Systematic characterization of regulatory variants of blood pressure genes

High blood pressure (BP) is the major risk factor for cardiovascular disease. Genome-wide association studies have identified genetic variants for BP, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4,608 genetic variants in linkage with 135 BP loci in vascular smooth muscle cells and cardiomyocytes by massively parallel reporter assays. High densities of regulatory variants at BP loci (i.e., ULK4, MAP4, CFDP1, PDE5A) indicate that multiple variants drive genetic association. Regulatory variants are enriched in repeats, alter cardiovascular-related transcription factor motifs, and spatially converge with genes controlling specific cardiovascular pathways. Using heuristic scoring, we define likely causal variants, and CRISPR prime editing finally determines causal variants for KCNK9, SFXN2, and PCGF6, which are candidates for developing high BP. Our systems-level approach provides a catalog of functionally relevant variants and their genomic architecture in two trait-relevant cell lines for a better understanding of BP gene regulation.We thank the Melé and Maass labs for intellectual input, Dr. Steven Erwood from Dr. Ronald Cohn’s lab for guidance in applying CRISPR prime editing, The Centre for Applied Genomics, The Structural & Biophysical Core Facility, and The Imaging Facility, The Hospital for Sick Children, Toronto, Canada, for assistance with high-throughput sequencing, luminescence detection, and imaging. We thank Dovetail Genomics, LLC, 100 Enterprise Way, Suite A101, Scotts Valley, CA 95066, USA, for generating Omni-C libraries and for the collaborative support throughout the project. W.O. was supported by a Fundació la Marató grant (ref. 321/C/2019), K.K. was supported by an OGS fellowship, J.W.L.B. was supported by a CGS-D fellowship, and D.F.L. was supported by an Ontario Genomics-CANSSI Ontario Postdoctoral Fellowship in Genome Data Science. This project was supported by Canada’s New Frontiers in Research Fund (NFRFE-2018-01305), the Canadian Institutes of Health Research (CIHR PJT 173542 [P.G.M.] and PJT 175034 [S.M., J.E.]), CIHR ENP 161429 under the frame of ERA PerMed (S.M.), the Ted Rogers Centre for Heart Research (S.M., J.E.), and the Heart and Stroke Foundation of Canada. J.E. holds a Canada Research Chair Tier 1 in Stem Cell Models of Childhood Disease, S.M. holds the Heart and Stroke Foundation of Canada & Robert M. Freedom Chair in Cardiovascular Science, M. Melé was supported by a Ramon y Cajal fellowship (RYC-2017-22249), and P.G.M. holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms.Peer Reviewed"Article signat per 14 autors/es: Winona Oliveros, Kate Delfosse, Daniella F. Lato , Katerina Kiriakopulos, Milad Mokhtaridoost, Abdelrahman Said, Brandon J. McMurray, Jared W.L. Browning, Kaia Mattioli, Guoliang Meng, James Ellis, Seema Mital, Marta Melé, Philipp G. Maass"Postprint (published version

Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10−7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7–58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.This project was supported by the Ted Rogers Centre for Heart Research (SM, JE), the Canadian Institutes of Health Research (PJT 175034) (SM, JE) and by the Canadian Institutes of Health Research (ENP 161429), under the frame of ERA PerMed (SM). SM holds the Heart and Stroke Foundation of Canada & Robert M Freedom Chair in Cardiovascular Science. SWS holds the GlaxoSmithKline Endowed Chair in Genome Sciences at the Hospital for Sick Children and the University of Toronto. PGM holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms. PGM acknowledges the support of the Government of Canada’s New Frontiers in Research Fund (NFRF), [NFRFE-2018-01305]. EO holds the Bitove Family Professorship of Adult Congenital Heart Disease. MM holds a Ramon y Cajal grant from the Spanish Ministry of Science and Innovation (RYC-2017-22249). WO is supported by funding from Fundació La Marató (321/C/2019). JB is funded by a Frans Van de Werf fellowship for clinical cardiovascular research, and by a senior clinical investigator fellowship of the FWO Flanders. KM was a National Science Foundation Graduate Research Fellow under grant no. DGE1144152 during the majority of the project. CS is the recipient of a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (1154992). JI is the recipient of an NHMRC Career Development Fellowship (1162929). RDB is the recipient of a New South Wales Health Cardiovascular Disease Senior Scientist Grant. PSD is supported by the DBT/Wellcome Trust- Indian Alliance. We acknowledge the Labatt Family Heart Centre Biobank at the Hospital for Sick Children for access to DNA samples, and The Centre for Applied Genomics at the Hospital for Sick Children for performing WGS. We thank Xiucheng Cui and Emanuela Pannia for performing the zebrafish experiments at the SickKids Zebrafish Genetics and Disease Models Core (CRISPR-Cas9 and gRNA syntheses, zebrafish embryo microinjections, gRNA PCR validation, qRT-PCR, cardiac imaging). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. We thank members of the ICGC/PCAWG working groups for generating the variant calls used in our case-control burden analyses.Peer Reviewed"Article signat per 38 autors/es: Robert Lesurf, Abdelrahman Said, Oyediran Akinrinade, Jeroen Breckpot, Kathleen Delfosse, Ting Liu, Roderick Yao, Gabrielle Persad, Fintan McKenna, Ramil R. Noche, Winona Oliveros, Kaia Mattioli, Shreya Shah, Anastasia Miron, Qian Yang, Guoliang Meng, Michelle Chan Seng Yue, Wilson W. L. Sung, Bhooma Thiruvahindrapuram, Jane Lougheed, Erwin Oechslin, Tapas Mondal, Lynn Bergin, John Smythe, Shashank Jayappa, Vinay J. Rao, Jayaprakash Shenthar, Perundurai S. Dhandapany, Christopher Semsarian, Robert G. Weintraub, Richard D. Bagnall, Jodie Ingles, Genomics England Research Consortium, Marta Melé, Philipp G. Maass, James Ellis, Stephen W. Scherer & Seema Mital"Postprint (published version

Human metastatic cholangiocarcinoma patient-derived xenografts and tumoroids for preclinical drug evaluation

Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30\\%–50\\% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA.We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA\_PDX). The CCA\_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA\_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA\_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues.This collection of CCA\_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA\_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA\_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD.Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA\_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.The authors would like to thank the patients and their families for their support. This work was supported by grants from the Fundaci o Marat o TV3 awarded to T. Macarulla, M. Mel e, and S. Peir o; BeiGene research grant awarded toT. Macarulla and S. Peir o; AECC (INVES20036TIAN), Ram on y Cajal investigator program (RYC2020-029098-I), Proyecto de IþDþi (PID2019-108008RJ-I00), and FERO Foundation grant awarded to T.V. Tian; Proyecto de Investigaci on en Salud from the Instituto de Salud Carlos III (ISCIII) (PI20/00898) awarded to T. Macarulla; FIS/FEDER from the Instituto de Salud Carlos III (ISCIII) (PI12/01250; CP08/00223; PI16/00253 and CB16/12/00449) awarded to S. Peir o; and Ram on y Cajal investigator program (RYC-2017-22249) awarded to M. Mel e. Q. Serra-Camprubí is a recipient of the Ph.D. fellowship from La Caixa Foundation (LCF/PR/PR12/51070001). A. LlopGuevara was supported by the AECC (INVES20095LLOP) and V. Serra by the ISCIII (CPII19/00033). E.J. Arenas was funded by the AECC (POSTD211413AREN).J. Arribas is funded by the Instituto de Salud Carlos III (AC15/00062, CB16/12/00449, and PI22/00001). This publication is based upon the work of COST Action CA18122, European Cholangiocarcinoma Network, supported by the COST (European Cooperation in Science and Technology, www.cost.eu), a funding agency for research and innovation networks. The authors would like to thank Dr. V.A. Raker for manuscript editing and Drs. N. Herranz and J. Mateo for scientific discussions. The authors acknowledge the infrastructure and support of the FERO Foundation, La Caixa Foundation, and the Cellex Foundation.Peer Reviewed"Article signat per 31 autors/es: Queralt Serra-Camprubí; Helena Verdaguer; Winona Oliveros; Núria Lupión-Garcia; Núria Lupión-Garcia;Alba Llop-Guevara; Cristina Molina; Maria Vila-Casadesús; Anthony Turpin; Cindy Neuzillet; Joan Frigola; Jessica Querol; Mariana Yáñez-Bartolomé; Florian Castet; Carles Fabregat-Franco; Carmen Escudero-Iriarte; Marta Escorihuela; Enrique J. Arenas; Cristina Bernadó-Morales; Noemí Haro; Francis J. Giles; Óscar J. Pozo; Josep M. Miquel ; Paolo G. Nuciforo; Ana Vivancos; Marta Melé; Violeta Serra ; Joaquín Arribas; Josep Tabernero; Sandra Peiró; Teresa Macarulla; Tian V. Tian"Postprint (published version

