Multigene testing of moderate-risk genes: be mindful of the missense.

BACKGROUND: Moderate-risk genes have not been extensively studied, and missense substitutions in them are generally returned to patients as variants of uncertain significance lacking clearly defined risk estimates. The fraction of early-onset breast cancer cases carrying moderate-risk genotypes and quantitative methods for flagging variants for further analysis have not been established. METHODS: We evaluated rare missense substitutions identified from a mutation screen of ATM, CHEK2, MRE11A, RAD50, NBN, RAD51, RINT1, XRCC2 and BARD1 in 1297 cases of early-onset breast cancer and 1121 controls via scores from Align-Grantham Variation Grantham Deviation (GVGD), combined annotation dependent depletion (CADD), multivariate analysis of protein polymorphism (MAPP) and PolyPhen-2. We also evaluated subjects by polygenotype from 18 breast cancer risk SNPs. From these analyses, we estimated the fraction of cases and controls that reach a breast cancer OR≥2.5 threshold. RESULTS: Analysis of mutation screening data from the nine genes revealed that 7.5% of cases and 2.4% of controls were carriers of at least one rare variant with an average OR≥2.5. 2.1% of cases and 1.2% of controls had a polygenotype with an average OR≥2.5. CONCLUSIONS: Among early-onset breast cancer cases, 9.6% had a genotype associated with an increased risk sufficient to affect clinical management recommendations. Over two-thirds of variants conferring this level of risk were rare missense substitutions in moderate-risk genes. Placement in the estimated OR≥2.5 group by at least two of these missense analysis programs should be used to prioritise variants for further study. Panel testing often creates more heat than light; quantitative approaches to variant prioritisation and classification may facilitate more efficient clinical classification of variants.

The enhancement of stress-related memory by glucocorticoids depends on synapsin-Ia/Ib.

The activation of glucocorticoid receptors (GR) by glucocorticoids increases stress-related memory through the activation of the MAPK signaling pathway and the downstream transcription factor Egr-1. Here, using converging in vitro and in vivo approaches, respectively, GR-expressing cell lines, culture of hippocampal neurons, and GR genetically modified mice (GR(NesCre)), we identified synapsin-Ia/Ib as one of the effectors of the glucocorticoid signaling cascade. Stress and glucocorticoid-induced activation of the GR modulate synapsin-Ia/Ib through two complementary mechanisms. First, glucocorticoids driving Egr-1 expression increase the expression of synapsin-Ia/Ib, and second, glucocorticoids driving MAPK activation increase its phosphorylation. Finally, we showed that blocking fucosylation of synapsin-Ia/Ib in the hippocampus inhibits its expression and prevents the glucocorticoid-mediated increase in stress-related memory. In conclusion, our data provide a complete molecular pathway (GR/Egr-1/MAPK/Syn-Ia/Ib) through which stress and glucocorticoids enhance the memory of stress-related events and highlight the function of synapsin-Ia/Ib as molecular effector of the behavioral effects of stress.

[Parathyroidectomy in end-stage renal disease: perioperative management of calcium-phosphorus balance]. / Parathyroïdectomie en insuffisance rénale terminale: prise en charge péri-opératoire de l'équilibre phosphocalcique.

The management of metabolic problems following parathyroidectomy in end stage renal disease remains poorly defined. Hypocalcemia is a common and serious complication in the post-operative period. The objective of the present study was to develop a protocol for the management of patients during the immediate perioperative period based on the best available data from the literature, and to verify its effectiveness and safety in three patients on chronic hemodialysis. A patient management protocol was developed based on data reported in the literature and was subsequently tested on three chronic dialysis patients suffering from tertiary hyperparathyroidism with an indication of parathyroidectomy. According to the literature, the risk of hypocalcemia following parathyroidectomy can be decreased by tight surveillance of calcium levels and preventive administration of calcium and vitamin D analogue to patients at high risk of hypocalcemia. By applying this protocol, profound hypocalcemia was avoided and the immediate post-operative period was uneventful in the three patients under study. In summary, the proposed protocol is safe and effective for the peri-parathyroidectomy management of patients on chronic hemodialysis.

