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Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur
The Egyptian Journal of Medical Human Genetics (2012) 13, 323–330
Ain Shams University
The Egyptian Journal of Medical Human Genetics www.ejmhg.eg.net www.sciencedirect.com
ORIGINAL ARTICLE
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur Ahsana Shah, Ruqaiya Hussain, Mohd Fareed, Mohammad Afzal
*
Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India Received 3 March 2012; accepted 3 April 2012 Available online 4 May 2012
KEYWORDS Sickle cell trait; Malaria; P. falciparum; Red blood cells; Allele frequency; Heterozygosity
Abstract Background: Sickle cell anaemia is an important genetic and public health problem in Manipur, which is a small hilly state, situated at the north eastern extreme corner of India sharing an international boundary with Myanmar. Our present study provides a comprehensive database on the occurrence of sickle cell trait in the Manipuri population especially with regard to Manipuri Muslims, as till date no work has been done on Manipuri Muslims with different castes. Aim: To study the gene frequency and heterozygosity condition of sickle cell trait in different populations of Manipur. Subjects and methods: We have undertaken a survey of the frequencies of sickle cell gene among Muslims with different castes, viz., Sheikh, Syed, Pathan and Moghul; Hindu (Meitei) and tribe (Naga). The blood samples were collected by finger-prick method and were tested for sickle cell trait by the wet sealed method using 2% freshly prepared sodium metabisulphite solution following the method of Daland and Castle. Results: The Moghul population shows the highest allele frequency of sickle cell gene, i.e., 16.96% while Naga shows the least, i.e., 8.6%. Females show a higher allele frequency of the sickle cell gene, i.e., 29.83% than males who show a frequency of 21.99%. The heterozygosity condition was found to be higher among Mughals, i.e., 28.12% while the least is exhibited by Naga, i.e., 15.72%. Higher percentage of sickle cell trait in this region might be resulting from the natural selection process in response to the high prevalence of malaria (Plasmodium falciparum) in Manipur to protect the inhabitants from the lethal effects of malaria. Ó 2012 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +91 5712700920, +91 9897839601; fax: +91 571 2707944, +91 571 2701239. E-mail address: [email protected] (M. Afzal). Peer review under responsibility of Ain Shams University.
Production and hosting by Elsevier 1110-8630 Ó 2012 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejmhg.2012.04.001
324 1. Introduction India is a store house of varied geographic landscape, consisting of 28 states to which are included the seven north eastern states popularly known as seven sisters. The north east is inhabited by a diverse population of tribes, castes, religious and migrant populations. The region is particularly interesting as represented by several tribal populations of mongoloid origin, who have migrated from eastern and south east Asian regions and settled in different parts during several waves of migration [1,2]. They share similar physical features but speak different languages and show differences in cultural, anthropological and genetical traits [3,4]. The topography of North East India, flanked in the north by the Himalayas and in the south by the Bay of Bengal, constitutes a unique narrow passage that connects the Indian subcontinent to East Asia and South East Asia through a 2000 km eastern border, shared with five neighbouring countries. Manipur (Fig. 1), one of the states of this region, is a small hilly state, situated in the north eastern extreme corner of India that connects the Indian subcontinent to East Asia and South East Asia as a unique narrow passageway [5]. Manipur is situated between 23.83° north latitude to 25.68° north latitude and 93.03° east longitude to 94.78° east longitude. The territory of Manipur includes an area of 22,327 km2 in which the hilly area (92% of the land) surrounds the centre of the valley area (8%) [6]. The hilly region occupies a large area of 20,571 km2. It has a wonderful balance of the flora and fauna by a combination of wet forest and temperate forest. This state, however small, is an abode of a number of Mongoloid groups, who speak different dialects of the Tibeto-Burman linguistic family. There are also a few groups who possess Caucasoid and Australoid racial elements [7]. Malaria is a serious public health problem in Laos [8], which is a land-locked country bordering Thailand, Combodia, Myanmar, Vietnam and China. Malaria is also the most common infectious cause of morbidity and mortality in Vietnam as it is in many developing countries in the tropics [9]. These reports can confirm the importance and high prevalence of malaria in Thailand and nearby Indo-China countries which of course includes Manipur [10]. As natural selection works on the whole individual, heritable traits will become more common in the next generation. Given enough time, this passive process can result in progressive adaptation and speciation. Sickle cell trait (SCT) occurs in approximately 300 million people worldwide, with high prevalence in Africa, the Arabian Peninsula, India, the Mediterranean, and the southern United States [11]. The trait is characterised by the inheritance of a normal haemoglobin gene (HbA) from one parent and an abnormal mutated beta globin gene, the sickle haemoglobin gene (HbS) from the other parent. In sickle cell disease, two abnormal allelomorphic haemoglobin genes are inherited, of which at least one must be the sickle haemoglobin. In the homozygous sickle cell disease (HbSS), both abnormal haemoglobins are HbS. Normal adult haemoglobin is made from a combination of two beta and two alpha globin chains and a haeme. The beta1-globin gene is located on the short arm of chromosome 11. Approximately 150 diseases have been linked to the same chromosome 11 [12]. The sickle gene is multicentric in origin, and four main haplotypes, representing four different mutations, have been identified [13].
A. Shah et al. The genetic change yields the red blood cells (RBC), to have a short life span of 10–20 days in comparison to the normal RBC life span of 120 days. Homozygote for this allele suffers from a severe form of anaemia in which haemoglobin crystallises in the blood, distorting the shape of the RBC. Sometimes the cells appear sickle shaped giving the disease the name Sickle cell anaemia. These sickle shaped cells also clog small blood vessels and cut off oxygen transport to various tissues. The heterozygous carriers of the mutant allele do not show disease symptoms unless they travel to high altitudes, where the oxygen concentration is low. These individuals, therefore, are said to possess the sickle cell trait [14]. Epidemiological studies have shown the wild type homozygotes to be more susceptible to infection by malarial parasites than heterozygotes. As a result, both kinds of homozygotes in this system are less fit than the heterozygotes. The 20 million people of India are known to suffer from sickle cell disease [15]. The highest frequency of sickle cell gene in India is reported from Orissa, followed by Assam, Madhya Pradesh, Uttar Pradesh, Tamil Nadu and Gujarat [16]. The average frequency of sickle gene is 4.3%, while that in Orissa is 9.1% [17]. The population diversity of sickle cell haemoglobinopathy in India mainly is the result of the admixture of genes between different populations, e.g. intruders or invading groups in and around the Gulf region, from Middle Eastern countries and Africa [18–21]. Epidemiologically, children comprise 52% of sickle cell patients. The three predominant forms (SS, SB and SD) of the disease are clinically and haematologically indistinguishable [22]. The range of the disease varies from 0% to 44%. It is 0–18.5% in north east zone, 0–33.5% in the west zone, 22.5–44.4% in the central zone and 1–40% in the southern zone [23].
