|Year : 2022 | Volume
| Issue : 3 | Page : 684-689
Association of genetic polymorphism in Vitamin D receptor and Paraoxonase I genes with type II diabetes mellitus patients in rural South Western Maharashtra
Pratik P Durgawale1, Kailas D Datkhile1, Virendra C Patil2, Vasant V Devkar2, Satish V Kakade3, Sarjerao A Dabane4, Vijaykumar S Wader5
1 Department of Molecular Biology and Genetics, KIMSDU, Karad, Maharashtra, India
2 Department of Medicine, KIMSDU, Karad, Maharashtra, India
3 Department of Preventive and Social Medicine, KIMSDU, Karad, Maharashtra, India
4 Sanjeevan Hospital and Medical Research Centre, Islampur, Maharashtra, India
5 Modern Diagnostic Laboratory, Islampur, Maharashtra, India
|Date of Submission||15-Oct-2020|
|Date of Decision||28-Nov-2020|
|Date of Acceptance||10-Dec-2020|
|Date of Web Publication||2-Nov-2022|
Dr. Kailas D Datkhile
Department of Molecular Biology and Genetics, KIMSDU, Karad, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Type II diabetes mellitus (T2DM) is increasing at an alarming rate in urban as well as rural parts of India. Change in life-style, dietary habits, aging, environmental factors contribute to developing this disorder, T2DM is a polygenic disorder which involves large number of genes interacting with each other and the environment to manifest itself phenotypically. Aims and Objectives: In the present study, we investigated the association of Vitamin D receptor (VDR) and Paraoxonase I (PON I) gene polymorphisms with T2DM patients among rural population in South western Maharashtra. Materials and Methods: The study groups included healthy non-diabetic control group (n = 120), non-obese diabetic group (n = 120), obese diabetic group (n = 120) from the same geographical region. The single nucleotide polymorphisms (SNPs) amongst these groups were studied using Polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) for VDR and PON I genes. Results and Conclusion: Amongst the studied genes VDR SNP rs1544410 [OR 0.24 (0.1-0.63); P 0.005] and Paraoxonase I SNP rs854560 (OR 0.47 (0.26-0.84); P 0.015) had a protective effect against T2DM for non-obese individuals. Whereas only VDR SNP rs1544410 [OR 0.025 (0.003-0.91); P 0.001] had a protective effect in case of obese individuals. The findings suggest greater gene-gene interaction and gene-environment interactions influence the phenotypic outcomes of genetic polymorphisms.
Keywords: Paraoxonase I, polymerase chain reaction-reaction-restriction fragment length polymorphism, type II diabetes mellitus, Vitamin D receptor
|How to cite this article:|
Durgawale PP, Datkhile KD, Patil VC, Devkar VV, Kakade SV, Dabane SA, Wader VS. Association of genetic polymorphism in Vitamin D receptor and Paraoxonase I genes with type II diabetes mellitus patients in rural South Western Maharashtra. J Datta Meghe Inst Med Sci Univ 2022;17:684-9
|How to cite this URL:|
Durgawale PP, Datkhile KD, Patil VC, Devkar VV, Kakade SV, Dabane SA, Wader VS. Association of genetic polymorphism in Vitamin D receptor and Paraoxonase I genes with type II diabetes mellitus patients in rural South Western Maharashtra. J Datta Meghe Inst Med Sci Univ [serial online] 2022 [cited 2023 Feb 8];17:684-9. Available from: http://www.journaldmims.com/text.asp?2022/17/3/684/360214
| Introduction|| |
Type II diabetes mellitus (T2DM) is a multifactorial disorder characterized by lowered insulin secretion by β cells of Islets of Langerhans More Details in the liver and increased insulin resistance by various tissues. Number of cases of diabetes in India has been increasing at an alarming rate. T2DM is also the most commonly occurring form of diabetes in India. In 1990, the number of cases was 26 million and grew to 65 million by 2016. The fact that India is leading the global surge in diabetes incidence puts an enormous burden on the socioeconomic development of the country. It has been noted that the changing lifestyle, migration to urban areas, low birth weight, aging, environmental and genetic factors may have mostly contributed to this rise in diabetes cases. However, considering the diversity in ethnicity, socioeconomic factors in India, few studies from the rural population of Western India are reported. Hence, there is a need to investigate the genetic and environmental factors of this population more closely.
