| Background: Diabetes mellitus(DM) is a popular chronic disease which destroys human healthy severely at present.As the main type DM, it is known thatβcell dysfunction and impaired insulin-stimulated glycogen synthesis of the peripheral tissue are the most consistent pathophogenesis characteristics of type 2 diabetes mellitus. T2DM is a heterogeneous and multifactorial inheritance disease,the mode of inheritance and pathogenesy of which is very complicated.CAPN10 is positional cloning gene relates to T2DM.It maps at 2q37-3 with the total length of 31 kb ,contains 15 extrons and 14 introns.CAPN10 is Ca2+ dependent cysteine proteinase in cell,it represents superfamily concerned with Ca2+ proteinum in structure , expresses in somatic muscles ,liver and pancreat。CAPN10 may participate in adjust of the cystoskeleton,modify of the enzyme and many cell procedure. It relates to architecture insulin sensibility and blood sugar level.CAPN10 has many polymorphism situs such as SNP43, SNP19 and SNP64.All of which associate with diabetes mellitus. Different races have different allele frequency, so there are differences in relationship with the invasion of diabetes mellitus.But up till now there is no research on the relationship between CAPN10 gene three SNPs and T2DM .Objectives(1)To study the genotype frequency, the allelic frequency and the haplotype frequency distribution of three SNPs including SNP 43,SNP44 and SNP 56 in CAPN10 gene.(2)To study the relationship between CAPN10 gene three SNPs and T2DM by Bi-PASA PCR .Methods(1)We collected the information about CAPN10 gene nucleotide sequences and three SNPs from GenBank of National Center for Biotechnology Information (NCBI). The primers were designed by Primer Premier 5.0 software and their specificities were tested by NCBI Blast 2.0 software.(2)The genotypes of three SNPs (SNP 43,SNP44 and SNP 56 ) were determined by Bi-PASA PCR technique.(3)The genotypes combination distribution of three SNPs of CAPN10 gene was analyzed by clustering analysis technique. There was considered significant difference when p value was 0.05 or less statistically.Results(1)We successfully studied three SNPs in CAPN10 gene by using Bi-PASA PCR technique. The superiority and stability of Bi-PASA PCR technique on determining gene SNPs had been tested.(2)The allele 1 frequencies of SNP43,SNP44 and SNP56 in T2DM cases were 0.9306,0.1910 and 0.6007 respectively while in control subjects were 0.8622 0.0969 and 0.4496 respectively. There were significant differences between every two groups(All p values<0.05).(3)G/G, A/G and A/A genotypes frequencies of SNP43 in T2DM cases and control subjects were 0.8611/0.7347, 0.1389/0.2551, 0.0000/0.0102 respectively. T/T, C/T and C/C genotypes frequencies of SNP44 in T2DM cases and control subjects were 0.6250/0.8061, 0.3681/0.1939 and 0.0069/0.0000 respectively. Genotypes frequencies of SNP 56 in T2DM cases and control subjects were G/G 0.1597/0.2857,A/G 0.4791/0.5306 and A/A 0.3611/0.1836 respectively. There were significant differences between the two groups in three SNPs(All p values<0.05).(4)The most three common haplotypes of CAPN10 gene among Chinese Han population were GCA,GTA and GTG, frequencies of which in both groups were 0.110/0.037, 0.449/0.378 and 0.294/0.388 respectively. P values were 8.2997, 2.4416 and 0.25000 respectively. The haplotype frequencies of ATG*,GCA* and GTG* were 0.024/0098,0.110/0.037 and 0.294/0.388 respectively.P values were 0.000377,0.003984 and 0.038156 respectively. There were significant differences between T2DM cases and control subjects. Conclusively CAPN10 gene polymorphisms might be associated with T2DM.Conclusions(1)The Bi-PASA PCR technique is a novel method by which we can conveniently determine SNP with more specificity, less expenditure, especially in detecting the zygosity of known mutations.(2)We found the features of genotypes combination distribution and frequencies distribution of three SNPs of CAPN10 gene and provided basic laboratory biological information which would facilitate further exploration into the etiopathogenesis, precaution and treatment of T2DM.(3)We found CAPN10 gene haplotypes might be associated with T2DM in southern Chinese Han population. |
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