Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study

In spite the close association of the triple-negative breast cancer immunophenotype with hereditary breast cancers and the BRCA1 pathway, there is a lack of population studies that determine the frequency of BRCA1 mutations among triple-negative breast cancer patients. To address this, we have screened a large sample of 403 women diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations. Median age at diagnosis was 50 years (range 20-83). The overall prevalence of triple-negative cases among the initial patient group with invasive breast cancer was 8%. BRCA1 was screened by direct DNA sequencing in all patients, including all exons where a mutation was previously found in the Greek population (exons 5, 11, 12, 16, 20, 21, 22, 23, 24-77% of the BRCA1 coding region), including diagnostic PCRs to detect the three Greek founder large genomic rearrangements. Sixty-five deleterious BRCA1 mutations were identified among the 403 triple-negative breast cancer patients (16%). Median age of onset for mutation carriers was 39 years. Among a total of 106 women with early-onset triple-negative breast cancer (<40 years), 38 (36%) had a BRCA1 mutation, while 27% of women with triple-negative breast cancer diagnosed before 50 years (56/208) had a BRCA1 mutation. A mutation was found in 48% (50/105) of the triple-negative breast cancer patients with family history of breast or ovarian cancer. It is noteworthy, however, that of the 65 carriers, 15 (23%) had no reported family history of related cancers. All but one of the carriers had grade III tumors (98%). These results indicate that women with early-onset triple-negative breast cancer, and ideally all triple-negative breast cancer patients, are candidates for BRCA1 genetic testing even in the absence of a family history of breast or ovarian cancer.     

BRCA1 and BRCA2 germline testing in Cretan isolates reveals novel and strong founder effects

Germline BRCA1 and BRCA2 loss-of-function variants have been linked to increased breast and ovarian cancer risk, with more than 5,000 distinct pathogenic variants being reported worldwide. Among individuals of Greek descent, the BRCA1/2 variant spectrum is heterogeneous, but characterized by strong founder effects. As patients from certain geographical regions of Greece (like Crete) were underrepresented in previous studies, we hypothesized that isolated Cretans, a southern Greece islanders' population with distinct demographic, cultural and genetic features, could harbor founder BRCA1/2 mutations. A total of 304 breast or/and ovarian cancer patients of Cretan descent, fulfilling NCCN criteria for genetic testing, were tested by NGS or Sanger sequencing, followed by MLPA. Haplotype analysis was subsequently performed to investigate potential founder effects of recurrent alleles. Overall, 16.5% (50/304) of the tested patients carried 22 different pathogenic variants; 48% in BRCA1, 52% in BRCA2. Three variants, namely two in BRCA2 (Δexons 12 and 13 and c.7806-2A>T) and one in BRCA1 (c.5492del), constituting approximately half (48%) of all detected pathogenic variants, were shown to have a founder effect, with all carriers sharing common haplotypes. Remarkably, these variants were confined to Cretans and have not been identified in other regions of Greece. The high prevalence of specific BRCA1/2 pathogenic variants among Cretans, provides the possibility of cost- and time-efficient screening of the Cretan population. Integrating this knowledge in local public health services may have a significant impact on cancer prevention, and may serve as a starting point for the implementation of testing on a population level.     

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern,North African,and South European countries

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population‐specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.     

Resistance to fosfomycin: Mechanisms,Frequency and Clinical Consequences

Fosfomycin has been used for the treatment of infections due to susceptible and multidrug-resistant (MDR) bacteria. It inhibits bacterial cell wall synthesis through a unique mechanism of action at a step prior to that inhibited by β-lactams. Fosfomycin enters the bacterium through membrane channels/transporters and inhibits MurA, which initiates peptidoglycan (PG) biosynthesis of the bacterial cell wall. Several bacteria display inherent resistance to fosfomycin mainly through MurA mutations. Acquired resistance involves, in order of decreasing frequency, modifications of membrane transporters that prevent fosfomycin from entering the bacterial cell, acquisition of plasmid-encoded genes that inactivate fosfomycin, and MurA mutations. Fosfomycin resistance develops readily in vitro but less so in vivo. Mutation frequency is higher among Pseudomonas aeruginosa and Klebsiella spp. compared with Escherichia coli and is associated with fosfomycin concentration. Mutations in cAMP regulators, fosfomycin transporters and MurA seem to be associated with higher biological cost in Enterobacteriaceae but not in Pseudomonas spp. The contribution of fosfomycin inactivating enzymes in emergence and spread of fosfomycin resistance currently seems low-to-moderate, but their presence in transferable plasmids may potentially provide the best means for the spread of fosfomycin resistance in the future. Their co-existence with genes conferring resistance to other antibiotic classes may increase the emergence of MDR strains. Although susceptibility rates vary, rates seem to increase in settings with higher fosfomycin use and among multidrug-resistant pathogens.     

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1-30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.     