2. Subjects and methods 2.1. Populations The Muslims of India comprise more than 13% of the population, and they belong to various castes, based on linguistic and ethnic groups besides, having a few tribes [24]. Manipuri Muslims comprise 8.32% of the total population, according to the 2001 census. They are mostly the migrants who started coming to the state in the middle of the 16th century [25]. They have been given different clan names which in Manipuri is called Yumnak or Sagei. The term ‘‘Sagei’’ is a corrupted word of Shaqzi which is an Urdu terminology. About 74 clans are reported in Manipur in the present times. Manipuri Muslims belong to Sheikh, Syed, Pathan or Mughal castes [26], they belong to Sunni sect only. The Meiteis represent the major tribal group, having 60% of the total population [27] and they follow Hindu religion. Meiteis are presumably formed by the admixture of Koomal, Looang, Moirang and Meitei, all of whom are reported to have arrived at different periods of time, coming from different directions and now represent the clans of the community [25]. While the Naga are the indigenous tribal population of Manipur, they belong to the Naga–Kuki–Chin group of the Tibeto-Burman linguistic family and are believed to have migrated to Manipur from Burma probably 300–400 years ago [28].
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur
Figure 1
325
Map of Manipur.
2.2. Sample collection Blood samples of five hundred and seventy-seven (577) individuals were collected from unrelated individuals belonging to both sexes from the following population groups; Muslims having castes: Sheikh, Syed, Pathan and Mughal, besides a Hindu (Meitei) and a tribal group (Naga), through taking prior informed consent from the individuals, taking into consideration the factors like caste, consanguinity, age, religion, etc. The survey was conducted among healthy individuals from the area of Imphal East and Imphal West districts during house to house visit, which were selected at random on a priori basis during the day time with the help of volunteers. Some samples were also collected from the schools by taking prior permission from the respective headmasters. Blood samples were also collected from Manipuri students studying in Aligarh Muslim University, Aligarh. The blood samples were collected by finger-prick method and were tested for sickle cell trait by the wet sealed method using 2% freshly prepared sodium metabisulphite solution following the method of Daland and Castle [29]. 2.3. Genetic data analysis The phenotypes were recorded for the sickle trait for each sample, and the allele frequencies were calculated according to the
Hardy–Weinberg law [30] using a gene counting method. The level of heterozygosity was calculated using the formula: X Heterozygosity ¼ 1 Ho P where Ho is the homozygosity of the allele, Ho = Pi2. Chi-square test: It is used for the measurement of the size of the discrepancy between the observed and expected values at particular degrees of freedom: v2 ¼
X ðObserved ExpectedÞ2 Expected
3. Results 3.1. Phenotypic frequency The Mughal population shows the highest frequency of sickle cell trait, i.e., 31.03% while Naga shows the least frequency of 16.46%. Among males Meitei shows the highest frequency of 30.43%, while Pathan shows the least, i.e., 7.7%. While among females Mughal population shows the highest frequency of 36.36%, and the Meitei shows the least frequency, i.e., 26.09%. The chi-square (v2) value of normal individuals is 10.993 (df = 5, p = 0.0515) while for carrier individuals it is 14.574 (df = 5, p = 0.0123) (Tables 1–3), the difference is statistically significant.
326 Table 1
A. Shah et al. Phenotypic distribution of sickle cell trait in Manipur.
Populations
Normal
Sheikh Syed Pathan Mughal Meitei Naga Total
Sickle carrier
Total sample
M
F
C
M
F
C
M
F
C
78 30 24 26 32 30 220
56 45 22 14 34 36 207
134 75 46 40 66 66 427
28 5 2 10 14 3 62
28 18 12 8 12 10 88
56 23 14 18 26 13 150
106 35 26 36 46 33 282
84 63 34 22 46 46 295
190 98 60 58 92 79 577
M, F and C represent male, female and combined, respectively.
Table 2
Phenotypic frequency of sickle cell trait in Manipur.
Populations
Sheikh Syed Pathan Mughal Meitei Naga Mean ± S.E.
Normal
Carrier
Total sample
M
F
C
M
F
C
M
F
C
73.58 85.71 92.31 72.22 69.57 90.91 80.72 ± 4.13
66.7 71.43 64.71 63.64 73.91 78.26 69.78 ± 2.34
70.53 76.53 76.67 68.97 71.74 83.54 74.66 ± 2.19
26.42 14.29 7.7 27.78 30.43 9.1 19.29 ± 4.12
33.3 28.57 35.29 36.36 26.09 27.14 31.13 ± 1.80
29.47 23.47 23.3 31.03 28.26 16.46 25.33 ± 2.19
55.79 35.71 43.3 62.07 50.00 41.77 48.11 ± 3.98
44.21 64.29 56.6 37.93 50.00 58.23 51.88 ± 3.97
32.93 16.98 10.4 10.5 15.94 13.69 16.74 ± 3.42
S.E. represents the standard error. M, F and C represent the male, female and combined, respectively.
3.2. Allele frequency The highest allele frequency of sickle cell gene was found among Mughals, being 16.96%, and the least among Nagas 8.6%. Among males, Meiteis show the highest allele frequency of sickle cell gene, i.e., 16.59% and the least was shown by Pathan, i.e., 3.9%. Among females, Mughals show the highest allele frequency, i.e., 20.23% and the least was shown by Naga, i.e., 11.53% (Table 4, Fig. 2). 3.3. Heterozygosity The heterozygosity condition was found to be highest among Mughals, i.e., 28.12%, while least frequency was shown by Naga, i.e., 15.72%. Among males, again highest heterozygosity condition was shown by Meitei, i.e., 27.68% and the least by Pathan, i.e., 7.49%. Among females, the highest heterozygosity condition was found among Mughals, i.e., 32.27% and the least was among the Nagas, i.e., 20.40% (Tables 5 and 6, Fig. 3). 4. Discussion Our present study provides a comprehensive database on the occurrence of sickle cell trait in the Manipuri population especially with regard to Manipuri Muslims. The knowledge of the prevalence of the trait in this religion is important, since they perform endogamous marriage among themselves and marriages between the carriers of this disease might result in the birth of children with sickle cell disorder. In the present study, the percentage of sickle cell carrier was found to be 25.99%. The percentages of sickle cell carriers were found to be 11.7% in a community based study of inmates of the Central jail, Raipur [31]. In Central India, the
prevalence of sickle cell trait (SCT) has been reported to be 11.1% [32]. The Indian Council of Medical Research conducted a multicentric study of tribal people in four states (Maharashtra, Gujarat, Orissa, and Tamil Nadu) focusing on nutritional status, anaemia and frequency of the sickle cell and b-thalassaemia traits. The result shows sickle cell trait to be 29% [33–35]. In a study by Balgir [36] it was seen that the most common haemoglobin disorders observed among 1015 cases of sickle cell trait were found to be 29.8%. The females overall show a higher percentage of frequency for sickle cell carrier, i.e., 29.83% when compared to males, who shows around 21.99%. Heterozygosity condition for the trait is found to be high, i.e., 24.05%, with females showing 41.86% while males have 34.31%. With regard to the sex distribution of the disorder, Wintrobe [37] stated that the sickle cell trait is more common among females, though some other authors did not find any such correlation [38,39]. In the present study, this correlation exists. Among Muslims, the Mughal caste shows the highest percentage of sickle cell carrier while Pathan shows the least, again among the six populations studied, Mughal population shows the highest phenotypic frequency, allelic frequency as well as heterozygosity condition. The Plasmodium falciparum causes a malignant form of malaria, the much virulent form which is highly prevalent in Manipur. Chishti et al. [40] confirmed the prevalence of severe and complicated P. falciparum malaria in Manipur and documented the necessity of malarial research in this region. Higher percentage of sickle trait carrier in this region might have resulted mainly from the natural selection process in response to the high prevalence of P. falciparum malaria in the region. Studies have shown that heterozygotes are less susceptible to infection by malarial parasites in comparison to the homozygotes, which are more prone to the parasite. The infected cells tend to sickle and
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur Table 3
327
Observed and expected values of sickle cell trait in Manipur.
