Indian J Med Res 140, August 2014, pp 231-237
Alpha thalassaemia in tribal communities of coastal Maharashtra, India
Madhav G. Deo* & Prakash V. Pawar*
Moving Academy of Medicine & Biomedicine, Pune, India
Received August 22, 2013
Background & objectives: In a routine community health survey conducted in adult Adivasis of the costal
Maharashtra, microcytosis and hyprochromia were observed in more than 80 per cent of both males
and females having normal haemoglobin levels suggesting the possibility of α-thalassaemia in these
communities. We conducted a study in Adivasi students in the same region to find out the magnitude of
α-thalessaemia.
methods: The participants (28 girls and 23 boys) were 14-17 yr old studying in a tribal school. Fasting
venous blood samples (5 ml) were subjected to complete blood count (CBC), Hb-HPLC and DNA
analysis using gap-PCR for deletion of – α3.7 and – α4.2, the two most common molecular lesions observed
in α-thalassaemia in India.
Results: Microcytic hypochromic anaemia was observed 50 and 35 per cent girls and boys, respectively.
Iron supplementation improved Hb levels but did not correct microcytois and hypochromia. more than
80 per cent non-anaemic students of both sexes showed microcytois and hypochromia. DNA analysis
confirmed that the haematological alterations were due to α-thalassaemia trait characterized by
deletion of – α3.7. Majority (> 60%) of the affected students had two deletions (-α3.7/-α3.7) genotype α+
thalassaemia.
Interpretation & conclusions: This is perhaps the first report on the occurrence of α-thalassaemia in
tribal communities of coastal Maharashtra. Very high (78.4%) haplotype frequency of -α3.7 suggests that
the condition is almost genetically fixed. These preliminary observations should stimulate well planned
large scale epidemiological studies on α-thalassaemia in the region.
Key words Alpha thalassaemia - tribals - western Maharashtra, India
a survey of anaemia was recently conducted in
June-July, 2012 (unpulished data) in a few congregations
of tribal communities’ in Mangoan taluka, Raigad
district, Maharashtra about 150 km south of Mumbai,
Maharashtra, India. A total of 143 tribal adults (88
females and 55 males) participated in the study.
*
Microcytic hypochromia anaemia was observed in 70
and 50 per cent of females and males, respectively.
Further, an important observation was that more than 80
per cent adults both males and females, who had normal
Hb level, also showed microcytosis and hypochromia
(unpublished data). In these subjects, RBC count was
Both authors contributed equally
231
232 INDIAN J MED RES, august 2014
raised and, despite low mean corpuscular volume
(MCV), the haematocrit was normal. These findings
were suggestive of α-or β-thalassaemia trait. High
frequency of clinically silent forms of either of the two
thalassaemias has not been reported in this region so
far. The probability of this being α-thalassaemia was
more likely as its high frequency has been reported
in other tribal populations in India1. This study was
planned to ascertain the existence and magnitude of
α-thalassaemia and its molecular biology in the Adivasis
in the region. Because of uncertainty of the life pattern,
the tribal people travel long distances in search of jobs.
Loss to follow up would have been, therefore, a major
confounding factor. Hence, it was decided to conduct
further studies in tribal school students.
Material & Methods
The study was conducted on 14-17 yr old healthy
class IX – X students of both sexes of Vanvashi
Aharam Shala (tribal school) between December 2012
and May 2013. The school was exclusively for tribal
children. A total of 51 healthy students (28 girls and 23
boys) participated in this preliminary cross-sectional
study. As no background information was available to
determine the sample size, it was decided to follow the
alternative strategy of expressing the statistical power
of the study in terms of confidence interval which turned
out to be ± 9% at 95% confidence limits. The study
design including the clinical protocol and the informed
consent form, were approved by the Institution Review
Board (IRB) of the Moving Academy of Medicine and
Biomedicine (MAMB), Pune, India. The study was
conducted with the approval of the school authorities.
Informed consent was obtained from the participants
and their parents. each class had about 25-30 students.
Participation was entirely voluntary. No one was
excluded and any one could withdraw anytime without
affecting his/her school privileges.
