Functional Study Of Single Nucleotide Polymorphism Rs10811660 In Predisposing The Risk Of Type 2 Diabetes

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease characterized by hyperglycemia leading to chronic damage and dysfunction of various tissues.Diabetes not only brings a huge economic burden to patients,but also brings mental and physical pain to patients.Complications of diabetes are more likely to lead to death of people with diabetes.At present,the situation of diabetes epidemic is serious,and understanding its pathogenesis is very important for the treatment and prevention of diabetes.Genome-wide association analysis(GWAS)is an effective method to reveal complex disease susceptibility genes and genetic variations affecting complex traits.In previous studies,genome-wide association analysis has found a large numbers of single nucleotide polymorphisms(SNPs)significantly associated with T2 DM.However,these SNPs are merely statistically associated with the risk of T2 DM,lack of functional and causal relationship.In the early stage,our group identified 11079 SNPs that affect the binding affinity of transcription factors with the high-throughput SNP-SELEX method and revealed 305 significantly related loci with T2 DM containing these SNPs through regression analysis using the genotyping data of T2 DM patients and non-diabetic controls in the UK biobank.In this study,we collected genotyping data of T2 DM patients and non-diabetic controls in Shaanxi Province to cross-validate the significant locus containing SNP rs10811660.Then we conducted further functional studies on this locus.SNP rs10811660 has two alleles,A and G.We found that the binding affinity of the two alleles of SNP rs10811660 to the transcription factor MAFG was significantly different.The G-allele of SNP rs10811660 was more enriched in MAFG Ch IP pulldown DNA than the A-allele.This means that MAFG preferentially binds to the G allele of SNP rs10811660.We speculate that this difference will affect the expression of downstream genes.To test it,we carried out a luciferase based reporter assay and found that the genomic fragment with G-allele displayed significantly stronger signal than A-allele although both allele-containing fragments presented mild enhancer activity.Hi-C analysis showed that CDKN2 A,CDKN2B and CDKN2B-AS1 genes were in the same topological associating domain as SNP rs10811660.When MAFG was knocked down by si RNA method,the transcription levels of CDKN2 A,CDKN2B and CDKN2B-AS1 were significantly decreased.The transcriptional levels of CDKN2 B and CDKN2B-AS1 were also significantly decreased after the SNP rs10811660 containing fragment was deleted by CRISPR/Cas9 mediated genome editing.These results suggest that CDKN2 B and CDKN2B-AS1 may be the target of the putative enhancer harboring SNP rs10811660,and these genes may contribute to the occurrence and development of T2 DM.In summary,we confirm that SNP rs10811660 is a risk-related SNP of T2 DM,which is located on the enhancer and regulates the expression of CDKN2 B and CDKN2B-AS1 genes by affecting the binding of transcription factor MAFG.This study identified genes that might affect the occurrence of T2 DM,and laid an important foundation for further study of its pathogenesis.This and the follow-up study can help understand the genetic mechanism of T2 DM,and lay the foundation for early screening and clinical diagnosis and treatment.