Populations
Normal
Sickle carrier
Total sample
M
F
C
M
F
C
M
F
C
Sheikh Obs Exp
78 69.0
56 64.9
134
28 23.1
28 32.8
56
106 92.86
84 97.14
190
Syed Obs Exp
30 38.6
45 36.3
75
5 9.51
18 13.4
23
35 47.19
63 50.10
98
Pathan Obs Exp
24 23.7
22 22.3
46
2 5.79
12 8.21
14
26 29.32
34 30.68
60
Mughal Obs Exp
26 20.6
14 19.3
40
10 7.44
8 10.5
18
36 28.35
22 29.65
58
Meitei Obs Exp
32 34
34 31
66
14 10.7
12 15.2
26
46 44.96
46 47.04
92
Naga Obs Exp
30 34
36 31
66
3 5.37
10 7.63
13
33 38.61
46 40.39
79
Total Chi-square value
220 10.993
207
427
62 14.574
88
150
282 16.850
295
577
M, F and C represent male, female and combined, respectively. Obs and Exp represent observed and expected values.
are selectively destroyed by body’s immune system. Thus having sickle cell gene in heterozygous condition promotes better survival and hence might have resulted in the occurrence of a higher percentage of sickle cell carriers in Manipur. Heterozygous condition (HbAS) provides significant protection against all such mortality, i.e., due to severe malarial anaemia, and high density parasitaemia. Significant reduction in mortality was detected between the ages 2 and 16 months, the highest risk period for severe malaria, showing the role of malaria in selection and maintenance of sickle cell gene [41]. Allison [42] first suggested the relative protection of sickle cell trait against P. falciparum malaria. He noted that whereas 14 out of 15 individuals without sickle cell trait developed malaria when inoculated with P. falciparum, only 2 of 15 individuals with sickle cell trait developed malaria when similarly inoculated. Subsequently, numerous studies have confirmed this high degree of resistance to severe P. falciparum malaria [43]. Whereas the rate of severe or complicated P. falciparum malaria requires acute hospitalisation, the hospitalisation decreased by approximately 90% in individuals with sickle cell trait, there was no significant reduction in the prevalence of asymptomatic parasitaemia [43]. The protection appears to increase with age from only 20% in the first 2 years of life to a maximum of 56% by the age of 10 years and decreasing to 30% in those aged more than 10 years [43]. Proposed protective mechanisms include reduced parasite growth and enhanced removal of parasitised cells through innate and acquired immune processes [44]. The protection is, however, specific for P. falciparum malaria and, not for any of the other three types of malaria (P. vivax, P. ovale, and P. malariae) [43]. More recent studies in West Africa suggest that the greatest impact of HbS is mainly protection against either death or se-
Table 4
Allele frequency of sickle cell trait in Manipur.
Populations
Hb(p)
Hbs (q)
M
F
C
M
F
C
Sheikh Syed Pathan Mughal Meitei Naga
0.8578 0.9258 0.9608 0.8498 0.8341 0.9535
0.8165 0.8452 0.8044 0.7977 0.8597 0.8847
0.8398 0.8748 0.8756 0.8304 0.8469 0.914
0.1422 0.0742 0.039 0.1502 0.1659 0.047
0.1835 0.1548 0.1956 0.2023 0.1403 0.1153
0.1602 0.1252 0.1244 0.1696 0.1531 0.086
Total
0.7801
0.7017
0.8602
0.2199
0.2983
0.1398
M, F and C represent male, female and combined. Hb(p) and Hbs(q) are dominant and recessive alleles, respectively.
vere disease, i.e., profound anaemia or cerebral malaria, while having less effect on infection per se [41,45,46]. Indeed, casecontrol studies suggest that HbS heterozygotes have a level of protection of approximately 60–80% against the severe complications of malaria [47]. A number of mechanisms have been proposed to explain as to how HbAS gene may result in the accelerated acquisition of malaria-specific immunity. As common with other red cell genetic defects, enhanced phagocytosis of HbAS erythrocytes has been demonstrated in vitro when they are infected with ring stage of P. falciparum [48], which is a process that appears to be mediated by a mechanism essentially similar to that involved in the phagocytosis of senescent or damaged normal erythrocytes. Experimental data suggest this process is initiated by enhanced oxidant damage to the erythrocyte membrane and that this leads to the aggregation of band 3 protein and the binding of
328
A. Shah et al. 1.2
1
0.8
0.6
Hb(p) Hbs(q)
0.4
0.2
Male
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
Female Figure 2
Table 5
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
0
Combined
Graph showing allele frequency of sickle cell trait in Manipur.
Genotypic frequency of sickle cell trait in Manipur.
Populations
Male 2
Female 2
2
Combined 2
p
2pq
q
p
2pq
q
p2
2pq
q2
Sheikh Syed Pathan Mughal Meitei Naga
0.7358 0.8571 0.9231 0.722 0.6957 0.9092
0.2439 0.1374 0.0749 0.2553 0.2768 0.0896
0.0203 0.0055 0.0015 0.0226 0.0275 0.0022
0.667 0.7144 0.6471 0.6363 0.7391 0.7827
0.2997 0.2617 0.3147 0.3227 0.2412 0.2040
0.0337 0.024 0.0383 0.0409 0.0147 0.0133
0.7052 0.7653 0.7667 0.6895 0.7172 0.8354
0.2691 0.2190 0.2178 0.28117 0.2593 0.1572
0.0256 0.0157 0.0155 0.0288 0.0234 0.0074
Total
0.6086
0.3431
0.0484
0.4924
0.4186
0.0889
0.7399
0.2405
0.0195
p2, 2pq and q2 are dominant, heterozygous and recessive genotypic frequencies.
