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Variation of Abnormal Hemoglobins Concentrated in Durg, Chhattisgarh: A Brief Note Based on Cross-Sectional Study

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Lingojwar et al., J Genet Disor Genet Rep 2016, 5:2 Journal of Genetic
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Variation of Abnormal
Hemoglobins Concentrated in
Durg, Chhattisgarh: A Brief
Note Based on Cross-Sectional
Devendra Lingojwar1,2,4*, Pramod Gupta2, Savita Bhutoria4,
Sarita Lingojwar1,2, Nikhil Mishra3 and Anil Kumar3
*Corresponding author: Dr. Devendra Lingojwar, Hematology Division,
Department of Medicine, Albert Einstein College of Medicine, Bronx NY, USA 10461,
Tel: +1 7169512776; E-mail:
Received: April 25, 2016 Accepted: May 19, 2016 Published: May 19, 2016
more specically in the tropical countries where malaria is endemic.
Mutation in some of the of genes encoding hemoglobin, red cell
enzymes and membrane proteins are being extensively studied with
reference to protection from Plasmodium falciparum [1]. Hemoglobin
S is very common in tribal Indian states upto 35% carrier prevalence
in states of Maharashtra [2].
HbE 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. HbE trait and Hb EE are mild
disorders [3]. e Hb E β26 (Glu to Lys) is concentrated in parts of
South East Asia where malaria is endemic and HbE carrier status has
been shown to confer some protection against P. falciparum malaria
[4]. Pathogenesis study suggested that patients who co-inherit a mild
β- thalassemia 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 aected [5]. First reported in Assam
with 23% carrier prevalence, HbE 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 hemoglobin 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 hemoglobin i.e. HbS
which is other than normal adult hemoglobin (Hb A), is earlier reported
and its prevalence pattern is being studied by dierent groups [7-10].
Prevalence of this abnormal hemoglobin in Central India specically in
Durg, Chhattisgarh is incompletely understood. Attempts were made to
analyze urban samples during sickle cell camp at Durg for nding Hb E
in the same population group.
ere are more than 50 hemoglobin J variants described in the
literature [11]. ey all have an electrophoretic mobility “faster” than
HbA towards anode in common. All are classied under “variants
of the alpha or beta chains” (single or multiple base changes) or
“hemoglobins with more than one amino acid substitution in the
alpha chain.” Hemoglobin J, depending on its type, have dierent
characteristics and functions. Indian variant of Hemoglobin J was
reported earlier in North Indian region [12]. Previously Hemoglobin
J has been noted by many researchers in various countries. e case of
HbJ-Rajappen was reported by Hyde et. al. and later by Henthorn et.
Al. in their results of a 10-year program in an English Health region
[13,14]. HbJ Baltimore was rst described in 1963 in an African-
American family. Since then, several cases have been reported in
distinct racial groups and also incidentally during the study of other
entities, such as thalassemia [15]. More recently, the increasingly
frequent determination of HbA1c in diabetic persons has contributed
to the appearance of cases of Hb J-Baltimore associated with
anomalous HbA1c values [16,17].
In brief, separation of hemoglobin composition was carried
out by cellulose acetate membrane electrophoresis method aer
solubility tests. Briey, peripheral blood samples (N=44) aer
red cell washing and solubility testing for screening HbS were
processed for hemolysate preparation by osmotic shock method.
Pure hemoglobin from hemolysate was subjected to cellulose acetate
membrane electrophoresis in Tris-Glycine buer (pH 8.6). Aer 45
minutes, resultant hemoglobin pattern was stained by Ponceau S red
Prevalence of different abnormal hemoglobins (Hb) in Indian
tribal and nontribal population groups is well established. Sickle
cell hemoglobin (HbS) is mostly concentrated in Central and
South Indian states, whereas HbD and Hb E is mostly found in
North-North-West and North-North-East states respectively. HbJ,
an alpha globin gene variant, is earlier reported in North India
whereas; its presence in the tribal Chhattisgarh state is not well
understood. HbE, a beta-globin gene variant was earlier reported
in North Eastern states of India. Prevalence of both these abnormal
hemoglobins in the Central India specically in Durg, Chattisgarh
is incompletely understood. In this study attempts were made to
analyze the presence of abnormal hemoglobins during screening
for sickle cell anemia. Briey, blood samples (N=44) were analyzed
for sickle cell anemia screening and conrmatory tests by solubility
tests and cellulose acetate membrane electrophoresis at alkaline
pH. Two samples showed an abnormal pattern of separation
on cellulose acetate membrane other than HbS. Out of total 44
tested samples, ve were sickle cell carriers (HbAS), one was
heterozygous HbAJ and the another one was homozygous for
HbE while remaining other were normal genotypes i.e. HbAA. In
brief, a case of homozygous Hemoglobin E from Kurmi caste of
other backward community (OBC) and a different heterozygote
pattern, i.e. Hb AJ from Brahmin community is reported from Durg,
Chattisgarh, Central India. This study provides possible indication
of variation of different abnormal hemoglobins, other than HbS,
present in the tribal state of Chhattisgarh.
