Molecular genetic testing for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is offered worldwide and is of importance for differential diagnosis, carrier detection and adequate genetic counseling, particularly for family planning. In 2008 the European Molecular Genetics Quality Network (EMQN) for the first time offered a European-wide external quality assessment scheme for CAH (due to 21-OH deficiency). The interest was great and over the last years at about 60 laboratories from Europe, USA and Australia regularly participated in that scheme. These best practice guidelines were drafted on the basis of the extensive knowledge and experience got from those annually organized CAH-schemes. In order to obtain the widest possible consultation with practicing laboratories the draft was therefore circulated twice by EMQN to all laboratories participating in the EQA-scheme for CAH genotyping and was updated by that input. The present guidelines address quality requirements for diagnostic molecular genetic laboratories, as well as criteria forCYP21A2 genotyping (including carrier-testing and prenatal diagnosis). A key aspect of that article is the use of appropriate methodologies (e.g., sequencing methods, MLPA (multiplex ligation dependent probe amplification), mutation specific assays) and respective limitations and analytical accuracy. Moreover, these guidelines focus on classification of variants, and the interpretation and standardization of the reporting ofCYP21A2genotyping results. In addition, the article provides a comprehensive list of common as well as so far unreportedCYP21A2-variants.

Based on newborn screening data, the carrier frequency of congenital adrenal hyperplasia (CAH) in the general population has been estimated to be 1:55. The higher CAH frequency (particularly of milder forms of the disease) reported for certain populations including Yugoslavs (1.6%) relates to population genetic and hormonal data. However, so far, true carrier frequency for CAH due to 21-OH deficiency has not been determined by comprehensive mutation analysis of the 21-OH gene (CYP21A2) in an unselected European population. This study used CYP21A2 genotyping (sequence/Southern blot analysis) to determine CAH carrier frequency in a middle European (Austrian) population. The study included 100 migrants from the former Yugoslavia and 100 individuals of non-Yugoslavian origin. None of these individuals showed clinical hyperandrogenism or had a family history of CAH. Genotyping 400 unrelated alleles from 200 clinically unaffected individuals, this study revealed a carrier frequency of 9.5%, including so-called “classic” (5.5%) and “nonclassic” (4%) CYP21A2-gene aberrations. The observed heterozygosity for CAH in Yugoslavs was not different (P = 0.8095) from that in non-Yugoslavs. In conclusion, the observed CAH carrier frequency of 9.5% suggests a higher prevalence of CAH heterozygosity in a middle European population than hitherto estimated independently of the individuals’ Yugoslav or non-Yugoslav origin.

Context: Single-nucleotide polymorphisms (SNPs) of the RET protooncogene (RET) could modify disease susceptibility and clinical phenotype in patients with sporadic or familial medullary thyroid carcinoma (FMTC). Objective/Design of the Study: Because frequencies of RET SNPs have not yet been evaluated in patients with elevated serum concentrations of calcitonin (hCt), a biochemical marker for medullary thyroid carcinoma (MTC), we studied RET SNPs in patients with FMTC (n = 22), patients with sporadic MTC (n = 45), and 71 subjects presenting with moderately elevated hCt concentrations (basal, >10 pg/ml; pentagastrin stimulated, > 50 < 100 pg/ml) in comparison with an age- and gender-matched control group (n = 79) with basal hCt concentrations in the normal range (<5 pg/ml). Methods: After DNA extraction from citrated whole blood, RET exons 10, 11, 13, 14, 15, and 16 and exon/intron boundaries were analyzed by PCR-based cycle sequencing for RET germ line mutations, exonic (G691S, L769L, S836S, S904S) and intronic (IVS13+158; NCBI rs2472737 = IVS14–24) SNPs. Results: In FMTC patients, the F791Y mutation was found to be associated (P = 0.001) with the L769L SNP. The exonic SNPs (G691S, L769L, S836S, and S904S) were not different among the four groups. The intron 14 SNP (IVS14–24), however, was more frequent in individuals with elevated hCt serum concentrations (P = 0.016) and patients with sporadic MTC (P < 0.001) when compared with the control group. Conclusions: These data suggest that the exon 13 (L769L) and the intron 14 (IVS14–24) SNPs could act as genetic modifiers in the development of some forms of hereditary and sporadic MTC, respectively.

Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-κB, STAT, and SP1 binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study we investigated the impact of 1,25D3, tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 on CaSR expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNFα was accompanied by a 134-fold induction of CaSR in Coga1A (p<0.01). In Caco2/AQ cells the expression of CaSR was upregulated also by IL-6 (3.5-fold). Our data demonstrated transcriptional and translational activation of the CaSR by 1,25D3, TNFα, and IL-6 in a time- and cell line-dependent manner. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

This study evaluated the hypothesis that peroxisome proliferator-activated receptor-γ (PPARγ) agonists, including thiazolidinediones (TZDs) and the rexinoid LG100268 (LG), directly affect human vascular cell function (proliferation, cell cycle, protein expression, lactate release) independently of (1) their PPARγ-activating potential and (2) the cells’ vascular origin. Human umbilical vein endothelial cells (HUVECs), human adult vein endothelial cells (HAVECs), human retinal endothelial cells (HRECs) and human retinal pericytes (HRPYCs) were incubated (48 h) with 2–50 μmol/l rosiglitazone (RSG), RWJ241947 (RWJ), pioglitazone (PIO), troglitazone (TRO), 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) and LG. Proliferation, cell cycle distribution, protein expression, peroxisome proliferator-activated receptor responsive element (PPRE) transcriptional activity and mitochondrial effects were determined by [3H]thymidine incorporation, FACS analyses, western blots, reporter assays and lactate release respectively. In HUVECs, RSG, RWJ, PIO, TRO, PGJ2 and LG reduced (p<0.01) proliferation (due to a G0/G1 cell cycle arrest) by up to 23%, 36%, 38%, 86%, 99% and 93% respectively. The antiproliferative response was similar in HRPYCs and HAVECs, but was attenuated in HRECs. Whereas p21WAF-1/Cip1 and p27Kip were differently affected in HUVECs, all agents reduced (p<0.05) expression of cyclins (D3, A, E, B), cyclin-dependent kinase-2 and hyperphosphorylated retinoblastoma protein. The rank order of the antiproliferative effects of TZDs in HUVECs (RSG≈PIO≈RWJ