Background Genomic rearrangements in cancer cells can create fusion genes that encode chimeric proteins or alter the expression of coding and non-coding RNAs. In some cancer types, fusions involving specific kinases are used as targets for therapy. Fusion genes can be detected by whole genome sequencing (WGS) and targeted fusion panels, but RNA sequencing (RNA-Seq) has the advantageous capability of broadly detecting expressed fusion transcripts. Results We developed a pipeline for validation of fusion transcripts identified in RNA-Seq data using matched WGS data from The Cancer Genome Atlas (TCGA) and applied it to 910 tumors from 11 different cancer types. This resulted in 4237 validated gene fusions, 3049 of them with at least one identified genomic breakpoint. Utilizing validated fusions as true positive events, we trained a machine learning classifier to predict true and false positive fusion transcripts from RNA-Seq data. The final precision and recall metrics of the classifier were 0.74 and 0.71, respectively, in an independent dataset of 249 breast tumors. Application of this classifier to all samples with RNA-Seq data from these cancer types vastly extended the number of likely true positive fusion transcripts and identified many potentially targetable kinase fusions. Further analysis of the validated gene fusions suggested that many are created by intrachromosomal amplification events with microhomology-mediated non-homologous end-joining. Conclusions A classifier trained on validated fusion events increased the accuracy of fusion transcript identification in samples without WGS data. This allowed the analysis to be extended to all samples with RNA-Seq data, facilitating studies of tumor biology and increasing the number of detected kinase fusions. Machine learning could thus be used in identification of clinically relevant fusion events for targeted therapy. The large dataset of validated gene fusions generated here presents a useful resource for development and evaluation of fusion transcript detection algorithms.

Genomic rearrangements in cancer cells can create gene fusions where the juxtaposition of two different genes leads to the production of chimeric proteins or altered gene expression through promoter-swapping. We have previously shown that fusion transcripts involving microRNA (miRNA) host genes contribute to deregulation of miRNA expression regardless of the protein-coding potential of these transcripts. Many different genes can also be used as 5' partners by a miRNA host gene in what we named recurrent miRNA-convergent fusions. Here, we have explored the properties of 5' partners in fusion transcripts that involve miRNA hosts in breast tumours from The Cancer Genome Atlas (TCGA). We hypothesised that firstly, 5' partner genes should belong to pathways and transcriptional programmes that reflect the tumour phenotype and secondly, there should be a selection for fusion events that shape miRNA expression to benefit the tumour cell through the known hallmarks of cancer. We found that the set of 5' partners in miRNA host fusions is non-random, with overrepresentation of highly expressed genes in pathways active in cancer including epithelial-to-mesenchymal transition, translational regulation and oestrogen signalling. Furthermore, many miRNAs were upregulated in samples with host gene fusions, including established oncogenic miRNAs such as mir-21 and the mir-106b~mir-93~mir-25 cluster. To the list of mechanisms for deregulation of miRNA expression, we have added fusion transcripts that change the promoter region. We propose that this adds material for genetic selection and tumour evolution in cancer cells and that miRNA host fusions can act as tumour 'drivers'.

The mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism (PA) and are characterized by constitutively producing aldosterone, which leads to hypertension. Several mutations have been identified in ion channels or ion channel-associated genes that result in APAs. To date, no studies have used a genome-wide association study (GWAS) approach to search for predisposing loci for APAs. Thus, we investigated Scandinavian APA cases (n = 35) and Swedish controls (n = 60) in a GWAS and discovered a susceptibility locus on chromosome Xq13.3 (rs2224095, OR = 7.9, 95% CI = 2.8–22.4, P = 1 × 10 –7 ) in a 4-Mb region that was significantly associated with APA. Direct genotyping of sentinel SNP rs2224095 in a replication cohort of APAs (n = 83) and a control group (n = 740) revealed persistently strong significance (OR = 6.1, 95% CI = 3.5–10.6, p < 0.0005). We sequenced an adjacent gene, MAGEE1 , of the sentinel SNP and identified a rare variant in one APA, p.Gly327Glu, which is complementary to other mutations in our primary cohort. Expression quantitative trait loci (eQTL) were investigated on the X-chromosome, and 24 trans-eQTL were identified. Some of the genes identified by trans-eQTL point towards a novel mechanistic explanation for the association of the SNPs with APAs. In conclusion, our study provides further insights into the genetic basis of APAs.