Cis and trans effects differentially contribute to the evolution of promoters and enhancers

Background Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants located on the same DNA molecule as the target gene, trans effects are due to genetic variants that affect diffusible elements. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we use massively parallel reporter assays to directly measure the transcriptional outputs of thousands of individual regulatory elements in embryonic stem cells and measure cis and trans effects between human and mouse. Results Our approach reveals that cis effects are widespread across transcribed regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we find that trans effects are rare but stronger in enhancers than promoters and are associated with a subset of transcription factors that are differentially expressed between human and mouse. While we find that cis-trans compensation is common within promoters, we do not see evidence of widespread cis-trans compensation at enhancers. Cis-trans compensation is inversely correlated with enhancer redundancy, suggesting that such compensation may often occur across multiple enhancers. Conclusions Our results highlight differences in the mode of evolution between promoters and enhancers in complex mammalian genomes and indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.K.M. was a National Science Foundation Graduate Research Fellow under grant no. DGE1144152 during the majority of the project. M.M. was a Gilead Fellow of the Life Sciences Research Foundation during part of the project and is currently supported by the Spanish Ministry of Science and Innovation with a Ramon y Cajal grant (RYC-2017-22249). J.L.R. is an HHMI faculty scholar.Peer ReviewedPostprint (published version

The landscape of expression and alternative splicing variation across human traits

Understanding the consequences of individual transcriptome variation is fundamental to deciphering human biology and disease. We implement a statistical framework to quantify the contributions of 21 individual traits as drivers of gene expression and alternative splicing variation across 46 human tissues and 781 individuals from the Genotype-Tissue Expression project. We demonstrate that ancestry, sex, age, and BMI make additive and tissue-specific contributions to expression variability, whereas interactions are rare. Variation in splicing is dominated by ancestry and is under genetic control in most tissues, with ribosomal proteins showing a strong enrichment of tissue-shared splicing events. Our analyses reveal a systemic contribution of types 1 and 2 diabetes to tissue transcriptome variation with the strongest signal in the nerve, where histopathology image analysis identifies novel genes related to diabetic neuropathy. Our multi-tissue and multi-trait approach provides an extensive characterization of the main drivers of human transcriptome variation in health and disease.This study was funded by the HumTranscriptom project with reference PID2019-107937GA-I00. R.G.-P. was supported by a Juan de la Cierva fellowship (FJC2020-044119-I) funded by MCIN/AEI/10.13039/501100011033 and ‘‘European Union NextGenerationEU/PRTR.’’ J.M.R. was supported by a predoctoral fellowship from ‘‘la Caixa’’ Foundation (ID 100010434) with code LCF/BQ/DR22/11950022. A.R.-C. was supported by a Formación Personal Investigador (FPI) fellowship (PRE2019-090193) funded by MCIN/AEI. R.C.-G. was supported by an FPI fellowship (PRE2020-092510) funded by MCIN/AEI. M.M. was supported by a Ramon y Cajal fellowship (RYC-2017-22249).Peer ReviewedPostprint (published version

Human Metastatic Cholangiocarcinoma Patient-Derived Xenografts and Tumoroids for Preclinical Drug Evaluation

