Somatic mutations render human exome and pathogen DNA more similar

Immunotherapy has recently shown important clinical successes in a substantial number of oncology indications. Additionally, the tumor somatic mutation load has been shown to associate with response to these therapeutic agents, and specific mutational signatures are hypothesized to improve this association, including signatures related to pathogen insults. We sought to study in silico the validity of these observations and how they relate to each other. We first addressed whether somatic mutations typically involved in cancer may increase, in a statistically meaningful manner, the similarity between common pathogens and the human exome. Our study shows that common mutagenic processes increase, in the upper range of biologically plausible frequencies, the similarity between cancer exomes and pathogen DNA at a scale of 12-16 nucleotide sequences and established that this increased similarity is due to the specific mutation distribution of the considered mutagenic processes. Next, we studied the impact of mutation rate and showed that increasing mutation rate generally results in an increased similarity between the cancer exome and pathogen DNA, at a scale of 4-5 amino acids. Finally, we investigated whether the considered mutational processes result in amino-acid changes with functional relevance that are more likely to be immunogenic. We showed that functional tolerance to mutagenic processes across species generally suggests more resilience to mutagenic processes that are due to exposure to elements of nature than to mutagenic processes that are due to exposure to cancer-causing artificial substances. These results support the idea that recognition of pathogen sequences as well as differential functional tolerance to mutagenic processes may play an important role in the immune recognition process involved in tumor infiltration by lymphocytes

Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization

Background: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. Results: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. Conclusions: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/

College of American Pathologists\u27 Laboratory Standards for Next-Generation Sequencing Clinical Tests

Context.-The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology. Objective.-To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or \u27\u27 dry bench\u27\u27 analyses. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests. Design.-To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results.-A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP\u27s molecular pathology checklist (MOL). Conclusions.-This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality

Tumor Antigens Revealed by Exome Seqeuncing Drive Editing of Tumor Immunogenicity

Accumulated data from animal models and human cancer patients strongly support the concept that immunity cannot only function as an extrinsic tumor suppressor, but also shape tumor immunogenicity. These observations led to the development of the cancer immunoediting hypothesis that stresses the dual host-protective and tumor-sculpting actions of immunity on developing cancers. We previously demonstrated important roles for lymphocytes and type I: IFN-α/β) and type II: IFN-γ) interferons in cancer immunoediting. In the present work, we confirmed the role of IFN-γ in sculpting tumor immunogenicity and provide evidence that antigens expressed by tumors drive the destructive or sculpting actions of immunity on cancers. Initial studies confirmed the finding that IFN-γ is a critical mediator of cancer immunoediting. Wild type mice treated with antibodies that neutralize IFN-γ developed more sarcomas than control mice. Furthermore, a subset of sarcomas generated in IFN-γ neutralized mice spontaneously reject when transplanted into wild type mice. Finally, these unedited tumors had differential requirements for IFN-γ responsiveness at the level of the host and the tumor to mediate tumor rejection. Although many immune components that participate in cancer immunoediting are known: e.g. IFN-γ), its underlying mechanisms remain poorly defined. We used massively parallel sequencing to characterize the expressed mutations in a highly immunogenic sarcoma, d42m1, and identified mutant spectrin-β2 as the major rejection antigen. Moreover, we demonstrate that editing of d42m1 tumor cells occurs via a T cell-dependent immunoselection process that promotes outgrowth of variants lacking mutant spectrin-β2. Thus, the strongly immunogenic characteristic of an unedited tumor can be ascribed to expression of a highly antigenic mutant protein. Subsequent studies established that antigen loss variants of d42m1 and edited sarcomas from wild type mice exhibit residual immunogenicity and respond to checkpoint blockade immunotherapy: anti-CTLA-4). Exome sequencing of these tumors has laid the groundwork for the eventual identification of the antigens targeted for destruction by this form of cancer immunotherapy. Taken together, these studies demonstrate that antigens drive the cancer immunoediting process and point to the future potential that cancer genome sequencing may have on the fields of tumor immunology and cancer immunotherapy

The Intrinsic Apoptotic Cascade in Hematopoiesis and Susceptibility to Therapy-Related Leukemia

