Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

First-line single-agent pembrolizumab for PD-L1-positive (tumor proportion score ≥ 50%) advanced non-small cell lung cancer in the real world: impact in brain metastasis: a national French multicentric cohort (ESCKEYP GFPC study).

BACKGROUND: Few real-world data are available in patients with advanced metastatic non-small cell lung cancer (NSCLC) treated with first-line immunotherapy, particularly in those with brain metastases at treatment initiation. METHODS: This was a national, retrospective, multicenter study that consecutively included all patients with PD-L1-positive (tumor proportion score ≥ 50%) advanced NSCLC who initiated first-line treatment with pembrolizumab as a single agent between May 2017 (date of availability of pembrolizumab in this indication in France) to November 22, 2019 (approval of the pembrolizumab-chemotherapy combination). Data were collected from medical records with local response assessment. RESULTS: The cohort included 845 patients and 176 (20.8%) had brain metastases at diagnosis. There were no significant differences in outcomes for patients with and without brain metastases: 9.2 (95% CI 5.6-15) and 8 (95% CI 6.7-9.2, p = 0.3) months for median progression-free survival (PFS) and, 29.5 (95% CI 17.2-NA) and 22 (95% CI 17.8-27.1, p = 0.3) months for median overall survival (OS), respectively. Overall response rates were 47% and 45% in patients with and without cerebral metastases. In multivariate analysis, performance status 2-4 vs. 0-1 and neutrophil-to-lymphocyte ratio ≥ 4 vs. < 4 were the main independent negative factors for OS; brain metastasis was not an independent factor for OS. CONCLUSION: In this large multicenter cohort, nearly 20% of patients initiating pembrolizumab therapy for advanced NSCLC had cerebral metastases. There was no significant difference in response rates, PFS and OS between patients with and without brain metastases.

Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Safety of oral hydration after cisplatin infusion in an outpatient lung cancer unit.

PURPOSE: Hydration is needed before and after cisplatin infusion for reducing the risk of nephrotoxicity. Even though there is no standard regimen, patients receive mostly intravenous hydration before and after cisplatin leading hospitalization during at least one night. Since the feasibility has been published, oral hydration after cisplatin was implemented in our practice. The safety of this new way of hydration needs to be assessed in clinical practice. METHODS: We collected medical records from patients treated by cisplatin for lung cancer in our unit between 2010 and 2016. We retrospectively analyzed the incidence of cisplatin induced nephrotoxicity between after and before the change of hydration regimen. RESULTS: Our patient cohort included 241 patients hydrated by intravenous regimen (IV/IV group) and 276 patient hydrated by intravenous and oral regimen (IV/PO group). Grade ≥ 1 nephrotoxicity occurred in 39.4 and 25.7% in the IV/IV and IV/PO groups respectively (p = 0.001). Age over 70 at baseline was a predictive factor for nephrotoxicity, but not estimated glomerular filtration rate nor cisplatin-associated drugs. After a multivariate analysis, age remained a predictive factor for nephrotoxicity and IV/PO hydration associated with a decrease in nephrotoxic risk. CONCLUSION: The implementation of oral hydration in our practice was not associated with an increase in nephrotoxicity. Our observation based on large data from clinical practice shows that oral hydration after cisplatin is safe.

Clinical and Molecular Epidemiology of Staphylococcal Toxic Shock Syndrome in the United Kingdom.

Staphylococcal toxic shock syndrome (TSS) was originally described in menstruating women and linked to TSS toxin 1 (TSST-1)-producing Staphylococcus aureus. Using UK national surveillance data, we ascertained clinical, molecular and superantigenic characteristics of TSS cases. Average annual TSS incidence was 0.07/100,000 population. Patients with nonmenstrual TSS were younger than those with menstrual TSS but had the same mortality rate. Children <16 years of age accounted for 39% of TSS cases, most caused by burns and skin and soft tissue infections. Nonmenstrual TSS is now more common than menstrual TSS in the UK, although both types are strongly associated with the tst+ clonal complex (CC) 30 methicillin-sensitive S. aureus lineage, which accounted for 49.4% of all TSS and produced more TSST-1 and superantigen bioactivity than did tst+ CC30 methicillin-resistant S. aureus strains. Better understanding of this MSSA lineage and infections in children could focus interventions to prevent TSS in the future.

