Trans-ethnic and Ancestry-Specific Blood-Cell Genetics in 746,667 Individuals from 5 Global Populations.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10-9, including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

The Polygenic and Monogenic Basis of Blood Traits and Diseases.

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation.

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells.

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.

Variation and impact of polygenic hematologic traits in monogenic sickle cell disease.

Several of the complications observed in sickle cell disease (SCD) are influenced by variation in hematologic traits (HT), such as fetal hemoglobin (HbF) level and neutrophil count. Previous large-scale genome-wide association studies carried out in largely healthy individuals have identified thousands of variants associated with HT, which have then been used to develop multi-ancestry polygenic trait scores (PTS). Here, we tested whether these PTS associate with HT in SCD patients and if they can improve statistical models associated with SCD-related complications. In 2,056 SCD patients, we found that the PTS predicted less HT variance than in non-SCD individuals of African ancestry. This was particularly striking at the Duffy/DARC locus, where we observed an epistatic interaction between the SCD genotype and the Duffy null variant (rs2814778) that led to a two-fold weaker effect on neutrophil count. PTS for these HT which are measured as part of routine practice were not associated with complications in SCD. In contrast, we found that a simple PTS for HbF that includes only six variants explained a large fraction of the phenotypic variation (20.5-27.1%), associated with acute chest syndrome and stroke risk, and improved the statistical modeling of the vaso-occlusive crisis rate. Using Mendelian randomization, we found that increasing HbF by 4.8% reduces stroke risk by 39% (P=0.0006). Taken together, our results highlight the importance of validating PTS in large diseased populations before proposing their implementation in the context of precision medicine initiatives.

Rare and low-frequency coding variants alter human adult height.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1-4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, AR and CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2 (giving an increase of 1-2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RA and NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells.

BACKGROUND: Genome-wide association studies (GWAS) have identified a variant (rs9349379) at the phosphatase and actin regulator 1 (PHACTR1) locus that is associated with coronary artery disease (CAD). The same variant is also an expression quantitative trait locus (eQTL) for PHACTR1 in human coronary arteries (hCA). Here, we sought to characterize PHACTR1 splicing pattern in atherosclerosis-relevant human cells. We also explored how rs9349379 modulates the expression of the different PHACTR1 splicing isoforms. METHODS: We combined rapid amplification of cDNA ends (RACE) with next-generation long-read DNA sequencing to discover all PHACTR1 transcripts in many human tissues and cell types. We measured PHACTR1 transcripts by qPCR to identify transcript-specific eQTLs. RESULTS: We confirmed a brain-specific long transcript, a short transcript expressed in monocytes and four intermediate transcripts that are different due to alternative splicing of two in-frame exons. In contrast to a previous report, we confirmed that the PHACTR1 protein is present in vascular smooth muscle cells. In 158 hCA from our collection and the GTEx dataset, rs9349379 was only associated with the expression levels of the intermediate PHACTR1 transcripts. CONCLUSIONS: Our comprehensive transcriptomic profiling of PHACTR1 indicates that this gene encodes six main transcripts. Five of them are expressed in hCA, where atherosclerotic plaques develop. In this tissue, genotypes at rs9349379 are associated with the expression of the intermediate transcripts, but not the immune-specific short transcript. This result suggests that rs9349379 may in part influence CAD by modulating the expression of intermediate PHACTR1 transcripts in endothelial or vascular smooth muscle cells found in hCA.

Whole-genome sequencing in French Canadians from Quebec.

