Biochemical properties of human dehydrogenase/reductase (SDR family) member 7.

Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group

Human Mutation METHODS Diagnostic Guidelines for High-Resolution Melting Curve (HRM) Analysis: An Interlaboratory Validation of BRCA1 Mutation Scanning Using the 96-Well LightScanner TM For the Focus Section on HRMA Technology

ABSTRACT: Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High-resolution melting curve (HRM) analysis is a promising and economical method for high-throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and highstandard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScanner TM (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1-specific mutation screening test using the 96-well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient-derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs)

A common haplotype of protamine 1 and 2 genes is associated with higher sperm counts

P gt Sperm chromatin compaction in the sperm head is achieved when histones are replaced by protamines during spermatogenesis. Haploinsufficiency of the protamine 1 (PRM1) or PRM2 gene causes infertility in mice. However, the published data remain inconclusive about a role of PRM1/2 variants in male infertility and their association with semen parameters. By full sequence analysis, we assessed the frequency of sequence variations in PRM1 and PRM2 in three groups of Caucasian patients with idiopathic teratozoospermia and normal (n = 88) or reduced sperm concentration (n = 83) and in men with a high percentage of normal sperm morphology and normal concentrations (n = 77). Two rare (c.54G gt A and c.102G gt T) and one common SNP (c.230A gt C) were identified in PRM1. In PRM2, some rare heterozygous mutations and the two common intronic SNPs 298G gt C and 373C gt A were detected. None of the PRM1/2 variants was associated with teratozoospermia or individually with other semen parameters. However, significant linkage disequilibrium was detected between the common SNPs of PRM1 and PRM2 which formed haplotypes. Analysis of the pooled group (n = 248) revealed that homozygous carriers of the common haplotype ACC had a twofold higher sperm concentration and count than men lacking this haplotype, with sperm counts of heterozygotes for ACC being midway between the homozygotes. This markedly decreased sperm output might either be caused by spermatozoa lacking the ACC haplotype not being viable, or subject to negative selection. In addition, a significant deviation from Hardy-Weinberg-Equilibrium of these SNPs might indicate natural selection in favour of the ACC allele which leads to higher sperm output and therefore better fertility. In conclusion, for the first time we describe an association of a common haplotype formed by PRM1 and PRM2 with sperm output in a large group of men

EuroGentest: A Collaborative Network Aimed at Improving the Quality of Genetic Testing in Europe and Beyond

The EuroGentest network aims to improve and harmonize the quality of genetic testing services in Europe, from test development through to information for patients. The network encompasses Biochemical, Clinical, Cyto- and Molecular Genetics, Genetic counselling and patients groups. the Unit 1 of the network has disseminted information on laboratory accreditation, created a database on the current QA programmes and status in European genetic testing services, and reference materials producers and providers.JRC.D.2-Reference material