Artificial light is an important and necessary part of urban environments, but light can have substantial direct and indirect effects on populations of various organisms. Urban areas are often situated close to water and thus organisms dependent on water could be especially vulnerable. Trichoptera is one of the most abundant insect orders in freshwater, but its attraction to light has not been analysed in detail. We contrasted catches in light traps and passive traps at three locations in Sweden. The results showed that artificial light can affect Trichoptera populations. Attraction to light varied between Trichoptera species and females were more attracted than males. Day‐, evening‐ and especially night‐active species were all attracted to light. Light catches of day‐ and evening‐active Trichoptera could partly be a consequence of atypical flight activity, i.e. they are deceived to take flight when a lamp is lit during night. In all, artificial light can alter Trichoptera populations, sex ratios and species composition. This impact should be considered when erecting and managing light sources near waterways.

Abstract Background and Aims The genome of every cell accumulates somatic mutations while aging. Somatic mutation data can be used to track a cell´s exposure to mutagens, thereby allowing the discovery of cell types that are more susceptible to mutate and become cancer and the underling mechanisms. Method To detect somatic mutations in healthy, human kidney, we set up a protocol for whole genome DNA sequencing of single non-cancer cells. The protocol requires in vitro clonal expansion prior to sequencing, a step that restricts the analysis to cells able to proliferate in vitro (progenitors), but allows a gene expression analysis in addition to genome sequencing. Cells were obtained from six living kidney donors undergoing surgery. In addition to the kidney cortex biopsy, multiple control tissues (skin, subcutaneous fata and visceral fat) were obtained from each donor, allowing a well-controlled comparison of mutation landscapes in different cell types. Donors´ age spanned from 30 to 69. Results Somatic mutation and gene expression data showed that we were able to culture two different populations of CD133/CD24 positive, tubular cells. One population showed a low amount of somatic mutations and a mutation profile similar to progenitors from other tissues (fat, skeletal muscle and blood), consistent with a lack of exposure to mutagens. Conversely, the other population showed high mutation burden and a unique mutation landscape, characterized by mutation enrichment in active chromatin, regulatory, and transcribed regions. Accumulation of potential, cancer-driver mutations was 6-fold faster in these compared to control cells. The mutation profile was similar to that of the most common kidney cancer subtypes (clear cell- and papillary cell-carcinoma) and indicated that these cells originated from the proximal tubule, in agreement with gene expression data. Conclusion Our somatic mutation data from single genomes support the existence of two different populations of proliferating tubule cells in healthy, human kidney. One is protected from mutagen exposure, similar to stem cells from other organs. The other population is derived from damaged proximal tubule cells and shows a high mutation rate between 30 and 70 years of age. Mutations are enriched in transcribed genes and regulatory regions, thus enhancing the chances of tumorigenic transformation and suggesting conditions that predispose to cancer in the kidney proximal tubule.

Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.

Background: The lifelong accumulation of somatic mutations underlies age-related phenotypes and cancer. Mutagenic forces are thought to shape the genome of aging cells in a tissue-specific way. Whole genome analyses of somatic mutation patterns, based on both types and genomic distribution of variants, can shed light on specific processes active in different human tissues and their effect on the transition to cancer. Results: To analyze somatic mutation patterns, we compile a comprehensive genetic atlas of somatic mutations in healthy human cells. High-confidence variants are obtained from newly generated and publicly available whole genome DNA sequencing data from single non-cancer cells, clonally expanded in vitro. To enable a well-controlled comparison of different cell types, we obtain single genome data (92% mean coverage) from multi-organ biopsies from the same donors. These data show multiple cell types that are protected from mutagens and display a stereotyped mutation profile, despite their origin from different tissues. Conversely, the same tissue harbors cells with distinct mutation profiles associated to different differentiation states. Analyses of mutation rate in the coding and non-coding portions of the genome identify a cell type bearing a unique mutation pattern characterized by mutation enrichment in active chromatin, regulatory, and transcribed regions. Conclusions: Our analysis of normal cells from healthy donors identifies a somatic mutation landscape that enhances the risk of tumor transformation in a specific cell population from the kidney proximal tubule. This unique pattern is characterized by high rate of mutation accumulation during adult life and specific targeting of expressed genes and regulatory regions.

