The use of whole exome sequencing data to identify candidate genes involved in cancer and benign tumour predisposition

The development of whole exome sequencing has transformed the study of disease predisposition. The sequencing of both large disease sets and smaller rare disease families enables the identification of new predisposition variants and potentially provide clinical insight into disease management. There is no standard protocol for analysing exome sequencing data. Outside of extremely large sequencing studies including thousands of individuals, statistical approaches are often underpowered to detect rare disease associated variants. Aggregation of variants into functionally related regions, including genes, gene clusters, and pathways, allows for the detection of biological processes that, when interrupted, may impact disease risk. In silico functional studies can also be utilised to further understand how variants disrupt biological processes and identify genotype-phenotype relationships. This study describes the exploration of sequencing datasets from cancers and benign tumour diseases including: i) hereditary diffuse gastric cancer, ii) sweat duct proliferation tumours, iii) adrenocortical carcinoma, and iv) breast cancer. Each set underwent germline whole exome sequencing followed by additional tumour or targeted sequencing to identify associated predisposition genes. Variants within a cluster of risk genes that are involved in double strand break repair were identified as associated with hereditary diffuse gastric cancer risk via gene ontology enrichment analysis. This cluster included PALB2 within which, using externally collated data, loss of function variants were identified as significantly associated with hereditary diffuse gastric cancer risk. Germline protein-affecting variants in the myosin gene MYH9 were identified in all individuals with a rare sweat duct proliferative syndrome, suggesting a role for MYH9 in skin development, regulation and tumorigenesis. These MYH9 variants were analysed in silico to identify a genotype-phenotype relationship between the clinical presentation and variants in the ATP binding pocket of the protein. Tumour matched normal sequence data from adrenocortical carcinoma cases was used to elucidate the role of Lynch syndrome genes in disease pathogenesis. Within the breast cancer set, candidate genes were selected to undergo targeted sequencing in a larger set of cases to further explore their role in breast cancer risk. Risk associated genes identified within this study may ultimately aid in diagnosis and management of disease. This thesis has also generated multiple novel tools and sequencing analysis techniques that may be of use for further studies by aiding in the prioritisation of candidate variants. The described techniques will provide support to researchers working on rare, statistically underpowered datasets and to provide standard analysis pipelines for a range of dataset sizes and types, including familial data and unrelated individuals

Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants. Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19. Results: We observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions. Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity.</p

Germline pathogenic variants in PALB2 and other cancer-predisposing genes in families with hereditary diffuse gastric cancer without CDH1 mutation: a whole-exome sequencing study.

BACKGROUND: Germline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants. METHODS: We did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer. FINDINGS: Protein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant. INTERPRETATION: The results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families. FUNDING: UK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007-13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene.

BACKGROUND: Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. METHODS: Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. RESULTS: In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7-19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. CONCLUSIONS: This is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families

Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling

IntroductionSARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants.MethodsWe combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19.ResultsWe observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions.DiscussionWe propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity

INVESTIGANDO EL CANCER DE PIEL: NUEVAS HERRAMIENTAS

Las mutaciones ocurren en nuestras c&eacute;lulas durante toda la vida. Aunque la mayor&iacute;a de las mutaciones son inofensivas, se acumulan en n&uacute;mero en nuestros tejidos a medida que envejecemos y, si surgen en genes clave, pueden alterar el comportamiento celular y encaminar a las c&eacute;lulas hacia el c&aacute;ncer. Tambi&eacute;n se especula que las mutaciones som&aacute;ticas (aquellas en tejidos no reproductivos) podr&iacute;an contribuir al envejecimiento y a enfermedades no relacionadas con el c&aacute;ncer. Sin embargo, las dificultades t&eacute;cnicas para detectar las mutaciones presentes en un peque&ntilde;o n&uacute;merode c&eacute;lulas, o incluso en c&eacute;lulas individuales, han obstaculizado la investigaci&oacute;n y limitado el progreso en la comprensi&oacute;n de los primerospasos en el desarrollo del c&aacute;ncer y el impacto de la mutaci&oacute;n som&aacute;tica en el envejecimiento y la enfermedad informan un trabajo que super&oacute;algunas de estas limitaciones experimentales para explorar las mutaciones som&aacute;ticas y la selecci&oacute;n en melanocitos individuales, el tipo de c&eacute;lula de la piel que puede dar lugar al c&aacute;ncer de melanoma

The Contribution of Germline Pathogenic Variants in Breast Cancer Genes to Contralateral Breast Cancer Risk in BRCA1/BRCA2/PALB2-Negative Women.

BACKGROUND: Contralateral breast cancer (CBC) is associated with younger age at first diagnosis, family history and pathogenic germline variants (PGVs) in genes such as BRCA1, BRCA2 and PALB2. However, data regarding genetic factors predisposing to CBC among younger women who are BRCA1/2/PALB2-negative remain limited. METHODS: In this nested case-control study, participants negative for BRCA1/2/PALB2 PGVs were selected from the WECARE Study. The burden of PGVs in established breast cancer risk genes was compared in 357 cases with CBC and 366 matched controls with unilateral breast cancer (UBC). The samples were sequenced in two phases. Whole exome sequencing was used in Group 1, 162 CBC and 172 UBC (mean age at diagnosis: 42 years). A targeted panel of genes was used in Group 2, 195 CBC and 194 UBC (mean age at diagnosis: 50 years). Comparisons of PGVs burdens between CBC and UBC were made in these groups, and additional stratified sub-analysis was performed within each group according to the age at diagnosis and the time from first breast cancer (BC). RESULTS: The PGVs burden in Group 1 was significantly higher in CBC than in UBC (p = 0.002, OR = 2.5, 95CI: 1.2-5.6), driven mainly by variants in CHEK2 and ATM. The proportions of PGVs carriers in CBC and UBC in this group were 14.8% and 5.8%, respectively. There was no significant difference in PGVs burden between CBC and UBC in Group 2 (p = 0.4, OR = 1.4, 95CI: 0.7-2.8), with proportions of carriers being 8.7% and 8.2%, respectively. There was a significant association of PGVs in CBC with younger age. Metanalysis combining both groups confirmed the significant association between the burden of PGVs and the risk of CBC (p = 0.006) with the significance driven by the younger cases (Group 1). CONCLUSION: In younger BRCA1/BRCA2/PALB2-negative women, the aggregated burden of PGVs in breast cancer risk genes was associated with the increased risk of CBC and was inversely proportional to the age at onset.ER

Genomic profiling of acute myeloid leukaemia associated with ataxia telangiectasia identifies a complex karyotype with wild-type TP53 and mutant KRAS, G3BP1 and IL7R.

Ataxia Telangiectasia (A-T) is an autosomal recessive disease, characterised by progressive neurodegeneration with cerebellar ataxia, oculo-cutaneous telangiectasia, immunodeficiency and cancer predisposition. It is caused by biallelic mutations in ATM (Ataxia Telangiectasia Mutated, chromosome 11q22.3) encoding a serine/threonine kinase essential in cell cycle control, DNA double-strand break repair and haematopoietic stem cell maintenance 1,2. Individuals with A-T classically develop lymphoid malignancies, a broad spectrum of other malignancies 3 and rarely, acute myeloid leukaemia (AML) 4-7. Previous AT-AML reports have described the karyotype and dismall clinical course (Supplementray Table S1). We performed sequential sample genomic profiling of an AML that developed in a 17-year-old female with A-T, to identify secondary genetic events towards myeloid malignancy (full clinical case history in Supplementary Information).MDT is funded by the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n.310018. SM is supported by the CCLG and Bloodwise