Changes in macrophage transcriptome associate with systemic sclerosis and mediate GSDMA contribution to disease risk.

OBJECTIVES: Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc. METHODS: We carried out RNA sequencing and genome-wide genotyping in MDMs from 57 patients with SSc and 15 controls. Our differential expression and expression quantitative trait locus (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes. RESULTS: We identified 602 genes upregulated and downregulated in SSc macrophages that were significantly enriched for genes previously implicated in SSc susceptibility (P=5×10-4), and 270 cis-regulated genes in MDMs. Among these, GSDMA was reported to carry an SSc risk variant (rs3894194) regulating expression of neighbouring genes in blood. We show that GSDMA is upregulated in SSc MDMs (P=8.4×10-4) but not in the skin, and is a significant eQTL in SSc macrophages and lipopolysaccharide/interferon gamma (IFNγ)-stimulated monocytes. Furthermore, we identify an SSc macrophage transcriptome signature characterised by upregulation of glycolysis, hypoxia and mTOR signalling and a downregulation of IFNγ response pathways. CONCLUSIONS: Our data further establish the link between macrophages and SSc, and suggest that the contribution of the rs3894194 risk variant to SSc susceptibility can be mediated by GSDMA expression in macrophages.

Characteristics of Lung Cancer in Patients Younger than 40 Years: A Prospective Multicenter Analysis in France.

OBJECTIVES: The aim of this study was to describe the demographic and clinico-pathological characteristics of lung cancer in patients younger than 40 years. MATERIALS AND METHODS: This was a prospective study performed within the Groupe Français de Pneumo-Cancérologie. Consecutive patients diagnosed with lung cancer before the age of 40 years were eligible. Data on demographics, medical history, clinico-pathological characteristics, treatment and overall survival were analysed. RESULTS: In total, 146 patients were included from January 2011 to December 2013. Median age was 38 years (IQR: 34-40). Women accounted for 41%. Main histological type was adenocarcinoma (77%). Only 3% had a prior history of cancer, but a family history (first- or second-degree relatives) of cancer was reported in 80 (55%) patients; 85 and 50% were current or past smokers of tobacco and cannabis, respectively; 82% had stage IIIB/IV at diagnosis. Median overall survival was 15.3 (95% CI: 8.1-24.0) months in the whole population, 10.3 (95% CI: 12.5-14.2) months in stage IV and 15 (95% CI: 8.7-35.2) months in stage III. One- and two-year overall survival rates were 57% (95 CI: 49-65) and 31.5% (95 CI: 27-43), respectively. Compared to smokers, non-smokers were significantly younger and more often females. Median overall survival was not statistically different between smokers and non-smokers.

Genetic analysis of the cardiac methylome at single nucleotide resolution in a model of human cardiovascular disease.

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.

Genomic pathology of SLE-associated copy-number variation at the FCGR2C/FCGR3B/FCGR2B locus.

