Discovery And Functional Characterization Of KLF11(C.1381 C>T,p.R461X) Mutant In Diabetic Patient

Background:With the development of economy,people’s daily life has changed dramatically,and the prevalence of diabetes among young people in the world is on the rise.There are many types of diabetes,and the progress of science and technology in recent years has made the differential diagnosis between diabetes types easier.Among them,adolescent onset adult diabetes(MODY),as a single gene diabetes,accounts for a certain proportion in young people with diabetes.There are multiple subtypes of MODY,and each subtype has related unique pathogenic genes.The pathogenic gene of MODY 7 diabetes is a zinc finger transcription factor KLF11.The expression product of KLF11 can combine with the insulin promoter to promote the expression of insulin gene.KLF11 deficiency is the cause of adult diabetes 7(MODY7)in young people.As of today,there is no relevant report on the clinical characteristics and functional studies of the mutation c.1381C>T at the new site of KLF11.This study aims to analyze the clinical characteristics of the family and verify the gene function of this mutation,provide a new starting point for clinical diagnosis and treatment of diabetes,and appropriately expand the known functions of KLF11.Methods:Collect the clinical data of the proband and some members of his/her family,and conduct full exon gene sequencing on the proband,his/her parents,and a brother of the proband with diabetes to generally obtain the mutant gene and mutation site in the family.At the same time,conduct site verification to determine the source of the mutant gene carried by the proband.Wild-type and mutant plasmids were constructed according to the key suspected pathogenic mutant genes,and the plasmids were transfected.Protein imprinting and real-time PCR were used to analyze the expression changes of the mutant genes themselves;The effect of gene mutation on the activity of insulin promoter was analyzed by double luciferase reporter gene experiment;The glucose-stimulated insulin secretion test(GSIS)is used to analyze the expression of KLF11 wild-type or mutant plasmid under the stimulation of high or low concentrations of glucose β The difference of insulin secretion in cell lines.Use ImageJ software to measure the gray value of protein electrophoresis strip and get the corresponding value;Prism9 software was used to analyze and chart all cell experimental data,and P<0.05 was considered statistically significant.Results:The whole exon gene sequencing showed that the proband had a heterozygous mutation of KLF11(c.1381C>T)gene,which was a new site mutation and a nonsense mutation,resulting in the codon encoding arginine at position 461 becoming a termination codon,and its clinical significance was not clear.The mother of the proband also has a heterozygous mutation of this gene,and the father of the proband and his brother have not found this mutation.In addition,the mother and brother of the proband also found HNF-1 a Gene mutation;The proband and the mother of the proband also have CCR5 gene mutations,and the proband,the mother of the proband,and the brother of the proband with diabetes also have NPHP4 gene mutations.Transfection of MIN6 cells(mouse islets)with plasmid carrying wild-type and mutant KLF11 gene β Cell tumor cells),and found that KLF11 protein synthesis decreased.Further plasmid transfection with concentration gradient showed that this phenomenon still existed.The plasmid carrying wild-type and mutant KLF11 gene was used to transfect 293A cells for double luciferase gene report test.It was found that the transcriptional activation of KLF11(c.1381C>T)on insulin promoter was lower than that of KLF11 wild-type.In addition,the use of islets β The cells studied whether the amount of insulin secreted by KLF11(c.1381C>T β Insulin secretion in cells decreased more significantly.C onclusions:This study involved the mutation of c.1381C>T at the new locus of KLF11,which was related to the occurrence of diabetes in the family(the proband and the mother).Cell experiments showed that the mutation reduced the transcription promotion of KLF11 on insulin gene;In the presence of this mutation,islets β The ability of cells to secrete insulin decreases,leading to the occurrence of diabetes in the proband.