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J Clin Invest ; 131(17)2021 09 01.
Article En | MEDLINE | ID: mdl-34623332

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

Genes, rel , Primary Immunodeficiency Diseases/genetics , Primary Immunodeficiency Diseases/immunology , Proto-Oncogene Proteins c-rel/deficiency , Proto-Oncogene Proteins c-rel/genetics , Adaptive Immunity/genetics , Adaptive Immunity/immunology , Child , Consanguinity , Female , Hematopoietic Stem Cell Transplantation , Homozygote , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Lymphocyte Activation , Lymphocytes/classification , Lymphocytes/immunology , Mutation , Myeloid Cells/immunology , Primary Immunodeficiency Diseases/therapy , Protein Isoforms
Genome Med ; 13(1): 169, 2021 10 28.
Article En | MEDLINE | ID: mdl-34706766

BACKGROUND: Québec was the Canadian province most impacted by COVID-19, with 401,462 cases as of September 24th, 2021, and 11,347 deaths due mostly to a very severe first pandemic wave. In April 2020, we assembled the Coronavirus Sequencing in Québec (CoVSeQ) consortium to sequence SARS-CoV-2 genomes in Québec to track viral introduction events and transmission within the province. METHODS: Using genomic epidemiology, we investigated the arrival of SARS-CoV-2 to Québec. We report 2921 high-quality SARS-CoV-2 genomes in the context of > 12,000 publicly available genomes sampled globally over the first pandemic wave (up to June 1st, 2020). By combining phylogenetic and phylodynamic analyses with epidemiological data, we quantify the number of introduction events into Québec, identify their origins, and characterize the spatiotemporal spread of the virus. RESULTS: Conservatively, we estimated approximately 600 independent introduction events, the majority of which happened from spring break until 2 weeks after the Canadian border closed for non-essential travel. Subsequent mass repatriations did not generate large transmission lineages (> 50 sequenced cases), likely due to mandatory quarantine measures in place at the time. Consistent with common spring break and "snowbird" destinations, most of the introductions were inferred to have originated from Europe via the Americas. Once introduced into Québec, viral lineage sizes were overdispersed, with a few lineages giving rise to most infections. Consistent with founder effects, the earliest lineages to arrive tended to spread most successfully. Fewer than 100 viral introductions arrived during spring break, of which 7-12 led to the largest transmission lineages of the first wave (accounting for 52-75% of all sequenced infections). These successful transmission lineages dispersed widely across the province. Transmission lineage size was greatly reduced after March 11th, when a quarantine order for returning travellers was enacted. While this suggests the effectiveness of early public health measures, the biggest transmission lineages had already been ignited prior to this order. CONCLUSIONS: Combined, our results reinforce how, in the absence of tight travel restrictions or quarantine measures, fewer than 100 viral introductions in a week can ensure the establishment of extended transmission chains.

COVID-19/transmission , COVID-19/epidemiology , COVID-19/virology , Canada/epidemiology , Europe/epidemiology , Genome, Viral , Humans , Molecular Epidemiology , Pandemics , Phylogeny , Public Health , Quebec/epidemiology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Travel
Cell ; 184(14): 3812-3828.e30, 2021 07 08.
Article En | MEDLINE | ID: mdl-34214472

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

CD28 Antigens/deficiency , Inheritance Patterns/genetics , Papillomaviridae/physiology , Skin/virology , T-Lymphocytes/immunology , Adult , Amino Acid Sequence , Animals , Base Sequence , CD28 Antigens/genetics , CD28 Antigens/metabolism , CD4-Positive T-Lymphocytes/immunology , Child , Endopeptidases/metabolism , Female , Genes, Recessive , HEK293 Cells , Homozygote , Humans , Immunity, Humoral , Immunologic Memory , Jurkat Cells , Keratinocytes/pathology , Male , Mice, Inbred C57BL , Oncogenes , Papilloma/pathology , Papilloma/virology , Pedigree , Protein Sorting Signals , RNA, Messenger/genetics , RNA, Messenger/metabolism
Nat Med ; 27(9): 1646-1654, 2021 09.
Article En | MEDLINE | ID: mdl-34183838

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.