Reduced FCGR3B copy number is associated with increased risk of systemic lupus erythematosus (SLE). The five FCGR2/FCGR3 genes are arranged across two highly paralogous genomic segments on chromosome 1q23. Previous studies have suggested mechanisms for structural rearrangements at the FCGR2/FCGR3 locus and have proposed mechanisms whereby altered FCGR3B copy number predisposes to autoimmunity, but the high degree of sequence similarity between paralogous segments has prevented precise definition of the molecular events and their functional consequences. To pursue the genomic pathology associated with FCGR3B copy-number variation, we integrated sequencing data from fosmid and bacterial artificial chromosome clones and sequence-captured DNA from FCGR3B-deleted genomes to establish a detailed map of allelic and paralogous sequence variation across the FCGR2/FCGR3 locus. This analysis identified two highly paralogous 24.5 kb blocks within the FCGR2C/FCGR3B/FCGR2B locus that are devoid of nonpolymorphic paralogous sequence variations and that define the limits of the genomic regions in which nonallelic homologous recombination leads to FCGR2C/FCGR3B copy-number variation. Further, the data showed evidence of swapping of haplotype blocks between these highly paralogous blocks that most likely arose from sequential ancestral recombination events across the region. Functionally, we found by flow cytometry, immunoblotting and cDNA sequencing that individuals with FCGR3B-deleted alleles show ectopic presence of FcγRIIb on natural killer (NK) cells. We conclude that FCGR3B deletion juxtaposes the 5'-regulatory sequences of FCGR2C with the coding sequence of FCGR2B, creating a chimeric gene that results in an ectopic accumulation of FcγRIIb on NK cells and provides an explanation for SLE risk associated with reduced FCGR3B gene copy number.

Health-related quality of life in elderly patients with advanced non-small cell lung cancer comparing carboplatin and weekly paclitaxel doublet chemotherapy with monotherapy.

In the Intergroupe Francophone de Cancérologie Thoracique 0501 trial the carboplatin-paclitaxel chemotherapy increased toxicity (most frequent, decreased neutrophil count, asthenia). We longitudinally compared health-related quality of life (HRQoL) of the two treatment arms.In total, 451 patients aged 70-89 years with advanced non-small cell lung cancer (NSCLC) were randomly assigned to receive carboplatin plus paclitaxel or vinorelbine or gemcitabine. HRQoL was assessed by means of the European Organisation for Research and Treatment of Cancer QLQ-C30 questionnaire at baseline, week 6 and week 18.Using a five-point decrease as the minimal clinically important difference, patients treated with the chemotherapy doublet exhibited a significant longer time until definitive deterioration (TUDD) for two HRQoL dimensions: physical functioning (median TUDD: 2.04 for the doublet versus 1.71 months for monotherapy; log-rank p=0.01) and nausea and vomiting (median: not reached versus 4.83, respectively; log-rank p=0.046). Cox multivariate analysis revealed the carboplatin and paclitaxel arm to be independently associated with longer TUDD for these two HRQoL dimensions. In addition, TUDD didn't significantly differ between the two arms for all the other HRQoL dimensions.The chemotherapy doublet did not reduce TUDD in elderly patients with advanced NSCLC. Moreover, TUDD was prolonged for two HRQoL dimensions, namely physical functioning and nausea and vomiting.

Erlotinib versus carboplatin and paclitaxel in advanced lepidic adenocarcinoma: IFCT-0504.

The IFCT-0504 phase II trial evaluated the efficacy of erlotinib versus carboplatin-paclitaxel (CP) as first-line treatment in 130 cases of advanced lepidic-predominant adenocarcinoma (ADC).The primary objective of the study was treatment efficacy, evaluated based on an end-point of disease control at 16 weeks.The primary objective was met, with a disease control in 35 (53%) out of 66 patients treated with CP and in 25 (39.1%) out of 64 patients treated with erlotinib. Median progression-free survival (PFS) for the total population was 3.6 months. The disease control rate did not differ between either the therapeutic arms or pathological subtypes, whereas there was a strong interaction between treatment arms and tumour pathological subtypes for PFS (p=0.009). Mucinous tumour patients treated with erlotinib exhibited an increased progression risk (hazard ratio 3.4, 95% CI 1.7-6.5; p≤0.001). The PFS for nonmucinous tumour patients was similar in both arms. Median overall survival was 20.1 months and did not differ between therapeutic arms. These findings were not further elucidated by molecular analyses and the toxicity profiles were as expected.Our study demonstrated the dominant role of CP alongside erlotinib in the management of advanced lepidic ADC. Based on these findings, erlotinib should not be administered in first-line therapy to patients with lepidic ADC in the absence of an epidermal growth factor receptor mutation.

Second-line therapy in elderly patients with advanced nonsmall cell lung cancer.