Apoptosis and the DNA damage response have been implicated in hematopoietic development and differentiation, as well as in the pathogenesis of myelodysplastic syndrome (MS) and leukemia. However, the specific roles of direct mediators of apoptosis, such as caspases, in hematopoiesis and leukemogenesis have not been elucidated. In order to address this, we studied the effects of loss of Caspase-9, the initiator caspase of the intrinsic apoptotic cascade, and Apaf1, the key component of the apoptosome, on fetal and adult hematopoiesis. We first found that loss of these key regulators has significant effects on the hematopoietic stem and progenitor compartment, with decreases in erythroid and B-cell progenitor abundance and impaired function of hematopoietic stem cells after transplantation. Long term adult hematopoiesis is also altered in bone marrow chimeras lacking Casp9 and Apaf1. Counter-intuitively, mice lacking these cell death components show low white blood cell counts, decreased B cell abundance and anemia. Ultimately, they die early due to bone marrow failure. Defects in apoptosis have also been previously implicated in susceptibility to therapy-related leukemia, a disease caused by exposure to DNA-damaging chemotherapeutics used as treatment for other malignancies. Decreased apoptosis could allow cells to inappropriately survive exposure to DNA damaging agents, giving rise to a population of cells with increased DNA damage that was more prone to clonal outgrowth or malignant transformation. We examined whether loss of Casp9 or Apaf1 altered response to N-ethyl-nitrosurea (ENU), an alkylator similar to those used in chemotherapy, and found that loss of these genes did result in increased DNA damage in surviving cells after ENU-treatment. Furthermore, exome sequencing revealed that loss of Casp9 when combined with alkylator treatment gives rise to oligoclonal hematopoiesis, a precursor to diseases such as MDS and acute myeloid leukemia. Taken together these findings suggest that loss of apoptosis could be a key step in the pathogenesis of therapy-related disease

The Genetics of Primary Immunodeficiency in Children

Studies of children with recurrent infection demonstrate that primary immunodeficiency (PID) has a significant genetic component. In PID, over 300 genes of high penetrance inherited mostly in autosomal recessive manner have already been identified. However, many children, including those with early onset immunodeficiency have not received a genetic diagnosis, despite use of targeted sequencing methods. I performed bioinformatic analysis in whole genome sequencing data in patients with immunodeficiency. These analyses were initially in a small cohort of affected children at Great Ormond Street in whom a genetic diagnosis was not known. I devised and utilised bioinformatic programs to identify novel genetic variants in this cohort. I evaluated the performance of whole genome sequence analyses with targeted gene panel analyses, which is the most utilised method of genetic diagnosis. To expand my analysis, I looked at a larger cohort of young people and adults with immunodeficiency as part of the large national collaborative project NIHR Bioresource Rare Diseases BRIDGE-PID project. I quantified the burden of rare coding variation in a case cohort compared to controls and used rare variant association analysis to identify potential novel candidate genes in primary immunodeficiency. The final chapter focuses on 2 novel genetic variants found in the cohort and our initial functional testing to verify genetic diagnosis. The work presented in this thesis demonstrates novel genetic causes of immunodeficiency and their functional implications. The results of my work have improved understanding of the genetic architecture of primary immunodeficiencies and has clinical utility in the diagnosis and subsequent treatment of immunodeficiency