Five ml of fasting venous blood was collected in
EDTA evacuated tubes and subjected to (i) complete
blood count comprising measurements of Hb, RBC,
haemtocrit, MCV, mean corpuscular haemoglobin
(MCH), mean corpuscular haemoglobin concentration
(MCHC) and red cell distribution width (RDW), using
an electronic haematological analyzer (Sysmax POCH100i, USA), (ii) Hb- HPLC on “Bio-rad Variant 2” using
a β-thalassaemai kit (USA) and (iii) for deletion -α3.7 and
-α4.2, the commonest genetic lesions in α-thalassaemia
in India1-3. DNA was extracted from whole peripheral
blood using Qiagen spin column (QIAamp DNA kit,
Germany) using manufacturer’s protocol and subjected
to gap-PCR4. For studies on iron metabolism 3 ml
of venous blood was separately collected in plain
tubes. Serum iron (Fe), total iron binding capacity
(TIBC) were estimated using Ferrozine Chromogen/
Magnesium carbonate colorimetry (Abbott Diagnostics,
USA), and ferritin by chemiluminescent microparticle
immunoassay (Siemens Diagnostic, Germany).
More than 85 per cent of boys and girls (95%
confidence interval ± 11%) showed both microcytosis
and hypochromia, which are important features of
iron deficiency5. Tribal children could suffer from iron
deficiency because of malnutrition and/or intestinal
worm infestations due to poor hygienic living
conditions. However, students are regularly given deworming therapy in the school eliminating the second
possibility. Another important contributing cause for
iron deficiency in adolescent girls is the blood loss
during menstruation. Serum iron profile was, therefore,
studied only in boys. To study the contribution of the
iron deficiency in the aetiology of the anaemia all
participants were given iron supplementation in the
form of supervised oral tablets of ferrous fumarate
(300 mg/day/per person) after the breakfast daily for 2
months. Haematological investigations were repeated
both in boys and girls. However, for the reasons
mentioned above, post-iron- supplementation serum
iron profile studies were conducted only in boys.
STATA version 13 software (STATA Corp. LP,
USA) was used for statistical analysis. Student t test
was applied for comparison.
Results
Results of haematological parameters before and
two months after iron supplementation are given in
Table I. Anaemia was defined as Hb <12 g/dl in females
and <13 g/dl in males. Fifty per cent of girls (14 out of
28) were anaemic before the treatment. This figure was
reduced to 30 per cent after the iron supplementation.
Severity of anaemia was also reduced. Seven out of
14 had Hb <11 g/dl before iron supplementation. On
the other hand, no girl had Hb <11 g/dl in the post
treatment phase. Although Hb levels had improved
after the iron supplementation, there were no changes
in MCV and MCH. Microcytosis was observed
not only in all 14 anaemic girls even after the iron
supplementation but also in 10 of the 14 girls with
normal Hb. MCV was normal (>80fl) in the remaining
four girls. Thus microcytosis was observed in 85
per cent of the girls even after iron supplementation.
Similar picture was observed in the MCH. However,
Category
8
15
Anaemic
Normal
14
14
No.
7.8-11.5
10.8
Median
12.0-13.9
12.8
Range
Median
12
Median
13.1-15.5
14.1
Range
Median
P<
14.1
Mean
13.0
0.69
13.9
12.9-15.6
14.0
13
11.7-14.7
13.1 1)**
0.06
12.8
12.1-14.3
0.005
10.7-12.8
Range
P<
11.9 (4)*
Mean
P<
12.9
11.8
11.1-12.9
0.001
Mean
P<
After
10.5 (7)* 11.9 (nil)**
Basal
Range
Mean
Hb (g/dl)
6.05
5.17-6.69
6.00
5.75
0.16
0.01
0.03
5.59
After
5.95
5.23-6.47
5.91
6.04
5.08-6.99
6.05
5.64