Abnormal hemoglobins; Hemoglobin E; Hemoglobin J; Sickle cell
carriers; Indian tribe; Chhattisgarh; Endemic malaria
Case Study
e origin of hemoglobinopathies and prevalence of endemic
malaria are inter-linked. Natural protection from malaria
pathogenesis aer infection might be the most likely and highly
accepted hypothesis of the origin of dierent alleles of normal adult
hemoglobin in dierent population groups throughout the world,
Citation: Lingojwar D, Gupta P, Bhutoria S, Lingojwar S, Mishra N, et al. (2016) Variation of Abnormal Hemoglobins Concentrated in Durg, Chhattisgarh: A
Brief Note Based on Cross-Sectional Study. J Genet Disor Genet Rep 5:2.
Page 2 of 3
Volume 5 • Issue 2 • 1000135
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 electrophoretic
separation of samples.
Out of 44 samples as shown in Table 1, ve samples showed
sickle cell carrier status with one fast migrating (HbA) and one slow
migrating band (HbS). Two samples i.e. #17 and #27 (Figures 1 and
2) showed mobility pattern dierent than HbS. Based on mobility
pattern, both samples were compared with normally fast migrating
HbA and slow migrating HbS. Homozygous Hemoglobin E formed
Gender Mean age in
Hemoglobin electrophoresis on cellulose
acetate membrane in alkaline conditions
Male (n=09) 26.77 09 0 0 0
Female (n=35) 22.08 28 5 1 1
Total (n=44) 23.04 37 5 1 1
prevalence 84.09 11.36 2.27 2.27
Table 1: All three abnormal hemoglobin’s along with normal pattern (Hb A+A)
shown, around 15% abnormal hemoglobin’s (Hb A+S, Hb E+E and Hb A+J) were
found in the study.
Figure 1: The family tree was shown. The man pointed by an arrow is the proband. The man A had a renal disease but the detail is unclear. The woman B suffers
from recurrent self-limiting febrile episodes and arthralgia but the diagnosis is not made. The man C is on hemodialysis. The woman D had a renal transplant.
Figure 2: Hemoglobin electrophoresis for sample # 17 and 27. Known A+S sample was applied in between #17 and #27 as appropriate control for referring
position of HbA, and HbS on the gel. One homozygous sickle cell disease sample (HbS+S) available in the lab from earlier studies* was also applied below
sample #17 as a second positive control for examining position of HbE+E and rst positive control HbA+S.
Citation: Lingojwar D, Gupta P, Bhutoria S, Lingojwar S, Mishra N, et al. (2016) Variation of Abnormal Hemoglobins Concentrated in Durg, Chhattisgarh: A
Brief Note Based on Cross-Sectional Study. J Genet Disor Genet Rep 5:2.
Page 3 of 3
Volume 5 • Issue 2 • 1000135
a single band and was very slow during electrophoresis and resolved
before HbS whereas, heterozygous HbAJ formed one band with
similar mobility pattern like HbA for its majority of fractions and
second fast migrating band faster than HbA. Separation pattern
of sample no. 17 was dierent from that of HbA and HbS. Aer
comparison with these abnormal hemoglobins, known mobility
of Hb E from review of literature and repeated application during
cellulose acetate membrane electrophoresis, it is conrmed that this
is Hb E.
In brief, in the present study, a case of homozygous HbE from
Kurmi caste of OBC community is found in one 21 year old normal
healthy female in Durg Chhattisgarh, Central India. Along with this one
abnormal heterozygous case of abnormal hemoglobin variant, HbAJ
was also found in one 20 year old healthy female from the Brahmin
community without any associated clinical manifestations. As this
study was based on very small sample size, the presence of dierent
hemoglobin variants such as HbE and HbJ in single study provides
possible indication of variation of dierent abnormal hemoglobins,
other than HbS, present in the tribal state of Chhattisgarh. is initial
nding of the presence of abnormal hemoglobin variant in the tribal
Chhattisgarh state may prove a signicant step for further careful
observation of routine sickle cell anemia screening.
Authors are thankful to the staff of ATG LAB for their kind help and support
for lab studies and Department of Biotechnology, Government V.Y.T. PG.
Autonomous College, Durg for their local hospitality and arrangement during the
diagnostic eld work.
Author contributions
Devendra Lingojwar (DL) contributed to the conception and design of the
project, prepared the manuscript and revised it critically for intellectual content.
PG, SL and NM has acquired, analyzed and interpreted the data, contributed to
data collection and analysis. DL, SB and AK has contributed in image analysis
and manuscript preparation. All authors approved the nal version of the article.
Grant information
This project was conducted as a part of an intramural project of ATG LAB
and no specic grant was used for this project from any funding agency.
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Author Afliation Top
1ATG LAB, Biotechnology Research Laboratory, Ganesh Nagar, Pimple Nilakh,
Pune, India
2RESEARCH (Regional Society for Education and Research in Community
Health), Pune, India
3Department of Biotechnology, Govt. V.Y.T. PG. Autonomous College, G.E.
Road, Durg Chhattisgarh, India
4Hematology Division, Department of Medicine, Albert Einstein College of
Medicine, Bronx NY, USA
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Sickle cell disease is a major genetic disorder amongst Scheduled Caste (SC), Scheduled Tribe (ST), and Other Backward Communities (OBC) population groups of Maharashtra. We modified diagnosis technique and developed simple laboratory technology to identify carrier (Hb SS) and sufferer (Hb AS) suitable for field work. In order to find out prevalence for sickle cell disorder we screened major communities from the state and found high prevalence amongst SC, ST and OBC. The overall prevalence amongst SC, ST and OBC is 10%. Severe joint pains and milder type of jaundice are peculiar symptoms amongst sicklers from the state of Maharashtra.
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