Daly, Mark J.

Abstract Inflammatory bowel diseases (IBD), principally Crohn’s disease (CD) and ulcerative colitis (UC), are common chronic disorders involving inflammation and often progressive tissue damage. Genome-wide association studies have mapped many risk signals, but the causal variants, effector genes and relevant cellular contexts remain difficult to resolve, limiting mechanistic interpretation and therapeutic translation. Here we performed a multi-ancestry GWAS meta-analysis of 125,992 individuals with IBD and more than 1.2 million controls, identifying 619 independent association signals (374 novel) at 420 IBD regions that account for 77–80% of SNP-based heritability. Fine-mapping resolved 81 high-confidence variants, 41 not previously reported. Although most signals were shared between CD and UC, 39% showed IBD subtype specificity, with UC signals showing stronger enrichment in functional annotations from intestinal epithelial, secretory and enteroendocrine cells, and CD showing stronger genetic correlations with circulating inflammatory biomarkers, including C-reactive protein and glycoprotein acetylation. Latent causal modelling supported a causal effect of decreased high-density lipoprotein on CD risk. By integrating bulk and single-cell eQTL and pQTL resources using colocalisation and Mendelian randomisation, together with coding-variant evidence from exome sequencing, we prioritised 664 candidate effector genes across 341 signals, including 390 newly implicated IBD genes, revealing new biological mechanisms and candidate therapeutic targets supported by human genetics.

Abstract Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder of the gastrointestinal tract whose genetic basis is only partly resolved because most risk variants identified by genome-wide association studies (GWAS) lie in non-coding regions, limiting direct gene assignment and biological interpretation 1,2 . Here we analysed whole-exome and whole-genome sequencing data from 86,213 IBD cases and 478,363 controls of European ancestry. We identified 68 IBD genes directly implicated by conditionally independent protein-coding associations across the allele frequency spectrum. Many newly implicated IBD genes are supported by orthogonal genomic or pleiotropic evidence, pointing to disease-related pathways and nominating targets with therapeutic relevance. We further identified allelic series and non-additive effects at key loci such as NOD2 and TYK2. These results show that large-scale sequencing can resolve disease genes and pathways that remain ambiguous from non-coding association alone, providing a more direct route from human genetics to biological insight and therapeutic hypotheses.

Abstract Rare Mendelian disorders affect 300-400 million people globally. Although genetic testing has become widely adopted, gene-specific evidence for tailored variant interpretation remains scattered across resources. We present Gene Portals, a framework for gene-centered multimodal knowledge bases that co-localize expert-harmonized clinical data, functional assays, population variation, structural annotations and gene-specific ACMG/AMP specifications within a single resource. A modular interface integrates this unified evidence with VCEP-refined ACMG specifications to enable automated gene-specific variant classification, infer molecular mechanisms, and support cross-gene analyses. We demonstrate the framework’s utility across five Gene portals spanning eleven neurodevelopmental disorder-associated genes, integrating data from 4,423 individuals with 2,838 unique variants, 36,149 ClinVar submissions, and 1,044 expert-curated molecular readouts. By organizing evidence that is otherwise dispersed across multiple sources into a unified, queryable framework, the SCN, GRIN, CACNA1A, SATB2 and SLC6A1 Gene Portals became widely used community resources and provide an extensible template for standardized rare-disease variant interpretation and mechanism-aware discovery.

