The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Targets of genome copy number reduction in primary breast cancers identified by integrative genomics

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBNI, and LZTSI, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4LI, and TBCID5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (> 85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http:// www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2006 Wiley-Liss, Inc.

Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants

We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases and 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations1-4 (9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (>1% allele frequency) and none met the pre-specified threshold for replication (P < 10-3). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk.

Discovering subgroups of patients from DNA copy number data using NMF on compacted matrices

In the study of complex genetic diseases, the identification of subgroups of patients sharing similar genetic characteristics represents a challenging task, for example, to improve treatment decision. One type of genetic lesion, frequently investigated in such disorders, is the change of the DNA copy number (CN) at specific genomic traits. Non-negative Matrix Factorization (NMF) is a standard technique to reduce the dimensionality of a data set and to cluster data samples, while keeping its most relevant information in meaningful components. Thus, it can be used to discover subgroups of patients from CN profiles. It is however computationally impractical for very high dimensional data, such as CN microarray data. Deciding the most suitable number of subgroups is also a challenging problem. The aim of this work is to derive a procedure to compact high dimensional data, in order to improve NMF applicability without compromising the quality of the clustering. This is particularly important for analyzing high-resolution microarray data. Many commonly used quality measures, as well as our own measures, are employed to decide the number of subgroups and to assess the quality of the results. Our measures are based on the idea of identifying robust subgroups, inspired by biologically/clinically relevance instead of simply aiming at well-separated clusters. We evaluate our procedure using four real independent data sets. In these data sets, our method was able to find accurate subgroups with individual molecular and clinical features and outperformed the standard NMF in terms of accuracy in the factorization fitness function. Hence, it can be useful for the discovery of subgroups of patients with similar CN profiles in the study of heterogeneous diseases.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome.

METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone.

FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions.

INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

The mitochondrial and autosomal mutation landscapes of prostate cancer

BACKGROUND: Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression.

OBJECTIVE: To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected.

DESIGN, SETTING, AND PARTICIPANTS: Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature.

RESULTS AND LIMITATIONS: Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.

CONCLUSIONS: The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

Inhibitors of alpha-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson's disease and related disorders.

An abundance of genetic, histopathological, and biochemical evidence has implicated the neuronal protein, alpha-synuclein (alpha-syn) as a key player in the development of several neurodegenerative diseases, the so-called synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. Development of disease appears to be linked to events that increase the intracellular concentration of alpha-syn or cause its chemical modification, either of which can accelerate the rate at which it forms aggregates. Examples of such events include increased copy number of genes, decreased rate of degradation via the proteasome or other proteases, or altered forms of alpha-syn, such as truncations, missense mutations, or chemical modifications by oxidative reactions. Aggregated forms of the protein, especially newly formed soluble aggregates, are toxic to cells, so that one therapeutic strategy would be to reduce the rate at which such oligomerization occurs. We have therefore designed several peptides and also identified small molecules that can inhibit alpha-syn oligomerization and toxicity in vitro. These compounds could serve as lead compounds for the design of new drugs for the treatment of PD and related disorders in the future.

RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle.

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications in target-directed therapeutics

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could. be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P

O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

Genome-Wide Association Study of Multiplex Schizophrenia Pedigrees

OBJECTIVE The authors used a genome-wide association study (GWAS) of multiply affected families to investigate the association of schizophrenia to common single-nucleotide polymorphisms (SNPs) and rare copy number variants (CNVs). METHOD The family sample included 2,461 individuals from 631 pedigrees (581 in the primary European-ancestry analyses). Association was tested for single SNPs and genetic pathways. Polygenic scores based on family study results were used to predict case-control status in the Schizophrenia Psychiatric GWAS Consortium (PGC) data set, and consistency of direction of effect with the family study was determined for top SNPs in the PGC GWAS analysis. Within-family segregation was examined for schizophrenia-associated rare CNVs. RESULTS No genome-wide significant associations were observed for single SNPs or for pathways. PGC case and control subjects had significantly different genome-wide polygenic scores (computed by weighting their genotypes by log-odds ratios from the family study) (best p=10-17, explaining 0.4% of the variance). Family study and PGC analyses had consistent directions for 37 of the 58 independent best PGC SNPs (p=0.024). The overall frequency of CNVs in regions with reported associations with schizophrenia (chromosomes 1q21.1, 15q13.3, 16p11.2, and 22q11.2 and the neurexin-1 gene [NRXN1]) was similar to previous case-control studies. NRXN1 deletions and 16p11.2 duplications (both of which were transmitted from parents) and 22q11.2 deletions (de novo in four cases) did not segregate with schizophrenia in families. CONCLUSIONS Many common SNPs are likely to contribute to schizophrenia risk, with substantial overlap in genetic risk factors between multiply affected families and cases in large case-control studies. Our findings are consistent with a role for specific CNVs in disease pathogenesis, but the partial segregation of some CNVs with schizophrenia suggests that researchers should exercise caution in using them for predictive genetic testing until their effects in diverse populations have been fully studied.

Genomewide linkage scan of schizophrenia in a large multicenter pedigree sample using single nucleotide polymorphisms

A genomewide linkage scan was carried out in eight clinical samples of informative schizophrenia families. After all quality control checks, the analysis of 707 European-ancestry families included 1615 affected and 1602 unaffected genotyped individuals, and the analysis of all 807 families included 1900 affected and 1839 unaffected individuals. Multipoint linkage analysis with correction for marker-marker linkage disequilibrium was carried out with 5861 single nucleotide polymorphisms (SNPs; Illumina version 4.0 linkage map). Suggestive evidence for linkage ( European families) was observed on chromosomes 8p21, 8q24.1, 9q34 and 12q24.1 in nonparametric and/or parametric analyses. In a logistic regression allele-sharing analysis of linkage allowing for intersite heterogeneity, genomewide significant evidence for linkage was observed on chromosome 10p12. Significant heterogeneity was also observed on chromosome 22q11.1. Evidence for linkage across family sets and analyses was most consistent on chromosome 8p21, with a one-LOD support interval that does not include the candidate gene NRG1, suggesting that one or more other susceptibility loci might exist in the region. In this era of genomewide association and deep resequencing studies, consensus linkage regions deserve continued attention, given that linkage signals can be produced by many types of genomic variation, including any combination of multiple common or rare SNPs or copy number variants in a region. Molecular Psychiatry (2009) 14, 786-795; doi:10.1038/mp.2009.11; published online 17 February 2009

Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy

Background: Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.

Methods: Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.

Results: A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P< 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.

Conclusions: These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.© 2013 Kavanagh et al.; licensee BioMed Central Ltd.

CNV analysis in a large schizophrenia sample implicates deletions at 16p12.1 and SLC1A1 and duplications at 1p36.33 and CGNL1

Large and rare copy number variants (CNVs) at several loci have been shown to increase risk for schizophrenia. Aiming to discover novel susceptibility CNV loci, we analysed 6,882 cases and 11,255 controls genotyped on Illumina arrays, most of which have not been used for this purpose before. We identified genes enriched for rare exonic CNVs among cases, and then attempted to replicate the findings in an additional 14,568 cases and 15,274 controls. In a combined analysis of all samples, 12 distinct loci were enriched among cases with nominal levels of significance (P500kb), rare CNVs showed a 1.2% excess in cases after excluding known schizophrenia-associated loci, suggesting that additional susceptibility loci exist. However, even larger samples are required for their discovery.