Effectiveness of twice-weekly pyrimethamine-sulfadoxine as primary prophylaxis of Pneumocystis carinii pneumonia and toxoplasmic encephalitis in patients with advanced HIV infection.

The safety and efficacy of a fixed 25 mg pyrimethamine-500 mg sulfadoxine combination supplemented with 15 mg folinic acid twice a week as primary prophylaxis of Pneumocystis carinii pneumonia (PCP) and toxoplasmic encephalitis was evaluated in 106 patients infected with the human immunodeficiency virus. All patients had a CD4+ T-lymphocyte count of less than 100 cells/microl at study entry. Efficacy in this single-arm open-label prospective study was analyzed on an as-treated basis. No patient received highly active antiretroviral treatment, including protease inhibitors or non-nucleoside reverse transcriptase inhibitors, while on study medication. PCP developed in four patients, one of whom had been noncompliant. No PCP episode occurred in the first year. Probabilities of freedom from PCP were 0.97 (95%CI, 0.92-1) after 24 months and 0.93 (95%CI, 0.84-1) after 36 months. Of 74 (69.8%) patients positive for anti-toxoplasma IgG antibodies, one noncompliant patient developed toxoplasmic encephalitis after 24 months. Allergic reactions were observed in 18 (17%) patients and resulted in permanent discontinuation in 7 (6.6%) patients. One (0.9%) patient who had continued prophylaxis despite progressive hypersensitivity reactions developed a serious adverse reaction (Stevens-Johnson syndrome). The median survival of study participants was 29 months, with relentless progression of AIDS accounting for most deaths. The prophylaxis regimen studied appeared safe and effective for primary prophylaxis of PCP and toxoplasmic encephalitis. Severe adverse events can likely be prevented by discontinuation of prophylaxis at the time allergic reactions are noted. Rechallenge frequently results in tolerance. Efficacy and safety compare favorably with previously studied regimens. This simple prophylactic regimen may provide a convenient alternative for patients failing or intolerant to approved regimens.

Isolation and characterization of the monkey UDP-glucuronosyltransferase cDNA clone monUGT1A01 active on bilirubin and estrogens.

Although enzymes that catalyze the formation of steroids are well known, less information is available about the enzymes involved in the metabolism of these hormones. Steroid glucuronidation, by UDP-glucuronosyltransferase enzymes, is one mechanism by which steroid hormones can be metabolized and eliminated from a tissue. Previous results suggest that the monkey represents the most appropriate animal model for studying the physiologic relevance of steroid glucuronidating enzymes. The monkey UGT1A01 cDNA clone was isolated by RT-PCR amplification of the liver RNA. The cDNA contains an open reading frame of 1599 bp encoding a protein of 533 residues. The primary structure of monkey UGT1A01 is 95% identical to human UGT1A1. To compare monkey and human UGT1A1 enzymes, both cDNA clones were transfected into HK293 cells and stable cell lines expressing each UGT1A1 protein were established. Western blot analysis of the monUGT1A01-HK293 and hUGT1A1-HK293 cell lines using a anti-UGT1A polyclonal antibody (RC-71) revealed expression of exogenous 55 kDa UGT1 proteins. The transferase activities of monkey and human UGT1A1 proteins were tested with over 60 compounds and were demonstrated to be active on the same compounds. For endogenous compounds only bilirubin and C18 steroids were glucuronidated by these enzymes. Using microsome preparation (from HK293 cell expressing monkey UGT1A01), the apparent K(m) values were 13, 5 and 6 microM for the conjugation of estradiol, 2-hydroxyestradiol and 2-hydroxyestrone, respectively, and were very similar to the values obtained with human UGT1A1. Specific RT-PCR analysis demonstrated the expression of monkey and human UGT1A1 transcripts in several tissues including liver, kidney, intestine, prostate, testis and ovary suggesting a contribution of this isoenzyme to estrogen metabolism in the cynomolgus monkey as in human.