Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course

Sex- and age-related differences in the management and outcomes of chronic heart failure: an analysis of patients from the ESC HFA EORP Heart Failure Long-Term Registry

Aims: This study aimed to assess age- and sex-related differences in management and 1-year risk for all-cause mortality and hospitalization in chronic heart failure (HF) patients. Methods and results: Of 16 354 patients included in the European Society of Cardiology Heart Failure Long-Term Registry, 9428 chronic HF patients were analysed [median age: 66 years; 28.5% women; mean left ventricular ejection fraction (LVEF) 37%]. Rates of use of guideline-directed medical therapy (GDMT) were high (angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, beta-blockers and mineralocorticoid receptor antagonists: 85.7%, 88.7% and 58.8%, respectively). Crude GDMT utilization rates were lower in women than in men (all differences: P\ua0 64 0.001), and GDMT use became lower with ageing in both sexes, at baseline and at 1-year follow-up. Sex was not an independent predictor of GDMT prescription; however, age >75 years was a significant predictor of GDMT underutilization. Rates of all-cause mortality were lower in women than in men (7.1% vs. 8.7%; P\ua0=\ua00.015), as were rates of all-cause hospitalization (21.9% vs. 27.3%; P\ua075 years. Conclusions: There was a decline in GDMT use with advanced age in both sexes. Sex was not an independent predictor of GDMT or adverse outcomes. However, age >75 years independently predicted lower GDMT use and higher all-cause mortality in patients with LVEF 6445%

Novel 18-gene signature for predicting relapse in ER-positive, HER2-negative breast cancer.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were madeBACKGROUND: Several prognostic signatures for early oestrogen receptor-positive (ER+) breast cancer have been established with a 10-year follow-up. We tested the hypothesis that signatures optimised for 0-5-year and 5-10-year follow-up separately are more prognostic than a single signature optimised for 10 years. METHODS: Genes previously identified as prognostic or associated with endocrine resistance were tested in publicly available microarray data set using Cox regression of 747 ER+/HER2- samples from post-menopausal patients treated with 5 years of endocrine therapy. RNA expression of the selected genes was assayed in primary ER+/HER2- tumours from 948 post-menopausal patients treated with 5 years of anastrozole or tamoxifen in the TransATAC cohort. Prognostic signatures for 0-10, 0-5 and 5-10 years were derived using a penalised Cox regression (elastic net). Signature comparison was performed with likelihood ratio statistics. Validation was done by a case-control (POLAR) study in 422 samples derived from a cohort of 1449. RESULTS: Ninety-three genes were selected by the modelling of microarray data; 63 of these were significantly prognostic in TransATAC, most similarly across each time period. Contrary to our hypothesis, the derived early and late signatures were not significantly more prognostic than the 18-gene 10-year signature. The 18-gene 10-year signature was internally validated in the TransATAC validation set, showing prognostic information similar to that of Oncotype DX Recurrence Score, PAM50 risk of recurrence score, Breast Cancer Index and IHC4 (score based on four IHC markers), as well as in the external POLAR case-control set. CONCLUSIONS: The derived 10-year signature predicts risk of metastasis in patients with ER+/HER2- breast cancer similar to commercial signatures. The hypothesis that early and late prognostic signatures are significantly more informative than a single signature was rejected.This work was supported by Breast Cancer Now working in partnership with Walk the Walk, as well as by the National Institute for Health Research Royal Marsden/ICR Biomedical Research Centre. ARB was funded by Cancer Research UK (grant number C569/A16891). The study was supported by funds from Skåne County Council’s Research and Development Foundation, Governmental Funding of Clinical Research within the National Health Service (grant number ALFSKANE-350191 [to MK]), the Swedish Breast Cancer Association (BRO), the Mrs Berta Kamprad Foundation and The Inger Persson Research Foundation. IS and JC were supported by Cancer Research UK (programme grant C569/A10404)

A mutation (N177S) in the structurally conserved helix initiation peptide motif of keratin 5 causes a mild EBS phenotype

Epidermolysis bullosa simplex (EBS) is a group of predominantly autosomal dominant hereditary disorders of the skin, which manifest as superficial skin blisters after minimal mechanical trauma. Three subtypes have been defined, based on clinical severity. Mutations affecting the genes encoding the epidermal keratins 5 (K5) and 14 (K14) have been linked to the disease, and generally those affecting the helix initiation and termination peptide motifs have been linked to severe EBS phenotypes. We report here a novel mutation in the helix initiation peptide of K5, N177S, that causes only a mild EBS-Weber Cockayne phenotype (EBS-WC). The mutation was identified by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encoding the exons of the KRT5 and KRT14 genes, and confirmed by mismatch allele-specific PCR, followed by restriction enzyme digestion with Tsp509 I. The patient is heterozygous for a mutation affecting codon 177, changing a conserved asparagine residue (N) to serine (S). Asparagine 177 is a highly conserved residue among all type II keratins. This is also the first report of a mutation at position 9 of 1A helix (1A:N9S) in a type II keratin. Unlike mutations affecting residues 4, 5, 7, 8, 10, and 11 of the 1A helix of K5 and K14, which were all previously linked to more severe (EBS) phenotypes, K5 1A:N9S produces only a mild EBS-WC phenotype

Steroid biotransforming strains designated Cochliobolus lunatus m118 and Curvularia lunata AT46 are both Curvularia lunata var lunata

The two strains designated ''Cochliobolus lunatus m118'' (MUCL 38696) and ''Curvularia lunata AT46'' (MUCL 38697) are both important for production of corticosteroids. The strain Cochliobolus lunatus m118 developed mycelium only; no conidia nor ascomata were observed Curvularia lunata AT46 produced some conidia on current growth media and conditions. We present for the first time the successful conidiation of ''m118'' on the special growth media and light. Although the strains did not cross nor did with other MUCL Curvularia lunata strains, remaining both asexual, they produced identical conidiation on specific media and under near UV light. Both strains gave the same results with API ZYM 20 (Biomerieux) test and they hybridized well with a randomly cloned Cochliobolus lunatus genomic probe. RAPD profiles with different primers showed some identical (species specific) markers and several nonidentical (strain specific) markers. Results demonstate that strains m118 and AT46 which have been widely used in biochemical and molecular biology studies but did not have a documented taxonomical classification, are two non-compatible Curvularia lunata var. lunata strains

Erratum: Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease (PLoS ONE (2014) 9, 6 (e99398) DOI: 10.1371/journal.pone.0099398)

10.1371/journal.pone.0104122PLoS ONE97e10412

Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease

10.1371/journal.pone.0099398PLoS ONE96e9939