Demographic and clinical parameters
A total of 1584 samples were included in this study, comprising of 344 DN cases, 1240 non-diabetic nephropathic controls (NDN) [970 healthy controls which are non-nephropathic and non-T2D (NDNT) and 270 were T2D without Nephropathy disease controls]. Baseline characteristics of the DN cases and control groups are shown in Table 1. It was observed that males were more affected with DN as compared to females. The family history of T2D was observed to be the highest among the T2D without Nephropathy controls. Alcohol consumption and smoking were observed to be highest in T2D without Nephropathy (Data not shown) controls while, BMI was observed to be highest in T2D without Nephropathy controls and lowest in DN cases.
The clinical parameters- BMI, SBP and RBS showed significant difference in all the three comparisons except DBP which showed the significant difference only in DN cases vs. T2D without Nephropathy controls. In case of biochemical parameters- CHO, LDL, urea and creatinine showed significant difference in all three comparisons, whereas, TriG and VLDL showed non-significant difference when DN cases were compared with NDNT controls. In univariate analysis for continuous parameters, it was observed that except for age, DBP and HDL, all other parameters (BMI, SBP, CHO, TriG, VLDL, LDL, serum urea, serum creatinine and RBS) were significantly associated with DN. However, when multivariate analysis was performed on the significantly associated parameters, only TriG, LDL and serum creatinine showed significant association with DN. The significance level was retained even after Bonferroni correction (corrected p-value = 0.0055, Supplementary Table S1). Univariate analysis and multivariate regression of binary parameters revealed that alcohol, physical inactivity, diet and gender retained their significant association with the disease after Bonferroni correction, (corrected p-value = 0.01, Supplementary Table S2).
Principal Component Analysis (pca)
Demographic, anthropometric and biochemical parameters constitute a major component responsible for the development of the disease. Thus, principal component analysis (PCA) was performed to highlight the role of demographic (age, gender, alcohol, smoking and dietary patterns), anthropometric (BMI) and biochemical (CHO, TriG, HDL, VLDL, LDL, urea, creatinine, RBS) factors in the development and progression of DN (Supplementary Table S3). The contribution of each factor in the development of the disease is shown in Supplementary Table S4. PCA revealed five factors i.e. Factor 1 (triglyceride and VLDL); Factor 2 (CHO and LDL); Factor 3 (SBP and DBP), Factor 4 (Serum Urea and Creatinine) and Factor 5 (BMI), contribute (~ 70%) to the DN development (Supplementary Table S3).
Genotype and allele distribution of ELMO1 SNPs
The allele and genotype frequencies of five SNPs in ELMO1 gene and their association with DN are shown in the Table 2.
rs741301 (T > C). The comparison of DN cases vs. NDN controls revealed that rs741301 (T > C) was significantly associated (p-value = 1.27 × 10− 5) with DN. A significant association [p-value = 1.92 × 10− 6, OR = 1.52 (1.28–1.81)] was observed for C-allele distribution, which conferred 1.52 fold risk towards DN. rs741301 showed significant association (p-value = 1.49 × 10− 6) with DN, when DN cases were compared with NDNT controls. The allelic distribution also showed significant association with DN [p-value = 1.74 × 10− 7, OR = 1.61(1.35–1.93)] and conferred 1.61 fold risk towards DN. Further, the comparison in DN cases with T2D without Nephropathy rs741301 did not show any significant association with DN. The SNP rs741301 followed the recessive model and C-allele was observed to be significantly associated with DN when compared with NDN and NDNT control groups in the studied population, respectively. It revealed that recessive model genotype, CC conferred 1.83 fold risk [p-value = 7.03 × 10− 5, OR = 1.83(1.35–2.47)] and 1.97 fold risk [p-value = 2.01 × 10− 5, OR = 1.97(1.44–2.71)] towards DN when compared DN vs. NDN controls and NDNT controls respectively. However, no significant association of the SNP with DN was observed when the DN cases were compared with T2D without Nephropathy (Table 2).
rs7799004 (T > C). On comparing DN cases vs. NDN controls, rs7799004 was observed to be significantly associated (p-value = 7.06 × 10− 12) with DN. The distribution of C-allele frequency also showed significant association and conferred 1.55-fold risk [p-value = 1.33 × 10− 6, OR = 1.55(1.30–1.85)] towards DN development. The comparison of DN cases with NDNT controls revealed that rs7799004 was significantly associated (p-value = 1.50 × 10− 13) with DN. The allele frequency distribution also showed significant association with DN [p-value = 8.51 × 10− 8, OR = 1.65(1.37–1.98)] and C-allele was observed to confer 1.65-fold risk towards DN. The comparison of genotypes in DN cases and T2D without Nephropathy controls revealed that rs7799004 was significantly associated (p-value = 0.0071) with DN. Model analyses revealed that, CC genotype showed 3.13 fold risk [p-value = 9.37 × 10− 13, OR = 3.13(2.26–4.34)], 3.76 fold risk [p-value = 1.39 × 10− 14, OR = 3.76(2.64–5.36)] and 1.92 fold risk [p-value = 0.0033, OR = 1.92(1.24–2.97)] towards DN development respectively.
rs1882080 (G > A). On comparing DN cases with NDN controls a significant association was observed (p-value = 6.79 × 10− 6) with DN. The A-allele distribution also observed to be significantly associated with DN [p-value = 0.001, OR = 1.34(1.13–1.58)] and provided 1.34-fold risk towards DN. On comparing DN cases with NDNT controls it was observed that rs1882080 was significantly associated (p-value = 1.72 × 10− 5), with DN. The allele frequency distribution also reflected a significant risk associated [(p-value = 0.0007, OR = 1.35(1.14–1.62)] with DN. On comparing genotypes of DN cases and T2D without Nephropathy controls it was observed that rs1882080 was significantly associated with DN (p-value = 4.35 × 10− 7). The model analyses showed that genotype AA showed approximately 2 folds risk in all the three comparisons viz. [p-value = 1.33 × 10− 6, OR = 2.01(1.51–2.68)], [p-value = 3.13 × 10− 6, OR = 2.01(1.49–2.72)] and [p-value = 5.08 × 10− 5, OR = 2.0(1.43–2.81)] respectively.
rs11769038 (G > T). The comparison of DN cases vs. NDN controls revealed that rs11769038 was significantly associated (p-value = 0.0006) with DN. The G-allele frequency distribution also showed significant association [p-value = 0.022, OR = 1.27(1.03–1.56)] and provided 1.27-fold risk towards DN. The comparison of DN cases vs. NDNT controls revealed that rs11769038 was significantly associated (p-value = 1.19 × 10− 5) with DN. The allele frequency distribution showed significant association with DN [p-value = 1.12 × 10− 5, OR = 1.59(1.29–1.95)] and G-allele provided 1.59-fold risk towards DN. The genotypic comparison of DN cases vs. T2D without Nephropathy controls revealed that rs11769038 was significantly associated with DN (p-value = 5.92 × 10− 11). The distribution of allele showed significant association [p-value = 1.22 × 10− 7, OR = 2.48(1.76–3.51)] and T-allele was observed to confer 2.48-fold risk towards DN development. The model analyses revealed that genotype GG conferred 1.40-fold risk [p-value = 0.0028, OR = 1.40(1.14–1.92)] towards DN development only when compared DN cases vs. NDNT controls.