Abstract Background: Mutational processes generate unique patterns of base substitutions generally referred to as mutational signatures. Mutational signatures can give insights into fundamental mutational processes underlying somatic mutations in tumors and have been shown to provide a starting point from which to evaluate therapeutic options. Neuroblastoma is clinically heterogeneous ranging from tendency of spontaneous regression to frequent formation of metastases. Earlier studies have identified several recurrent genetic alterations, including mutations in genes such as ALK and ATRX, as well as chromosomal rearrangements such as MYCN amplification, 11q deletion and 17 gain. Despite recent progress in the treatment of high-risk neuroblastoma, patients in this subgroup still have a poor prognosis, underscoring the need to further stratify patients in order to help inform clinical decision making. Methods: Here, we sought to characterize high-risk patients and their underlying pattern of aberrations by conducting whole genome sequencing of 30 neuroblastoma patients. For each tumor/normal pair, the mutational landscape comprising single nucleotide and structural variants including copy number alterations was investigated. Non-negative matrix factorization was used to decompose single nucleotide variants to discover mutational signatures. Results: De novo mutational signature analysis identified two mutational profiles (signature A and signature B) that were compared to known signatures of the pan-cancer study by Alexandrov et al. (2013). The landscape of signature A is closest to their signature 18 (0.97 cosine similarity) and revealed a strong bias towards C>A substitutions mainly found in neuroblastoma, and signature B is closest to signature 5 (0.82 cosine similarity) identified in most of the cancers. Out of 13 patients with a clear dominance of signature A, 12 patients were classified with MYCN amplification or 11q deletion. Furthermore, 5 out of 6 patients with a main contribution of signature B were metastasis or relapse cases. Conclusions: Our results suggest that what is currently considered to be the typical neuroblastoma signature is mainly present in primary high-risk patients whereas the mutational landscape is altered in more advanced stages of the disease. To better understand the connection between the identified signatures and the high-risk subgroup, we are currently investigating the genome wide landscape of the observed C>A substitutions. Citation Format: Susanne E. Reinsbach, Malin Larsson, Angela Martinez-Monleon, Rose-Marie Sjöberg, Niloufar Javanmardi, Anna Djos, Per Kogner, Susanne Fransson, Erik Larsson Lekholm, Tommy Martinsson. Identification of mutational signatures in high-risk neuroblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2246.

Human aging is associated with a decline in skeletal muscle (SkM) function and a reduction in the number and activity of satellite cells (SCs), the resident stem cells. To study the connection between SC aging and muscle impairment, we analyze the whole genome of single SC clones of the leg muscle vastus lateralis from healthy individuals of different ages (21-78 years). We find an accumulation rate of 13 somatic mutations per genome per year, consistent with proliferation of SCs in the healthy adult muscle. SkM-expressed genes are protected from mutations, but aging results in an increase in mutations in exons and promoters, targeting genes involved in SC activity and muscle function. In agreement with SC mutations affecting the whole tissue, we detect a missense mutation in a SC propagating to the muscle. Our results suggest somatic mutagenesis in SCs as a driving force in the age-related decline of SkM function.