Autoimmunity/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Programmed Cell Death 1 Receptor/genetics , STAT3 Transcription Factor/genetics , Tuberculosis/immunology , Autoimmunity/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD56 Antigen/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Child , Humans , Immune Checkpoint Inhibitors/adverse effects , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy/adverse effects , Interleukin-23/genetics , Interleukin-6/genetics , Intraepithelial Lymphocytes/immunology , Intraepithelial Lymphocytes/pathology , Male , Mycobacterium tuberculosis/pathogenicity , Neoplasms/complications , Neoplasms/drug therapy , Neoplasms/mortality , Programmed Cell Death 1 Receptor/deficiency , Tuberculosis/genetics , Tuberculosis/mortality
J Clin Invest ; 131(14)2021 07 15.
Article En | MEDLINE | ID: mdl-34043590

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,864 COVID-19 cases (713 with severe and 1,151 with mild disease) and 15,033 ancestry-matched population controls across 4 independent COVID-19 biobanks. We tested whether rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only 1 rare pLOF mutation across these genes among 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We found no evidence of association of rare LOF variants in the 13 candidate genes with severe COVID-19 outcomes.

COVID-19/genetics , COVID-19/immunology , Interferon Type I/genetics , Interferon Type I/immunology , Loss of Function Mutation , SARS-CoV-2 , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Child, Preschool , Cohort Studies , Female , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Infant , Infant, Newborn , Interferon Regulatory Factor-7/genetics , Male , Middle Aged , Severity of Illness Index , Toll-Like Receptor 3/genetics , Whole Exome Sequencing , Whole Genome Sequencing , Young Adult
Nat Commun ; 12(1): 2627, 2021 05 11.
Article En | MEDLINE | ID: mdl-33976190

The kidney and upper urinary tract develop through reciprocal interactions between the ureteric bud and the surrounding mesenchyme. Ureteric bud branching forms the arborized collecting duct system of the kidney, while ureteric tips promote nephron formation from dedicated progenitor cells. While nephron progenitor cells are relatively well characterized, the origin of ureteric bud progenitors has received little attention so far. It is well established that the ureteric bud is induced from the nephric duct, an epithelial duct derived from the intermediate mesoderm of the embryo. However, the cell state transitions underlying the progression from intermediate mesoderm to nephric duct and ureteric bud remain unknown. Here we show that nephric duct morphogenesis results from the coordinated organization of four major progenitor cell populations. Using single cell RNA-seq and Cluster RNA-seq, we show that these progenitors emerge in time and space according to a stereotypical pattern. We identify the transcription factors Tfap2a/b and Gata3 as critical coordinators of this progenitor cell progression. This study provides a better understanding of the cellular origin of the renal collecting duct system and associated urinary tract developmental diseases, which may inform guided differentiation of functional kidney tissue.

Nephrons/embryology , Organogenesis/genetics , Stem Cells/physiology , Animals , Cell Differentiation/genetics , Embryo, Mammalian , Female , GATA3 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Male , Mice , Mice, Transgenic , Models, Animal , RNA-Seq , Single-Cell Analysis , Transcription Factor AP-2/metabolism
Med (N Y) ; 2(4): 411-422.e5, 2021 04 09.
Article En | MEDLINE | ID: mdl-33521749

