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Indian Journal of Human Genetics, Vol. 17, No. 3, September-December, 2011, pp. 164-168 Original Article Genetic analysis of OCT1 gene polymorphisms in an Indian population Gurusamy Umamaheswaran1, Ramakrishnan G Praveen2, Annan S Arunkumar1, Ashok K Das2, Deepak G Shewade1, Chandrasekaran Adithan1 1 ICMR Centre for Advance Research in Pharmacogenomics, Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India Code Number: hg11036 DOI: 10.4103/0971-6866.92094 Abstract Background: Genetic variants of the organic cation transporter (OCT1) gene could influence interindividual variation in clinical response to metformin therapy. The genetic basis for the single-nucleotide polymorphism (SNP) of OCT1 gene has been established in other populations, but it remains to be elucidated in the Indian population. This study is focused on OCT1 gene variants rs2282143 (P341L, 1022C>T), rs628031 (M408V, 1222A>G) and rs622342 (1386C>A) frequency distributions in the South Indian Tamilian population. Keywords: Diabetes, metformin, OCT1, polymorphism Introduction Polyspecific organic cation transporters (OCTs) belong to the solute carrier SLC22 superfamily of transporters, and they translocate a wide variety of endogenous and exogenous substances of cationic nature. There are three isoforms of OCTs, namely OCT1, OCT2 and OCT3, with similar membrane topology consisting of 12 transmembrane domains. [1] Of these, OCT1 is one of the most abundantly expressed transporters in the liver and plays a major role in the hepatic uptake and renal transport of the biguanide agent, metformin. [2] Metformin is an oral antidiabetic drug, widely used for treating type 2 diabetes mellitus as first-line monotherapy. The main action of metformin is the suppression of hepatic gluconeogenesis. [3] In vivo studies have shown that the concentration of metformin in liver was greatly decreased in OCT1 gene knockout mice than in mice with normal OCT1 transporter activity. [4] The gene encoding human OCT1, also termed SLC22A1, is mapped onto chromosome 6q25.3 and consists of 11 exons spanning 37 kb. Human OCT1 gene is highly polymorphic, and numerous polymorphisms have been described in various populations leading to differences in transporter function. [2],[4] Among the many variants, nonsynonymous variants rs2282143 in exon 6 (Pro341Leu, 1022C>T) and rs628031 in exon 7 (Met408Val, 1222A>G) were the most extensively explored in relation to metformin response. More recently, it has been reported that a novel polymorphism rs622342 (1386C>A) located in an intron between exon 8 and exon 9 was associated with the glucose-lowering effect of metformin. [5] Several studies have evaluated the relationship between genetic variations of OCT1 gene and the pharmacokinetics and clinical consequences of metformin with conflicting results. [5],[6],[7] Further available data indicate substantial differences in the allele frequencies of the OCT1 gene polymorphisms between the different ethnic groups. Tamilians are Dravidians living in the southern parts of India and north eastern part of Sri Lanka. They are ethnically, linguistically and culturally related to the other Dravidian people of the subcontinent. An estimated 77 million Tamilians reside in India and around the world (Tamil people available at http://en.wikipedia.org, accessed on December 10, 2010). There are studies reporting the allele and genotype frequency of OCT1 gene polymorphism in other populations, but so far there is no data available for Indians. Hence, the present study was aimed to determine the genotype and allele frequency of OCT1 gene polymorphism in the South Indian Tamilian population. Materials and Methods Subjects: The study was carried out in 112 unrelated healthy volunteers of South Indian Tamilian origin, aged between 18 and 60 years, of either sex. This includes 62 (55.4%) women and 50 (44.6%) men. All of them were residents of South India (Tamilnadu and Pondicherry) for at least three generations. Written informed consent was obtained from all the volunteers and this protocol was approved by the Institute Ethics Committee, JIPMER. For all subjects, 5 ml of venous blood was collected using EDTA as an anticoagulant. Genomic DNA was extracted from peripheral blood leucocytes using the standard phenol-chloroform method, and the samples were stored at 4°C. Genotyping rs2282143 and rs622342 Genotyping for rs2282143 and rs622342 was carried out using a Real Time Thermocycler (ABI Prism 7300, Foster city, CA, USA) using the TaqMan SNP genotyping assay method. C__11764545_20 was used as the SNP genotyping assay ID (Applied Biosystems, Foster City, CA, USA) for rs2282143 and TaqMan custom genotyping assay was used for rs622342 polymorphism. The primers 5′-GGGTTAATAGAGAGAGAAATAATGCTCAAAA-3′ (forward) and 5′-GCCTTTCTTGATGTTTTTTTGTGGTTAGTATT-3′ (reverse) and probes VIC-TGATGAAAACTTCAAATACA-MGB and FAM-TGATGAAAACTTCCAATACA-MGB were used to detect OCT1 rs622342 SNP. The polymerase chain reaction (PCR) reaction was carried out in duplicates using 20 μl final volume that contained 10 μl of TaqMan Universal PCR master mix (2X), 0.75 μl of 20 X working stock of SNP genotyping assay and 4.5 μl of genomic DNA diluted in DNase-free water and 4.75 μl of deionised water. The thermocycler conditions included one cycle at 50°C for 2 min; one cycle at 95°C for 10 min to activate the AmpliTaq Gold polymerase followed by 40 cycles of denaturation at 92°C for 15 s and annealing/extension at 60°C for 1 min. The allelic discrimination analysis was performed using 7300 SDS software version 1.3.1. rs628031 Genotyping of rs628031 was determined by PCR followed by restriction fragment length polymorphism. PCR was performed using the following primers: 5′-TTTCTTCAGTCTCTGACTCATGCC-3′ and 5′-AAAAAACTTTGTAGACAAAGGTAGCACC-3′, respectively. The PCR conditions used for amplification were as follows. An initial denaturation at 94°C for 5 min followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 45 s and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. The amplified products (397 bp) were analyzed on 1% agarose gel followed by restriction digestion using MscI, incubated overnight at 37°C. The digested PCR products were analyzed on 8% polyacrylamide gel and identified by ethidium bromide staining. Digestion of the 397 bp amplicon resulted in either the retention of the 397 bp amplicon or complete digestion to 210 bp and 187 bp fragments, corresponding to individuals homozygous for Val/Val or Met/Met genotypes, respectively. The presence of all three fragments (397 bp, 210 bp and 187 bp) corresponded to heterozygous for Met/Val genotypes [Figure - 1]. The accuracy and reliability of all the genotyping methods were further confirmed by direct DNA sequencing (Macrogen, South Korea). Statistical analysis: Data analysis was performed using Instat Graph pad 3. The direct gene counting method was used to determine the frequency of genotypes and alleles. The genotype data was analyzed using Chi-square and Fischer′s exact test. The observed frequencies were compared with the expected frequencies and tested for the Hardy-Weinberg equilibrium. Differences in allele frequencies between Tamilian and other ethnic populations were measured using the Chi-square test and Fisher′s exact test. Linkage disequilibrium (LD) was calculated using Haploview software version 4.1. A P-value <0.05 was considered statistically significant. Results [Table - 1] illustrates the genotype and allele frequencies of OCT1 gene obtained in the study population. The genotype frequencies of rs2282143 homozygote wild type (C/C), heterozygote (C/T) and homozygote variant (T/T) were 83.0%, 16.1% and 0.9%, respectively. Forty-four individuals (39.3%) were found to be heterozygous (A/G) carriers and 68 individuals (60.7%) were homozygous for mutant type (GG), whereas no homozygous wild type (A/A) was found in the studied population regarding rs628031, while the distribution of the rs622342 genotypes was 59.0% for AA, 33.0% for AC and 8.0% for CC. The variant allele frequencies for rs2282143, rs628031 and rs622342 were observed at 8.9%, 80.3% and 24.5%, respectively. The studied polymorphisms were in Hardy-Weinberg equilibrium except the SNP rs628031. LD analysis showed that the OCT1 gene polymorphisms were not in LD. The comparisons among OCT1 allele frequencies in worldwide populations show different patterns between Asians, Africans and Europeans [Table - 2]. Discussion In recent years, OCT1 gene polymorphisms have gained considerable attention due to their role in the differential response of metformin. In this study, we addressed the frequency of three previously described polymorphisms of the OCT1 gene in 112 healthy volunteers of South Indian Tamilian population, and the data was compared with other published studies. OCT1 gene polymorphisms were found to be common, but the distribution of the variant allele frequencies showed significant interethnic variations [Table - 2]. To the best of our knowledge, this is the first study to document the