Human metastatic cholangiocarcinoma; Xenografts; TumoroidsColangiocarcinoma metastàtic humà; Xenoempelts; TumoroidesColangiocarcinoma metastásico humano; Xenoinjertos; TumoroidesPurpose: Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30%–50% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA. Experimental Design: We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA_PDX). The CCA_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues. Results: This collection of CCA_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD. Conclusions: Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials.This work was supported by grants from the Fundació Marató TV3 awarded to T. Macarulla, M. Melé, and S. Peiró; BeiGene research grant awarded to T. Macarulla and S. Peiró; AECC (INVES20036TIAN), Ramón y Cajal investigator program (RYC2020-029098-I), Proyecto de I+D+i (PID2019-108008RJ-I00), and FERO Foundation grant awarded to T.V. Tian; Proyecto de Investigación en Salud from the Instituto de Salud Carlos III (ISCIII) (PI20/00898) awarded to T. Macarulla; FIS/FEDER from the Instituto de Salud Carlos III (ISCIII) (PI12/01250; CP08/00223; PI16/00253 and CB16/12/00449) awarded to S. Peiró; and Ramón y Cajal investigator program (RYC-2017-22249) awarded to M. Melé. Q. Serra-Camprubí is a recipient of the Ph.D. fellowship from La Caixa Foundation (LCF/PR/PR12/51070001). A. Llop-Guevara was supported by the AECC (INVES20095LLOP) and V. Serra by the ISCIII (CPII19/00033). E.J. Arenas was funded by the AECC (POSTD211413AREN). J. Arribas is funded by the Instituto de Salud Carlos III (AC15/00062, CB16/12/00449, and PI22/00001). This publication is based upon the work of COST Action CA18122, European Cholangiocarcinoma Network, supported by the COST (European Cooperation in Science and Technology, www.cost.eu), a funding agency for research and innovation networks. The authors would like to thank Dr. V.A. Raker for manuscript editing and Drs. N. Herranz and J. Mateo for scientific discussions. The authors acknowledge the infrastructure and support of the FERO Foundation, La Caixa Foundation, and the Cellex Foundation

Epigenetic characterization of cholangiocarcinomas

Cholangiocarcinoma (CCA) is a rare type of cancer and accounts for 10-20% of primary liver diagnosed cancers, being the second most common hepatobiliary malignancy.The only prospect of a long-term cure for these malignancies is offered by surgical resection of the tumor. However, the prognosis of all patients remains poor due to the high rate of recurrence of these tumors. In addition, the majority of patients are not diagnosed until the disease is nonresectable, thus only being suitable for palliative chemotherapy or supportive care1. Recently, some groups have published data on CCA describing its complex pathogenesis involving many different molecular pathways, with some of them being potential therapeutic targets.Among the potential therapeutic targets, mutations on the IDH1/2 gene are present in around 20% of CCA patients2. This mutation generates what is called an “oncometabolite” (D2HG) that is responsible for many of the biological effects associated to IDH1 mutations in cancer. The main effect of D2HG is to competitively inhibit a family of alpha-KGdependent enzymes, such as TET and JmJC, leading to a global increase of DNA and histone methylation. Now, there is the need to establish a comprehensive understanding on how IDH1 mutations leads to the alteration of chromatin states in CCA. To this end, here we analyze the epigenome of CCA PDXs in terms of DNA accessibility, DNA methylation and transcriptome profiling in IDH1 wildtype and mutant samples. Our main goal is to shed some light on the relation between the genetic and epigenetic architecture of this type of cancer by performing an integrative analysis to characterize this unique set of CCA patient-derived models

The landscape of expression and alternative splicing variation across human traits

Understanding the consequences of individual transcriptome variation is fundamental to deciphering human biology and disease. We implement a statistical framework to quantify the contributions of 21 individual traits as drivers of gene expression and alternative splicing variation across 46 human tissues and 781 individuals from the Genotype-Tissue Expression project. We demonstrate that ancestry, sex, age, and BMI make additive and tissue-specific contributions to expression variability, whereas interactions are rare. Variation in splicing is dominated by ancestry and is under genetic control in most tissues, with ribosomal proteins showing a strong enrichment of tissue-shared splicing events. Our analyses reveal a systemic contribution of types 1 and 2 diabetes to tissue transcriptome variation with the strongest signal in the nerve, where histopathology image analysis identifies novel genes related to diabetic neuropathy. Our multi-tissue and multi-trait approach provides an extensive characterization of the main drivers of human transcriptome variation in health and disease.This study was funded by the HumTranscriptom project with reference PID2019-107937GA-I00. R.G.-P. was supported by a Juan de la Cierva fellowship (FJC2020-044119-I) funded by MCIN/AEI/10.13039/501100011033 and “European Union NextGenerationEU/PRTR.” J.M.R. was supported by a predoctoral fellowship from “la Caixa” Foundation (ID 100010434) with code LCF/BQ/DR22/11950022. A.R.-C. was supported by a Formación Personal Investigador (FPI) fellowship (PRE2019-090193) funded by MCIN/AEI. R.C.-G. was supported by an FPI fellowship (PRE2020-092510) funded by MCIN/AEI. M.M. was supported by a Ramon y Cajal fellowship (RYC-2017-22249). Figures 4A and S1A and the graphical abstract were created with BioRender.com. We thank the donors and their families for their generous gifts of organ donation for transplantation and tissue donations for the GTEx research project and the GTEx consortium members