Alpha thalassaemia in tribal communities of coastal 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more than 80 per cent adults both males and females, who had normal Hb level, also showed microcytosis and hypochromia (unpublished data).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 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 POCH-100i, USA), (ii) Hb- HPLC on “Bio-rad Variant 2” using a β-thalassaemai kit (USA) and (iii) for deletion -α^3.7 and -α^4.2, the commonest genetic lesions in α-thalassaemia in India123. 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 de-worming 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, 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.

Table I Haematological parameters before and after two months oral iron supplementation

Serum iron profile was studied in only 17 of the 23 boys (Table II). In the remaining six either the pre-or 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).

Table II Serum iron, (TIBC) and ferritin before and after iron supplementation in boys (n=17)

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 [-α^3.7/-α^3.7] and 10 single [-α^3.7/αα] deletions (Figure; Table III). One sample showed compound heterozygous deletion -α^3.7/-α^4.2. 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 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).

Close

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 (-α^3.7/-α^3.7) and lane 4: compound heterozygous α-globin gene deletions (-α^3.7/-α^4.2) showing 2022 bp (α^3.7) and 1628 bp (α^4.2) bands.

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Table III Genotype distribution in relation to sex and tribes

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 – α^3.7. Further, more than 60 per cent of the affected students had homozygous two deletions (-α^3.7/-α^3.7) genotype. All students with two deletion genotype (-α^3.7/-α^3.7) exhibited microcytosis and hypochromia which was not the case with one deletion type (-α^3.7/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 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 -α^3.7 deletion, which was observed in more than 90 per cent of the participants. Only one student had compound homozygous -α^3.7 and -α^4.2 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 -α^3.7, -α^4.2, -α^SEA, -α^FIL, -α^MED and -α^20.5. Rightward -α^3.7 and leftward -α^4.2 are the two most common deletions observed in α-thalassaemia in India123. However, there are regional differences. For example, tribal communities of Orissa and Andhra Pradesh showed even proportion of -α^3.7 and -α^4.2 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 seen in South East Asia910. 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) β^A (normal) and β^S (mutant) chains, have to compete with the smaller pool of α-chains. Because of lower avidity of β^S 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 trait1213. 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 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 (-α^3.7) 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.