Table 6 Homozygosity and heterozygosity of sickle cell trait in Manipur. Populations
Male
Female
Combined
Ho
Ht
Ho
Ht
Ho
Ht
Sheikh Syed Pathan Mughal Meitei Naga
0.761 0.8626 0.9251 0.7447 0.7232 0.9104
0.2439 0.1374 0.0749 0.2553 0.2768 0.0896
0.7003 0.7383 0.6853 0.6773 0.7588 0.796
0.2997 0.2617 0.3147 0.3227 0.2412 0.2040
0.7309 0.781 0.7822 0.7188 0.7407 0.8428
0.2691 0.2190 0.2178 0.2812 0.2593 0.1572
Total
0.6569
0.3431
0.5814
0.4186
0.7595
0.2405
Ho and Ht represent homozygosity and heterozygosity, respectively.
autologous IgG and complement system [48], a mechanism similar to that previously proposed for a thalassaemia [49]. It, therefore, seems plausible that enhanced immunity could be mediated by the accelerated acquisition of antibodies to altered host antigens expressed on the parasite-infected red cell surface, such as band 3 protein [50]. On the other hand, parasite-derived proteins such as the variant surface antigen P. falciparum eryth-
rocyte membrane protein-1 might represent an alternative target, a hypothesis supported by the raised titres of antibodies directed towards variant antigens seen in HbAS children living in Gambia [51]. These mechanisms need not be mutually exclusive. As an alternative explanation, it seems possible that by controlling parasite densities, during malarial infections, innate processes might paradoxically increase the chronicity of individual infection [52]. The heterozygosity condition might have increased in Mughal population in response to the high occurrence of sickle cell disease, and these results shows a clear example of natural selection occurring in the population, if this condition had not arisen it would have led to wiping up of the population from the region. In our present study, however, no case of disease has been reported. These might be probably due to the early death of individuals having homozygous state for sickle cell gene. High prevalence of malaria in this region has caused a major health problem for the population. Large scale screening of the population is very much needed since the region shows high occurrence of the trait. The parasite carrier may serve as endemic carrier for the source of infection for a long time since malaria in such individuals is rarely identified and they receive no treatment. Yet, the infectivity of gametocytes in the
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur
329
1 0.9 0.8 0.7 0.6 0.5 Ho
0.4
Ht
0.3 0.2 0.1
Male Figure 3
Female
NA GA
M EI TE I
SY ED PA TH AN M OG HU L
SH EI KH
NA GA
M EI TE I
SY ED PA TH AN M OG HU L
SH EI KH
M EI TE I NA GA
PA TH AN M OG HU L
SY ED
SH EI KH
0
Combined
Graph showing homozygosity and heterozygosity of sickle cell trait in Manipur.
immune individuals appears to be less marked than in the nonimmune individuals [52]. Sickle cell disorder has remained a neglected field of research in this country and the magnitude of problem has never been appreciated in spite of the fact that the sickled RBCs were detected in the blood of Indian patients as early as 1952. This was largely because most of the subsequent reports spread a misconception that the sickle gene in India was confined to the tribal population or some scheduled castes only [53]. In the present study, only six population groups have been taken up, but the results show that even non-tribal groups show the presence of sickle cell gene. However as the region is inhabited by a large number of tribal populations, screening for this trait must be encouraged in Manipur. Since the tribal populations are mostly confined to hilly regions of Manipur and they get limited health service, and education, screening and prenatal counselling for the disease in these areas can help a lot in minimizing the occurrence of the sickle cell disease and, thus can help them to make informed choices and avoid the birth of sickle cell children. The awareness of the disease and its implication for the public health problem, though have been increasing in recent days, yet this awareness is mostly confined to the urban regions of Manipur valley areas and, therefore the people residing in remote areas also need to be made aware of the disease. 5. Conclusion The study highlights the prevalence of the sickle cell trait among six populations from different regions of the state. Since our present finding shows the high occurrence of the trait in the region, effective large scale screening in other populations is the need of the hour. The disease is not curable and causes high degree of morbidity and mortality among the individuals suffering from the disease. Thus there is an urgent need for special intervention strategies for prevention and control of the disease in the areas supposed to be affected with endemic malaria. Conflict of interest The authors declare no conflict of interest.
Acknowledgements Thanks are due to the Department of Science & Technology (DST), New Delhi, for awarding INSPIRE Fellowship (No. IF10378) to the first author (Ahsana Shah) and to the Chairman, Department of Zoology, A.M.U., Aligarh (U.P), India, for laboratory facilities. My special thanks are due to the headmasters, teachers, students of various schools and also the individuals who voluntarily donated blood samples for this study. References [1] Rapson EJ. People and languages. In: Rapson EJ, editor. Cambridge history of India, vol. 1. Delhi: S. Chand and Co.; 1995. p. 33–57. [2] Dani AH. Prehistory and protohistory of Eastern India. Calcutta: Firma K.L. Mukhopadhyaya; 1960. [3] Roychoudhury AK. Genetic relationships of the populations in Eastern India. Ann Hum Bio 1992;19:489–501. [4] Bhasin MK, Walter H. Genetics of caste and tribe of India. Delhi: Kamla Raj Enterprises; 2001. [5] Cordaux R, Weiss G, Saha N, Stoneking M. The North East Indian Passageway: a barrier or corridor for human migrations. Mol Biol Evol 2004;21(8):1525–33. [6] Vedaja S. Manipur geography and regional development of Manipur. New Delhi: Rajesh Publication; 1998. [7] Singh MR, Singh TS. Genetic Polymorphism at three loci in two populations of Manipur, India. Anthropol Anz 2008;66(2):191–8. [8] Pholsena K. The malarial situation and antimalaria program in Laos. Southeast Asian J Trop Med Public Health 1992;23 (Suppl. 4):394–402. [9] Hein TT, VinhChau NV, Vinh NN, Hung NT, Phung MQ, Toan LM, et al. Management of multiple drug-resistant malaria in Vietnam. Ann Acad Med Singapore 1997;26(5):6596–603. [10] Wiwanitkit V. Genetic disorder and malaria in Indo-China region. J Vector Borne Dis 2008;45(2):98–104. [11] World Health Organization. Sickle cell anemia. Report by the secretariat. 59th World Health, Assembly; 2006. [12] Geoffrey T, Amma OA, Freda OB, Yaw AA. Complications associated with sickle cell trait: a brief narrative review. Am J Med 2009;122:507–12 [Review]. [13] Powars DR, Meiselman HJ, Fisher TC, Hiti A, Johnson C. Beta-S gene cluster haplotypes modulate hematologic and hemorheologic expression in sickle cell anemia. Use in predicting clinical severity. Am J Pediatr Hematol Oncol 1994;16(1):55–61.
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A. Shah et al. [37] Wintrobe MM. Clinical haematology, 9th ed. Philadelphia, London: Lea and Febiger; 1993. p. 1061. [38] Samal GC. Sickle cell anaemia with acute myeloid leukemia, a case report. Indian Pediatr 1979;16(5):453–4. [39] Deshmukh P, Garg BS, Garg N, Prajapati NC, Bharambe MS. Prevalence of Sickle cell disorder in rural Wardha. Indian J Community Med 2006;31(1):26–7. [40] Chishti SA, Duidang L, Kasar A, Ramam M, Luikham A. Severe falciparum malarial infection in Ukhrul, Manipur. J Indian Med Assoc 2000;98(10):619–22. [41] Aidoo M, Telouw DJ, Kolczak MS, Alexander ND, Weatherall DJ, Wambua S, et al. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 2002;359(9314):1311–2. [42] Allison AC. Protection afforded by sickle cell trait against subtertian malarial infection. Br Med J 1954;1(4857):290–329. [43] Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Kortok M, Snow RW, et al. Sickle cell trait and the risk of plasmodium malaria and other childhood diseases. J Infect Dis 2005;192(1):178–86. [44] Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, Wambua S, et al. An immune basis for malaria protection by the sickle cell trait. PLos Med 2005;2(5):128. [45] Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, et al. Common West African HLA antigens are associated with protection from severe malaria. Nature 1991;352(6336):595–600. [46] Williams TN. Red blood cell defects and malaria. Mol Biochem Parasitol 2006;149:121–7 [Review]. [47] Weatheral DJ. Genetic variation and susceptibility to infection: the red cell and malaria. Br J Haematol 2008;141(3):276–86 [Review]. [48] Ayi K, Turrini F, Piga A, Arese P. Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum-malaria in sickle-trait and beta-thalassemia-trait. Blood 2004;104(10):3364–71. [49] Williams TN, Weatherall DJ, Newbold CI. The membrane characteristics of Plasmodium falciparum-infected and uninfected heterozygous alpha (0) thalassaemic erythrocytes. Br J Haematol 2002;118(2):663–70. [50] Hebbel RP. Sickle hemoglobin instability: a mechanism for malaria protection. Redox Rep 2003;8(5):238–40 [Review]. [51] Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 1989;83(3):293–303. [52] Roberts DJ, Williams TN. Haemoglobinopathies and resistance to malaria. Redox Rep 2003;8(5):304–10 [Review]. [53] Parmar D. Sickle cell anaemia: review and remedial hope. Egypt J Med Hum Genet 2009;10(2) [Review].
Ain Shams University
The Egyptian Journal of Medical Human Genetics www.ejmhg.eg.net www.sciencedirect.com
ORIGINAL ARTICLE
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur Ahsana Shah, Ruqaiya Hussain, Mohd Fareed, Mohammad Afzal
*
Section of Genetics, Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India Received 3 March 2012; accepted 3 April 2012 Available online 4 May 2012
KEYWORDS Sickle cell trait; Malaria; P. falciparum; Red blood cells; Allele frequency; Heterozygosity
Abstract Background: Sickle cell anaemia is an important genetic and public health problem in Manipur, which is a small hilly state, situated at the north eastern extreme corner of India sharing an international boundary with Myanmar. Our present study provides a comprehensive database on the occurrence of sickle cell trait in the Manipuri population especially with regard to Manipuri Muslims, as till date no work has been done on Manipuri Muslims with different castes. Aim: To study the gene frequency and heterozygosity condition of sickle cell trait in different populations of Manipur. Subjects and methods: We have undertaken a survey of the frequencies of sickle cell gene among Muslims with different castes, viz., Sheikh, Syed, Pathan and Moghul; Hindu (Meitei) and tribe (Naga). The blood samples were collected by finger-prick method and were tested for sickle cell trait by the wet sealed method using 2% freshly prepared sodium metabisulphite solution following the method of Daland and Castle. Results: The Moghul population shows the highest allele frequency of sickle cell gene, i.e., 16.96% while Naga shows the least, i.e., 8.6%. Females show a higher allele frequency of the sickle cell gene, i.e., 29.83% than males who show a frequency of 21.99%. The heterozygosity condition was found to be higher among Mughals, i.e., 28.12% while the least is exhibited by Naga, i.e., 15.72%. Higher percentage of sickle cell trait in this region might be resulting from the natural selection process in response to the high prevalence of malaria (Plasmodium falciparum) in Manipur to protect the inhabitants from the lethal effects of malaria. Ó 2012 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +91 5712700920, +91 9897839601; fax: +91 571 2707944, +91 571 2701239. E-mail address: [email protected] (M. Afzal). Peer review under responsibility of Ain Shams University.
Production and hosting by Elsevier 1110-8630 Ó 2012 Ain Shams University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejmhg.2012.04.001
324 1. Introduction India is a store house of varied geographic landscape, consisting of 28 states to which are included the seven north eastern states popularly known as seven sisters. The north east is inhabited by a diverse population of tribes, castes, religious and migrant populations. The region is particularly interesting as represented by several tribal populations of mongoloid origin, who have migrated from eastern and south east Asian regions and settled in different parts during several waves of migration [1,2]. They share similar physical features but speak different languages and show differences in cultural, anthropological and genetical traits [3,4]. The topography of North East India, flanked in the north by the Himalayas and in the south by the Bay of Bengal, constitutes a unique narrow passage that connects the Indian subcontinent to East Asia and South East Asia through a 2000 km eastern border, shared with five neighbouring countries. Manipur (Fig. 1), one of the states of this region, is a small hilly state, situated in the north eastern extreme corner of India that connects the Indian subcontinent to East Asia and South East Asia as a unique narrow passageway [5]. Manipur is situated between 23.83° north latitude to 25.68° north latitude and 93.03° east longitude to 94.78° east longitude. The territory of Manipur includes an area of 22,327 km2 in which the hilly area (92% of the land) surrounds the centre of the valley area (8%) [6]. The hilly region occupies a large area of 20,571 km2. It has a wonderful balance of the flora and fauna by a combination of wet forest and temperate forest. This state, however small, is an abode of a number of Mongoloid groups, who speak different dialects of the Tibeto-Burman linguistic family. There are also a few groups who possess Caucasoid and Australoid racial elements [7]. Malaria is a serious public health problem in Laos [8], which is a land-locked country bordering Thailand, Combodia, Myanmar, Vietnam and China. Malaria is also the most common infectious cause of morbidity and mortality in Vietnam as it is in many developing countries in the tropics [9]. These reports can confirm the importance and high prevalence of malaria in Thailand and nearby Indo-China countries which of course includes Manipur [10]. As natural selection works on the whole individual, heritable traits will become more common in the next generation. Given enough time, this passive process can result in progressive adaptation and speciation. Sickle cell trait (SCT) occurs in approximately 300 million people worldwide, with high prevalence in Africa, the Arabian Peninsula, India, the Mediterranean, and the southern United States [11]. The trait is characterised by the inheritance of a normal haemoglobin gene (HbA) from one parent and an abnormal mutated beta globin gene, the sickle haemoglobin gene (HbS) from the other parent. In sickle cell disease, two abnormal allelomorphic haemoglobin genes are inherited, of which at least one must be the sickle haemoglobin. In the homozygous sickle cell disease (HbSS), both abnormal haemoglobins are HbS. Normal adult haemoglobin is made from a combination of two beta and two alpha globin chains and a haeme. The beta1-globin gene is located on the short arm of chromosome 11. Approximately 150 diseases have been linked to the same chromosome 11 [12]. The sickle gene is multicentric in origin, and four main haplotypes, representing four different mutations, have been identified [13].
A. Shah et al. The genetic change yields the red blood cells (RBC), to have a short life span of 10–20 days in comparison to the normal RBC life span of 120 days. Homozygote for this allele suffers from a severe form of anaemia in which haemoglobin crystallises in the blood, distorting the shape of the RBC. Sometimes the cells appear sickle shaped giving the disease the name Sickle cell anaemia. These sickle shaped cells also clog small blood vessels and cut off oxygen transport to various tissues. The heterozygous carriers of the mutant allele do not show disease symptoms unless they travel to high altitudes, where the oxygen concentration is low. These individuals, therefore, are said to possess the sickle cell trait [14]. Epidemiological studies have shown the wild type homozygotes to be more susceptible to infection by malarial parasites than heterozygotes. As a result, both kinds of homozygotes in this system are less fit than the heterozygotes. The 20 million people of India are known to suffer from sickle cell disease [15]. The highest frequency of sickle cell gene in India is reported from Orissa, followed by Assam, Madhya Pradesh, Uttar Pradesh, Tamil Nadu and Gujarat [16]. The average frequency of sickle gene is 4.3%, while that in Orissa is 9.1% [17]. The population diversity of sickle cell haemoglobinopathy in India mainly is the result of the admixture of genes between different populations, e.g. intruders or invading groups in and around the Gulf region, from Middle Eastern countries and Africa [18–21]. Epidemiologically, children comprise 52% of sickle cell patients. The three predominant forms (SS, SB and SD) of the disease are clinically and haematologically indistinguishable [22]. The range of the disease varies from 0% to 44%. It is 0–18.5% in north east zone, 0–33.5% in the west zone, 22.5–44.4% in the central zone and 1–40% in the southern zone [23].
2. Subjects and methods 2.1. Populations The Muslims of India comprise more than 13% of the population, and they belong to various castes, based on linguistic and ethnic groups besides, having a few tribes [24]. Manipuri Muslims comprise 8.32% of the total population, according to the 2001 census. They are mostly the migrants who started coming to the state in the middle of the 16th century [25]. They have been given different clan names which in Manipuri is called Yumnak or Sagei. The term ‘‘Sagei’’ is a corrupted word of Shaqzi which is an Urdu terminology. About 74 clans are reported in Manipur in the present times. Manipuri Muslims belong to Sheikh, Syed, Pathan or Mughal castes [26], they belong to Sunni sect only. The Meiteis represent the major tribal group, having 60% of the total population [27] and they follow Hindu religion. Meiteis are presumably formed by the admixture of Koomal, Looang, Moirang and Meitei, all of whom are reported to have arrived at different periods of time, coming from different directions and now represent the clans of the community [25]. While the Naga are the indigenous tribal population of Manipur, they belong to the Naga–Kuki–Chin group of the Tibeto-Burman linguistic family and are believed to have migrated to Manipur from Burma probably 300–400 years ago [28].
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur
Figure 1
325
Map of Manipur.
2.2. Sample collection Blood samples of five hundred and seventy-seven (577) individuals were collected from unrelated individuals belonging to both sexes from the following population groups; Muslims having castes: Sheikh, Syed, Pathan and Mughal, besides a Hindu (Meitei) and a tribal group (Naga), through taking prior informed consent from the individuals, taking into consideration the factors like caste, consanguinity, age, religion, etc. The survey was conducted among healthy individuals from the area of Imphal East and Imphal West districts during house to house visit, which were selected at random on a priori basis during the day time with the help of volunteers. Some samples were also collected from the schools by taking prior permission from the respective headmasters. Blood samples were also collected from Manipuri students studying in Aligarh Muslim University, Aligarh. The blood samples were collected by finger-prick method and were tested for sickle cell trait by the wet sealed method using 2% freshly prepared sodium metabisulphite solution following the method of Daland and Castle [29]. 2.3. Genetic data analysis The phenotypes were recorded for the sickle trait for each sample, and the allele frequencies were calculated according to the
Hardy–Weinberg law [30] using a gene counting method. The level of heterozygosity was calculated using the formula: X Heterozygosity ¼ 1 Ho P where Ho is the homozygosity of the allele, Ho = Pi2. Chi-square test: It is used for the measurement of the size of the discrepancy between the observed and expected values at particular degrees of freedom: v2 ¼
X ðObserved ExpectedÞ2 Expected
3. Results 3.1. Phenotypic frequency The Mughal population shows the highest frequency of sickle cell trait, i.e., 31.03% while Naga shows the least frequency of 16.46%. Among males Meitei shows the highest frequency of 30.43%, while Pathan shows the least, i.e., 7.7%. While among females Mughal population shows the highest frequency of 36.36%, and the Meitei shows the least frequency, i.e., 26.09%. The chi-square (v2) value of normal individuals is 10.993 (df = 5, p = 0.0515) while for carrier individuals it is 14.574 (df = 5, p = 0.0123) (Tables 1–3), the difference is statistically significant.
326 Table 1
A. Shah et al. Phenotypic distribution of sickle cell trait in Manipur.
Populations
Normal
Sheikh Syed Pathan Mughal Meitei Naga Total
Sickle carrier
Total sample
M
F
C
M
F
C
M
F
C
78 30 24 26 32 30 220
56 45 22 14 34 36 207
134 75 46 40 66 66 427
28 5 2 10 14 3 62
28 18 12 8 12 10 88
56 23 14 18 26 13 150
106 35 26 36 46 33 282
84 63 34 22 46 46 295
190 98 60 58 92 79 577
M, F and C represent male, female and combined, respectively.
Table 2
Phenotypic frequency of sickle cell trait in Manipur.
Populations
Sheikh Syed Pathan Mughal Meitei Naga Mean ± S.E.