Functional characterization of CHEK2 variants in a Saccharomyces cerevisiae system

Genetic testing for cancer predisposition leads to the identification of a number of variants with uncertain significance. To some extent, variants of BRCA1/2 have been classified, in contrast to variants of other genes. CHEK2 is a typical example, in which a large number of variants of unknown clinical significance were identified and still remained unclassified. Herein, the CHEK2 variant assessment was performed through an in vivo, yeast‐based, functional assay. In total, 120 germline CHEK2 missense variants, distributed along the protein sequence, and two large in‐frame deletions were tested, originating from genetic test results in breast cancer families, or selected from the ClinVar database. Of these, 32 missense and two in‐frame deletions behaved as non‐functional, 73 as functional, and 15 as semi‐functional, after comparing growth rates of each strain with positive and negative controls. The majority of non‐functional variants were localized in the CHK2 kinase and forkhead‐associated domains. In vivo results from the non‐functional variants were in agreement with in silico predictions, and, where available, with strong breast cancer family history, to a great extent. The results of the largest, to date, yeast‐based assay, evaluating CHEK2 variants, can complement and assist in the classification of rare CHEK2 variants with unclear clinical significance.     

Weight Loss May Reverse Blunted Sympathetic Neural Responsiveness to Glucose Ingestion in Obese Subjects With Metabolic Syndrome

OBJECTIVE

The purpose of this study was to examine the effects of weight loss on sympathetic nervous system responsiveness to glucose ingestion in obese subjects with metabolic syndrome, in whom such responses are reportedly blunted.

RESEARCH DESIGN AND METHODS

Thirty four subjects, 19 insulin resistant and 15 insulin sensitive and aged 55 ± 1 years (mean ± SE) with BMI 31.6 ± 0.6 kg/m², who fulfilled the Adult Treatment Panel III criteria for metabolic syndrome participated. Simultaneous measurements of whole-body norepinephrine spillover rate, calf blood flow, and intra-arterial blood pressure were made at times 0, 30, 60, 90, and 120 min postglucose (75 g). The experiment was repeated after a 3-month hypocaloric diet with or without an exercise program.

RESULTS

Body weight decreased by 8.1 ± 0.9 and 8.4 ± 1.1 kg and resting norepinephrine spillover by 94 ± 31 and 166 ± 58 ng/min (all P ≤ 0.01) in insulin-resistant and insulin-sensitive subjects, respectively. Weight loss was accompanied by a marked increase in sympathetic responsiveness after glucose but only in insulin-resistant subjects. In this subgroup, comparative increases in norepinephrine spillover rates at baseline and after weight loss averaged −3 ± 25 versus 73 ± 24 ng/min at 30 min (P = 0.039), 36 ± 21 versus 115 ± 28 ng/min at 60 min (P = 0.045), 9 ± 21 versus 179 ± 50 ng/min at 90 min (P < 0.001), and 40 ± 48 versus 106 ± 39 ng/min at 120 min (P = 0.24).

CONCLUSIONS

Weight loss reverses blunted sympathetic responsiveness to glucose ingestion in insulin-resistant subjects with metabolic syndrome, which is relevant to postprandial energy utilization and body weight homeostasis.There is much evidence to indicate that the sympathetic nervous system (SNS) is activated by food intake and particularly by carbohydrate ingestion (1–3). Oral glucose elicits a marked and sustained increase in SNS activity over a 2-h period in healthy humans, as assessed by measurements of plasma norepinephrine concentration, by regional norepinephrine spillover, and by direct microneurographic recordings of muscle sympathetic nerve activity (MSNA) (1–3). At least two mechanisms are thought to contribute to this neurophysiological response: the postprandial increase in plasma insulin and the entrance of a nutrient into the gastrointestinal tract (4). Insulin exerts its sympathoexcitatory effects either directly by facilitating central sympathetic outflow or indirectly by activation of the baroreceptor reflex in response to its peripheral vasodilatory effects (5). The observation that C-peptide–negative diabetic patients retain a blunted MSNA responsiveness to oral glucose, even in the absence of endogenous insulin (6), suggests that insulin-independent factors such as hemodynamic adjustment to splanchnic vasodilation and gastrointestinal distension may also contribute to sympathetic activation after carbohydrate ingestion (4). Postprandial SNS stimulation is physiologically important in the regulation of facultative thermogenesis or the rise in energy expenditure after dietary intake (7).A growing body of data suggests that obesity and the insulin-resistant state are accompanied by impaired sympathetic neural responsiveness to physiological hyperinsulinemia, glucose ingestion, and changing energy states. Vollenweider et al. (8) showed that in lean young subjects MSNA increased by 94% in response to euglycemic hyperinsulinemia, whereas in age-matched obese subjects the increase was only 9%. MSNA responses to oral glucose are diminished in insulin-resistant Pima Indians (9), and short-term under- and overfeeding is accompanied by blunted sympathetic responsiveness in obese subjects (10). The metabolic syndrome is an increasingly prevalent multidimensional risk factor for cardiovascular disease and type 2 diabetes. Several indexes of SNS activity, such as norepinephrine spillover from sympathetic nerves and MSNA at rest, are known to be increased in subjects with the metabolic syndrome even in the absence of hypertension (11,12). We have recently demonstrated that obese insulin-resistant subjects with metabolic syndrome have both blunted and delayed norepinephrine spillover and MSNA responses to oral glucose compared with obese insulin-sensitive subjects with metabolic syndrome (13). Sympathetic responsiveness related inversely to measures of central adiposity and the insulin response and positively to fitness level (13).Weight loss and exercise are recommended as first-line treatments for the metabolic syndrome, and both lifestyle modalities are known to reduce SNS activity and improve metabolic syndrome components (11). No study to date has examined whether weight loss alters the dynamic sympathetic neural response to glucose ingestion. The aims of the present investigation were twofold: 1) to test the hypothesis that weight loss would reverse the blunted SNS response to glucose in obese individuals with metabolic syndrome and 2) to compare the benefits of weight loss in insulin-resistant and insulin-sensitive subjects. To accomplish these aims, we simultaneously measured plasma glucose, insulin, leptin, whole-body norepinephrine kinetics, calf blood flow, intra-arterial blood pressure, and heart rate during a standard oral glucose tolerance test (OGTT) at baseline and after a 12-week weight loss program.     