Obesity is known to be a risk factor for diabetes, especially T2DM. T2DM is characterized by insulin resistance and reduced insulin secretion from the pancreas. As mentioned above, the changing life-style of the Indian population has contributed to rise in obesity amongst Indians., Several metabolic impairments may contribute to obesity such as genetic variations in metabolic genes. These metabolic genes may also play a role in the development of diabetes. One such gene is the Vitamin D receptor (VDR) gene, which codes for the VDR protein. This protein belongs to the nuclear hormone receptor superfamily and is expressed in the β cells of Islets of Langerhans. Upon activation by the metabolically active form of Vitamin D (Vitamin D (25[OH] D)), VDR activates the transcriptional complex that regulates the expression of insulin. VDR gene is present on chromosome 12 and is flanked by an upstream promoter region and a 3' untranscribed region (3' UTR), which houses several polymorphisms. These polymorphisms enable the expression of different forms of VDR protein, thus affecting its functionality. In the present study, three single-nucleotide polymorphisms (SNPs) (rs7975232, rs1544410, rs731236) of the VDR gene were analyzed by polymerase chain reaction reaction-restriction fragment length polymorphism (PCR-RFLP).
The Paraoxonase1 gene is located on chromosome region 7q21.3–22.1. The protein encoded by this gene PON1 is secreted mostly in the liver and is bound to the extracellular high-density lipoprotein (HDL). Its esterase activity primarily helps protect the HDL and low-density lipoprotein (LDL) cholesterol from oxidation and also protects other biomolecules from oxidative stress caused by oxidized phospholipids.,, As such greater activity of PON1 may help prevent oxidative damage in disease states such as diabetes mellitus.,,, An important SNP in this regard is the PON1-L55M (rs854560) present in the coding region.,,, This SNP rs854560 was analyzed in the present study using PCR-RFLP. The purpose of the present study was to study the association of these SNPs with obese, nonobese T2DM patients and nondiabetic controls.
| Materials and Methods|| |
Clinically diagnosed T2DM patients attending the Out-patient department and in-patient department for the Department of Medicine at the Krishna Hospital and Medical Research Centre, Karad were enrolled for the study. Based on body mass index (BMI), patient groups were classified into two groups, namely obese and nonobese (n = 120 individuals each) were enrolled in this study. One hundred and twenty individuals were enrolled as control from KMRC.
The individuals with hypothyroidism, hyperthyroidism, tuberculosis, malignancy, pregnancy, Cushing's syndrome, hypolipidemic drug were excluded from the study. After receiving informed consent from the patients and controls, 3 ml whole blood was collected in Ethylenediaminetetraacetic acid-containing vacutainer (make: BD vacutainer).
Genomic DNA extraction
Genomic DNA was extracted using blood DNA extraction kit (Make: Qiagen) following the manufacturer's instructions. The extracted DNA and blood samples were stored at −80°C until further use.
Polymerase chain reaction restriction fragment length polymorphism
Analysis of rs7975232, rs1544410, rs731236 SNPs of VDR gene
The SNPs rs7975232 and rs1544410 located in intron 8 and SNP rs731236 located in exon 9 of the VDR gene coding region were amplified by PCR (Make: Eppendorf). Following oligonucleotides and conditions were used for PCR-RFLP analysis of rs7975232 and rs731236:, Forward primer 5' CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA3,' reverse primer 5' CAC TTC GAG CAC AAG GGG CGT TAG C3.' The PCR conditions used were: 95°C for 5 min (min); 35 cycles of 95°C for 30 s (s), 60°C for 1 min, 72°C for 1 min; followed by final extension at 72°C for 10 min. After confirmation of PCR amplification, the 740 bp amplicon was digested with ApaI (rs7975232) at 37°C for 16 h (h) and analyzed on 2% agarose gel electrophoresis. The homozygous wild-type allele was undigested (740 bp), homozygous variant-type allele resulted in bands of 530 bp, 210 bp; while the heterozygous allele was digested into 740 bp, 530 bp, 210 bp fragments. For analysis of rs731236, the amplicon of 740 bp was digested with TaqI at 65°C for 16 h and analyzed on 2% agarose gel electrophoresis. The homozygous wild-type allele showed 290 bp, 245 bp, 205 bp fragments; the homozygous variant-type allele resulted in bands of 495 bp, 245 bp; while the heterozygous allele was digested into 495 bp, 290 bp, 245 bp, 205 bp fragments.