rs1345365 (G > A). The evaluation of DN cases with NDN controls revealed that rs1345365 showed significant association (p-value = 1.31 × 10− 9) with DN development. Allele frequency was also observed to be associated with DN [p-value = 7.45 × 10− 13, OR = 2.02(1.66–2.46)] and G-allele provided 2.02-fold risk towards DN. The comparison of DN cases vs. NDNT controls revealed that rs1345365 showed significant association (p-value = 5.61 × 10− 20) with DN development. The allele frequency distribution showed significant association [p-value = 3.84 × 10− 24, OR = 2.73(2.24–3.33)] and A-allele provided an increased risk of 2.73-fold towards DN. The comparison of genotypes in DN cases and T2D without Nephropathy controls revealed significant association (p-value = 7.29 × 10− 4) with DN development. Allelic association revealed significant association [p-value = 2.38 × 10− 15, OR = 5.02(3.27–7.71)] with DN and A-allele conferred 5.02-fold risk towards the development of DN. Under model analyses recessive model genotype GG conferred 2.02 fold risk [p-value = 5.82 × 10− 6, OR = 2.02(1.56–2.61)], [p-value = 1.28 × 10− 18, OR = 3.24(2.48–4.24)] conferred 3.24 fold risk and 3.83 fold increased risk [p-value = 0.0015, OR = 3.83(1.58–9.30)] towards DN development respectively towards the development of DN. In this group ELMO1 polymorphisms significantly associated with DN and retained the level of significance even after Bonferroni correction (p-value = 0.0033), while rs7799004, lost its significance after correction.
Haplotype Analyses
ELMO1 gene haplotype frequencies (rs11769038, rs1882080, rs741301, rs7799004, and rs1345365) for all the compared groups are given in the Table 3. The sequence of haplotypes is in the direction of rs11769038, rs1882080, rs741301, and rs7799004. The polymorphism rs1345365 was not in LD with any of the other polymorphism thus was omitted from the haplotype analysis. The haplotypes G-G-C-C and G-A-T-T provided 2.40 fold [OR = 2.40(2.0-2.87)] and 2.17 fold [OR = 2.17(1.76–2.68)] risk with p-value of 1.09 × 10− 22 and 1.21 × 10− 13 for DN, respectively. In the comparison of DN cases vs. NDNT controls the haplotype G-G-T-T [p-value = 5.31 × 10− 26, OR = 0.089(0.05–0.15)] attributed protection towards DN whereas, haplotype T-A-T-T (p-value = 0.21) did not show any association with DN. It was observed that haplotypes G-G-C-C and G-A-T-T were significantly associated with high risk for DN with odds ratio of 2.54 95%CI = 2.11–3.06) and 3.38 (95%CI = 2.67–4.27) with p-value of 1.49 × 10− 23 and 2.48 × 10− 26 respectively when compared with NDNT control group. However, the haplotypes T-A-T-T [p-value = 6 × 10− 4, OR = 0.70(0.57–0.86)] and G-G-T-T [p-value = 3.59 × 10− 27, OR = 0.08 (0.05–0.15)] attributed protection towards DN (DN vs. NDNT). While comparing DN cases with T2D without Nephropathy controls, haplotypes G-G-C-C [p-value = 1.8 × 10− 8, OR = 2.05(1.59–2.64)] and T-A-T-T [p-value = 8.3 × 10− 9, OR = 3.27(2.15–4.98)] presented a 2.05 fold and 3.27 fold increased risk towards the development of DN, respectively. The haplotype G-A-T-T [p-value = 6 × 10− 4, OR = 0.70(0.57–0.86)] was attributing protection against DN. Haplotype G-G-T-T did not show significant association [p-value = 0.31, OR = 0.87(0.68–1.13)] with DN (Fig. 2).
Rna Secondary Structure Prediction
Elucidation of RNA structure and the access to correctly annotated RNA structure is of great importance, especially in the predictions of its secondary and 3D structures 28. Protein secondary structure and initiation codon in the mRNA are known to influence the translation efficiency 29. Therefore, in the present study, the RNA secondary structure of the wild allele and mutant allele of the ELMO1 gene polymorphisms with reference allele were analysed. There was a slight reduction in the energy of the wild type allele as compared to the variant type allele as shown in the Fig. 3. The changes in their structure of wild type and mutant are shown in the enclosed circle. There is a slight decrease in the free energy for each polymorphism, thus causing the change in the secondary structure of RNA after folding.