Association of ACE DD genotype with Hypertension among the tribal populations of South India.

The Renin-Angiotensin System (RAS) is an important regulator of the blood pressure(BP). The level of the vasoactive peptide Angiotensin-II, is mainly determined by the RAS enzyme, angiotensin converting enzyme-1 (ACE-1). Polymorphisms in ACE gene is reported to be associated with hypertension in various populations worldwide. We investigated the association of ACE I/D polymorphisms with hypertension among the tribal populations of South India. Samples were collected from hypertensive patients (n = 33) and healthy controls (n = 37). Genotyping was performed using Polymerase chain reaction (PCR) with allele specific primers. The DD genotype is significantly observed among the cases (


Introduction
The Indian population has been an enigmatic subject for archaeologists, historians, anthropologists and geneticists for many decades, by virtue of its enormous linguistic, historic and cultural diversity [1].The dominant and most powerful key cultural factor of the indian subcontinent is the caste system.Dating back to time immemorial, this system is characterized by its well stratified, endogamous and close-cultural values, which could strongly prohibit any exchange from outside.Genetically, this restriction significantly control the gene flow between the population subgroups, which creates specific genetic patterns, that could make the groups either susceptible or resistant to various diseases [2][3].
The angiotensin-I converting enzyme (ACE) gene is widely studied for its crucial roles in the Renin-Angiotensin system (RAS) and reported to be associated with Hypertension and other cardio and cerebro vascular diseases [22 -23].Many studies showed that polymorphisms of certain genes related to metabolism, including that of ACE, are also associated with the onset and development of Type 2-Diabetes [22, 24 -25].There is a 287 bp DNA fragment insertion (I) and /or deletion(D) polymorphism in the intron 16 of ACE gene [22,26], which was recently studied [27][28][29][30][31] in the context of its association with Hypertension and Diabetes [22, 28 -30, 32-35] in various populations worldwide.In this study, we examined the distribution of ACE I/D polymorphisms in their association with Hypertension among the tribal populations of Southern India.

Subjects and methods:
A total of 33 hypertensive patients and 37 healthy controls were included in this study, with blood pressure (BP) measured with standard mercury manometer.Subjects with BP 120/80 mmHg (SBP/DBP) were considered normal, and those with higher values (SBP ≥ 140 mmHg and DBP ≥ 90mmHg) were considered hypertensive.The selected tribal communities are Mudhuvar, Pulaiyan, Mannan and Kaniyan.Blood samples (5 ml) were collected from the subjects between the age of 15-85 years, with proper informed consents obtained.Genomic DNA was extracted from the whole blood using the Salting out method [ 36 ].The isolated DNA was suspended and stored in 10mm Tris and 0.1mM EDTA for genotyping.The polymorphic loci were genotyped (Table : 1) using the standard 30-cycle PCR.Appropriate annealing temperatures and additives were optimized for each system.The PCR protocols were followed as reported earlier [37][38].The amplicons were separated by electrophoresis.Later, the EtBr stained gels were visualized under UV.

Statistical analysis:
The software package SPSS 16.0 was used for the analysis of odds ratio (OR), involving both the genotype and allele frequencies.The analyses of allelic associations using the χ 2 test with P values <0.05 as significant, were performed using the population genetics package, POPGENE [93] Results: Association studies between genetic polymorphisms and diseases are the hallmarks for unravelling the genetic pattern of complex diseases.Studying the relationship between allelic and genotype frequencies of candidate genes among both affected and healthy subjects, to understand the genetic etiology of complex human traits, is an efficient method to elucidate their disease pathogenesis .
Henceforth , we tried to elucidate the possible association of the ACE I/D polymorphisms with Hypertension among the tribal communities of South India.The D allele was observed to be more prevalent among cases than controls, as shown in Table 2 and 3. Genotype distributions, allelic frequencies and the corresponding odds ratios (OR) were calculated for each variants as shown in Table : 4.
No significant association was observed in the ungrouped data.However, when segregated the subjects into male and female, we found that the DD homozygous genotype has a significant prevalence upon females than in males.An increase in the frequency of DD (63.6%) homozygous genotype was observed among the patients than controls (32.4%)(Table :3).As shown in Table : 4, the distribution of the DD genotype is more dominant, when compared to the ID and II genotypes.