Tumours displaying differentiation towards normal fat constitute the most common subgroup of soft tissue neoplasms. A series of such tumours was investigated by whole‐exome sequencing followed by targeted ultra‐deep sequencing. Eighty per cent of angiolipomas, but not any other tumour type, displayed mutations in the protein kinase D2 (PRKD2) gene, typically in the part encoding the catalytic domain. The absence of other aberrations at the chromosome or RNA level suggests that PRKD2 mutations are critical for angiolipoma development. Consistently, the mutated PRKD2 alleles were present at low (3–15%) frequencies, indicating that only a subset of the tumour cells is affected. Indeed, by sequencing mature fat cells and other cells separately, the former typically showed the highest mutation frequencies. Thus, we hypothesize that altered PRKD2 signalling in the adipocytic cells drives tumourigenesis and, in agreement with its pivotal role in angiogenesis, induces the vessel formation that is characteristic for angiolipoma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The EPH and ephrins function as both receptor and ligands and the output on their complex signaling is currently investigated in cancer. Previous work shows that some EPH family members have clinical value in breast cancer, suggesting that this family could be a source of novel clinical targets. Here we quantified the mRNA expression levels of EPH receptors and their ligands, ephrins, in 65 node positive breast cancer samples by RT-PCR with TaqMan® Micro Fluidics Cards Microarray. Upon hierarchical clustering of the mRNA expression levels, we identified a subgroup of patients with high expression, and poor clinical outcome. EPHA2, EPHA4, EFNB1, EFNB2, EPHB2 and EPHB6 were significantly correlated with the cluster groups and particularly EPHB2 was an independent prognostic factor in multivariate analysis and in four public databases. The EPHB2 protein expression was also analyzed by immunohistochemistry in paraffin embedded material (cohort 2). EPHB2 was detected in the membrane and cytoplasmic cell compartments and there was an inverse correlation between membranous and cytoplasmic EPHB2. Membranous EPHB2 predicted longer breast cancer survival in both univariate and multivariate analysis while cytoplasmic EPHB2 indicated shorter breast cancer survival in univariate analysis. Concluding: the EPH/EFN cluster analysis revealed that high EPH/EFN mRNA expression is an independent prognostic factor for poor survival. Especially EPHB2 predicted poor breast cancer survival in several materials and EPHB2 protein expression has also prognostic value depending on cell localization.

One of the central proteins in B-lymphocyte development is the transcription factor early B-cell factor 1 (Ebf1). Ebf1 is critical for the activation of B-lineage restricted genes in the earliest B-lineage progenitors and for restriction of lineage fate options. The activity is highly dependent on functional Ebf1 dose because mice carrying a heterozygous deletion of the Ebf1 gene display reduced numbers of CD19CD43 B-cell progenitors, whereas the CD19CD43 proB cell compartment remains intact. Ebf1 levels are also of relevance in leukemia because mutations resulting in reduced functional EBF1 dose and increased expression of post-transcriptional inhibitors of EBF1, ZNF521, or ZNF423 are found in B-cell acute lymphoblastic leukemia (B-ALL).Adirect role forEbf1dose inmalignant transformation was supported by the findings that combined expression of constitutively active Stat5 (caStat5) and heterozygous loss of eitherEbf1 or Pax5 results in B-cell leukemia inmice. Heterozygote deletion of PAX5 is a rather common genetic alteration in human B-ALL and a genetic polymorphism causing reduced functional PAX5 activity has been found in families with a high incidence of leukemia. The finding that the developmental block observed in Pax5-deficient leukemia cells can be reversed on restoration of Pax5 expression suggests that the reduction in Pax5 function results in a reversible disruption of differentiation. A similar mechanism of action has been proposed for Ebf1; however, reduced amounts of Ebf1 in normal cells appear to result in reduced proliferation and expansion of B-cell progenitors, indicating that the involvement of EBF1 in malignant transformation is more complex. To increase our understanding of the functions of Ebf1 inmalignant transformation, we identified dose-dependent processes regulated by Ebf1 in early B-cell development, revealing changes in DNA repair and cell survival. Because these data suggested that Ebf1 functions differently than what has been reported for Pax5 in the transformation process, we investigated the functional collaboration between Ebf1 and Pax5 in leukemogenesis, revealing a strong functional synergy on the development of leukemia. Together, our data suggest that reduced levels of Ebf1 may contribute to malignant transformation by a combination of impairedDNArepair and increased cell survival rather than simply by a differentiation block.