Genetics of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is one the most common forms of non-Hodgkin lymphoma and one of the most aggressive B-cell neoplasms. Although most patients respond to current standard treatments, a significant number of them relapse and become refractory to treatment. Hence, there is a need for new approaches in the management of DLBCL. In recent years, a number of studies using next-generation sequencing (NGS) have contributed to the characterization of the disease and have revealed a set of deregulated cellular mechanisms in DLBCL. However, DLBCL is a very heterogeneous disease and additional work is required in order to achieve a comprehensive understanding of the mechanisms underlying its lymphomagenesis. The work described in this thesis aimed to further characterize the genome of DLBCL and identify novel genomic alterations by employing a number of NGS methods. Furthermore, this thesis sought to functionally describe the effect of some of these alterations and explore how they could be meaningful from a therapeutic perspective. We first embarked on a targeted sequencing approach in order to assess the impact of mutations in a set of key DNA repair genes. DNA repair is critical in B-cell development as it ensures the regulation of antibody diversification processes. The results showed that half of the tumors analyzed carry mutations in DNA repair genes and the most frequently targeted mechanisms are mismatch repair, DNA damage response, homologous recombination and non-homologous end-joining. Moreover, functional analysis enabled the association of a number of those mutations to specific phenotypes. Whole exome sequencing identified a number or previously unreported somatic mutation targets in DLBCL. In addition, it revealed that some genes known to be involved in DLBCL were mutated at a much higher frequency in Chinese patients as compared to patients from Western populations. The impact of mutations in DTX1, a gene encoding for a negative regulator of Notch signaling, was functionally assessed and a number of mutations were found to be deleterious. Finally, whole genome sequencing enabled the identification of structural variations such as translocations in the genome of DLBCL. IGH translocations are a hallmark of DLBCL and the locus is often found juxtaposed to proto-oncogenes which leads to their upregulation. We identified PD-L1, a common culprit of immune suppression in cancer, as a novel IGH translocation partner in DLBCL. Translocations involving the PD-L1 locus were found to impact patient survival and were associated with the more aggressive non-germinal center-like subtype of the diseas

Unravelling novel genetic and functional mechanisms of primary immunodeficiencies