BACKGROUND: Coronavirus disease 2019 (COVID-19) primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell-, tissue-, and organ-specific injury due to COVID-19. METHODS: Our test leverages genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. FINDINGS: We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cell-free DNA correlated with the World Health Organization (WHO) ordinal scale for disease progression and was significantly increased in patients requiring intubation. CONCLUSIONS: This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19. FUNDING: This work was supported by NIH grants 1DP2AI138242 (to I.D.V.), R01AI146165 (to I.D.V., M.P.C., F.M.M., and J.R.), 1R01AI151059 (to I.D.V.), K08-CA230156 (to W.G.), and R33-AI129455 to C.Y.C., a Synergy award from the Rainin Foundation (to I.D.V.), a SARS-CoV-2 seed grant at Cornell (to I.D.V.), a National Sciences and Engineering Research Council of Canada fellowship PGS-D3 (to A.P.C.), and a Burroughs-Wellcome CAMS Award (to W.G.). D.C.V. is supported by a Fonds de la Recherche en Sante du Quebec Clinical Research Scholar Junior 2 award. C.Y.C. is supported by the California Initiative to Advance Precision Medicine, and the Charles and Helen Schwab Foundation.

COVID-19 , Cell-Free Nucleic Acids , Virus Diseases , Humans , Methylation , SARS-CoV-2/genetics
Front Genet ; 11: 612515, 2020.
Article En | MEDLINE | ID: mdl-33335541

Population sequencing often requires collaboration across a distributed network of sequencing centers for the timely processing of thousands of samples. In such massive efforts, it is important that participating scientists can be confident that the accuracy of the sequence data produced is not affected by which center generates the data. A study was conducted across three established sequencing centers, located in Montreal, Toronto, and Vancouver, constituting Canada's Genomics Enterprise ( Whole genome sequencing was performed at each center, on three genomic DNA replicates from three well-characterized cell lines. Secondary analysis pipelines employed by each site were applied to sequence data from each of the sites, resulting in three datasets for each of four variables (cell line, replicate, sequencing center, and analysis pipeline), for a total of 81 datasets. These datasets were each assessed according to multiple quality metrics including concordance with benchmark variant truth sets to assess consistent quality across all three conditions for each variable. Three-way concordance analysis of variants across conditions for each variable was performed. Our results showed that the variant concordance between datasets differing only by sequencing center was similar to the concordance for datasets differing only by replicate, using the same analysis pipeline. We also showed that the statistically significant differences between datasets result from the analysis pipeline used, which can be unified and updated as new approaches become available. We conclude that genome sequencing projects can rely on the quality and reproducibility of aggregate data generated across a network of distributed sites.

Cell ; 183(7): 1826-1847.e31, 2020 12 23.
Article En | MEDLINE | ID: mdl-33296702

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αß T and non-classic CD4+ αß TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αß T, and CD4+ αß TH1∗ cells unable to compensate for this deficit.

Adaptive Immunity , Immunity, Innate , Interferon-gamma/immunology , Mycobacterium/immunology , T-Box Domain Proteins/metabolism , Amino Acid Sequence , Base Sequence , Cell Lineage , Child, Preschool , Chromatin/metabolism , CpG Islands/genetics , DNA Methylation/genetics , Dendritic Cells/metabolism , Epigenesis, Genetic , Female , Homozygote , Humans , INDEL Mutation/genetics , Infant , Interferon-gamma/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Loss of Function Mutation/genetics , Male , Mycobacterium Infections/genetics , Mycobacterium Infections/immunology , Mycobacterium Infections/microbiology , Pedigree , T-Box Domain Proteins/chemistry , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , T-Lymphocytes, Helper-Inducer/immunology , Transcriptome/genetics
medRxiv ; 2020 Jul 29.
Article En | MEDLINE | ID: mdl-32766608

COVID-19 primarily affects the lungs, but evidence of systemic disease with multi-organ involvement is emerging. Here, we developed a blood test to broadly quantify cell, tissue, and organ specific injury due to COVID-19, using genome-wide methylation profiling of circulating cell-free DNA in plasma. We assessed the utility of this test to identify subjects with severe disease in two independent, longitudinal cohorts of hospitalized patients. Cell-free DNA profiling was performed on 104 plasma samples from 33 COVID-19 patients and compared to samples from patients with other viral infections and healthy controls. We found evidence of injury to the lung and liver and involvement of red blood cell progenitors associated with severe COVID-19. The concentration of cfDNA correlated with the WHO ordinal scale for disease progression and was significantly increased in patients requiring intubation. This study points to the utility of cell-free DNA as an analyte to monitor and study COVID-19.

