Ethylmalonic encephalopathy ETHE1 R163W/R163Q mutations alter protein stability and redox properties of the iron centre.

ETHE1 is an iron-containing protein from the metallo β-lactamase family involved in the mitochondrial sulfide oxidation pathway. Mutations in ETHE1 causing loss of function result in sulfide toxicity and in the rare fatal disease Ethylmalonic Encephalopathy (EE). Frequently mutations resulting in depletion of ETHE1 in patient cells are due to severe structural and folding defects. However, some ETHE1 mutations yield nearly normal protein levels and in these cases disease mechanism was suspected to lie in compromised catalytic activity. To address this issue and to elicit how ETHE1 dysfunction results in EE, we have investigated two such pathological mutations, ETHE1-p.Arg163Gln and p.Arg163Trp. In addition, we report a number of benchmark properties of wild type human ETHE1, including for the first time the redox properties of the mononuclear iron centre. We show that loss of function in these variants results from a combination of decreased protein stability and activity. Although structural assessment revealed that the protein fold is not perturbed by mutations, both variants have decreased thermal stabilities and higher proteolytic susceptibilities. ETHE1 wild type and variants bind 1 ± 0.2 mol iron/protein and no zinc; however, the variants exhibited only ≈ 10% of wild-type catalytically activity. Analysis of the redox properties of ETHE1 mononuclear iron centre revealed that the variants have lowered reduction potentials with respect to that of the wild type. This illustrates how point mutation-induced loss of function may arise via very discrete subtle conformational effects on the protein fold and active site chemistry, without extensive disruption of the protein structure or protein-cofactor association

ETHE1-p.Arg163Gln and p.Arg163Trp mutations affect the mononuclear iron site reduction potential.

<p><b>A</b>. EPR spectra of ETHE1 proteins at 1 mg.ml⁻¹ in 150 mM Tris-HCl pH 7.5 and 300 mM NaCl. <b>B</b>. Redox titrations followed by EPR spectroscopy. Solid and dashed lines are Nernst Curves obtained for the different ETHE1 proteins one-electron reduction processes (n = 1) and the oxidation-reduction potentials are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107157#pone-0107157-t001" target="_blank">Table 1</a>. EPR spectra were recorded at 9.39 GHz microwave frequency, 2 mW microwave power, 1 mT modulation amplitude, 8 K temperature.</p

Biochemical and conformational properties of ETHE1.

<p>The values presented for the different properties correspond to an average of at least three independent determinations (n = 3).</p><p>Biochemical and conformational properties of ETHE1.</p

ETHE1-p.Arg163Gln and p.Arg163Trp are folded but have decreased stability.

<p><b>A</b>. Far-UV CD spectra of purified ETHE1 proteins at 0.1 mg.ml⁻¹ in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Spectra are offset for clarity. <b>B</b>. Molecular model of human ETHE1 highlighting the position of Arg163 in respect to the Fe centre, molecular modeling was made based in the <i>Arabidopsis thaliana</i> ETHE1-like protein structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107157#pone.0107157-McCoy1" target="_blank">[18]</a>. <b>C</b>. Thermal denaturation curves of ETHE1 proteins, followed by ellipticity variation at 222 nm from which the fraction of unfolded protein is determined. <b>D</b>. Time course of limited proteolysis experiment at 37°C in 0.1 M Tris-HCl, pH 8.5 monitored by SDS-PAGE. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107157#s2" target="_blank">Materials and Methods</a> for details.</p

The impact of a breast cancer diagnosis on health-related quality of life. A prospective comparison among middle-aged to elderly women with and without breast cancer

<p><b>Background</b> The improved survival after breast cancer has prompted knowledge on the effect of a breast cancer diagnosis on health-related quality of life (HQoL). This study compared changes in HQoL among women from before to after breast cancer diagnosis with longitudinal changes among women who remained breast cancer-free.</p> <p><b>Material and methods</b> The Danish Diet, Cancer and Health study included 57 053 cancer-free persons aged 50–64 years at baseline (1993–1997). We used data from first follow-up (1999–2002) and second follow-up (2010–2012) on HQoL [Medical Outcomes Survey, short form (SF-36)] obtained from 542 women aged 64–82 years with primary breast cancer (stages I–III) and a randomly matched sample of 729 women who remained breast cancer-free. Linear regression models were used to estimate the differences in changes in HQoL between women with and without breast cancer; the analyses were repeated with stratification according to age, comorbidity, partner support and time since diagnosis.</p> <p><b>Results</b> Women with breast cancer reported significantly larger decreases in HQoL from before to after diagnosis than those who remained breast cancer-free (physical component summary, −2.0; 95% CI −2.8; −1.2, mental component summary, −1.5, 95% CI −2.3; −0.6). This association was significantly modified by comorbidity and time since diagnosis.</p> <p><b>Conclusions</b> Women with breast cancer reported significantly larger HQoL declines than breast cancer-free women. Breast cancer diagnosis seems to have the greatest impact on HQoL closest to diagnosis and in women with comorbidity indicating that this group should be offered timely and appropriate follow-up care to prevent HQoL declines.</p

A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome

PURPOSE: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. METHODS: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. RESULTS: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. CONCLUSION: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants. KEYWORDS: Lynch syndrome; assay calibration; functional assay; variant classification; variants of uncertain significanc