frequencies of OCT1 gene polymorphisms in any of the Indian populations. The polymorphism associated with decreased transporter activity rs2282143 was detected at 8.9% in this study, which was comparable to the data obtained from Chinese (11%), Vietnamese (5.5%), [8] Asians residing in USA (11.7%) and African-Americans (8.2%). [7] On the other hand, it was lower than those observed in North East Asians from Japan (16.8%) [9] and Korea (16.7%). [8] Likewise, it was significantly higher than those reported in European Caucasians (0-2%). [7],[8] In the present study, the frequency of rs628031 genotypes was significantly (P < 0.001) lower in the Tamilian population than expected based on the Hardy-Weinberg calculations. The rs628031, Met408Val polymorphism might have accumulated over generations, which perhaps led to substantial changes in Tamilian population. Nevertheless, the frequency of 80.3% obtained for the rs628031 G allele was similar to other Asian populations previously described in Koreans (74%), [8] Japanese (81%), [9] Asians living in the USA (76.2%) and African-Americans (73.5%). [7] However, it was significantly higher than European Caucasians from the USA (59.8%), [7] Germany (57.4%) [10] and the Netherlands (60.3%). [5] The occurrence of relatively low frequency of rs2282143 and rs628031 OCT1 gene polymorphisms in Caucasians suggests that the impaired OCT1 transport activity may vary between Asians, Africans and Caucasians. The SNP rs622342 was first identified by Becker et al. in Dutch diabetic patients with a frequency of about 37%, which was higher than the Tamilians (24.5%) observed in the present study. They assessed the association of OCT1 gene polymorphism with the glucose-lowering effect of metformin, and found that the metformin therapy was less-effective in individuals with heterozygous (AC) and homozygous (CC) mutant carriers of rs622342 OCT1 gene polymorphism. [5] Later on, in another study interaction between OCT1 (rs622342) and MATE1 (rs2289669), genes were observed in relation to metformin response. [11] Furthermore, a study conducted in healthy Caucasian male subjects has shown that the polymorphisms of OCT1 were associated with increased renal clearance of metformin. [10] Recently, studies have correlated the expression level of OCT1 gene with the intracellular concentration of imatinib, an anticancer drug, and have found that chronic myeloid leukemia patients who had higher baseline expression levels of OCT1 showed greater progression-free survival rates. [12],[13] More recently, a population-based cohort study has suggested that patients with AC and CC genotypes of rs622342 polymorphism will have reduced response to anti-Parkinsonian drugs. [14] This explains that the polymorphisms of OCT1 will also show variable response to imatinib and anti-Parkinsonian drugs. Indians are more prone to diabetes, and its prevalence is becoming an enormous health burden in the country, which has gone beyond epidemic to pandemic proportions. According to the International Diabetes Federation, it has been estimated that the number of diabetic patients in India will increase from 50.8 million to 79.4 million by 2030 (Prevalence of Diabetes available at www.idf.org, assessed on December 23, 2010). In the context of India being the "diabetic capital" of the world and metformin is the most commonly used anti-diabetic drug in India, the pattern and frequency of OCT1 gene polymorphisms observed in this study becomes important indicating the prevalence of variant alleles in this region. However, it does not reveal a direct evidence for genotype-phenotype correlation. Further, information on the prevalence of OCT1 gene polymorphisms in populations of different ethnic origin may be essential in explaining the interpatient and interethnic differences in the disposition and distribution of organic cationic drugs such as metformin, imatinib and levodopa. In conclusion, our observation provides a framework for future correlation studies on the relevance of OCT1 variants in response to drugs that are substrates of OCT1. Acknowledgments The study was funded by the Indian Council of Medical Research (ICMR), New Delhi. We thank Mrs. Yogalakshmi Kathir for collecting blood samples. We are grateful to Mrs. M. Aarthi for her assistance in language editing. References
Copyright 2011 - Indian Journal of Human Genetics The following images related to this document are available:Photo images[hg11036t1.jpg] [hg11036t2.jpg] [hg11036f1.jpg] |
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