4.83-6.32
5.55
5.48
4.87-6.36
0.003
4.82-6.67
5.75
5.60
4.24-6.3
5.41
5.29
5.45-5.84
5.19
Basal
RBC (x 106/µl)
0.4
44.8
41.2
38.4
0.44
43.9
40.9-48.9
44.3
42.5
37.9-47.4
42.5
40.0
39.5-43.1
0.01
40.5-49.0
44.6
39.8
36.7-43.1
39.7
40.5
39.0
After
36-42.2
0.002
39.0-44.2
40.9
36.5
28.8-38.6
35.9
Basal
Haematocrit (%)
74.9
75.0
70.3
0.25
74.5
68.8-82.2
0.88
68.6-81.0
74.6
70.1
70.8
71.7
61.5-81.5
0.04
60.1-81.1
69.7
72.3
74.9
69.3
65.2-78
70.0
After
68.2-84.7
0.52
70.2-92.0
76.1
69.1
57.2-79.7
69.3
Basal
MCV (fl)
21.4
After
29.4
Basal
30.5
After
MCHC (g/dl)
18.4
Basal
16.2
After
RDW (%)
0.06
23.5
21.2
31.6
29.6
0.003
32.1
30.7
15.5
16.6
0.02
14.9
16.0
0.007
21.8
22.4
30.0
31.4
0.48
30.8
31.3
17.2
15.3
0.004
16.7
14.8
0.02
23.8
21.7
31.6
30.6
0.97
31.8
30.6
16.1
16.3
0.27
16.1
15.6
23.7
0.42
23.0
31.4
0.53
31.5
15.9
0.96
14.7
21.0-27.0 21.3-26.5 30.0-34.8 30.3-34.6 13.1-19.1 13.5-17.8
23.6
20.3
17.7-24.9 18.3-26.4 27.6-31.2 29.6-32.4 14.0-22.2 13.3-21.4
20.9
22.7
21.4-30.2 21.0-28.2 30.4-33.3 30.0-39.9 13.1-19.1 12.4-17.2
24.1
21
15.6-23.7 19.5-24.3 27.1-31.3 29.5-31.2 14.4-26.7 13.7-21.5
20.4
Basal
MCH (pg)
Anaemia is defined as Hb < 12g/dl and <13 g/dl in females and males, respectively; Number of students below 11.0 gHb in females and 12.0 g in males *before and **after iron supplementation
Hb, haemoglobin; RBC, red blood cells, mcV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; RDW, red cell distribution width
Male
Normal
Females Anaemic
Sex
Table I. Haematological parameters before and after two months oral iron supplementation
Deo & Pawar: Alpha thalassaemia in coastal Maharashtra 233
234 INDIAN J MED RES, august 2014
the RDW was reduced slightly after the treatment in
anaemic girls. Although anaemia was observed in only
35 per cent boys, haematological picture before and
after iron supplementation was more or less similar to
that observed in the girls (Table I). in the anaemic boys
also Hb improved but there were no changes in MCV
and MCH after the iron supplementation.
Serum iron profile was studied in only 17 of the
23 boys (Table II). In the remaining six either the preor post-iron supplementation serum samples could
not be obtained. Average basal serum iron, TIBC and
ferritin were 95.9 µg/dl, 393.9 µg/dl and 26.05 ng/ml,
respectively. No changes were observed in serum iron
after iron supplementation which, however, resulted
in significant reduction in TIBC and increase in the
ferritin (Table II).
Abnormal HPLC pattern was observed in four of
the 51 (7.8%) students. Two (all males) were sickle cell
carriers (HbAS) and one was HbD trait. One boy had
raised HbA2 (5.9%) indicative of β-thalasssaemia trait.
In the remaining, the pattern was more or less normal.
None of the samples showed high levels of HbF. In
two students, who were carriers of sickle cell disease,
HbS levels were 27.1 and 26.2 per cent. The latter
also showed a marginal rise in HbA2 (3.8%). In our
laboratory 3.5 per cent is the upper limit of normal for
HbA2.
of the 51 samples, 45 (88.2%) showed gene
deletions.Thirty four (66.7%) had double [-α3.7/-α3.7]
and 10 single [-α3.7/αα] deletions (Figure; Table III).
One sample showed compound heterozygous deletion
-α3.7/-α4.2. The remaining six samples had normal (αα/αα)
genotype. Proportion of boys and girls in each category
was more or less the same. The participants belonged
mostly to two tribal communities namely Takkars and
Katkari (Table III). The former accounted for 64 per
Table II. Serum iron, (TIBC) and ferritin before and after iron supplementation in boys (n=17)
Serial no.