Early B-cell factor 1 (Ebf1) is a transcription factor with documented dose-dependent functions in normal and malignant B-lymphocyte development. To understand more about the roles of Ebf1 in malignant transformation, we investigated the impact of reduced functional Ebf1 dosage on mouse B-cell progenitors. Gene expression analysis suggested that Ebf1 was involved in the regulation of genes important for DNA repair and cell survival. Investigation of the DNA damage in steady state, as well as after induction of DNA damage by UV light, confirmed that pro-B cells lacking 1 functional allele of Ebf1 display signs of increased DNA damage. This correlated to reduced expression of DNA repair genes including Rad51, and chromatin immunoprecipitation data suggested that Rad51 is a direct target for Ebf1. Although reduced dosage of Ebf1 did not significantly increase tumor formation in mice, a dramatic increase in the frequency of pro-B cell leukemia was observed in mice with combined heterozygous mutations in the Ebf1 and Pax5 genes, revealing a synergistic effect of combined dose reduction of these proteins. Our data suggest that Ebf1 controls DNA repair in a dose-dependent manner providing a possible explanation to the frequent involvement of EBF1 gene loss in human leukemia.

Background Plasma adiponectin levels have previously been inversely associated with carotid intima‐media thickness ( IMT ), a marker of subclinical atherosclerosis. In this study, we used a sex‐stratified Mendelian randomization approach to investigate whether adiponectin has a causal protective influence on IMT . Methods and Results Baseline plasma adiponectin concentration was tested for association with baseline IMT , IMT progression over 30 months, and occurrence of cardiovascular events within 3 years in 3430 participants (women, n=1777; men, n=1653) with high cardiovascular risk but no prevalent disease. Plasma adiponectin levels were inversely associated with baseline mean bifurcation IMT after adjustment for established risk factors (β=−0.018, P <0.001) in men but not in women (β=−0.006, P =0.185; P for interaction=0.061). Adiponectin levels were inversely associated with progression of mean common carotid IMT in men (β=−0.0022, P =0.047), whereas no association was seen in women (0.0007, P =0.475; P for interaction=0.018). Moreover, we observed that adiponectin levels were inversely associated with coronary events in women (hazard ratio 0.57, 95% CI 0.37 to 0.87) but not in men (hazard ratio 0.82, 95% CI 0.54 to 1.25). A gene score of adiponectin‐raising alleles in 6 loci, reported recently in a large multi‐ethnic meta‐analysis, was inversely associated with baseline mean bifurcation IMT in men (β=−0.0008, P =0.004) but not in women (β=−0.0003, P =0.522; P for interaction=0.007). Conclusions This report provides some evidence for adiponectin protecting against atherosclerosis, with effects being confined to men; however, compared with established cardiovascular risk factors, the effect of plasma adiponectin was modest. Further investigation involving mechanistic studies is warranted.