Abstract Given the frequent and largely incurable occurrence of multiple myeloma, identification of germline genetic mutations that predispose cells to multiple myeloma may provide insight into disease etiology and the developmental mechanisms of its cell of origin, the plasma cell (PC). Here, we identified familial and early-onset multiple myeloma kindreds with truncating mutations in lysine-specific demethylase 1 (LSD1/KDM1A), an epigenetic transcriptional repressor that primarily demethylates histone H3 on lysine 4 and regulates hematopoietic stem cell self-renewal. In addition, we found higher rates of germline truncating and predicted deleterious missense KDM1A mutations in patients with multiple myeloma unselected for family history compared with controls. Both monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma cells have significantly lower KDM1A transcript levels compared with normal PCs. Transcriptome analysis of multiple myeloma cells from KDM1A mutation carriers shows enrichment of pathways and MYC target genes previously associated with myeloma pathogenesis. In mice, antigen challenge followed by pharmacologic inhibition of KDM1A promoted PC expansion, enhanced secondary immune response, elicited appearance of serum paraprotein, and mediated upregulation of MYC transcriptional targets. These changes are consistent with the development of MGUS. Collectively, our findings show that KDM1A is the first autosomal-dominant multiple myeloma germline predisposition gene providing new insights into its mechanistic roles as a tumor suppressor during post-germinal center B-cell differentiation. Significance: KDM1A is the first germline autosomal dominant predisposition gene identified in multiple myeloma and provides new insights into multiple myeloma etiology and the mechanistic role of KDM1A as a tumor suppressor during post-germinal center B-cell differentiation. Cancer Res; 78(10); 2747–59. ©2018 AACR.

Abstract Background: Known gene mutations account for approximately 50% of the risk for breast cancer. However, a considerable fraction of heritable risk remains unexplained. Fifteen percent of the risk is accounted for by BRCA1/2 and another 3% by TP53, PTEN, LKB1 and CDH1. CHEK2, ATM, PALB2, BRIP1, RAD51C, RAD51D and BARD1 account for 4%, while SNPs discovered from large multicenter genome-wide association studies explain another 14% of the heritable risk. Founder mutations in the DNA repair pathway genes such as BRCA1 and BRCA2 account for the majority of AJ breast cancer mutations. Methods: We performed exome sequencing of 49 early onset (age <35) breast cancer cases, and 85 BRCA wild type familial breast cancer cases, all of Ashkenazi Jewish (AJ) from the New York City area. A recurrent truncating mutation was then analyzed in 3131 breast cancer cases and 2716 unaffected women of Ashkenazi ancestry from New York and Israel. Using CRISPR and overexpression systems on the human mammary epithelial cell line HMLE, transcript and protein levels were assayed for the mutant and compared to wild-type. Treatment with IlludinS and UVC were performed to assess DNA damage response. Finally, clonogenic survival assay was also performed. Results: Amongst the DNA repair pathway genes, exome sequencing revealed a heterozygous recurrent truncating mutation in ERCC3 (R109X) in 2 of 49 early onset breast cancer cases of AJ ancestry and 4 familial AJ probands. Taqman genotyping in a case control setting from New York and Israel revealed 54 mutation carriers in 3131 cases and 32 in the 2716 controls. In total, there were 60 heterozygotes detected in 3209 cases and 32 in 2716 controls [OR 1.59 (95% CI 1.01-2.50)]; p = 0.02 Fisher one- tailed). Functional studies using CRISPR and overexpression systems on human mammary epithelial cells, show that the mutation results in lower transcript levels and this reduction is effected by nonsense mediated decay of the mutant transcript. Western blotting showed that the mutation resulted in a smaller protein (∼12kDa). Clonogenic assays showed similar survival rate of mutant and wildtype under UVC exposure, however the mutant cell line showed significantly smaller colony size demonstrating a growth disadvantage that was further increased upon DNA damage. Treatment with fungal sesquiterpene IlludinS, a known sensitizer to mutant ERCC3 cell lines, showed drastically reduced survival when compared to the wild type human mammary epithelial cells. Conclusions: We demonstrate that ERCC3 is a moderate risk gene for breast cancer in individuals of Ashkenazi ancestry. ERCC3 is somatically mutated in multiple cancers including breast, ovarian and pancreatic cancers, however its role as a cancer susceptibility gene requires further elucidation. Additional functional and population genetic studies to further characterize this novel ERCC3 variant are underway. Citation Format: Joseph Vijai, Sabine Topka, Kara Maxwell, Vignesh Ravichandran, Tinu Thomas, Danylo Villano, Ann Maria, Pragna Gaddam, Anne Lincoln, Steven Hart, Susan Neuhausen, Mark Robson, Jeffrey Weitzel, Mark Daly, Katherine Nathanson, Fergus Couch, Gadi Rennert, Kenneth Offit. ERCC3 R109X is a moderate risk breast cancer risk variant in Ashkenazi Jews. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 796.

