Bhaskar V.K.S. Lakkakula

Functional PstI/RsaI Polymorphisms in the CYP2E1 Gene among South Indian Populations

Human cytochrome P4502E1 (CYP2E1) is a well-conserved xenobiotic-metabolizing enzyme expressed in liver, kidney, nasal mucosa, brain, lung, and other tissues. CYP2E1 is inducible by ethanol, acetone, and other low-molecular weight substrates and may mediate development of chemically-mediated cancers. CYP2E1 polymorphisms alter the transcriptional activity of the gene. This study was conducted in order to investigate the allele frequency variation in different populations of Andhra Pradesh. Two hundred and twelve subjects belonging to six populations were studied. Genotype and allele frequency were assessed through TaqMan allelic discrimination (rs6413419) and polymerase chain reaction-sequencing (-1295G>C and -1055C>T) after DNA isolation from peripheral leukocytes. The data were compared with other available world populations. The SNP rs6413419 is monomorphic in the present study, -1295G>C and -1055C>T are less polymorphic and followed Hardy-Weinberg equilibrium in all the populations studied. The -1295G>C and -1055C>T frequencies were similar and acted as surrogates in all the populations. Analysis of HapMap populations data revealed no significant LD between these markers in all the populations. Low frequency of CYP2E1 c2 could be useful in the understanding of south Indian population gene composition, alcohol metabolism, and alcoholic liver disease development. However, screening of additional populations and further association studies are necessary. The heterogeneity of Indian population as evidenced by the different distribution of CYP2E1 c2 may help in understanding the population genetic and evolutionary aspects of this gene.

Association Between the M268T Polymorphism in the Angiotensinogen Gene and Essential Hypertension in a South Indian Population

Essential hypertension is a complex multifactorial disease caused by interactions between genetic and environmental factors. It is an independent determinant of cardiovascular risk. The main aim of this study was to investigate the possible influence of angiotensinogen M268T polymorphisms on hypertension in two endogamous caste populations of South India. Systolic and diastolic blood pressure, anthropometric variables, and lipid profiles were assessed. Direct sequencing of PCR products was adopted for genotyping. This polymorphism was found to be in Hardy–Weinberg equilibrium in the patients and controls of both populations. Binary odds ratios showed significant association between the M268T polymorphism and hypertension in both populations. Multivariate analysis revealed significant differences in body mass index, chest girth, calf circumference, skinfold measurements, total cholesterol, and triglyceride levels between these genotypes in the Gavara and Vaishya populations. These data further support the hypothesis that hypertension is influenced by the AGT M268T polymorphism.

Gene Frequencies of the Human GSTT1 (Null Allele) and GSTP1 (Ile105Val) Polymorphisms among South Indian Populations

Background: Glutathione S-transferases (GSTs) are members of the phase II biotransformation enzymes that play a key role in cellular detoxification of chemical carcinogens and xenobiotics. Variations at GST genes have been reported in different human populations, and some association studies have reported increased risk for cancers and other disease end points. The present study was conducted to investigate the allele frequency variations in south Indian populations. Methods: GSTT1 null allele and GSTP1 Ile105Val polymorphisms were genotyped in two hundred and twelve subjects (aged 34 to 60 years old) belong to six populations using PCR and PCR-RFLP techniques respectively. Results: Both GSTT1 ins-del and GSTP1 Ile105Val are polymorphic in all populations. GSTP1 Ile105Val followed the Hardy-Weinberg equilibrium. The GSTT1 null allele frequencies ranged from 11.6% to 22.2% and GSTP1 Ile105Val “Val” allele frequency ranged from 20.0% to 38.2% in the study populations. HapMap data showed the highest frequency of Val105 allele in African populations followed by European populations. East Asian populations showed the lowest frequency of Val105 allele. Conclusion: The variations observed in allelic distribution of GST genes may presumably be due to the selective pressure exerted on populations of that region. In conclusion, the present study reports the frequency of GSTT1 null allele and GSTP1 Ile105Val polymorphisms in Indian populations which provides foundation for potential epidemiological and clinical studies.

A novel polymorphism in codon 25 of the KRAS gene associated with gallbladder carcinoma patients of the eastern part of India.

Gallbladder cancer (GBC) is more prevalent than other cancers in North India. The asymptomatic nature of the disease is a problem in the diagnosis and treatment. Analysis of oncogenes or tumor suppressor genes could be of importance in this regard. KRAS is the most frequently mutated member and is said to be one of the most activated oncogenes. The present study was aimed to determine the role of intragenic variants in the KRAS gene, in the progression of GBC in the eastern part of India. Sixty gallbladder carcinoma subjects (13 men and 47 women) with histologically proven diagnosis and 90 individuals (14 men and 76 women) who have no diagnosed cancer were included in the present study. All single-nucleotide polymorphisms present in exons 1 and 2 were analyzed by polymerase chain reaction followed by sequencing. We could not find the most frequently reported mutations at codons 12, 13, and 61 of the KRAS gene that occur in human malignancies. However, in this study, we detected one novel polymorphism at codon 25 (CAG>CAT; Gln25His) in exon 1 of the KRAS gene in both germline and tissue DNA. Multivariable logistic regression analysis with adjustment for age and sex revealed that the Gln25His variant of the KRAS gene was significantly associated with GBC. In silico analysis has validated the KRAS p.Q25H polymorphism as a disease-causing variant. Further, screening of the DNA samples in a cohort of ancestral tribal populations from various parts of the country without information on the phenotype, however, revealed the presence of the previously reported codon 12 and 25 polymorphisms, thereby indicating that the novel variant is population specific in the region.

NOS3 tagSNPs does not modify the chronic kidney disease progression in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary and progressive renal disorder. It is also recognised as the most frequent genetic cause of chronic kidney diseases (CKD). In the present study, four tagging SNPs and two more well studied polymorphisms (Intron 4 VNTR and Glu298Asp) the NOS3 gene were investigated to unravel the potential modifier effect of NOS3 gene on the progression of CKD in ADPKD.

NAT2 genetic variations among South Indian populations

The N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes involved in the metabolism of drugs, environmental toxins and the aromatic amine carcinogens present in cigarette smoke. Genetic variations in NAT2 have long been recognized as the cause of variable enzymatic activity or stability, leading to slow or rapid acetylation. In the present study, we genotyped three single-nucleotide polymorphisms (SNPs) from the NAT2 gene (rs1799929, rs1799930 and rs1799931), using TaqMan allelic discrimination, among 212 individuals from six major South Indian populations and compared the results with other available Indian and worldwide data. All three of the markers followed Hardy–Weinberg equilibrium and were highly polymorphic in the studied populations. The constructed haplotypes showed a high level of heterozygosity. All of the populations in the present study commonly shared only four haplotypes out of the eight possible three-site haplotypes. The haplotypes exhibited fairly high frequencies across multiple populations, where three haplotypes were shared by all six populations with a cumulative frequency ranging from 88.2% (Madiga) to 97.0% (Balija). We also observed a tribal-specific haplotype. A strong linkage disequilibrium (LD) between rs1799929 and rs1799930 was consistent in all of the studied populations, with the exception of the Madiga. A comparison of the genomic regions 20-kb up- and downstream of rs1799930 in a large number of worldwide samples showed a strong LD of this SNP with another NAT2 SNP, rs1112005, among the majority of the populations. Moreover, our lifestyle test (hunter–gatherer versus agriculturist) in comparison with the NAT2 variant suggested that two of the studied populations (Balija and Madiga) have likely shifted their diet more recently.

Lack of Association of EPHX1 Genotypes and Haplotypes with Oral Cancer in South Indians

As genetic variation is thought to contribute to the etiology of oral cancer, microsomal epoxide hydrolase (EPHX1) was chosen as a candidate gene. This study thus sought to investigate possible genetic associations between the rs1051740, rs2292566, and rs2234922 polymorphisms of EPHX1 and oral cancer. Oral cancer patients (n=157) and healthy control subjects (n=132) were screened for the genotypes using TaqMan allelic discrimination. The associations between genotypes, alleles, and haplotypes of the three mutations and oral cancer were then analyzed using a case-control study. All the three single-nucleotide polymorphisms were polymorphic, with minor allele frequencies of 0.368, 0.249, and 0.232, respectively, for rs1051740, rs2292566, and rs2234922. None of the polymorphic sites deviated from Hardy-Weinberg equilibrium. There were no significant differences in genotype or allele frequencies of three single-nucleotide polymorphisms between controls and cases with oral cancer. Of the three studied polymorphisms, two were in strong linkage disequilibrium and formed one haplotype block. None of the haplotypes showed significant association with oral cancer. EPHX1 gene polymorphisms and haplotypes were not involved in the susceptibility to oral cancer in South Indian subjects.

Evidence of the involvement of the polymorphisms near MSX1 gene in non-syndromic cleft lip with or without cleft palate.

OBJECTIVE Non-syndromic cleft lip, with or without cleft palate (NSCL/P) is a common craniofacial birth defect, characterised by an incomplete separation between nasal and oral cavities without any other congenital anomaly in humans. Several genes which play a role in cell differentiation, migration, growth and apoptosis, have been associated with clefting. The purpose of this study was to investigate the association between single-nucleotide polymorphisms (SNPs) near MSX1 gene and NSCL/P among South Indian population. METHODS A case-control analysis of five single nucleotide polymorphisms near MSX1 gene (rs11726039, rs868257, rs6446693, rs1907998 and rs6832405) was carried out in 173 patients with NSCL/P and 176 unaffected controls to determine their association with NSCL/P. RESULTS All SNPs were polymorphic in the study population. Comparisons of allele and genotype frequencies revealed that the C variant allele and the TC/CC genotypes of rs11726039 was significantly higher in controls than in the NSCL/P group (OR: 0.63; 95% CI: 0.41-0.097; p=0.037). However, neither of these findings remained significant after Bonferroni correction for multiple comparisons. The frequencies of rs868257, rs6446693, rs1907998 and rs6832405 minor alleles and genotypes were similar between the control and NSCL/P groups. No significant linkage disequilibrium (LD) was observed. Genotype-genotype interaction and the haplotype analysis did not reveal any significant association with NSCL/P. CONCLUSIONS The study results were suggestive of a positive association between MSX1 rs11726039 and NSCL/P in the South Indian population.

Two promoter polymorphisms in TBX22 are associated with the risk of NSCLP in Indian women.

The aetiology of nonsyndromic cleft lip with or without cleft palate (NSCLP) is complex and involves both genetic and environmental risk factors. Classical research has shown that growth and patterning of the developing palatal shelves depend on epithelial–mesenchymal interactions. Expression of several signalling molecules and transcription factors in the anterior palate during early palate development has been documented. TBX22 encodes a T-box containing transcription factor and mutations in this gene are responsible for X-linked cleft palate and ankyloglossia. In the present study, we analysed two TBX22 promoter rs7055763 and rs41307258 single-nucleotide polymorphisms (SNPs) in 173 patients with NSCLP and 176 normal controls of south Indian origin using Kbioscience KASPar chemistry, which is a competitive allele-specific PCR SNP genotyping system. As the SNPs are located on chromosome X, the association analysis was carried out separately in men and women. Significant associations of rs7055763 (P=0.034) and rs41307258 (P=0.022) with NSCLP were found only in women. Both polymorphisms increased the risk for NSCLP in the heterozygous and homozygous variant state, but this was not significant. Both SNPs were not associated with a risk for NSCLP in men. Pair-wise linkage disequilibrium between rs7055763 and rs41307258 was strong and significant (D′=0.97 and r2=0.77). Only three haplotypes were observed with an estimated frequency more than 5%. Haplotype AA, which carries both mutant alleles (rs7055763 A – rs41307258 A), was significantly associated with an increased risk of NSCLP in women, but not in men. Our study showed a significant role of TBX22 promoter polymorphisms (rs7055763 and rs41307258) in the pathogenesis of NSCLP and reinforces the previous findings of a strong link between X-linked genes and orofacial clefts.

Significant association of MTHFD1 1958G>A single nucleotide polymorphism with nonsyndromic cleft lip and palate in Indian population.

Objectives: Nonsyndromic cleft lip and palate (NSCLP) is genetically distinct from those with syndromic clefts, and accounts for ~70% of cases with Oral clefts. Folate, or vitamin B9, is an essential nutrient in our diet. Allelic variants in genes involved in the folate pathway might be expected to have an impact on risk of oral clefts. Given the key role of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) in folate metabolism, it would be of significant interest to assess its role in NSCLP etiology. Study Design: The present study aims at examining the association between MTHFD1 1958G>A polymorphism and NSCLP risk by conducting a case-control study in south Indian population. Our sample comprised of 142 cases with nonsyndromic clefts and 141 controls without clefts or family history of clefting. The MTHFD1 1958G>A polymorphism was genotyped using PCR-RFLP. Results: An increased risk was found for the heterozygous 1958GA (OR=2.44; P=0.020) and homozygous 1958AA (OR=2.45; P=0.012) genotypes in the children. When the dominant model (AG+AA vs GG) was applied the risk remained the same as co-dominant model, but the level of significance increased (OR=2.44; P=0.002). Conclusions: The results indicated the MTHFD1 1958G>A polymorphism to be one of the important genetic determinants of NSCLP risk in South Indian subjects. Key words:MTHFD1, orofacial cleft, SNP, genetics.