Objective: Experimental studies have suggested that autoimmunity is involved in atherosclerosis and provided evidence that both protective and pro-atherogenic immune responses exist. This concept has received support from small clinical studies implicating autoantibodies directed against apolipoprotein B-100 (apoB-100) in human atherosclerosis. We examined circulating autoantibodies directed against native and malondialdehyde (MDA)-modified epitope p210 of apoB-100 (IgG-p210(nat) and IgM-p210(MDA)) in relation to early atherosclerosis in a large, European longitudinal cohort study of healthy high-risk individuals.Approach and results: IgG-p210(nat) and IgM-p210(MDA) were quantified in baseline plasma samples of 3430 participants in the IMPROVE study and related to composite and segment-specific measures of severity and rate of progression of carotid intima-media thickness (cIMT) determined at baseline and after 30 months. IgM-p210(MDA) autoantibody levels were independently related to several cIMT measures both in the common carotid artery and in the carotid bulb, including measures of cIMT progression, higher levels being associated with lower cIMT or slower cIMT progression. Consistent inverse relationships were also found between plasma levels of IgG-p210(nat) and baseline composite measures of cIMT. These associations disappeared when adjusting for established and emerging risk factors, and there were no associations with rate of cIMT progression besides in certain secondary stratified analyses.Conclusions: The present study provides further evidence of involvement of autoantibodies against native and MDA-modified apoB-100 peptide 210 in cardiovascular disease in humans and demonstrates that these associations are present already at a subclinical stage of the disease. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Objective Vitamin D deficiency has been implicated in cardiovascular disease and is associated with multiple cardiovascular risk factors. We investigated the serum 25-hydroxyvitamin D (25(OH)D) concentration in relation to latitude, baseline carotid intima-media thickness (IMT), and IMT progression, the carotid IMT measures being surrogate markers of subclinical atherosclerosis and cardiovascular disease risk.Approach and Results Serum 25(OH)D concentration was related to high-resolution carotid IMT measures in 3430 middle-aged and elderly subjects with high cardiovascular risk but no prevalent disease, who were recruited at 7 centers in Finland, Sweden, The Netherlands, France, and Italy. Participants underwent carotid ultrasound examination at baseline and at months 15 and 30 after entry into the study, whereas blood samples, clinical data, and information about lifestyle were collected at baseline. Serum 25(OH)D levels were positively associated with latitude (Jonckheere-Terpstra =166.643; P<0.001) and, as previously reported, associated with a range of cardiovascular risk factors. There were no independent relationships between 25(OH)D and segment-specific or composite IMT measures in the entire cohort. In analyses stratified by sex, diabetes mellitus, and statin treatment, weak associations with some baseline and progression measures of carotid IMT were observed in males, diabetics, and nonstatin-treated individuals.Conclusions Levels of 25(OH)D differed across Europe, were highest in the North, showed multiple associations with established and emerging cardiovascular risk factors but were not consistently, independently related to measures of carotid IMT. This argues against a protective role of vitamin D against subclinical atherosclerosis in high-risk individuals.

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

Background— Carotid intima-media thickness (cIMT) is a widely accepted marker of subclinical atherosclerosis. To date, large-scale investigations of genetic determinants of cIMT are sparse. Methods and Results— To identify cIMT-associated genes and genetic variants, a discovery analysis using the Illumina 200K CardioMetabochip was conducted in 3430 subjects with detailed ultrasonographic determinations of cIMT from the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study. Segment-specific IMT measurements of common carotid, bifurcation, and internal carotid arteries, and composite IMT variables considering the whole carotid tree (IMT mean , IMT max , and IMT mean-max ), were analyzed. A replication stage investigating 42 single-nucleotide polymorphisms for association with common carotid IMT was undertaken in 5 independent European cohorts (total n=11 590). A locus on chromosome 16 (lead single-nucleotide polymorphism rs4888378, intronic in CFDP1 ) was associated with cIMT at significance levels passing multiple testing correction at both stages (array-wide significant discovery P =6.75×10 −7 for IMT max ; replication P =7.24×10 −6 for common cIMT; adjustments for sex, age, and population substructure where applicable; minor allele frequency 0.43 and 0.41, respectively). The protective minor allele was associated with lower carotid plaque score in a replication cohort ( P =0.04, n=2120) and lower coronary artery disease risk in 2 case-control studies of subjects with European ancestry (odds ratio [95% confidence interval] 0.83 [0.77–0.90], P =6.53×10 −6 , n=13 591; and 0.95 [0.92–0.98], P =1.83×10 −4 , n=82 297, respectively). Queries of human biobank data sets revealed associations of rs4888378 with nearby gene expression in vascular tissues (n=126–138). Conclusions— This study identified rs4888378 in the BCAR1-CFDP1-TMEM170A locus as a novel genetic determinant of cIMT and coronary artery disease risk in individuals of European descent.