European survey on the use of prophylactic fluconazole in neonatal intensive care units

Neonatal fungal infections are associated with substantial mortality and morbidity. Although prophylactic use of several antifungals has been proposed, this practice remains controversial. In order to evaluate the use of fluconazole prophylaxis in European NICUs, we conducted a cross-sectional survey by means of a structured questionnaire that was sent to European level II and III neonatal intensive care units, over a 9-month period, as part of a neonatal research FP7 European project. A total of 193 questionnaires from 28 countries were analysed. Use of antifungal prophylaxis was reported by 55% of the responders, and the most frequently used antifungal agent was fluconazole (92%). Main indications for prophylaxis were low gestational age (<28 weeks) and birth weight (<1,000 g). A dose of 3 mg/kg was used in 66% of NICUs using fluconazole, with an administration interval of 72 h in 52% of them. All responders acknowledged the need for additional trials on the efficacy of prophylactic fluconazole. Non-users of fluconazole prophylaxis were more likely to be influenced by the local incidence of candidiasis, the risk of increasing antifungal resistance and the absence of specific recommendations by paediatric societies. Conclusions: Major concerns about the use of fluconazole prophylaxis include its efficacy, the risk of emergence of resistant species and the absence of clear consensus to support routine use. Future studies that address these issues will contribute to a more rational use of fluconazole prophylaxis.     

Sympathetic activity in major depressive disorder: identifying those at increased cardiac risk?

BACKGROUND: Evidence exists linking major depressive disorder (MDD) with clinical cardiovascular events. The importance of the sympathetic nervous system in the generation of cardiac risk in other contexts is established. OBJECTIVE: To examine the importance of the sympathetic nervous system in the generation of cardiac risk in patients with major depressive disorder (MDD). METHODS: Studies were performed in 39 patients meeting the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) criteria for MDD and in 76 healthy subjects. Treatment for patients consisted of selective serotonin reuptake inhibition (SSRI) for 12 weeks. Whole body and cardiac sympathetic activity were examined using noradrenaline isotope dilution methodology and sympathetic nerve recording techniques. Measurement of the extraction of infused tritiated noradrenaline by the heart, and estimation of cardiac dihydroxyphenylglycol production provided direct quantification of neuronal noradrenaline reuptake. RESULTS: Sympathetic activity, particularly in the heart and for the whole body, in patients with MDD followed a bimodal distribution. Elevated values were observed in patients with co-morbid panic disorder (P = 0.006). Consistent with a defect in noradrenaline reuptake, the cardiac extraction of tritiated noradrenaline (0.80 +/- 0.01 versus 0.56 +/- 0.04%, P < 0.001) and cardiac dihydroxyphenylglycol overflow (109 +/- 8 versus 73 +/- 11, P = 0.01) were reduced in patients with MDD. SSRI therapy abolished the excessive sympathetic activation, with whole body noradrenaline spillover falling from 518 +/- 83 to 290 +/- 41 ng/min (P = 0.008). CONCLUSIONS: We have identified a subset of patients with MDD in whom sympathetic nervous activity is extraordinarily high, including in the sympathetic outflow to the heart. Treatment with an SSRI may reduce sympathetic activity in a manner likely to reduce cardiac risk.     

Prospective memory functioning among ecstasy/polydrug users: evidence from the Cambridge Prospective Memory Test (CAMPROMPT)

Rationale  

Prospective memory (PM) deficits in recreational drug users have been documented in recent years. However, the assessment of PM has largely been restricted to self-reported measures that fail to capture the distinction between event-based and time-based PM. The aim of the present study is to address this limitation.