PAX4 R192H and P321H polymorphisms in type 2 diabetes and their functional defects.

We have previously identified PAX4 mutations causing MODY9 and a recent genome-wide association study reported a susceptibility locus of type 2 diabetes (T2D) near PAX4. In this study, we aim to investigate the association between PAX4 polymorphisms and T2D in Thai patients and examine functions of PAX4 variant proteins. PAX4 rs2233580 (R192H) and rs712701 (P321H) were genotyped in 746 patients with T2D and 562 healthy normal control subjects by PCR and restriction-fragment length polymorphism method. PAX4 variant proteins were investigated for repressor function on human insulin and glucagon promoters and for cell viability and apoptosis upon high glucose exposure. Genotype and allele frequencies of PAX4 rs2233580 were more frequent in patients with T2D than in control subjects (P=0.001 and 0.0006, respectively) with odds ratio of 1.66 (P=0.001; 95% confidence interval, 1.22-2.27). PAX4 rs712701 was not associated with T2D but it was in linkage disequilibrium with rs2233580. The 192H/321H (A/A) haplotype was more frequent in T2D patients than in controls (9.5% vs 6.6%; P=0.009). PAX4 R192H, but not PAX4 P321H, impaired repression activities on insulin and glucagon promoters and decreased transcript levels of genes required to maintain ß-cell function, proliferation and survival. Viability of ß-cell was reduced under glucotoxic stress condition for the cells overexpressing either PAX4 R192H or PAX4 P321H or both. Thus these PAX4 polymorphisms may increase T2D risk by defective transcription regulation of target genes and/or decreased ß-cell survival in high glucose condition.

Detection of CAPN10 copy number variation in Thai patients with type 2 diabetes by denaturing high performance liquid chromatography and real-time quantitative polymerase chain reaction.

AIMS/INTRODUCTION: A combination of multiple genetic and environmental factors contribute to the pathogenesis of type 2 diabetes. Copy number variations (CNVs) are associated with complex human diseases. However, CNVs can cause genotype deviation from the Hardy-Weinberg equilibrium (HWE). A genetic case-control association study in 216 Thai diabetic patients and 192 non-diabetic controls found that, after excluding genotyping errors, genotype distribution of calpain 10 (CAPN10) SNP44 (rs2975760) deviated from HWE. Here, we aimed to detect CNV within the CAPN10 SNP44 region. MATERIALS AND METHODS: CNV within the CAPN10 SNP44 region was detected using denaturing high-performance liquid chromatography, and the results confirmed by real-time quantitative polymerase chain reaction with SYBR Green I. RESULTS: Both methods successfully identified CNV in the CAPN10 SNP44 region, obtaining concordant results. Correction of genotype calling based on the status of identified CNVs showed that the CAPN10 SNP44 genotype is in good agreement with HWE (P > 0.05). However, no association between CNV genotypes and risk of type 2 diabetes was observed. CONCLUSIONS: Identified CNVs for CAPN10 SNP44 genotypes lead to deviation from HWE. Furthermore, both denaturing high-performance liquid chromatography and real-time quantitative polymerase chain reaction are useful for detecting CNVs.

Identification of copy number variation of CAPN10 in Thais with type 2 diabetes by multiplex PCR and denaturing high performance liquid chromatography (DHPLC).

Copy number variations (CNVs) have been shown to be associated with several diseases. They can cause deviation of genotypes from Hardy-Weinberg Equilibrium (HWE). Genetic case-control association studies in Thais revealed that genotype distribution of CAPN10 Indel19 was deviated from HWE after correction of genotyping error. Therefore, we aim to identify CNVs within CAPN10 Indel19 region. The semi-quantitative denaturating high performance liquid chromatography (DHPLC) method was used to detect CNVs in the region of CAPN10 Indel19 marker in cohort of 305 patients with type 2 diabetes and 250 control subjects without diabetes. CNVs in the region of CAPN10 Indel19 was successfully detected by DHPLC. After correction of genotype calling based on the status of identified CNVs, CAPN10 Indel19 genotypes were well-fitted for HWE (p>0.05). However, we did not find association between CNV genotypes and risk of type 2 diabetes in our population. CNVs in CAPN10 have been identified in Thais. These CNVs lead to deviation from HWE of CAPN10 Indel19 genotypes. After excluding identified CNVs from the analysis, CAPN10 Indel19 was associated with type 2 diabetes. The information obtained from our study would be helpful for genotyping accuracies of SNPs residing in the CNVs region.

Novel adiponectin variants identified in type 2 diabetic patients reveal multimerization and secretion defects.

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect.