Nattachet Plengvidhya

Recent advances in the genetics of maturity-onset diabetes of the young and other forms of autosomal dominant diabetes

Much effort has been dedicated during the last decade to the identification of genes for maturity-onset diabetes of the young and other forms of autosomal dominant diabetes. The study of this monogenic syndrome has led to the identification of several genes that were not previously suspected to play a role in glucose homeostasis. All these genes code for transcription factors that are expressed in beta-cells, where they play a crucial role in cellular differentiation and metabolism. Work is in progress to define the exact role of these genes in glucose homeostasis, and to understand whether they are also involved in common type 2 diabetes. It is estimated that many other genes for these forms of diabetes remain to be discovered. Finding these loci will provide critical knowledge for identifying those cellular pathways that are involved in insulin secretion and action, with crucial implications for studies of the etiology of type 2 diabetes and the development of drugs to treat this disorder.

Mutations of maturity‐onset diabetes of the young (MODY) genes in Thais with early‐onset type 2 diabetes mellitus

Objective  Six known genes responsible for maturity‐onset diabetes of the young (MODY) were analysed to evaluate the prevalence of their mutations in Thai patients with MODY and early‐onset type 2 diabetes.

Defective PAX4 R192H transcriptional repressor activities associated with maturity onset diabetes of the young and early onset-age of type 2 diabetes

AIMS PAX4 R192H polymorphism was reported to be associated with maturity onset diabetes of the young (MODY) and early onset-age of type 2 diabetes (T2D). This study aimed to evaluate transcriptional repression activity of PAX4 R192H polymorphism on its target promoters comparing with wild-type PAX4. METHODS Wild-type PAX4 and PAX4 R192H proteins were expressed in vitro and the cell compartmentalization of each protein was examined after transfection of the plasmid constructs into βTC3 cells followed by Western-blot analysis. The plasmid containing wild-type PAX4 or PAX4 R192H was co-transfected into βTC3 and αTC-1.9 cells with insulin or glucagon promoter-reporter construct. Transcriptional repression activities were then determined by dual-luciferase reporter assay. RESULTS Wild-type PAX4 and PAX4 R192H, which were found to be equally expressed in vitro and transfection systems, were present in the nuclear compartment. Transcriptional repressor activities of PAX4 R192H on human insulin and glucagon promoters were reduced when they were compared with those of wild-type PAX4. CONCLUSIONS These results suggested that PAX4 R192H polymorphism generated a protein with defect in transcriptional repressor activities on its target genes, which may lead to β-cell dysfunction associated with MODY and early onset-age of T2D as reported in our previous study.

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.

Aberrant mRNA splicing of paired box 4 ( PAX4 ) IVS7-1G>A mutation causing maturity-onset diabetes of the young, type 9

AimsPaired box 4 (PAX4) mutations cause maturity-onset diabetes of the young, type 9 (MODY9). The molecular defect and alteration of PAX4 function associated with the mutation PAX4 IVS7-1G>A in a family with MODY9 and severe diabetic complications were studied.MethodsWe investigated the functional consequences of PAX4 IVS7-1G>A on mRNA splicing using minigene assays. Wild-type and mutant PAX4 were expressed in mouse pancreatic β- and α-cell lines, and protein levels and translocation of PAX4 into the nucleus were determined. We also examined transcriptional repression of PAX4 target-gene promoters and β-cell viability under diabetic-like (high-glucose) conditions.ResultsPAX4 IVS7-1G>A disrupts an acceptor splice site, causing an adjacent cryptic splice site within exon 8 to be used, resulting in a three-nucleotide deletion and glutamine deletion at position 250 (p.Q250del). Wild-type and PAX4 Q250del proteins were expressed at similar levels and could translocate normally into the nucleus in βTC3 and αTC1.9 cells. However, the repressor functions of PAX4 Q250del on human insulin and glucagon promoters in INS-1 832/13 and αTC1.9 cells were significantly decreased, compared with that of wild-type PAX4. Moreover, the rate of apoptosis was increased in INS-1 cells over-expressing PAX4 Q250del when cultured in high-glucose conditions.ConclusionsPAX4 IVS7-1G>A caused aberrant mRNA splicing and PAX4 Q250 deletion. The mutation impaired PAX4 repressor functions on target-gene promoters and increased susceptibility to apoptosis upon high glucose exposure. Thus, PAX4 IVS7-1G>A contributes to the pathogenesis of diabetes in this MODY9 family through β-cell dysfunction.

Transcription factor 7-like 2 (TCF7L2) variations associated with earlier age-onset of type 2 diabetes in Thai patients.

Type 2 diabetes (T2D) is a complex disorder caused by the interaction between genetic predisposition and environmental factors (Freeman and Cox 2006). Recent progress in genetic and genomic research of T2D has shown that genes involved in pancreatic beta-cell development and function are involved in pathogenesis of T2D. Identification of these genes will provide a better understanding of pathogenesis, which may lead to the improvement of diagnosis, treatment, and prevention of this increasingly prevalent and costly condition. Genomewide linkage analysis has revealed that a region on chromosome 10q contained a T2D susceptibility gene, which was later ascribed to possess intronic variations of the transcription factor 7-like 2 (TCF7L2). The variation of TCF7L2 was associated with a two-fold increase of T2D risk in the Icelandic population (Grant et al. 2006). This association has been replicated in cohorts of European, Asian and African descent. The precise mechanism by which variations of TCF7L2 predispose to T2D is not clear. It has been suggested that TCF7L2 encodes a transcription factor that is expressed in foetal pancreas and involved in Wnt signalling pathway through the regulation of glucagon-like peptide (GLP-1), which has a primary role in glucose homeostasis (Cauchi et al. 2006). Since there is no previous genetic study describing the TCF7L2 variation on T2D risk in Thais, we therefore investigated the association of five variants at rs7896340, rs7901695, rs7903146, rs12255372, and rs11196205 of TCF7L2 with T2D by high resolution melting analysis. The associations between SNP genotype,

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.

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 β-cell function, proliferation and survival. Viability of β-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 β-cell survival in high glucose condition.

Withdrawal of sulfonylureas from patients with type 2 diabetes receiving long-term sulfonylurea and insulin combination therapy results in deterioration of glycemic control: a randomized controlled trial

Background The benefit of sulfonylureas (SUs) to patients with type 2 diabetes mellitus receiving long-term insulin treatment is unclear. This study evaluated glycemic control and beta-cell function after SU withdrawal in these patients. Methods In this 8-week randomized controlled study, patients with type 2 diabetes who had been treated with insulin for at least 3 years plus moderate to high doses of SUs were randomly assigned to withdrawal (n=16) or continuation (n=16) of SUs. Clinical characteristics, glycemic control, hypoglycemic events, and insulin secretion, including homeostasis model assessment of beta-cell function (HOMA-B) score, C-peptide concentration, and Matsuda index, were evaluated at baseline and after 2 and 8 weeks. Results Thirty patients (16 in the SU withdrawal group and 14 in the SU continuation group) completed the study. Median duration of diabetes was 17 (range 5–40) years. Baseline clinical characteristics, glycemic control, and HOMA-B were similar in the two groups, but the mean fasting C-peptide concentration was higher in the SU withdrawal group. After 8 weeks, the SU withdrawal group showed a significant increase in mean glycosylated hemoglobin levels from 7.8%±0.5% (62±5 mmol/mol) to 8.6%±1.2% (71±13 mmol/mol; P=0.002), whereas the SU continuation group showed a slight but not significant increase from 7.7%±0.5% (61±5 mmol/mol) to 7.9%±1.2% (63±13 mmol/mol; P=0.37). Insulin secretion, as measured by C-peptide and HOMA-B, decreased by 18% and 36%, respectively, in the SU withdrawal group. Hypoglycemic events were significantly more frequent in the SU continuation group whereas body weight did not change significantly in either group. Conclusion Withdrawal of SU from patients with type 2 diabetes receiving long-term combination treatment with SU and insulin resulted in deterioration of glycemic control and insulin secretion.

Functional defect of truncated hepatocyte nuclear factor-1α (G554fsX556) associated with maturity-onset diabetes of the young

A novel frameshift mutation attributable to 14-nucleotide insertion in hepatocyte nuclear factor-1alpha (HNF-1alpha) encoding a truncated HNF-1alpha (G554fsX556) with 76-amino acid deletion at its carboxyl terminus was identified in a Thai family with maturity-onset diabetes of the young (MODY). The wild-type and mutant HNF-1alpha proteins were expressed by in vitro transcription and translation (TNT) assay and by transfection in HeLa cells. The wild-type and mutant HNF-1alpha could similarly bind to human glucose-transporter 2 (GLUT2) promoter examined by electrophoretic mobility shift assay (EMSA). However, the transactivation activities of mutant HNF-1alpha on human GLUT2 and rat L-type pyruvate kinase (L-PK) promoters in HeLa cells determined by luciferase reporter assay were reduced to approximately 55-60% of the wild-type protein. These results suggested that the functional defect of novel truncated HNF-1alpha (G554fsX556) on the transactivation of its target-gene promoters would account for the beta-cell dysfunction associated with the pathogenesis of MODY.