Similarly, the following oligonucleotides and conditions were used for PCR-RFLP analysis of rs 1544410:, Forward primer 5' CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA 3,' Reverse primer 5' AAC CAG CGG GAA GAG GTC AAG GG 3.' The PCR conditions were: 95°C for 5 min; 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min; followed by final extension at 72°C for 10 min. After confirmation of PCR amplification, the 825 bp amplicon was digested with BsmI at 65°C for 16 h and analyzed on 2% agarose gel electrophoresis. The homozygous wild-type allele was undigested (825 bp), homozygous variant-type allele resulted in bands of 649 bp, 176 bp; while the heterozygous allele was digested into 825 bp, 649 bp, 176 bp fragments.
Analysis of rs854560 of paraoxonase I gene
The following primers were used for PCR amplification of rs854560: Forward primer 5'-TTG AGG AAT AAG CTC TAG TCC A-3,' Reverse primer 5' GAA AGA CTT AAA CTG CCA GTC C-3' The PCR product was amplified using the following conditions: 95°C for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s; followed by final extension at 72°C for 10 min. PCR amplification resulted in 384 bp amplicon and was digested with NlaIII at 37°C for 16 h and analyzed on 2.5% agarose gel electrophoresis. The homozygous wild-type allele was undigested (384 bp), homozygous variant-type allele resulted in bands of 282 bp, 102 bp, while the heterozygous allele resulted in 384 bp, 282 bp, 102 bp fragments.
The demographic characteristics of the three study groups were analyzed with Chi-squared test by using Instat software (Make Graphpad version 3.06). The analysis of the distribution of alleles among the study groups was performed using analysis of variance and binary logistic regression modeling was used to calculate the odds ratio (OR), adjusted OR and 95% confidence intervals using SPSS Statistics software (Make IBM version 20 (IBM, Armonk, NY, USA)).
| Ethical clearance|| |
The Institutional Ethics Committee of KIMSDU, Karad, Maharashtra has approved the Research work proposed to be carried out at KIMSDU, Karad, Date : 14th Dec 2017 with Reference no KIMSDU/EIC/2017-18/338.
| Results and Discussion|| |
The demographic features of the patient and control group were analyzed using Chi-squared test and P < 0.05 was considered statistically significant. The patients and control groups were matched for age and gender. The results are represented in [Table 1].
|Table 1: Analysis of demographic features of patients and control using Chi-squared test|
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We observed that the education and economic status of the patient groups were significantly different from the control group. Vegetarian or mixed diet had similar distribution between the control group and diabetic patients. Family history of T2DM was found to be significantly different among patients and control. This observation is in accordance with the heredity of T2DM reported earlier, indicating the possibility of T2DM in offsprings is about 40% when one of the parents is diabetic and increases to about 70% when both the parents are diabetic. However, it must be noted that since T2DM is a polygenic occurrence, it doesn't follow Mendelian inheritance and is one of the challenges in determining its genetic susceptibility. Tobacco and alcohol consumption was also distributed differently and its higher occurrence in patients may be due to their mutagenic effects and impairment of liver function leading to unbalanced lipid metabolism and contribute to T2DM progression. The majority of controls were farmers who were used to rigorous work daily and may not be accustomed to exercising. Such a lifestyle was markedly different from diabetic patients who reported low physical work in their day-to-day schedule but had started exercising after physician recommendation. This may be one of the contributing factors to T2DM development and have been attributed to increase in diabetes across India.,, Other systemic diseases such as hypertension and ischemic heart disease were significantly more in T2DM patients. These associated ailments may be due to environmental factors, impaired lipid metabolism, atherosclerosis resulting from dysfunction of several common genes involved in all these disease states.
The distribution of SNPs, calculation of Odds ratio, and binary logistic regression modeling among the control group and nonobese diabetic was performed as below [Table 2].
|Table 2: Calculation of odds' ratio and adjusted odds' for SNPs among control group and non-obese diabetic patient group|
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The distribution of genotypes for rs7975232 among control and nonobese diabetics was not significantly different among control and nonobese diabetic patients. The distribution of rs731236 was similar among control and nonobese diabetic patients. rs1544410 was found to be differently distributed among control and nonobese diabetics with a negative association indicating a protective effect conferred by the variant genotype (OR 0.24 [0.095–0.63]; P 0.005). This SNP present in intron 8 of VDR is located toward the 3' UTR regulatory region of the gene. Polymorphisms in this regulatory region are known to affect mRNA stability and functional protein expression level., rs854560 was also found to be distributed differently among the control group and nonobese diabetic group and a negative association of the heterozygous genotype indicated a protective effect against T2DM (OR 0.47 [0.26–0.84]; P 0.015). This rs854560 SNP is present in the coding region of the Paraoxonase1 gene and has an effect on its activity. The variant genotype resulting in the nonsynonymous substitution of amino acid methionine by leucine has been shown to be translated into a more active form of the Paraoxonase1 enzyme. This increased activity may have a protective effect against oxidative stress in many disorders, including T2DM.
The distribution of SNPs, calculation of Odds ratio, and binary logistic regression modeling among control group and obese diabetic was as per [Table 3].
|Table 3: Calculation of odds' ratio and adjusted odds' for SNPs among control group and obese diabetic patient group|
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The SNPs rs7975232 and rs731236 were found to be similarly distributed among control group and obese diabetic patients. This indicates these two SNPs of the VDR are not associated with T2DM in obese individuals. On the other hand, rs1544410 was found to be significantly different in terms of distribution among control and obese diabetic individuals. A significant Odds ratio (OR 0.025 [0.003–0.91]; P 0.001) indicates a negative correlation between the variant genotype and T2DM among obese individuals. Since this SNP is located in intron 8 of VDR, it may affect the mRNA stability or protein expression level by altering the transcription factor binding site and provide a protective effect against T2DM for obese individuals., The SNP rs854560 was found to be similarly distributed among the control group and obese diabetic group and was not associated with the occurrence of T2DM.
An interesting point to note from the above observations is that the association of variant genotypes for SNPs rs7975232 and rs854560 were different for obese and nonobese groups. Their variant genotypes had a protective effect against the occurrence of T2DM for nonobese individuals but not for obese individuals. This effect might be seen due to gene-environment interactions, which dictate that gene expression involves a lot of other factors which have a collective phenotypic effect. It is possible that the gene products are involved in various pathways and the different factors which might be differently expressed in obese and nonobese conditions have differing outcomes.,
The VDR polymorphisms and their association with the prevalence of T2DM has been reported for a variety of populations. A North Indian study did not find any association between rs731236, rs854560 polymorphisms, and T2DM. A French study also failed to find any association between rs7975232, rs731236, rs854560 polymorphisms, and T2DM. Similarly, studies reported from Hungry, Poland, North America have not found any association between these polymorphisms and the prevalence of T2DM.,, However, a German study and South Asian study had reported a significant association of BsmI (rs854560) polymorphism and T2DM., These differences in observations may be due to population diversity. Another possibility arises due to the classification of diabetic individuals based on BMI in our study. Most of the reported studies have mentioned the mean BMI of individuals, which may include obese as well as nonobese individuals. This may lead to different outcomes as we observed the SNPs with varying association based on obesity of the subject group.
The Paraoxonase1 SNP rs854560 located in the coding region of the Paraoxonase1 gene results in substitution of the amino acid Leucine by Methionine. This alloenzyme has been found to more active in the body against oxidation of LDL and amelioration of oxidized LDL. A Japanese study has reported association of diabetic complications such retinopathy for the 55 L genotype and the protective effect of 55M genotype against such complications. This observation is in line with our results, which indicate the protective effect of the variant genotype (55M) against diabetes in nonobese individuals. However, a Swiss study reported no association of this variant in diabetic and nondiabetic individuals. Other studies from Europe indicate a protective effect of the 55M genotype and a meta-analysis study reported similar findings for its protective effect, which included 16 studies reported from Asia, Africa, and Europe.
| Conclusion|| |
It was observed that the variant genotype for VDR SNP rs1544410 and heterozygous genotype for Paraoxonase I rs854560 conferred a protective effect against T2DM for nonobese individuals; however, in the case of obese individuals only VDR SNP rs1544410 had a protective effect. Although these results are statistically significant, it must be remembered that T2DM is a multifactorial disease state involving myriad number of gene-gene and gene-environment interactions. As such, further studies on a larger cohort need to be conducted to generalize these findings.
The authors are thankful to the Krishna Institute of Medical Sciences, Deemed to be University for financial assistance. Technical support from staff members at the Department of Molecular Biology and Genetics is duly acknowledged.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]