Discussion:
The gene encoding angiotensin converting enzyme (ACE) of RAS is polymorphic and frequently reported to be associated with Hypertension [40].Epidemiological studies show that various genetic and environmental factors are involved in the pathogenesis of Hypertension.ACE is the key enzyme in the Renin-Angiotensin system, which can catalyze the conversion of Angiotensin-I to Angiotensin-II [22, 24,41].The ACE gene is located in chromosome 17q23 and contains 26 exons and 25 introns with a total length of 21 kb.The Insertion/Deletion (I/D) polymorphisms are defined according to presence or absence of the 287 bp fragment in intron 16.
Alu elements are mobile gentic elements [42].deriving their name from the Alu Ӏ restriction sites they comprise [43].ACE gene is one of the insertional polymorphisms of Alu elements [44][45].Many reports studied the Alu polymorphisms in the context of human genetics and evolution [46][47][48][49][50][51][52][53] .Recently, various studies reported the relationship between ACE gene polymorphism and Type 2 Diabetes.For example, the recently published meta-analysis [51], comprising a total of 41 studies with 4708 cases and 5368 controls, analyzed the association between ACE I/D polymorphisms and Type 2-Diabetes in Chinese population.Another report on Egyptian populations suggests that the DD genotype and the D allele are associated with hypertension and Type 2 Diabetes [55].The exact role of ACE gene in the onset of hypertension remains controversial.Woo et al, [56] reported that the ACE gene is not associated with hypertension .However, in other population-based studies, the D allele was found to be associated [56][57][58].The present study revealed the association of ACE genotypes with hypertension among the tribal population (n = 70) of the Southern region of India.In contrast to the urban populations, these tribal populations represent a genetically homogenous primitive population subgroups ,where the former is highly heterogenous in nature.Hence, studies with these demographically isolated communities could yield valuable results in the field of medical genetics and history and anthropology.In this study, we utilized the common Case-control design to analyze these associations.The DD homozygous genotype is found to be associated with hypertension.The same ACE DD genotype is also reported to be associated with diabetic nephropathy in north indian populations [59].
Since, the genetic matrix is a non-modifiable factor in the causation of diseases, greater attention should be given to modify the environmental factors.The clinicians could identify groups of patients, with the susceptible ACE genotypes that need intensive monitoring and correct the risk factors such as smoking, alcohol consumption and high BMI for an effective prevention of both onset and the development of hypertension .

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International Letters of Natural Sciences Vol. 52

Conclusion
In conclusion, a significant association of the DD genotype with hypertensive patients, especially, with females of the tribal populations was observed.With the observed association of II/DD genotype with hypertension, this study anticipates more studies with larger cohorts to extend and elucidate these associations.

Aknowledgement
Authors also acknowledge the UGC, New Delhi for finanacial support.

Table 2 : Distribution of allele frequencies in Cases and Controls stratified according to gender.
χ 2 : Chi-square with 1 degree of freedom; OR: odds ratio

Table 3 : Distribution of genotype frequencies of polymorphisms in Patients and Controls.
* Statistically significant.n: Sample size; OR: Odds ratio; CI: Confidence Interval; Ref: References.

Table 4 : Distribution of ACE I/D polymorphisms (dominant and recessive model) in Patients and Controls.
DOM:Dominant model: REC: Recessive model; n: sample size; OR: Odds ratio; CI: Confidence Interval: DOM model: Only when DD is present, the diseases would be caused, REC model: when D is in homozygous or heterozygous it will cause diseases.* Statistically significant.