Normal
Carrier
Total sample
M
F
C
M
F
C
M
F
C
73.58 85.71 92.31 72.22 69.57 90.91 80.72 ± 4.13
66.7 71.43 64.71 63.64 73.91 78.26 69.78 ± 2.34
70.53 76.53 76.67 68.97 71.74 83.54 74.66 ± 2.19
26.42 14.29 7.7 27.78 30.43 9.1 19.29 ± 4.12
33.3 28.57 35.29 36.36 26.09 27.14 31.13 ± 1.80
29.47 23.47 23.3 31.03 28.26 16.46 25.33 ± 2.19
55.79 35.71 43.3 62.07 50.00 41.77 48.11 ± 3.98
44.21 64.29 56.6 37.93 50.00 58.23 51.88 ± 3.97
32.93 16.98 10.4 10.5 15.94 13.69 16.74 ± 3.42
S.E. represents the standard error. M, F and C represent the male, female and combined, respectively.
3.2. Allele frequency The highest allele frequency of sickle cell gene was found among Mughals, being 16.96%, and the least among Nagas 8.6%. Among males, Meiteis show the highest allele frequency of sickle cell gene, i.e., 16.59% and the least was shown by Pathan, i.e., 3.9%. Among females, Mughals show the highest allele frequency, i.e., 20.23% and the least was shown by Naga, i.e., 11.53% (Table 4, Fig. 2). 3.3. Heterozygosity The heterozygosity condition was found to be highest among Mughals, i.e., 28.12%, while least frequency was shown by Naga, i.e., 15.72%. Among males, again highest heterozygosity condition was shown by Meitei, i.e., 27.68% and the least by Pathan, i.e., 7.49%. Among females, the highest heterozygosity condition was found among Mughals, i.e., 32.27% and the least was among the Nagas, i.e., 20.40% (Tables 5 and 6, Fig. 3). 4. Discussion Our present study provides a comprehensive database on the occurrence of sickle cell trait in the Manipuri population especially with regard to Manipuri Muslims. The knowledge of the prevalence of the trait in this religion is important, since they perform endogamous marriage among themselves and marriages between the carriers of this disease might result in the birth of children with sickle cell disorder. In the present study, the percentage of sickle cell carrier was found to be 25.99%. The percentages of sickle cell carriers were found to be 11.7% in a community based study of inmates of the Central jail, Raipur [31]. In Central India, the
prevalence of sickle cell trait (SCT) has been reported to be 11.1% [32]. The Indian Council of Medical Research conducted a multicentric study of tribal people in four states (Maharashtra, Gujarat, Orissa, and Tamil Nadu) focusing on nutritional status, anaemia and frequency of the sickle cell and b-thalassaemia traits. The result shows sickle cell trait to be 29% [33–35]. In a study by Balgir [36] it was seen that the most common haemoglobin disorders observed among 1015 cases of sickle cell trait were found to be 29.8%. The females overall show a higher percentage of frequency for sickle cell carrier, i.e., 29.83% when compared to males, who shows around 21.99%. Heterozygosity condition for the trait is found to be high, i.e., 24.05%, with females showing 41.86% while males have 34.31%. With regard to the sex distribution of the disorder, Wintrobe [37] stated that the sickle cell trait is more common among females, though some other authors did not find any such correlation [38,39]. In the present study, this correlation exists. Among Muslims, the Mughal caste shows the highest percentage of sickle cell carrier while Pathan shows the least, again among the six populations studied, Mughal population shows the highest phenotypic frequency, allelic frequency as well as heterozygosity condition. The Plasmodium falciparum causes a malignant form of malaria, the much virulent form which is highly prevalent in Manipur. Chishti et al. [40] confirmed the prevalence of severe and complicated P. falciparum malaria in Manipur and documented the necessity of malarial research in this region. Higher percentage of sickle trait carrier in this region might have resulted mainly from the natural selection process in response to the high prevalence of P. falciparum malaria in the region. Studies have shown that heterozygotes are less susceptible to infection by malarial parasites in comparison to the homozygotes, which are more prone to the parasite. The infected cells tend to sickle and
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur Table 3
327
Observed and expected values of sickle cell trait in Manipur.
Populations
Normal
Sickle carrier
Total sample
M
F
C
M
F
C
M
F
C
Sheikh Obs Exp
78 69.0
56 64.9
134
28 23.1
28 32.8
56
106 92.86
84 97.14
190
Syed Obs Exp
30 38.6
45 36.3
75
5 9.51
18 13.4
23
35 47.19
63 50.10
98
Pathan Obs Exp
24 23.7
22 22.3
46
2 5.79
12 8.21
14
26 29.32
34 30.68
60
Mughal Obs Exp
26 20.6
14 19.3
40
10 7.44
8 10.5
18
36 28.35
22 29.65
58
Meitei Obs Exp
32 34
34 31
66
14 10.7
12 15.2
26
46 44.96
46 47.04
92
Naga Obs Exp
30 34
36 31
66
3 5.37
10 7.63
13
33 38.61
46 40.39
79
Total Chi-square value
220 10.993
207
427
62 14.574
88
150
282 16.850
295
577
M, F and C represent male, female and combined, respectively. Obs and Exp represent observed and expected values.
are selectively destroyed by body’s immune system. Thus having sickle cell gene in heterozygous condition promotes better survival and hence might have resulted in the occurrence of a higher percentage of sickle cell carriers in Manipur. Heterozygous condition (HbAS) provides significant protection against all such mortality, i.e., due to severe malarial anaemia, and high density parasitaemia. Significant reduction in mortality was detected between the ages 2 and 16 months, the highest risk period for severe malaria, showing the role of malaria in selection and maintenance of sickle cell gene [41]. Allison [42] first suggested the relative protection of sickle cell trait against P. falciparum malaria. He noted that whereas 14 out of 15 individuals without sickle cell trait developed malaria when inoculated with P. falciparum, only 2 of 15 individuals with sickle cell trait developed malaria when similarly inoculated. Subsequently, numerous studies have confirmed this high degree of resistance to severe P. falciparum malaria [43]. Whereas the rate of severe or complicated P. falciparum malaria requires acute hospitalisation, the hospitalisation decreased by approximately 90% in individuals with sickle cell trait, there was no significant reduction in the prevalence of asymptomatic parasitaemia [43]. The protection appears to increase with age from only 20% in the first 2 years of life to a maximum of 56% by the age of 10 years and decreasing to 30% in those aged more than 10 years [43]. Proposed protective mechanisms include reduced parasite growth and enhanced removal of parasitised cells through innate and acquired immune processes [44]. The protection is, however, specific for P. falciparum malaria and, not for any of the other three types of malaria (P. vivax, P. ovale, and P. malariae) [43]. More recent studies in West Africa suggest that the greatest impact of HbS is mainly protection against either death or se-
Table 4
Allele frequency of sickle cell trait in Manipur.
