lower frequencies. In the present study, the HbF levels were higher in the AS and the SS patients, with one or two Arab-Indian haplotypes as compared to the other haplotypes. Conclusion: The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/ from Saudi-Arabia or India and it was associated with higher HbF levels and a milder disease severity
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Hemoglobin E Syndromes
Article
Full-text available
  • Feb 2007
Hemoglobin (Hb) E is one of the world's most common and important mutations. It results in a heterogeneous group of disorders whose phenotype range from asymptomatic to severe. Hb E trait and Hb EE are mild disorders. The combination of Hb E and Hb S (Hb SE) results in a sickle cell disease syndrome similar to sickle beta(+) thalassemia. It is important to distinguish Hb E disorders diagnostically because of this marked difference in clinical course among different genotypes. Screening tests, including hemoglobin electrophoresis and high-pressure liquid chromatography (HPLC), may suggest other mutations, unless one is familiar with the findings. E beta-thalassemia, the most serious form of E syndromes, affects a million people worldwide and is increasing in North America. Its phenotype ranges from mild anemia to severe transfusion-dependent thalassemia major. Several genetic modifiers affect the phenotype, including the type of beta-thalassemia mutation, Hb F levels, and co-inheritance of alpha-thalassemia. However, the cause of the phenotypic variability is largely unknown. A prospective natural history study of E beta-thalassemia in Sri Lanka suggests that environmental modifiers are prognostically important. The clinical course of E beta-thalassemia is punctuated by acute and chronic complications that may cause serious morbidity and mortality. Recent studies indicate these patients are at high risk for thromboembolism secondary to a hypercoagulable state increased by splenectomy. Morbidity from iron overload in nontransfused patients secondary to increased gastrointestinal iron absorption is common. Cardiopulmonary disease, including pulmonary hypertension, requires ongoing monitoring and is secondary to iron overload, thromboembolism, and hemolysis-induced nitric oxide deficiency. These patients are excellent candidates for Hb F-modulating agents because moderate changes in hemoglobin may result in marked improvement in phenotype. Recent studies with hydroxyurea indicate 40% of patients will clinically improve with hydroxyurea.
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Neonatal Screening of Sickle Cell Anemia: A Preliminary Report
Article
To evaluate feasibility of systematic neonatal screening for sickle cell disease in Chhattisgarh. A pilot study was done from February 2008 through January 2009 in Department of Pediatrics & Neonatology, Pt. J.N.M. Medical College & Dr.B.R.A.M. Hospital, Raipur (Chhattisgarh) on a total of 1,158 neonates. Blood samples from the neonates were taken after 48 h of birth on filter paper for detection of sickle cell anemia using Biorad hemoglobin variant Neonatal sickle cell short programme by high performance liquid chromatography (HPLC). On follow up, cases were analyzed by HPLC using Beta thalassemia short program to rule out false negative case and other hemoglobin variants. Of the 1,158 neonates screened, 628 were boys (54.2%) and 530 were girls (45.8%). Sickle cell disease was found in 3 cases (0.2%) (95%C.I 0.12-0.28), sickle cell trait was found in 68 cases (5.8%) (95%C.I 4.5-7.5). After 6-9 mo of age three cases of sickle cell diseases were reinvestigated, out of which one case turned out to be double heterozygous for sickle cell and beta thalassemia trait. Fourteen preterm neonates reported as normal in initial screening were called for follow up after 6 mo of age, 10 infants reported in OPD and 4 lost in follow up. These 10 infants were reinvestigated; 2 had sickle cell disease, 1 had sickle cell trait and 7 infants were normal. Sixty eight cases of sickle cell trait found with initial screening were also called for follow up after 6 mo of age; 61 cases reported in OPD between 6 mo to 1 y of age and 7 cases lost in follow up. Sixty one infants were reinvestigated; 60 had sickle cell trait and 1 had sickle cell disease which was reported earlier as Sickle cell trait (FAS). Thus on total follow up of cases, there were 5(0.4%) sickle cell disease, 61(5.26%) sickle cell trait, 1(0.08%) double heterozygous for sickle cell and beta thalassemia trait which needs mutation studies for thalassemia characterization (s/β(0) or s/β(+)). Early detection of sickle cell disease (SS) done by neonatal screening will help in early prevention and management of complications in postnatal period.
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Community Expansion and Gene Geography of Sickle Cell Trait and G6PD Deficiency, and Natural Selection against Malaria: Experience from Tribal Land of India
Article
  • Jan 2012
Malaria is globally endemic in tropical and subtropical regions and so is the hemoglobinopathies, thalassemias and glucose-6-phosphate dehydrogenase (G6PD) deficiency. This biological dogma of hyper-endemic all over the tribal land in India leads to high morbidity and mortality. The directed genetic abnormalities of human erythrocytes have found to decrease the susceptibility towards malaria parasites and the heterozygotes of abnormalities probably confer protection against the Plasmodium falciparum infection. A fascinating trend for an inverse relationship between sickle cell disorders and G6PD deficiency in scheduled caste and tribal communities of Central-Eastern India has been observed. When the frequency of sickle cell allele decreases in malaria endemic cross-section of the tribal population, the frequency of G6PD deficiency allele increases and vice versa. This medical aspect is important from an evolutionary biological background and could be an excellent point for molecular analyses to determine the signature of selection in the genomic regions of β- globin and G6PD genes. Since the selection favors the mutation with least cost to the population [as the clinical manifestations of G6PD deficiency are mild and do not result in a complete loss of enzyme activity against the sickle cell disease with high morbidity and mortality in the region] and the predominant frequency of G6PD deficiency over the sickle cell disorders in some tribal communities, it seems that the replacement of sickle cell allele for G6PD deficiency is occurring in the scheduled castes/tribes of Chhattisgarh, Madhya Pradesh, Maharashtra and Odisha states in Central India. These findings are consistent with our previous studies carried out in Central-Eastern India.
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Severity differences in β-thalassaemia/haemoglobin E syndromes: Implication of genetic factors
Article
  • May 1993
Genetic factors determining the difference in severity of anaemia in beta-thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co-inheritance of alpha-thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a beta-thalassaemia chromosome with Xmn I cleavage site at position -158 of the G gamma-globin gene which was linked to the haplotype -+-++ or ++-++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the G gamma-globin gene and higher production of haemoglobin F, which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and G gamma/A gamma ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.
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Extended Linkage Disequilibrium Surrounding the Hemoglobin E Variant Due to Malarial Selection
Article
  • Jul 2004
The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.
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