There is no dedicated study on second-line treatment for elderly patients with advanced nonsmall cell lung cancer (NSCLC). We report the results on second-line erlotinib therapy from our previously published phase III study comparing single-agent therapy with platinum-based doublet (carboplatin plus paclitaxel) therapy in 451 elderly patients. Erlotinib was given to patients exhibiting disease progression or experiencing excessive toxicity during first-line therapy, until further progression or unacceptable toxicity. In total, 292 (64.7%) patients received erlotinib as second-line therapy. Initial performance status 0-1, stage IV NSCLC and an Activities of Daily Living score of 6 were independent factors for receiving erlotinib. Median (95% CI) overall survival was 4 (3.2-6.7) versus 6.8 (5.0-8.3) months in the single-agent and doublet arms, respectively (p=0.089). Performance status 0-1, never having smoked, adenocarcinoma and weight loss ≤5% were favourable independent prognostic factors of survival, whereas the randomisation arm had no significant impact. Among the 292 patients who received erlotinib, 60 (20.5%) experienced grade 3-4 toxic effects, the most frequent being rash. Erlotinib as second-line therapy is feasible, leading to efficacy results similar to those obtained in a previous randomised study that was not dedicated to elderly patients, with acceptable toxicity.

Targeted genetic testing for familial hypercholesterolaemia using next generation sequencing: a population-based study.

BACKGROUND: Familial hypercholesterolaemia (FH) is a common Mendelian condition which, untreated, results in premature coronary heart disease. An estimated 88% of FH cases are undiagnosed in the UK. We previously validated a method for FH mutation detection in a lipid clinic population using next generation sequencing (NGS), but this did not address the challenge of identifying index cases in primary care where most undiagnosed patients receive healthcare. Here, we evaluate the targeted use of NGS as a potential route to diagnosis of FH in a primary care population subset selected for hypercholesterolaemia. METHODS: We used microfluidics-based PCR amplification coupled with NGS and multiplex ligation-dependent probe amplification (MLPA) to detect mutations in LDLR, APOB and PCSK9 in three phenotypic groups within the Generation Scotland: Scottish Family Health Study including 193 individuals with high total cholesterol, 232 with moderately high total cholesterol despite cholesterol-lowering therapy, and 192 normocholesterolaemic controls. RESULTS: Pathogenic mutations were found in 2.1% of hypercholesterolaemic individuals, in 2.2% of subjects on cholesterol-lowering therapy and in 42% of their available first-degree relatives. In addition, variants of uncertain clinical significance (VUCS) were detected in 1.4% of the hypercholesterolaemic and cholesterol-lowering therapy groups. No pathogenic variants or VUCS were detected in controls. CONCLUSIONS: We demonstrated that population-based genetic testing using these protocols is able to deliver definitive molecular diagnoses of FH in individuals with high cholesterol or on cholesterol-lowering therapy. The lower cost and labour associated with NGS-based testing may increase the attractiveness of a population-based approach to FH detection compared to genetic testing with conventional sequencing. This could provide one route to increasing the present low percentage of FH cases with a genetic diagnosis.

Activating transcription factor 1 directs Mhem atheroprotective macrophages through coordinated iron handling and foam cell protection.

RATIONALE: Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH. OBJECTIVE: This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. METHODS AND RESULTS: To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-ß). Heme-induced HO-1 and LXR-ß were suppressed by knockdown of ATF-1, and HO-1 and LXR-ß were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-ß in turn led to induction of other genes central to cholesterol efflux, such as LXR-α and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. CONCLUSIONS: These results show that ATF-1 mediates HO-1 induction by heme and drives macrophage adaptation to intraplaque hemorrhage. Our definition of an ATF-1-mediated pathway for linked protection from foam cell formation and oxidant stress may have therapeutic potential.

Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease.