Nat Commun ; 11(1): 3406, 2020 07 08.
Article En | MEDLINE | ID: mdl-32641768

Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.

Brain Neoplasms/genetics , Brain/metabolism , Glioblastoma/genetics , Neoplastic Stem Cells/metabolism , Sequence Analysis, RNA/methods , Transcriptome/genetics , Adult , Animals , Antineoplastic Agents, Alkylating/pharmacology , Brain/embryology , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Female , Fetus , Glioblastoma/pathology , Glioblastoma/therapy , Humans , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/drug effects , Single-Cell Analysis/methods , Temozolomide/pharmacology , Xenograft Model Antitumor Assays/methods
Sci Rep ; 10(1): 581, 2020 01 17.
Article En | MEDLINE | ID: mdl-31953485

While comparison of primary tumor and metastases has highlighted genomic heterogeneity in colorectal cancer (CRC), previous studies have focused on a single metastatic site or limited genomic testing. Combining data from whole exome and ultra-deep targeted sequencing, we explored possible evolutionary trajectories beyond the status of these mutations, particularly among patient-matched metastatic tumors. Our findings confirm the persistence of known clinically-relevant mutations (e.g., those of RAS family of oncogenes) in CRC primary and metastases, yet reveal that latency and interval systemic therapy affect the course of evolutionary events within metastatic lesions. Specifically, our analysis of patient-matched primary and multiple metastatic lesions, developed over time, showed a similar genetic composition for liver metastatic tumors, which were 21-months apart. This genetic makeup was different from those identified in lung metastases developed before manifestation of the second liver metastasis. These results underscore the role of latency in the evolutionary path of metastatic CRC and may have implications for future treatment options.

Colorectal Neoplasms/genetics , Gene Regulatory Networks , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Lung Neoplasms/genetics , Lung Neoplasms/secondary , DNA Copy Number Variations , DNA Mutational Analysis , Female , Gene Frequency , Genetic Heterogeneity , High-Throughput Nucleotide Sequencing , Humans , Male , Time Factors , Whole Exome Sequencing
medRxiv ; 2020 Dec 21.
Article En | MEDLINE | ID: mdl-33398295

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.

Mol Biol Evol ; 37(1): 2-10, 2020 Jan 01.
Article En | MEDLINE | ID: mdl-31504792

Recent reports have identified differences in the mutational spectra across human populations. Although some of these reports have been replicated in other cohorts, most have been reported only in the 1000 Genomes Project (1kGP) data. While investigating an intriguing putative population stratification within the Japanese population, we identified a previously unreported batch effect leading to spurious mutation calls in the 1kGP data and to the apparent population stratification. Because the 1kGP data are used extensively, we find that the batch effects also lead to incorrect imputation by leading imputation servers and a small number of suspicious GWAS associations. Lower quality data from the early phases of the 1kGP thus continue to contaminate modern studies in hidden ways. It may be time to retire or upgrade such legacy sequencing data.

Human Genome Project , Artifacts , Genome-Wide Association Study , Humans , Japan , Mutation
Gigascience ; 8(6)2019 06 01.
Article En | MEDLINE | ID: mdl-31185495

BACKGROUND: With the decreasing cost of sequencing and the rapid developments in genomics technologies and protocols, the need for validated bioinformatics software that enables efficient large-scale data processing is growing. FINDINGS: Here we present GenPipes, a flexible Python-based framework that facilitates the development and deployment of multi-step workflows optimized for high-performance computing clusters and the cloud. GenPipes already implements 12 validated and scalable pipelines for various genomics applications, including RNA sequencing, chromatin immunoprecipitation sequencing, DNA sequencing, methylation sequencing, Hi-C, capture Hi-C, metagenomics, and Pacific Biosciences long-read assembly. The software is available under a GPLv3 open source license and is continuously updated to follow recent advances in genomics and bioinformatics. The framework has already been configured on several servers, and a Docker image is also available to facilitate additional installations. CONCLUSIONS: GenPipes offers genomics researchers a simple method to analyze different types of data, customizable to their needs and resources, as well as the flexibility to create their own workflows.

Genomics/methods , Software , DNA Methylation , Epigenomics/methods , Humans , Metagenomics/methods , Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methods
Circ Res ; 124(4): 553-563, 2019 02 15.
Article En | MEDLINE | ID: mdl-30582441

RATIONALE: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. OBJECTIVE: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. METHODS AND RESULTS: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5×10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%-6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%-3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates, including RYR1, ZFPM1, CAMTA2, DLX6, and PCM1. CONCLUSIONS: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.

Exome , Mutation Rate , Tetralogy of Fallot/genetics , Autoantigens/genetics , Calcium-Binding Proteins/genetics , Cell Cycle Proteins/genetics , Homeodomain Proteins/genetics , Humans , Loss of Function Mutation , Mutation, Missense , Nuclear Proteins/genetics , Receptor, Notch1/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Vascular Endothelial Growth Factor Receptor-3/genetics
Cancer Cell ; 30(6): 891-908, 2016 Dec 12.
Article En | MEDLINE | ID: mdl-27960086

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.

Central Nervous System Neoplasms/genetics , Chromatin/genetics , Epigenomics/methods , Receptor, Platelet-Derived Growth Factor beta/genetics , Rhabdoid Tumor/genetics , SMARCB1 Protein/genetics , Teratoma/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Central Nervous System Neoplasms/drug therapy , DNA Methylation , Dasatinib/pharmacology , Dasatinib/therapeutic use , Epigenesis, Genetic/drug effects , Humans , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Rhabdoid Tumor/drug therapy , Teratoma/drug therapy
Methods Mol Biol ; 1458: 311-37, 2016.
Article En | MEDLINE | ID: mdl-27581031

The transcriptome is composed of different types of RNA molecules including mRNAs, tRNAs, rRNAs, and other noncoding RNAs that are found inside a cell at a given time. Analyzing transcriptome patterns can shed light on the functional state of the cell as well as on the dynamics of cellular behavior associated with genomic and environmental changes. Likewise, transcriptome analysis has been a major help in solving biological issues and understanding the molecular basis of many diseases including human cancers. Specifically, since targeted and whole genome sequencing studies are becoming more common in identifying the driving factors of cancer, a comprehensive and high-resolution analysis of the transcriptome, as provided by RNA-Sequencing (RNA-Seq), plays a key role in investigating the functional relevance of the identified genomic aberrations. Here, we describe experimental procedures of RNA-Seq and downstream data processing and analysis, with a focus on the identification of abnormally expressed transcripts and genes.

Neoplasms/genetics , Neoplasms/pathology , Tumor Microenvironment/genetics , Computational Biology/methods , Gene Expression Profiling/methods , Gene Library , Gene Ontology , High-Throughput Nucleotide Sequencing , Humans , Quality Control , Transcriptome
Nat Commun ; 5: 5135, 2014 Oct 29.
Article En | MEDLINE | ID: mdl-25351205

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

Carcinoma, Renal Cell/genetics , Genetic Variation , Genome, Human/genetics , Genomics , Adult , Aged , Aged, 80 and over , Cohort Studies , Europe , Female , Focal Adhesions/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Mutation , Mutation Rate , Oncogene Proteins, Fusion/genetics , Phosphatidylinositol 3-Kinases/genetics , RNA Splicing/genetics , Sequence Analysis, DNA , Signal Transduction/genetics