Age
(yr)
Iron (μg/dl)
(65-175)*
Ferritin (ng/ml)
(25-400)*
Before
After
Before
After
Before
After
1
17
115
77
322
295
24.29
34.96
2
15
110
72
344
335
13.56
31.33
3
15
41
85
428
350
3.37
10.6
4
15
55
77
408
317
7.75
28.89
5
15
93
151
431
364
16.99
30.04
6
15
97
106
381
321
9.73
19.61
7
14
112
92
454
370
6.73
16.54
8
15
103
137
379
348
75.67
89.83
9
16
126
31
384
330
83.61
77.45
10
14
95
79
406
340
7.66
29.93
11
13
99
84
349
306
61.54
45.49
12
14
142
107
383
321
13.04
37.68
13
15
53
163
561
477
4.77
6.43
14
17
112
117
367
257
21.07
79.51
15
16
46
102
363
315
5.57
13.72
16
16
142
79
400
379
49.4
79.32
17
16
90
107
332
310
49.75
65.11
95.9 ±31
98 ± 31.5
393.6 ± 56
337.4 ± 46.6
26.7 ± 26.05
41.0 ± 27.1
Mean ± SD
P<
Denote normal ranges in our laboratory
*
TIBC (μg/dl)
(250-450)*
0.87
0.001
0.002
-α3.7/-α4.2
1
-α3.7/-α3.7
M
-α3.7/αα
αα/αα
Deo & Pawar: Alpha thalassaemia in coastal Maharashtra Step up Ladder
2
3
4
235
LIS1/2350 bp
-α3.7/2022 bp
-α2/1800 bp
-α4.2/1628 bp
Fig. Gene deletion profile analysis in alpha thalassaemia using 1% agarose gel after multiplex gap PCR. Top band in each lane is the LIS1
2350 bp internal control. In addition band/bands in different lanes are as follows. Lane 1: 1800 bp α2-globin (αα/αα); lane 2: single 2022
bp α-globin gene deletion (-α 3.7) and 1800 bp (α2-globin gene) bands; lane 3: homozygous α-globin gene deletions (-α3.7/-α3.7) and lane 4:
compound heterozygous α-globin gene deletions (-α3.7/-α4.2) showing 2022 bp (α3.7) and 1628 bp (α4.2) bands.
cent and the latter for 34 per cent of the participants.
Although the number in each category was small, more
than 60 per cent students in both communities showed
homozygous two gene deletions (Table III).
Discussion
Microcytic hypochromic anaemia was observed
in 50 and 35 per cent girls and boys and though iron
supplementation improved Hb levels but could not
correct microcytosis and hypochromia. microcytosis
and hypchromia was also observed in children with
normal Hb. Hb-HPLC pattern was normal in 90 per
cent of the participants. These observations suggested
that the haematological alterations were probably
due to α+ thalassaemia, which was confirmed through
DNA studies in which the main lesion was deletion
– α3.7. Further, more than 60 per cent of the affected
students had homozygous two deletions (-α3.7/-α3.7)
genotype. All students with two deletion genotype
(-α3.7/-α3.7) exhibited microcytosis and hypochromia
which was not the case with one deletion type (-α3.7/
αα) in whom all haematological parameters were more
of less normal (data not shown). These results should
be, however, taken as indicative only as the sample size
in each group was small to draw any firm conclusions.
Our finding indicated existence of widespread iron
deficiency in our study subjects.
we have demonstrated, albeit on a small sample, a
high prevalence of α-thalassaemia (its silence variety
and trait) affecting about 90 per cent of students from
236 INDIAN J MED RES, august 2014
Table III. Genotype distribution in relation to sex and tribes
Categories
No.
Genotype [Number and (%)]
Haplotype frequencies (%)
(- α /- α )
(- α /- α )
(- α /αα)
(αα/αα)
αα
-α
1 (1.9)
10 (19.7)
6 (11.6)
21.6
78.4
3.7
3.7
3.7
4.2
3.7
Total
51
34 (66.7)
Females
28
20 (71.5)
-
5 (17.8)
3 (107)
18.1
81.9
Males
23
14 (60.9)
1 (4.3)
5 (21.7)
3 (13.1)
24.0
76.0
7 (21.2)
5 (15.2)
25.8
74.2
3 (18.7)
1 (6.3)
15.6
84.4
Genotype in relation to tribes
Thakkar
33
21 (63.6)
Katkari
16
11 (68.7)
1 (6.3)
The remaining two belonged to Kokana communities. Both showed two gene deletions (-α /-α )
3.7
tribal communities in coastal Maharashtra. Prevalence
of α-thalassaemia trait in India varies between 11 and
71 per cent and shows regional variation1. Very high
frequencies of α-thalassaemia have been reported
in tribals around Surat in West Central Gujarat and
Nilgiris in South6. tribals in the North-eastern states
of India (Assam and Arunachal Pradesh) show low
prevalence (below 4%)7 despite the fact that it is a
malaria endemic area. Very high frequencies have also
been seen in parts of Papua New Guinea8.
The most common molecular lesion in this study
was rightward -α3.7 deletion, which was observed in
more than 90 per cent of the participants. Only one
student had compound homozygous -α3.7 and -α4.2
deletions. No significant differences were seen in the
frequency of the deletions in the two sexes. More than
120 known molecular defects, both gene mutations
and deletions, cause α-thalassaemia. However, unlike
β-thalassaemia, 95 per cent of α-thalassaemias in the
world are due to deletion of α genes9. Six most common
deletions in different parts of the world are -α3.7, -α4.2,
-αSEA, -αFIL, -αMED and -α20.5. Rightward -α3.7 and leftward
-α4.2 are the two most common deletions observed in
α-thalassaemia in India1-3. However, there are regional
differences. For example, tribal communities of Orissa
and Andhra Pradesh showed even proportion of -α3.7
and -α4.2 deletions6. One of the limitations of this study
was that it was focussed on the two most common gene
mutations observed in α-thalassaemia in India. Other
molecular lesions were not investigated.
α-thalassaemia due to deletion of the two α genes
could have genotype of -α/-α (trans) or --/αα (cis). The
former pattern ensures that genetic recombination will
not cause 3 or 4 gene deletions. The latter, however,
will produce deletion of 3 or 4 α genes resulting in Hb
H disease or hydrops fetalis, respectively, as has been
3.7
seen in South East Asia9,10. The genotype in this study
appears to be -α/-α (trans). However, this needs to be
confirmed by gene mapping studies.
In pure sickle cell trait, HbS is around 45 per
cent of the total haemoglobin11. in this study, in two
students of α-thalassaemia who also had sickle cell
trait (HbAS), the HbS levels were less than 30 per
cent. α-thalassaemia is associated with reduced
synthesis of α-chains11. In co-morbidities (sickle cell
disease occurring in association with α thalassaemias)
βA (normal) and βS (mutant) chains, have to compete
with the smaller pool of α-chains. Because of lower
avidity of βS chains, the level of HbS is more affected
as compared to HbA resulting in lower proportion of
HbS in these cases12. In silent and trait varieties it is
between 25-35 per cent which has also the case in the
present study. This may explain why co-inheritance
of α-thalassaemias results in relatively milder clinical
picture in patients of sickle cell trait12,13. The same
reasons may explain the marginal rise in HbA2 observed
in one case of HbAS in this study.
Iron deficiency is the commonest cause of
microcytic hypochromic anaemia in India5. However,
microcytic hypochromic anaemia in the tribal
students in this study appeared to be only partly due
to iron deficiency superimposed on the background
of α-thalassaemia. Therefore, the anaemia would only
partially respond to iron therapy, as has been the case
in the present study. There is a danger that enthusiastic
long term iron supplementation may result in iron
overload in anaemic tribal individuals. This observation
should be useful to family physician practicing in tribal
areas.
In the present study, the students belonged to
Katkari and Thakkar tribes14. high prevalence of
α-thalassaemia with haplotype frequencies of 95 per
Deo & Pawar: Alpha thalassaemia in coastal Maharashtra cent for -α has been reported in Ganit and Kokna tribal
communities in Gujarat6. Our observations suggest
existence of a large continuous belt of alpha thalassaemia
in Western Maharashtra. However, this needs to be
confirmed by large scale epidemiological studies.
Such high prevalence of a common genetic deletion
(-α3.7) in different tribal communities geographically
separated by large distances should be of interests to
anthropologists in tracing ancestry, intermixing and
migrations in the tribal communities in India.
5.
Yadav D, Chandra J. Iron deficiency: beyond anemia. Indian J
Pediatr 2011; 78 : 65-72.
6.
Labie D, Rao S, Dunda O, Dode C, Lapoumeroulie C, Devi
V, et al. Haplotypes in tribals indians bearing the sickle gene:
evidence for the unicentric origin of the βs mutation and the
unicentric origin of the tribal population in India. Hum Biol
1989; 61 : 479-91.
7.
Sen R, Chakrabarti S, Sengupta B, De M, Haldar A, Poddar S,
et al. Alpha-thalassemia among tribal populations of Eastern
India. Hemoglobin 2005; 29 : 277-80.
8.
Yenchitsomanus PT, Summers KM, Bhatia KK, Cattani J,
Board PG. Extremely high frequencies of alpha-globin gene
deletion in Madang and on Kar Kar Island, Papua New Guinea.
Am J Hum Genet 1985; 37 : 778-84.
9.
Harteveld CL, Higgs DR. α-thalassaemia. Orphanet J Rare
Dis 2010; 5 : 13.
Acknowledgment
The project was supported by a grant from Sir Dorabji Tata
Trust, Mumbai, India. Authors acknowledge the Vanvashi Ashram
Shala authorities for providing the necessary local support for the
project, Dr Roshan Colah, Deputy Director and her colleagues at
the National Institute of Immunohematology (ICMR), Mumbai, for
training the second author (PVP) who also conducted part of the
gap-PCR in her laboratory.
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Reprint requests:Dr Madhav G. Deo, Moving Academy of Medicine & Biomedicine, 13, Swastishree Society
Ganesh Nagar, Pune 411 052, India
e-mail: [email protected]