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Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

Elek Z, Németh N, Nagy G, Németh H, Somogyi A, Hosszufalusi N, Sasvári-Székely M, Rónai Z - PLoS ONE (2015)

Bottom Line: The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively.Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types.Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3' UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

No MeSH data available.


Related in: MedlinePlus

Analysis of the effect of WFS1 rs9457 SNP on miR-185 binding by luciferase reporter assay.The entire 3’ UTR region of the WFS1 gene was subcloned into the pMIR luciferase riporter vector. Transient transfection were performed in HEK293 cells, for details, see Method section. Luciferase activity values normalized to β-galactosidase activity were measured, Panel A represents average ± SD values of three independent experiments. (*** p < 0.001). The “C” luciferase construct harbored the rs9457 C allele, whereas the “G” contained the rs9457 G variant. “Seed” construct was generated by changing all seven nucleotides in the core binding site of WFS1 3’ UTR region. “Control” construct contained a different DNA-insert with the same length lacking any sequence complementary to miR-185. Panel B demonstrates the sequence alignment of miR-185 and its binding site in the WFS1 3’ UTR. Mismatches are shown by the shifted nucleotides of the miRNA, the rs9457 locus is shown by dark background.
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pone.0139519.g002: Analysis of the effect of WFS1 rs9457 SNP on miR-185 binding by luciferase reporter assay.The entire 3’ UTR region of the WFS1 gene was subcloned into the pMIR luciferase riporter vector. Transient transfection were performed in HEK293 cells, for details, see Method section. Luciferase activity values normalized to β-galactosidase activity were measured, Panel A represents average ± SD values of three independent experiments. (*** p < 0.001). The “C” luciferase construct harbored the rs9457 C allele, whereas the “G” contained the rs9457 G variant. “Seed” construct was generated by changing all seven nucleotides in the core binding site of WFS1 3’ UTR region. “Control” construct contained a different DNA-insert with the same length lacking any sequence complementary to miR-185. Panel B demonstrates the sequence alignment of miR-185 and its binding site in the WFS1 3’ UTR. Mismatches are shown by the shifted nucleotides of the miRNA, the rs9457 locus is shown by dark background.

Mentions: Putative role of the rs9457 locus on miRNA binding was analyzed by in vitro luciferase assay, as according to the prediction of the PolymiRTS[14] database, this SNP is located in the binding site of the seed sequence of microRNA-185 (miR-185). The 3’ UTR region of the WFS1 gene was subcloned in a pMIR-Report luciferase reporter vector, construct with the “G” allele at the rs9457 locus was created by site directed mutagenesis. The same approach was used to generate the “seed mutant” construct, which lacked the entire binding site of the seed sequence of miR-185. Moreover a construct without any binding site of miR-185 (referred to as “Control”) was also used. Luciferase enzyme levels were normalized to β-galactosidase activities. The lowest relative luciferase activity (35% of the “Control”) could be measured with the construct containing a “C” allele of the rs9457 polymorphism (Fig 2). Interestingly a single mismatch caused by the SNP resulted in a more, than 1.7 times increase (p < 0.05) in luciferase level, and this value was practically identical with the activity of the “seed” construct. It might be related to the finding of the in silico sequence alignment, which showed only 6 complementary bases out of the 7 nucletotides of the seed sequence in case of the “C” allele, and the “G” variant adds a further mismatch into the region (Fig 2, Panel B), resulting in a hypothetically even weaker interaction between miRNA and mRNA. The putative role of miRNA-based regulation was further analyzed by investigating the haplotype containing the rs1046320 “A”–rs1046322 “A”–rs9457 “C” variants, which are the risk alleles of diabetes (Table 5) according to our association study. Earlier we demonstrated that the rs1046322 locus altered the binding efficiency of miR-668[8], now the combined effect of miR-185 and miR-668 was assessed. Relative luciferase activities were measured in the presence or absence of any or both of miR-185 and miR-668, respectively. Presence of both miRNAs resulted in a 0.71 relative luciferase activity (p = 0.013) compared to the reference sample (without miR-668 and miR-185). Application of only miR-185 caused a somewhat smaller effect (relative activity: 0.78, p = 0.036). On the other hand miR-668 did not result in a significant decrease (relative activity: 0.89, p = 0.251) in relative luciferase level. This observation might be explained by the fact that the rs1046320 “A” allele leads to a mismatch in the binding site of the seed region of miR-668, thus its weaker binding and milder effect can be expected.


Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

Elek Z, Németh N, Nagy G, Németh H, Somogyi A, Hosszufalusi N, Sasvári-Székely M, Rónai Z - PLoS ONE (2015)

Analysis of the effect of WFS1 rs9457 SNP on miR-185 binding by luciferase reporter assay.The entire 3’ UTR region of the WFS1 gene was subcloned into the pMIR luciferase riporter vector. Transient transfection were performed in HEK293 cells, for details, see Method section. Luciferase activity values normalized to β-galactosidase activity were measured, Panel A represents average ± SD values of three independent experiments. (*** p < 0.001). The “C” luciferase construct harbored the rs9457 C allele, whereas the “G” contained the rs9457 G variant. “Seed” construct was generated by changing all seven nucleotides in the core binding site of WFS1 3’ UTR region. “Control” construct contained a different DNA-insert with the same length lacking any sequence complementary to miR-185. Panel B demonstrates the sequence alignment of miR-185 and its binding site in the WFS1 3’ UTR. Mismatches are shown by the shifted nucleotides of the miRNA, the rs9457 locus is shown by dark background.
© Copyright Policy
Related In: Results  -  Collection

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pone.0139519.g002: Analysis of the effect of WFS1 rs9457 SNP on miR-185 binding by luciferase reporter assay.The entire 3’ UTR region of the WFS1 gene was subcloned into the pMIR luciferase riporter vector. Transient transfection were performed in HEK293 cells, for details, see Method section. Luciferase activity values normalized to β-galactosidase activity were measured, Panel A represents average ± SD values of three independent experiments. (*** p < 0.001). The “C” luciferase construct harbored the rs9457 C allele, whereas the “G” contained the rs9457 G variant. “Seed” construct was generated by changing all seven nucleotides in the core binding site of WFS1 3’ UTR region. “Control” construct contained a different DNA-insert with the same length lacking any sequence complementary to miR-185. Panel B demonstrates the sequence alignment of miR-185 and its binding site in the WFS1 3’ UTR. Mismatches are shown by the shifted nucleotides of the miRNA, the rs9457 locus is shown by dark background.
Mentions: Putative role of the rs9457 locus on miRNA binding was analyzed by in vitro luciferase assay, as according to the prediction of the PolymiRTS[14] database, this SNP is located in the binding site of the seed sequence of microRNA-185 (miR-185). The 3’ UTR region of the WFS1 gene was subcloned in a pMIR-Report luciferase reporter vector, construct with the “G” allele at the rs9457 locus was created by site directed mutagenesis. The same approach was used to generate the “seed mutant” construct, which lacked the entire binding site of the seed sequence of miR-185. Moreover a construct without any binding site of miR-185 (referred to as “Control”) was also used. Luciferase enzyme levels were normalized to β-galactosidase activities. The lowest relative luciferase activity (35% of the “Control”) could be measured with the construct containing a “C” allele of the rs9457 polymorphism (Fig 2). Interestingly a single mismatch caused by the SNP resulted in a more, than 1.7 times increase (p < 0.05) in luciferase level, and this value was practically identical with the activity of the “seed” construct. It might be related to the finding of the in silico sequence alignment, which showed only 6 complementary bases out of the 7 nucletotides of the seed sequence in case of the “C” allele, and the “G” variant adds a further mismatch into the region (Fig 2, Panel B), resulting in a hypothetically even weaker interaction between miRNA and mRNA. The putative role of miRNA-based regulation was further analyzed by investigating the haplotype containing the rs1046320 “A”–rs1046322 “A”–rs9457 “C” variants, which are the risk alleles of diabetes (Table 5) according to our association study. Earlier we demonstrated that the rs1046322 locus altered the binding efficiency of miR-668[8], now the combined effect of miR-185 and miR-668 was assessed. Relative luciferase activities were measured in the presence or absence of any or both of miR-185 and miR-668, respectively. Presence of both miRNAs resulted in a 0.71 relative luciferase activity (p = 0.013) compared to the reference sample (without miR-668 and miR-185). Application of only miR-185 caused a somewhat smaller effect (relative activity: 0.78, p = 0.036). On the other hand miR-668 did not result in a significant decrease (relative activity: 0.89, p = 0.251) in relative luciferase level. This observation might be explained by the fact that the rs1046320 “A” allele leads to a mismatch in the binding site of the seed region of miR-668, thus its weaker binding and milder effect can be expected.

Bottom Line: The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively.Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types.Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

ABSTRACT
The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3' UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3' UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

No MeSH data available.


Related in: MedlinePlus