Integration of genome-wide expression profiling with linkage analysis is a new approach to identifying genes underlying complex traits. We applied this approach to the regulation of gene expression in the BXH/HXB panel of rat recombinant inbred strains, one of the largest available rodent recombinant inbred panels and a leading resource for genetic analysis of the highly prevalent metabolic syndrome. In two tissues important to the pathogenesis of the metabolic syndrome, we mapped cis- and trans-regulatory control elements for expression of thousands of genes across the genome. Many of the most highly linked expression quantitative trait loci are regulated in cis, are inherited essentially as monogenic traits and are good candidate genes for previously mapped physiological quantitative trait loci in the rat. By comparative mapping we generated a data set of 73 candidate genes for hypertension that merit testing in human populations. Mining of this publicly available data set is expected to lead to new insights into the genes and regulatory pathways underlying the extensive range of metabolic and cardiovascular disease phenotypes that segregate in these recombinant inbred strains.

Safety and Patient-Reported outcomes of atezolizumab plus chemotherapy with or without bevacizumab in stage IIIB/IV non-squamous non-small cell lung cancer with EGFR mutation, ALK rearrangement or ROS1 fusion progressing after targeted therapies (GFPC 06-2018 study).

BACKGROUND: In an open-label multicenter non-randomized non-comparative phase II study in patients with stage IIIB/IV non-squamous non-small cell lung cancer (NSCLC), oncogenic addiction (EGFR mutation or ALK/ROS1 fusion), with disease progression after tyrosine-kinase inhibitor and no prior chemotherapy (NCT04042558), atezolizumab, carboplatin, pemetrexed with or without bevacizumab showed some promising result. Beyond the clinical evaluation, we assessed safety and patient-reported outcomes (PROs) to provide additional information on the relative impact of adding atezolizumab to chemotherapy with and without bevacizumab in this population. MATERIALS: Patients received platinum-pemetrexed-atezolizumab-bevacizumab (PPAB cohort) or, if not eligible, platinum-pemetrexed-atezolizumab (PPA cohort). The incidence, nature, and severity of adverse events (AEs) were assessed. PROs were evaluated using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-Core 30 and EORTC QLQ-Lung Cancer 13). RESULT: Overall, 68 (PPAB) and 72 (PPA) patients were evaluable for safety. Grade 3-4 AEs occurred in 83.8% (PPAB) and 63.9% (PPA). Grade 3-4 atezolizumab-related AEs occurred in 29.4% and 19.4%, respectively. Grade 3-4 bevacizumab-related AEs occurred in 36.8% (PPAB). Most frequent grade 3-4 AEs were neutropenia (19.1% in PPAB; 23.6% in PPA) and asthenia (16.2% in PPAB; 9.7% in PPA). In PPAB, we observed a global stability in global health security (GHS) score, fatigue and dyspnea with a constant tendency of improvement, and a significant improvement in cough. In PPA, we observed a significant improvement in GHS score with a significant improvement in fatigue, dyspnea and cough. At week 54, we observed an improvement from baseline in GHS score for 49.2% of patients. In both cohorts, patients reported on average no clinically significant worsening in their overall health or physical functioning scores. CONCLUSION: PPAB and PPA combinations seem tolerable and manageable in patients with stage IIIB/IV non-squamous NSCLC with oncogenic addiction (EGFR mutation or ALK/ROS1 fusion) after targeted therapies.

Combined ChIP-Seq and transcriptome analysis identifies AP-1/JunD as a primary regulator of oxidative stress and IL-1ß synthesis in macrophages.

BACKGROUND: The oxidative burst is one of the major antimicrobial mechanisms adopted by macrophages. The WKY rat strain is uniquely susceptible to experimentally induced macrophage-dependent crescentic glomerulonephritis (Crgn). We previously identified the AP-1 transcription factor JunD as a determinant of macrophage activation in WKY bone marrow-derived macrophages (BMDMs). JunD is over-expressed in WKY BMDMs and its silencing reduces Fc receptor-mediated oxidative burst in these cells. RESULTS: Here we combined Jund RNA interference with microarray analyses alongside ChIP-sequencing (ChIP-Seq) analyses in WKY BMDMs to investigate JunD-mediated control of macrophage activation in basal and lipopolysaccharide (LPS) stimulated cells. Microarray analysis following Jund silencing showed that Jund activates and represses gene expression with marked differential expression (>3 fold) for genes linked with oxidative stress and IL-1ß expression. These results were complemented by comparing whole genome expression in WKY BMDMs with Jund congenic strain (WKY.LCrgn2) BMDMs which express lower levels of JunD. ChIP-Seq analyses demonstrated that the increased expression of JunD resulted in an increased number of binding events in WKY BMDMs compared to WKY.LCrgn2 BMDMs. Combined ChIP-Seq and microarray analysis revealed a set of primary JunD-targets through which JunD exerts its effect on oxidative stress and IL-1ß synthesis in basal and LPS-stimulated macrophages. CONCLUSIONS: These findings demonstrate how genetically determined levels of a transcription factor affect its binding sites in primary cells and identify JunD as a key regulator of oxidative stress and IL-1ß synthesis in primary macrophages, which may play a role in susceptibility to Crgn.

Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA.

Bronchial colonisation in patients with lung cancer: a prospective study.

Bronchial colonisation is frequently reported in patients with lung cancer, and has a potential impact on therapeutic management and prognosis. We aimed to prospectively define the prevalence and nature of bronchial colonisation in patients at the time of diagnosing lung cancer. 210 consecutive patients with lung cancer underwent a flexible bronchoscopy for lung cancer. The type and frequency of bacterial, mycobacterial and fungal colonisation were analysed and correlated with the patients' and tumours' characteristics. Potential pathogens were found in 48.1% of samples: mainly the Gram-negative bacilli Escherichia coli (8.1%), Haemophilus influenzae (4.3%) and Enterobacter spp. (2.4%); Gram-positive cocci, Staphylococcus spp. (12.9%) and Streptococcus pneumoniae (3.3%); atypical mycobacteria (2.9%); Candida albicans (42.9%); and Aspergillus fumigatus (6.2%). Aged patients (p=0.02) with chronic obstructive pulmonary disease (p=0.008) were significantly more frequently colonised; however, tumour stage, atelectasis, bronchial stenosis and abnormalities of chest radiography were not associated with a higher rate of colonisation. Squamous cell carcinoma tended to be more frequently colonised than other histological subtypes. Airway colonisation was reported in almost half of patients presenting with lung cancer, mainly in fragile patients, and was significantly associated with worse survival (p=0.005). Analysing colonisation status of patients at the time of diagnosis may help improve the management of lung cancer.

The use of next-generation sequencing in clinical diagnosis of familial hypercholesterolemia.

PURPOSE: Familial hypercholesterolemia is a common Mendelian disorder associated with early-onset coronary heart disease that can be treated by cholesterol-lowering drugs. The majority of cases in the United Kingdom are currently without a molecular diagnosis, which is partly due to the cost and time associated with standard screening techniques. The main purpose of this study was to test the sensitivity and specificity of two next-generation sequencing protocols for genetic diagnosis of familial hypercholesterolemia. METHODS: Libraries were prepared for next-generation sequencing by two target enrichment protocols; one using the SureSelect Target Enrichment System and the other using the PCR-based Access Array platform. RESULTS: In the validation cohort, both protocols showed 100% specificity, whereas the sensitivity for short variant detection was 100% for the SureSelect Target Enrichment and 98% for the Access Array protocol. Large deletions/duplications were only detected using the SureSelect Target Enrichment protocol. In the prospective cohort, the mutation detection rate using the Access Array was highest in patients with clinically definite familial hypercholesterolemia (67%), followed by patients with possible familial hypercholesterolemia (26%). CONCLUSION: We have shown the potential of target enrichment methods combined with next-generation sequencing for molecular diagnosis of familial hypercholesterolemia. Adopting these assays for patients with suspected familial hypercholesterolemia could improve cost-effectiveness and increase the overall number of patients with a molecular diagnosis.