Populations
Hb(p)
Hbs (q)
M
F
C
M
F
C
Sheikh Syed Pathan Mughal Meitei Naga
0.8578 0.9258 0.9608 0.8498 0.8341 0.9535
0.8165 0.8452 0.8044 0.7977 0.8597 0.8847
0.8398 0.8748 0.8756 0.8304 0.8469 0.914
0.1422 0.0742 0.039 0.1502 0.1659 0.047
0.1835 0.1548 0.1956 0.2023 0.1403 0.1153
0.1602 0.1252 0.1244 0.1696 0.1531 0.086
Total
0.7801
0.7017
0.8602
0.2199
0.2983
0.1398
M, F and C represent male, female and combined. Hb(p) and Hbs(q) are dominant and recessive alleles, respectively.
vere disease, i.e., profound anaemia or cerebral malaria, while having less effect on infection per se [41,45,46]. Indeed, casecontrol studies suggest that HbS heterozygotes have a level of protection of approximately 60–80% against the severe complications of malaria [47]. A number of mechanisms have been proposed to explain as to how HbAS gene may result in the accelerated acquisition of malaria-specific immunity. As common with other red cell genetic defects, enhanced phagocytosis of HbAS erythrocytes has been demonstrated in vitro when they are infected with ring stage of P. falciparum [48], which is a process that appears to be mediated by a mechanism essentially similar to that involved in the phagocytosis of senescent or damaged normal erythrocytes. Experimental data suggest this process is initiated by enhanced oxidant damage to the erythrocyte membrane and that this leads to the aggregation of band 3 protein and the binding of
328
A. Shah et al. 1.2
1
0.8
0.6
Hb(p) Hbs(q)
0.4
0.2
Male
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
Female Figure 2
Table 5
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
M EI TE I NA GA
SY ED PA TH AN M OG HU L
SH EI KH
0
Combined
Graph showing allele frequency of sickle cell trait in Manipur.
Genotypic frequency of sickle cell trait in Manipur.
Populations
Male 2
Female 2
2
Combined 2
p
2pq
q
p
2pq
q
p2
2pq
q2
Sheikh Syed Pathan Mughal Meitei Naga
0.7358 0.8571 0.9231 0.722 0.6957 0.9092
0.2439 0.1374 0.0749 0.2553 0.2768 0.0896
0.0203 0.0055 0.0015 0.0226 0.0275 0.0022
0.667 0.7144 0.6471 0.6363 0.7391 0.7827
0.2997 0.2617 0.3147 0.3227 0.2412 0.2040
0.0337 0.024 0.0383 0.0409 0.0147 0.0133
0.7052 0.7653 0.7667 0.6895 0.7172 0.8354
0.2691 0.2190 0.2178 0.28117 0.2593 0.1572
0.0256 0.0157 0.0155 0.0288 0.0234 0.0074
Total
0.6086
0.3431
0.0484
0.4924
0.4186
0.0889
0.7399
0.2405
0.0195
p2, 2pq and q2 are dominant, heterozygous and recessive genotypic frequencies.
Table 6 Homozygosity and heterozygosity of sickle cell trait in Manipur. Populations
Male
Female
Combined
Ho
Ht
Ho
Ht
Ho
Ht
Sheikh Syed Pathan Mughal Meitei Naga
0.761 0.8626 0.9251 0.7447 0.7232 0.9104
0.2439 0.1374 0.0749 0.2553 0.2768 0.0896
0.7003 0.7383 0.6853 0.6773 0.7588 0.796
0.2997 0.2617 0.3147 0.3227 0.2412 0.2040
0.7309 0.781 0.7822 0.7188 0.7407 0.8428
0.2691 0.2190 0.2178 0.2812 0.2593 0.1572
Total
0.6569
0.3431
0.5814
0.4186
0.7595
0.2405
Ho and Ht represent homozygosity and heterozygosity, respectively.
autologous IgG and complement system [48], a mechanism similar to that previously proposed for a thalassaemia [49]. It, therefore, seems plausible that enhanced immunity could be mediated by the accelerated acquisition of antibodies to altered host antigens expressed on the parasite-infected red cell surface, such as band 3 protein [50]. On the other hand, parasite-derived proteins such as the variant surface antigen P. falciparum eryth-
rocyte membrane protein-1 might represent an alternative target, a hypothesis supported by the raised titres of antibodies directed towards variant antigens seen in HbAS children living in Gambia [51]. These mechanisms need not be mutually exclusive. As an alternative explanation, it seems possible that by controlling parasite densities, during malarial infections, innate processes might paradoxically increase the chronicity of individual infection [52]. The heterozygosity condition might have increased in Mughal population in response to the high occurrence of sickle cell disease, and these results shows a clear example of natural selection occurring in the population, if this condition had not arisen it would have led to wiping up of the population from the region. In our present study, however, no case of disease has been reported. These might be probably due to the early death of individuals having homozygous state for sickle cell gene. High prevalence of malaria in this region has caused a major health problem for the population. Large scale screening of the population is very much needed since the region shows high occurrence of the trait. The parasite carrier may serve as endemic carrier for the source of infection for a long time since malaria in such individuals is rarely identified and they receive no treatment. Yet, the infectivity of gametocytes in the
Gene frequency of sickle cell trait among Muslim populations in a malarial belt of India, i.e., Manipur
329
1 0.9 0.8 0.7 0.6 0.5 Ho
0.4
Ht
0.3 0.2 0.1
Male Figure 3
Female
NA GA
M EI TE I
SY ED PA TH AN M OG HU L
SH EI KH
NA GA
M EI TE I
SY ED PA TH AN M OG HU L
SH EI KH
M EI TE I NA GA
PA TH AN M OG HU L
SY ED
SH EI KH
0
Combined
Graph showing homozygosity and heterozygosity of sickle cell trait in Manipur.
immune individuals appears to be less marked than in the nonimmune individuals [52]. Sickle cell disorder has remained a neglected field of research in this country and the magnitude of problem has never been appreciated in spite of the fact that the sickled RBCs were detected in the blood of Indian patients as early as 1952. This was largely because most of the subsequent reports spread a misconception that the sickle gene in India was confined to the tribal population or some scheduled castes only [53]. In the present study, only six population groups have been taken up, but the results show that even non-tribal groups show the presence of sickle cell gene. However as the region is inhabited by a large number of tribal populations, screening for this trait must be encouraged in Manipur. Since the tribal populations are mostly confined to hilly regions of Manipur and they get limited health service, and education, screening and prenatal counselling for the disease in these areas can help a lot in minimizing the occurrence of the sickle cell disease and, thus can help them to make informed choices and avoid the birth of sickle cell children. The awareness of the disease and its implication for the public health problem, though have been increasing in recent days, yet this awareness is mostly confined to the urban regions of Manipur valley areas and, therefore the people residing in remote areas also need to be made aware of the disease. 5. Conclusion The study highlights the prevalence of the sickle cell trait among six populations from different regions of the state. Since our present finding shows the high occurrence of the trait in the region, effective large scale screening in other populations is the need of the hour. The disease is not curable and causes high degree of morbidity and mortality among the individuals suffering from the disease. Thus there is an urgent need for special intervention strategies for prevention and control of the disease in the areas supposed to be affected with endemic malaria. Conflict of interest The authors declare no conflict of interest.
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