Genome-wide association studies (GWAS) have had a tremendous success in the identification of common DNA sequence variants associated with complex human diseases and traits. However, because of their design, GWAS are largely inappropriate to characterize the role of rare and low-frequency DNA variants on human phenotypic variation. Rarer genetic variation is geographically more restricted, supporting the need for local whole-genome sequencing (WGS) efforts to study these variants in specific populations. Here, we present the first large-scale low-pass WGS of the French-Canadian population. Specifically, we sequenced at ~5.6× coverage the whole genome of 1970 French Canadians recruited by the Montreal Heart Institute Biobank and identified 29 million bi-allelic variants (31 % novel), including 19 million variants with a minor allele frequency (MAF) <0.5 %. Genotypes from the WGS data are highly concordant with genotypes obtained by exome array on the same individuals (99.8 %), even when restricting this analysis to rare variants (MAF <0.5, 99.9 %) or heterozygous sites (98.9 %). To further validate our data set, we showed that we can effectively use it to replicate several genetic associations with myocardial infarction risk and blood lipid levels. Furthermore, we analyze the utility of our WGS data set to generate a French-Canadian-specific imputation reference panel and to infer population structure in the Province of Quebec. Our results illustrate the value of low-pass WGS to study the genetics of human diseases in the founder French-Canadian population.

Myocardial Infarction-Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries.

OBJECTIVE: Coronary artery disease (CAD), including myocardial infarction (MI), is the main cause of death in the world. Genome-wide association studies have identified dozens of single nucleotide polymorphisms (SNPs) associated with CAD/MI. One of the most robust CAD/MI genetic associations is with intronic SNPs in the gene PHACTR1 on chromosome 6p24. How these PHACTR1 SNPs influence CAD/MI risk, and whether PHACTR1 itself is the causal gene at the locus, is currently unknown. APPROACH AND RESULTS: Using genetic fine-mapping and DNA resequencing experiments, we prioritized an intronic SNP (rs9349379) in PHACTR1 as causal variant. We showed that this variant is an expression quantitative trait locus for PHACTR1 expression in human coronary arteries. Experiments in endothelial cell extracts confirmed that alleles at rs9349379 are differentially bound by the transcription factors myocyte enhancer factor-2. We engineered a deletion of this myocyte enhancer factor-2-binding site using CRISPR/Cas9 genome-editing methodology. Heterozygous endothelial cells carrying this deletion express 35% less PHACTR1. Finally, we found no evidence that PHACTR1 expression levels are induced when stimulating human endothelial cells with vascular endothelial growth factor, tumor necrosis factor-α, or shear stress. CONCLUSIONS: Our results establish a link between intronic SNPs in PHACTR1, myocyte enhancer factor-2 binding, and transcriptional functions at the locus, PHACTR1 expression levels in coronary arteries and CAD/MI risk. Because PHACTR1 SNPs are not associated with the traditional risk factors for CAD/MI (eg, blood lipids or pressure, diabetes mellitus), our results suggest that PHACTR1 may influence CAD/MI risk through as yet unknown mechanisms in the vascular endothelium.

Hundreds of variants clustered in genomic loci and biological pathways affect human height.

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Deep resequencing of GWAS loci identifies rare variants in CARD9, IL23R and RNF186 that are associated with ulcerative colitis.

Genome-wide association studies and follow-up meta-analyses in Crohn's disease (CD) and ulcerative colitis (UC) have recently identified 163 disease-associated loci that meet genome-wide significance for these two inflammatory bowel diseases (IBD). These discoveries have already had a tremendous impact on our understanding of the genetic architecture of these diseases and have directed functional studies that have revealed some of the biological functions that are important to IBD (e.g. autophagy). Nonetheless, these loci can only explain a small proportion of disease variance (~14% in CD and 7.5% in UC), suggesting that not only are additional loci to be found but that the known loci may contain high effect rare risk variants that have gone undetected by GWAS. To test this, we have used a targeted sequencing approach in 200 UC cases and 150 healthy controls (HC), all of French Canadian descent, to study 55 genes in regions associated with UC. We performed follow-up genotyping of 42 rare non-synonymous variants in independent case-control cohorts (totaling 14,435 UC cases and 20,204 HC). Our results confirmed significant association to rare non-synonymous coding variants in both IL23R and CARD9, previously identified from sequencing of CD loci, as well as identified a novel association in RNF186. With the exception of CARD9 (OR = 0.39), the rare non-synonymous variants identified were of moderate effect (OR = 1.49 for RNF186 and OR = 0.79 for IL23R). RNF186 encodes a protein with a RING domain having predicted E3 ubiquitin-protein ligase activity and two transmembrane domains. Importantly, the disease-coding variant is located in the ubiquitin ligase domain. Finally, our results suggest that rare variants in genes identified by genome-wide association in UC are unlikely to contribute significantly to the overall variance for the disease. Rather, these are expected to help focus functional studies of the corresponding disease loci.