Abstract Germline mutations of BRCA1 and BRCA2 underlie joint susceptibility to breast and ovarian cancers, but do not account for all cases. Genome wide association studies show little overlap between common variants associated with breast and ovarian cancer susceptibility, whereas rare variants in genes initially identified as breast cancer susceptibility genes also confer susceptibility to ovarian cancer cases, unselected for breast cancer. Ovarian and breast tumors also are observed in Peutz-Jeghers and possibly in Lynch syndrome. To further elucidate rare germline susceptibilities for breast and ovarian cancer, we have ascertained a unique cohort of 90 BRCA1/2-negative probands with a personal history of breast cancer and ovarian/fallopian tube/peritoneal cancer, unselected for family history. Cases are from the clinical genetics services of Memorial Sloan-Kettering Cancer Center and University of Pennsylvania. Analysis is ongoing using both targeted and exome sequencing approaches. Germline DNA of 69 cases will be subjected to whole exome sequencing. A total of 21 additional cases will undergo targeted sequencing for 28 genes (8 cases) and an extended panel of 54 genes (13 cases). The majority of ovarian cancers are high-grade serous epithelial ovarian carcinoma (n=51), in combination with invasive ductal or DCIS (n=36), invasive lobular (n=4), or unspecified (n=11) breast cancers. The remaining ovarian subgroups comprise other epithelial subtypes; endometrioid (n=9), clear cell (n=3), mucinous and low-grade serous (n=3) and non-epithelial (n=4) or unclassified (n=20), in combination with invasive ductal or DCIS (n=21), invasive lobular (n=5) or unspecified (n=13) breast cancers. Preliminary analysis of the targeted set of genes in 69 cases, reveals 43 predicted pathogenic coding or splice site variants; 17 of which are within PALB2, MSH2, MSH6, BARD1, FANCE, CDKN2A, TP53, SETD2, SIRT1, BRIP1, RAD50, and RAD51D and are not seen in dbSNP138, 1000 Genomes or Exome Variant Server ESP6500 and 26 variants with reported frequencies in MUTYH, MSH6, MLH1, FAM175A, RAD50, UIMC1, JARID2, PHF3, SIRT1, MRE11A, ATM, BRCA2, TP53BP1, CDH1, RAD51D, BRCA1, BABAM1 of less than 1% allele frequency in public databases. Analysis of these variants across and within histologic subtypes is being undertaken to look for specific genotype-phenotype correlations. Analysis of novel candidate genes and pathways by co-segregation, functional analysis, as well as external replication is underway to determine whether shared rare variants, mutated genes, or altered pathways confer cancer susceptibility in this cohort. Germline susceptibility to breast and ovarian cancer is heterogenous and remains a critical research and clinical question. Improved understanding of the susceptibility genetics will allow further examination of the utility of targeted cancer prevention strategies in those who are found to be at risk. Citation Format: Kasmintan A. Schrader, Kara N. Maxwell, Joseph Vijai, Steven Hart, Tinu Thomas, Bradley Wubbenhorst, Lucia Guidugli, Robert Klein, Marina Corines, Liying Zhang, Susan Neuhausen, Jeffrey Weitzel, Namrata Gupta, Larry Norton, Clifford Hudis, Gad Getz, Mark Daly, Steven Lipkin, David Altshuler, Fergus Couch, Katherine Nathanson, Kenneth Offit. Determination of cancer susceptibility in probands with breast and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3282. doi:10.1158/1538-7445.AM2014-3282

Objective IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. Design We conducted a GWA study (GWAS) of IBS in a general population sample of 11 326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. Results One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31×10 −6 in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. Conclusions Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations.