Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

<p>Abstract</p> <p>Background</p> <p>Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the <it>HFE </it>gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for <it>HFE </it>mutations screening based on TaqMan technology and to determine the frequencies of <it>HFE </it>mutations in the Slovenian population.</p> <p>Methods</p> <p>Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of <it>HFE </it>mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing.</p> <p>Results</p> <p>The genotyping assay of the H63D, S65C and C282Y mutations in the <it>HFE </it>gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous <it>HFE </it>genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5 – 14.2%), 1.8% (95% CI 1.4 – 2.5%) and 3.6% (95% CI 3.0 – 4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions.</p> <p>Conclusion</p> <p>The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.</p

Genetic deficiency of aldose reductase counteracts the development of diabetic nephropathy in C57BL/6 mice

National Science Foundation of China [30770490]; 973 Program of China [2009CB941601]; Science Planning Program of Fujian Province [2009J1010]; Natural Science Foundation of Fujian Province [2009J01180]; Fujian Provincial Department of Science and TechnoloThe aim of the study was to investigate the effects of genetic deficiency of aldose reductase in mice on the development of key endpoints of diabetic nephropathy. A line of Ar (also known as Akr1b3)-knockout (KO) mice, a line of Ar-bitransgenic mice and control C57BL/6 mice were used in the study. The KO and bitransgenic mice were deficient for Ar in the renal glomeruli and all other tissues, with the exception of, in the bitransgenic mice, a human AR cDNA knockin-transgene that directed collecting-tubule epithelial-cell-specific AR expression. Diabetes was induced in 8-week-old male mice with streptozotocin. Mice were further maintained for 17 weeks then killed. A number of serum and urinary variables were determined for these 25-week-old mice. Periodic acid-Schiff staining, western blots, immunohistochemistry and protein kinase C (PKC) activity assays were performed for histological analyses, and to determine the levels of collagen IV and TGF-beta 1 and PKC activities in renal cortical tissues. Diabetes-induced extracellular matrix accumulation and collagen IV overproduction were completely prevented in diabetic Ar-KO and bitransgenic mice. Ar deficiency also completely or partially prevented diabetes-induced activation of renal cortical PKC, TGF-beta 1 and glomerular hypertrophy. Loss of Ar results in a 43% reduction in urine albumin excretion in the diabetic Ar-KO mice and a 48% reduction in the diabetic bitransgenic mice (p < 0.01). Genetic deficiency of Ar significantly ameliorated development of key endpoints linked with early diabetic nephropathy in vivo. Robust and specific inhibition of aldose reductase might be an effective strategy for the prevention and treatment of diabetic nephropathy

Functional annotations of diabetes nephropathy susceptibility loci through analysis of genome-wide renal gene expression in rat models of diabetes mellitus

<p>Abstract</p> <p>Background</p> <p>Hyperglycaemia in diabetes mellitus (DM) alters gene expression regulation in various organs and contributes to long term vascular and renal complications. We aimed to generate novel renal genome-wide gene transcription data in rat models of diabetes in order to test the responsiveness to hyperglycaemia and renal structural changes of positional candidate genes at selected diabetic nephropathy (DN) susceptibility loci.</p> <p>Methods</p> <p>Both Affymetrix and Illumina technologies were used to identify significant quantitative changes in the abundance of over 15,000 transcripts in kidney of models of spontaneous (genetically determined) mild hyperglycaemia and insulin resistance (Goto-Kakizaki-GK) and experimentally induced severe hyperglycaemia (Wistar-Kyoto-WKY rats injected with streptozotocin [STZ]).</p> <p>Results</p> <p>Different patterns of transcription regulation in the two rat models of diabetes likely underlie the roles of genetic variants and hyperglycaemia severity. The impact of prolonged hyperglycaemia on gene expression changes was more profound in STZ-WKY rats than in GK rats and involved largely different sets of genes. These included genes already tested in genetic studies of DN and a large number of protein coding sequences of unknown function which can be considered as functional and, when they map to DN loci, positional candidates for DN. Further expression analysis of rat orthologs of human DN positional candidate genes provided functional annotations of known and novel genes that are responsive to hyperglycaemia and may contribute to renal functional and/or structural alterations.</p> <p>Conclusion</p> <p>Combining transcriptomics in animal models and comparative genomics provides important information to improve functional annotations of disease susceptibility loci in humans and experimental support for testing candidate genes in human genetics.</p