Epigenetic mechanisms underlying arsenic-associated lung carcinogenesis.

Arsenic is an established human carcinogen, but the mechanisms through which it contributes to for instance lung cancer development are still unclear. As arsenic is methylated during its metabolism, it may interfere with the DNA methylation process, and is therefore considered to be an epigenetic carcinogen. In the present study, we hypothesize that arsenic is able to induce DNA methylation changes, which lead to changes in specific gene expression, in pathways associated with lung cancer promotion and progression. A549 human adenocarcinoma lung cells were exposed to a low (0.08 µM), intermediate (0.4 µM) and high (2 µM) concentration of sodium arsenite for 1, 2 and 8 weeks. DNA was isolated for whole-genome DNA methylation analyses using NimbleGen 2.1 M deluxe promoter arrays. In addition, RNA was isolated for whole-genome transcriptomic analysis using Affymetrix microarrays. Arsenic modulated DNA methylation and expression levels of hundreds of genes in a dose-dependent and time-dependent manner. By combining whole-genome DNA methylation and gene expression data with possibly involved transcription factors, a large molecular interaction network was created based on transcription factor-target gene pairs, consisting of 216 genes. A tumor protein p53 (TP53) subnetwork was identified, showing the interactions of TP53 with other genes affected by arsenic. Furthermore, multiple other new genes were discovered showing altered DNA methylation and gene expression. In particular, arsenic modulated genes which function as transcription factor, thereby affecting target genes which are known to play a role in lung cancer promotion and progression.

Strategies to fine-map genetic associations with lipid levels by combining epigenomic annotations and liver-specific transcription profiles.

Characterization of the epigenome promises to yield the functional elements buried in the human genome sequence, thus helping to annotate non-coding DNA polymorphisms with regulatory functions. Here, we develop two novel strategies to combine epigenomic data with transcriptomic profiles in humans or mice to prioritize potential candidate SNPs associated with lipid levels by genome-wide association study (GWAS). First, after confirming that lipid-associated loci that are also expression quantitative trait loci (eQTL) in human livers are enriched for ENCODE regulatory marks in the human hepatocellular HepG2 cell line, we prioritize candidate SNPs based on the number of these marks that overlap the variant position. This method recognized the known SORT1 rs12740374 regulatory SNP associated with LDL-cholesterol, and highlighted candidate functional SNPs at 15 additional lipid loci. In the second strategy, we combine ENCODE chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) data and liver expression datasets from knockout mice lacking specific transcription factors. This approach identified SNPs in specific transcription factor binding sites that are located near target genes of these transcription factors. We show that FOXA2 transcription factor binding sites are enriched at lipid-associated loci and experimentally validate that alleles of one such proxy SNP located near the FOXA2 target gene BIRC5 show allelic differences in FOXA2-DNA binding and enhancer activity. These methods can be used to generate testable hypotheses for many non-coding SNPs associated with complex diseases or traits.

A critical view on autoantibodies in lupus nephritis: Concrete knowledge based on evidence.

Deposition of autoantibodies in glomeruli is a key factor in the development of lupus nephritis (LN). For a long time, anti-dsDNA and anti-C1q antibodies were thought to be the main cause of the kidney damage. However, recent studies have shown that the list of autoantibidies that have renal tropism and deposit in the kidney in LN is increasing and the link between anti-dsDNA and renal pathology is weak due to potential confounders. Aspecific bindings of dsDNA with cationic antibodies and of anti-dsDNA with several renal antigens such as actinin, laminin, entactin, and annexinA2 raised doubts about the specific target of these antibodies in the kidney. Moreover, the isotype of anti-dsDNA in SLE and LN has never received adequate interest until the recent observation that IgG2 are preponderant over IgG1, IgG3 and IgG4. Based on the above background, recent studies investigated the involvement of anti-dsDNA IgG2 and of other antibodies in LN. It was concluded that circulating anti-dsDNA IgG2 levels do not distinguish between LN versus non-renal SLE, and, in patients with LN, their levels do not change over time. Circulating levels of other antibodies such as anti-ENO1 and anti-H2 IgG2 were, instead, higher in LN vs non-renal SLE at the time of diagnosis and decreased following therapies. Finally, new classes of renal antibodies that potentially modify the anti-inflammatory response in the kidney are emerging as new co-actors in the pathogenetic scenario. They have been defined as 'second wave antibodies' for the link with detoxifying mechanisms limiting the oxidative stress in glomeruli that are classically stimulated in a second phase of inflammation. These findings have important clinical implications that may modify the laboratory approach to LN. Serum levels of anti-ENO1 and anti-H2 IgG2 should be measured in the follow up of patients for designing the length of therapies and identify those patients who respond to treatments. Anti-SOD2 could help to monitor and potentiate the anti-inflammatory response in the kidney.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing.

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Discovery of anti-Formin-like 1 protein (FMNL1) antibodies in membranous nephropathy and other glomerular diseases.

Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.

Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans.

Red blood cell, white blood cell, and platelet measures, including their count, sub-type and volume, are important diagnostic and prognostic clinical parameters for several human diseases. To identify novel loci associated with hematological traits, and compare the architecture of these phenotypes between ethnic groups, the CARe Project genotyped 49,094 single nucleotide polymorphisms (SNPs) that capture variation in ~2,100 candidate genes in DNA of 23,439 Caucasians and 7,112 African Americans from five population-based cohorts. We found strong novel associations between erythrocyte phenotypes and the glucose-6 phosphate dehydrogenase (G6PD) A-allele in African Americans (rs1050828, P<2.0×10(-13), T-allele associated with lower red blood cell count, hemoglobin, and hematocrit, and higher mean corpuscular volume), and between platelet count and a SNP at the tropomyosin-4 (TPM4) locus (rs8109288, P=3.0×10(-7) in Caucasians; P=3.0×10(-7) in African Americans, T-allele associated with lower platelet count). We strongly replicated many genetic associations to blood cell phenotypes previously established in Caucasians. A common variant of the α-globin (HBA2-HBA1) locus was associated with red blood cell traits in African Americans, but not in Caucasians (rs1211375, P<7×10(-8), A-allele associated with lower hemoglobin, mean corpuscular hemoglobin, and mean corpuscular volume). Our results show similarities but also differences in the genetic regulation of hematological traits in European- and African-derived populations, and highlight the role of natural selection in shaping these differences.

Reexpression of LGI1 in glioma cells results in dysregulation of genes implicated in the canonical axon guidance pathway.

The LGI1 gene suppresses invasion in glioma cells and predisposes to epilepsy. In a gene expression array comparison between parental cells and T98G cell clones forced to express LGI1, we demonstrate that the canonical axon guidance pathway is the most significantly affected. In particular, aspects of axon guidance that involve reorganization of the actin cytoskeleton, which is also involved in cell movement and invasion, were affected. Analysis of actin fiber organization using fluorescence microscopy demonstrated that different T98G cell clones expressing the exogenous LGI1 gene show high levels of stress fibers compared with controls. Since stress fiber formation is associated with loss of cell mobility, we used scratch wound assays to demonstrate that LGI1-expressing clones show a significant reduction in cell mobility. LGI1 reexpression also resulted in loss of the PDGFRA and EGFR proteins, suggesting a rapid turnover of these receptors despite increased mRNA levels for PDGFRA. LGI1 suppression of invasion is associated with loss of ERK/MAPK1 activation. LGI1 is a secreted protein, and when the culture supernatant from cells expressing FLAG- and GFP-tagged proteins were applied to parental T98G cells, ERK/MAPK1 phosphorylation and cell mobility was suppressed, demonstrating that the LGI1 protein acts as a suppressive agent for cell movement in this assay. These observations support a previous suggestion that LGI1 can reduce cellular invasion in in vitro assays and, as a secreted agent, may be developed as a means of treating metastatic cancer. In addition, this observation provides a mechanistic link for LGI1's common role in metastasis and epilepsy development.

Interpreting aCGH-defined karyotypic changes in gliomas using copy number status, loss of heterozygosity and allelic ratios.

We have used SNP mapping arrays to simultaneously record copy number changes, loss of heterozygosity and allele ratios (ploidy) in a series of 13 gliomas. This combined analysis has defined novel amplification events in this tumor type involving chr1:241544532-243005121 and chr18:54716681-54917277 which contain the AKT3 and ZNF532 genes, respectively. The high resolution of this analysis has also identified homozygous deletions involving chr17:25600031-26490848 and Chr19:53883612-55061878. Throughout the karyotypes of these tumors, the combined analysis revealed counter intuitive relationships between copy number and LOH that requires reinterpretation of the significance of copy number gains and losses. It was not uncommon to observe copy number gains that were associated with loss of heterozygosity as well as copy number losses that were not. These events appeared to be related to ploidy status in the tumors as determined using allelic ratio calculations. Overall, this analysis of gliomas provides evidence for the need to perform more comprehensive interpretation of the CGH data beyond copy number analysis alone to evaluate the significance of individual events in the karyotypes.

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes.

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.

Comprehensive Profiling of the Rheumatoid Arthritis Antibody Repertoire.

OBJECTIVE: Autoantibodies against citrullinated proteins are found in 64-89% of rheumatoid arthritis (RA) patients, with 88-99% specificity. This study was undertaken to create an unbiased, comprehensive profile of serum antibodies against the human proteome, including the citrullinome and the homocitrullinome, in RA patients, using a high-density peptide array. METHODS: Our high-density peptide array, consisting of >4.6 million peptides, contained the entire annotated human proteome. The 20,246 proteins were represented as overlapping 16-mer peptides. In addition to native peptides, citrullinated and homocitrullinated peptides were included, as substitutions for arginine and lysine, and provided a comprehensive screen against all possible epitopes. Twenty-six serum samples (from 8 controls and 18 RA patients) were profiled on the high-density peptide array. Using RA-specific epitopes, we constructed an 8-epitope diagnostic biomarker on a Gyrolab xPlore instrument with a cohort of 92 serum samples (from 29 controls and 63 RA patients). The diagnostic biomarker was further validated with an independent cohort of 181 serum samples (from 54 controls and 127 RA patients). RESULTS: In the initial cohort the diagnostic performance of the 8-epitope biomarker yielded 96.6% specificity and 92.1% sensitivity. The overall diagnostic performance in the validation cohort was 94.4% specificity and 85% sensitivity. In both cohorts, the performance of the 8-epitope diagnostic biomarker compared favorably against the Abnova cyclic citrullinated peptide 2 (CCP2) assay. Using data from the peptide array, we identified novel RA-specific epitopes and formed the basis of a new RA diagnostic assay. CONCLUSION: Comprehensive antibody profiling using a high-density peptide array not only identified novel RA-specific epitopes but also allowed us to construct a novel diagnostic biomarker that is as specific as and more sensitive than the Abnova CCP2 assay.