RESUMO: As imunodeficiências primárias (IDP) constituem um grupo heterogéneo de doenças congénitas que podem afetar o desenvolvimento e/ou a função de qualquer componente do sistema imunitário. Isto pode conduzir à alteração de qualquer uma das funções do sistema imune e portanto os doentes com IDP podem apresentar infeções recorrentes ou susceptibilidade especifica a determinado micro-organismo , mas também podem apresentar auto-imunidade, linfoproliferação, híper-inflamação ou até neoplasias. A recente generalização da disponibilidade das técnicas de sequenciação de nova geração permitu não só a identificação de genes responsáveis por IDP previamente descritas, mas também a descoberta de novas IDP. É sabido que a grande maioria das IDP são defeitos monogénicos que podem apresentar expressão e penetrância incompletas. O reconhecimento que mutações no mesmo gene podem conduzir a diferentes fenótipos, mas também que mutações em múltiplos genes diferentes podem conduzir ao mesmo fenótipo, tornou mandatória a utilização de técnicas de sequenciação de nova geração para elucidar os quadros clínicos destes doentes ou grupos de doentes. Ao compreendermos o impacto destas experiências da Natureza no sistema imunitário, foi possível não só expandir o conhecimento, mas também mudar condutas terapêuticas e o aconselhamento das famílias. Esta mudança de paradigma conduziu a uma necessidade continua de redefinir os sinais de alarme para a suspeição de IDP. Além da clássica susceptibilidade infeciosa, as manifestações auto-imunes de início precoce, a poliautoimunidade, a hiper-inflamação ou a infalamação recorrente (sistémica ou específica de órgão), a alergia exuberante (especialmente associada a outras manifestações), e até algumas neoplasias (como a leucemia juvenil mieolomonocítica) podem ser manifestações reveladoras de IDP. Esta tese teve como objectivo revelar novos mecanismos genéticos e/ou funcionais de IDPs. Para isso foram investigados doentes seguidos na Unidade de Imunodeficiências Primárias do Hospital Dona Estefânia, Centro Hospitalar de Lisboa Central, que tivessem uma IDP com sintomas incomuns e fatais ou uma IDP com sintomas atípicos que não permitisse estabelecer um diagnóstico genético definitivo. A investigação destes doentes permitiu adicionar novos fenótipos a IDP previamente conhecidas: hiperplasia nodular regenerativa num doente com imunodeficiência combinada grave causada por uma nova mutação hipomórfica no gene IL2RG; síndrome hemofagocítico secundário a infeções por BCG e Citomegalovirus num doente com deficiência do receptor do IFNγ; doença inflamatória intestinal e um fenótipo assemelhando-se ao sindrome trico-enterohepático causado por uma mutação hipomórfica do gene TTC7A; cutis laxa num doente com síndrome de APLAID causado por uma nova mutação do gene PLCG2. A investigação de um conjunto de doentes com doença inflamatória intestinal de início infantil (IO-IBD) permitiu a identificação de uma causa monogénica para a doença em 54% dos doentes e em muitos desses casos, isto conduziu a alterações terapêuticas. Pudemos descrever uma nova causa de IO-IBD, imunodeficiência e surdez neurossensorial, causada por mutações em STXBP3, que não tinha sido previamente associada a doença humana. Adicionalmente, num doente com um fenótipo IPEX-like, identificamos uma mutação homozigótica do gene ZC3H12A, codificando uma proteína Regnase-1 disfuncional condicionando uma regulação pós-transcricional aberrante de IL-6. Finalmente, numa doente com fasceites graves recorrentes, identificamos uma variante de significado incerto no gene IRG1 e provamos que essa variante conduz a uma alteração do metabolismo de ácido itacónico nos macrófagos, causando incapacidade de detectar adequadamente a resolução da infeção e conduzindo a hiperinflamação.ABSTRACT: Primary immunodeficiencies (PIDs) constitute a heterogeneous group of congenital diseases that affect the development and/or the function of any component of the immune system. As any function of the immune system can be affected, patients with PIDs can present with recurrent infections or a higher specific susceptibility to certain microorganisms, but also with autoimmunity, lymphoproliferation, hyper-inflammation or even cancer. With the massive generalization of the next-generation sequencing (NGS) techniques, the discovery of new PIDs and novel genetic causes for previously known PIDs has been boosted. It is known that the vast majority of PIDs are monogenic, with variable expressivity and often with incomplete penetrance. The recognition that mutations in the same gene can present with several clinical phenotypes, but also that mutations in multiple different genes can manifest with the same phenotype, has made mandatory the use of NGS to elucidate the clinical picture of individual patients or patient groups. By understanding the impact of such nature's experiments on the immune system, it has been possible to expand knowledge, change therapeutic strategies and family counselling. This has led to a continuous change of the warning signs that can reveal PIDs. Adding to the infectious susceptibility, early onset autoimmune manifestations, polyautoimmunity, hyper-inflammation, or recurrent inflammation (systemic or organspecific), exuberant allergy (especially if associated with other manifestations), and even some neoplasms (such as juvenile myelomonocyte leukaemia), could be indications to investigate the presence of a PID. This thesis aimed to reveal new genetic and/or functional mechanisms of PIDs. Patients enrolled in this thesis were followed at the Primary Immunodeficiencies Unit of Hospital Dona Estefânia, Centro Hospitalar de Lisboa Central, having either a known PID that presented fatal and uncommon symptoms or an undiagnosed PID presenting atypical clinical symptoms. The investigation of these patients added new phenotypes to previously described PIDs: nodular regenerative hyperplasia in a patient with leaky X-linked severe combined immunodeficiency caused by a novel hypomorphic mutation in the IL2RG gene; hemophagocytic lymphohistiocytosis secondary to BCG and Cytomegalovirus infections in a patient with IFNγ receptor deficiency; inflammatory bowel disease (IBD) and a phenotype resembling tricho-entero-hepatic syndrome caused by a hypomorphic TTC7A mutation; cutis laxa in an APLAID patient caused by a novel mutation in the PLCG2 gene. The investigation of an Infantile-onset IBD cohort of patients allowed the identification of a monogenic cause for the disease in 54% of the patients and in many cases, this led to changes in the therapeutic approach. We were able to identify a novel cause of IO-IBD, immunodeficiency and sensoryneural immunodeficiency, caused by STXBP3 mutations, not previously associated with human disease. Moreover, in a patient presenting an IPEX-like phenotype we have identified a novel PID caused by homozygous mutations in the ZC3H12A gene, leading to a dysfunctional regnase-1 protein that caused aberrant post-transcriptional regulation of IL-6 production. Finally, in a patient with severe recurrent fasciitis we identified a variant of unknown significance in the IRG1 gene and aimed to prove that the IRG1 variant lead to altered itaconate metabolism in macrophages, thus resulting in inappropriate sensing of the resolution of the infection and hyperinflammation

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis