Germline Level Research Articles

Single cell long read whole genome sequencing reveals somatic transposon activity in human brain.

The advent of single cell DNA sequencing revealed astonishing dynamics of genomic variability, but failed at characterizing smaller to mid size variants that on the germline level have a profound impact. In this work we discover novel dynamics in three brains utilizing single cell long-read sequencing. This provides key insights into the dynamic of the genomes of individual cells and further highlights brain specific activity of transposable elements.

Germline Variants in DNA Interstrand-Cross Link Repair Genes May Contribute to Increased Susceptibility for Serrated Polyposis Syndrome.

Serrated polyposis syndrome (SPS) is characterized by the development of multiple colorectal serrated polyps and increased predisposition to colorectal cancer (CRC). However, the molecular basis of SPS, especially in cases presenting family history of SPS and/or polyps and/or CRC in first-degree relatives (SPS-FHP/CRC), is still poorly understood. In a previous study, we proposed the existence of two molecular entities amongst SPS-FHP/CRC families, proximal/whole-colon and distal SPS-FHP/CRC, according to the preferential location of lesions and somatic events involved in tumor initiation. In the present study, we aimed to investigate these distinct subgroups of SPS patients in a larger cohort at the germline level and to identify the genetic defects underlying an inherited susceptibility for these two entities. Next-generation sequencing was performed using multigene analysis with a custom-designed panel in a Miseq platform in 60 SPS patients (with and without/unknown FHP/CRC). We found germline pathogenic variants in 6/60 patients (ATM, FANCM, MITF, RAD50, RAD51C, and RNF43). We also found variants of unknown significance (VUS), with prediction of probable damaging effect in 23/60 patients (ATM, BLM, BRCA1, FAN1, ERCC2, ERCC3, FANCA, FANCD2, FANCL, MSH2, MSH6, NTHL1, PALB2, PDGFRA, PMS2, PTCH1, RAD51C, RAD51D, RECQL4, TSC2, WRN, and XRCC5 genes). Most variants were detected in gene coding for proteins of the Fanconi Anemia (FA) pathway involved in the DNA Interstrand-Cross Link repair (ICLR). Notably, variants in ICLR genes were significantly more frequent in the proximal/whole-colon than in the distal subgroup [15/44 (34%) vs 1/16 (6%), p = 0.025], as opposed to the non-ICLR genes that were slightly more frequent in the distal group [8/44 (18%) vs. 5/16 (31%), p > 0.05]. Germline defects in the DNA-ICLR genes may contribute to increased serrated colorectal polyps/carcinoma risk in SPS patients, particularly in proximal/whole-colon SPS. The inclusion of DNA-ICLR genes in the genetic diagnosis of SPS patients, mainly in those with proximal/whole-colon lesions, should be considered and validated by other studies. In addition, patients with germline defects in the DNA-ICLR genes may be more sensitive to treatment with platinum-based therapeutics, which can have implications in the clinical management of these patients.

Molecular Pathophysiology of Parathyroid Tumorigenesis-The Lesson from a Rare Disease: The "MEN1 Model".

Primary hyperparathyroidism represents the third most prevalent endocrine disease in the general population, consisting of an excessive secretion of parathyroid hormone from one or, more frequently, more of the parathyroid glands, leading to a dysregulation of calcium homeostasis. Schematically, its development occurs primarily by pathophysiological events with genetic mutation, at the germline and/or somatic level, that favor the neoplastic transformation of parathyroid cells and promote their aberrant proliferation, and mutations determining the shift in the PTH "set-point", thus interfering with the normal pathways of PTH secretion and leading to a "resetting" of Ca2+-dependent PTH secretion or to a secretion of PTH insensitive to changes in extracellular Ca2+ levels. Familial syndromic and non-syndromic forms of primary hyperparathyroidism are responsible for approximately 2-5% of primary hyperparathyroidism cases and most of them are inherited forms. The history of the genetic/molecular studies of parathyroid tumorigenesis associated with multiple endocrine neoplasia type 1 syndrome (MEN1) represents an interesting model to understand genetic-epigenetic-molecular aspects underlying the pathophysiology of primary hyperparathyroidism, both in relation to syndromic and non-syndromic forms. This minireview aims to take a quick and simplified look at the MEN1-associated parathyroid tumorigenesis, focusing on the molecular underlying mechanisms. Clinical, epidemiological, and observational studies, as well as specific guidelines, molecular genetics studies, and reviews, have been considered. Only studies submitted to PubMed in the English language were included, without time constraints.

Nodule-specific NRF2-targeted upregulation in patients with KEAP1 mutations and familial nontoxic multinodular goiter.

Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2 related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole transcript expression array and immunohistochemistry. We found five probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H), which had no meaningful concomitance with mutations of other goiter-associated genes in germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating < 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Atlas of telomeric repeat diversity in Arabidopsis thaliana

BackgroundTelomeric repeat arrays at the ends of chromosomes are highly dynamic in composition, but their repetitive nature and technological limitations have made it difficult to assess their true variation in genome diversity surveys.ResultsWe have comprehensively characterized the sequence variation immediately adjacent to the canonical telomeric repeat arrays at the very ends of chromosomes in 74 genetically diverse Arabidopsis thaliana accessions. We first describe several types of distinct telomeric repeat units and then identify evolutionary processes such as local homogenization and higher-order repeat formation that shape diversity of chromosome ends. By comparing largely isogenic samples, we also determine repeat number variation of the degenerate and variant telomeric repeat array at both the germline and somatic levels. Finally, our analysis of haplotype structure uncovers chromosome end-specific patterns in the distribution of variant telomeric repeats, and their linkage to the more proximal non-coding region.ConclusionsOur findings illustrate the spectrum of telomeric repeat variation at multiple levels in A. thaliana—in germline and soma, across all chromosome ends, and across genetic groups—thereby expanding our knowledge of the evolution of chromosome ends.

Contribution of Helios in Ets1-Deficient NK Cells

Abstract Natural killer (NK) cells mediate anti-viral and anti-cancer immune responses. However, the gene networks that regulate NK cell development and function remain poorly understood. One transcription factor critical for proper NK cell development is Ets1. Previously we showed that loss of Ets1 at the germline level resulted in a developmental block, a functional defect, as well as a phenotype reminiscent of chronic cytokine stimulation in the NK cell compartment. Ets1-deficient NK cells also overexpress Helios, an Ikaros family transcription factor encoded by Ikzf2. Ikaros family transcription factors regulate development in many hematopoietic cell types; therefore, we hypothesized that Helios may be acting downstream of Ets1 to mediate the observed developmental block or functional defects. To investigate this, we generated NK cell-driven conditional deletion of Ets1 only (Ets1 cKO) or Ets1 and Ikzf2 (2xKO). The data presented here confirmed that Helios is highly expressed at the protein level in Ets1 cKO NK cells. Further, overexpression of Helios in Ets1-deficient NK cells does not contribute to the block in maturation nor the cytokine production defect. However, activated NK cells show high Helios expression, suggesting that Helios expression in Ets1-/- NK cells may be a consequence of the chronic cytokine stimulation phenotype.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract

Co-Occurrence of Beckwith–Wiedemann Syndrome and Early-Onset Colorectal Cancer

CRC is an adult-onset carcinoma representing the third most common cancer and the second leading cause of cancer-related deaths in the world. EO-CRC (<45 years of age) accounts for 5% of the CRC cases and is associated with cancer-predisposing genetic factors in half of them. Here, we describe the case of a woman affected by BWSp who developed EO-CRC at age 27. To look for a possible molecular link between BWSp and EO-CRC, we analysed her whole-genome genetic and epigenetic profiles in blood, and peri-neoplastic and neoplastic colon tissues. The results revealed a general instability of the tumor genome, including copy number and methylation changes affecting genes of the WNT signaling pathway, CRC biomarkers and imprinted loci. At the germline level, two missense mutations predicted to be likely pathogenic were found in compound heterozygosity affecting the Cystic Fibrosis (CF) gene CFTR that has been recently classified as a tumor suppressor gene, whose dysregulation represents a severe risk factor for developing CRC. We also detected constitutional loss of methylation of the KCNQ1OT1:TSS-DMR that leads to bi-allelic expression of the lncRNA KCNQ1OT1 and BWSp. Our results support the hypothesis that the inherited CFTR mutations, together with constitutional loss of methylation of the KCNQ1OT1:TSS-DMR, initiate the tumorigenesis process. Further somatic genetic and epigenetic changes enhancing the activation of the WNT/beta-catenin pathway likely contributed to increase the growth advantage of cancer cells. Although this study does not provide any conclusive cause-effect relationship between BWSp and CRC, it is tempting to speculate that the imprinting defect of BWSp might accelerate tumorigenesis in adult cancer in the presence of predisposing genetic variants.

Molecular Genetic Characteristics of FANCI, a Proposed New Ovarian Cancer Predisposing Gene.

FANCI was recently identified as a new candidate ovarian cancer (OC)-predisposing gene from the genetic analysis of carriers of FANCI c.1813C>T; p.L605F in OC families. Here, we aimed to investigate the molecular genetic characteristics of FANCI, as they have not been described in the context of cancer. We first investigated the germline genetic landscape of two sisters with OC from the discovery FANCI c.1813C>T; p.L605F family (F1528) to re-affirm the plausibility of this candidate. As we did not find other conclusive candidates, we then performed a candidate gene approach to identify other candidate variants in genes involved in the FANCI protein interactome in OC families negative for pathogenic variants in BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and FANCI, which identified four candidate variants. We then investigated FANCI in high-grade serous ovarian carcinoma (HGSC) from FANCI c.1813C>T carriers and found evidence of loss of the wild-type allele in tumour DNA from some of these cases. The somatic genetic landscape of OC tumours from FANCI c.1813C>T carriers was investigated for mutations in selected genes, copy number alterations, and mutational signatures, which determined that the profiles of tumours from carriers were characteristic of features exhibited by HGSC cases. As other OC-predisposing genes such as BRCA1 and BRCA2 are known to increase the risk of other cancers including breast cancer, we investigated the carrier frequency of germline FANCI c.1813C>T in various cancer types and found overall more carriers among cancer cases compared to cancer-free controls (p = 0.007). In these different tumour types, we also identified a spectrum of somatic variants in FANCI that were not restricted to any specific region within the gene. Collectively, these findings expand on the characteristics described for OC cases carrying FANCI c.1813C>T; p.L605F and suggest the possible involvement of FANCI in other cancer types at the germline and/or somatic level.

Chickens, more than humans, focus the diversity of their immunoglobulin genes on the complementarity-determining region but utilise amino acids, indicative of a more cross-reactive antibody repertoire.

The mechanisms of B-cell diversification differ greatly between aves and mammals, but both produce B cells and antibodies capable of supporting an effective immune response. To see how differences in the generation of diversity might affect overall repertoire diversity, we have compared the diversity characteristics of immunoglobulin genes from domestic chickens to those from humans. Both use V(D)J gene rearrangement and somatic hypermutation, but only chickens use somatic gene conversion. A range of diversity analysis tools were used to investigate multiple aspects of amino acid diversity at both the germline and repertoire levels. The effect of differing amino acid usages on antibody characteristics was assessed. At both the germline and repertoire levels, chickens exhibited lower amino acid diversity in comparison to the human immunoglobulin genes, especially outside of the complementarity-determining region (CDR). Chickens were also found to possess much larger and more hydrophilic CDR3s with a higher predicted protein binding potential, suggesting that the antigen-binding site in chicken antibodies is more flexible and more polyreactive than that seen in human antibodies.

Germline-Encoded Positional Cysteine Polymorphisms Enhance Diversity in Antibody Ultralong CDR H3 Regions.

Ab "ultralong" third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the "knob" domain of the CDR H3, which is encoded by the DH gene segment and sits atop a β-ribbon "stalk" that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.

Genetic drivers of Cushing’s disease: Frequency and associated phenotypes

Cushing's disease (CD) is often explained by a single somatic sequence change. Germline defects, however, often go unrecognized. We aimed to determine the frequency and associated phenotypes of genetic drivers of CD in a large cohort. We studied 245 unrelated patients with CD (139 female, 56.7%), including 230 (93.9%) pediatric and 15 (6.1%) adult patients. Germline exome sequencing was performed in 184 patients; tumor exome sequencing was also done in 27 of them. A total of 43 germline samples and 92 tumor samples underwent Sanger sequencing of specific genes. Rare variants of uncertain significance, likely pathogenic (LP), or pathogenic variants in CD-associated genes, were identified. Germline variants (13 variants of uncertain significance, 8 LP, and 11 pathogenic) were found in 8 of 19 patients (42.1%) with positive family history and in 23 of 226 sporadic patients (10.2%). Somatic variants (1 LP and 7 pathogenic) were found in 20 of 119 tested individuals (16.8%); one of them had a coexistent germline defect. Altogether, variants of interest were identified at the germline level in 12.2% of patients, at the somatic level in 7.8%, and coexisting germline and somatic variants in 0.4%, accounting for one-fifth of the cohort. We report an estimate of the contribution of multiple germline and somatic genetic defects underlying CD in a single cohort.

Investigating the role of somatic sequencing platforms for phaeochromocytoma and paraganglioma in a large UK cohort.

ObjectivesPhaeochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumours with malignant potential and a hereditary basis in almost 40% of patients. Germline genetic testing has transformed the management of PPGL enabling stratification of surveillance approaches, earlier diagnosis and predictive testing of at‐risk family members. Recent studies have identified somatic mutations in a further subset of patients, indicating that molecular drivers at either a germline or tumour level can be identified in up to 80% of PPGL cases. The aim of this study was to investigate the clinical utility of somatic sequencing in a large cohort of patients with PPGL in the United Kingdom.Design and PatientsProspectively collected matched germline and tumour samples (development cohort) and retrospectively collected tumour samples (validation cohort) of patients with PPGL were investigated.MeasurementsClinical characteristics of patients were assessed and tumour and germline DNA was analysed using a next‐generation sequencing strategy. A screen for variants within ‘mutation hotspots’ in 68 human cancer genes was performed.ResultsOf 141 included patients, 45 (32%) had a germline mutation. In 37 (26%) patients one or more driver somatic variants were identified including 26 likely pathogenic or pathogenic variants and 19 variants of uncertain significance. Pathogenic somatic variants, observed in 25 (18%) patients, were most commonly identified in the VHL, NF1, HRAS and RET genes. Pathogenic somatic variants were almost exclusively identified in patients without a germline mutation (all but one), suggesting that somatic sequencing is likely to be most informative for those patients with negative germline genetic test results.ConclusionsSomatic sequencing may further stratify surveillance approaches for patients without a germline genetic driver and may also inform targeted therapeutic strategies for patients with metastatic disease.

Abstract 863: Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer

Abstract Introduction: In experimental and epidemiological studies, alterations in several core circadian genes at the germline and tumor level have been associated with prostate cancer. The aim of this study was to investigate mRNA expression of circadian related genes in men with metastatic castration-resistant prostate cancer (mCRPC), and the association with survival. Methods: We assessed whole exome and RNA sequencing data from 317 mCRPC patients from the Stand Up to Cancer-Prostate Cancer Foundation (SU2C-PCF) database. Data were obtained from six sites: metastasis to bone (n=107; n=65 deaths), lymph node (n=129; n=88 deaths), liver (n=42; n=35 deaths), lung (n=6; n=3 deaths), and other soft tissue (n=26; n=20 deaths), as well as primary prostate (n=7; n=5 deaths) over a median follow-up of 71.8 months. We evaluated expression of twelve core circadian genes (ARNTL, CLOCK, CRY1, CRY2, CSNK1E, NR1D1, NPAS2, PER1, PER2, PER3, RORA, TIMELESS) as transcripts per million (TPM). We used the correlation of correlations method to estimate inter-gene correlations between tissue. Unpaired Wilcoxon rank sum tests compared circadian expression differences with tumor mutations in AR and p53, two of the most common genomic alterations in mCRPC. We conducted multivariable Cox regression, overall and stratified by tissue type, to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) for expression (modeled continuously) and overall survival, adjusted for age and PSA at diagnosis, Gleason, treatment, and histology. Results: Many genes showed low or negative correlation across tissues, with the greatest discordance in CSNK1E (μICC=0.10), and greatest concordance in TIMELESS (μICC=0.55). Lower expression of ARNTL was found in patients with alterations to both p53 and AR. Similarly, higher expression of PER2 and RORA was found in AR-/p53+, compared to those AR+ and AR-/p53-. Higher expression of TIMELESS was associated with risk of death overall and across all tissue sites (HRoverall: 1.02, 95% CI: 1.01-1.03). In liver, higher expression of CLOCK (HR: 0.22, 95% CI: 0.07 - 0.71) and CSNK1E (HR: 0.87, 95% CI: 0.76 - 1.00), and lower expression of CRY1 (HR: 1.62, 95% CI: 1.16 - 2.26) was associated with a lower risk of death. Higher expression of CRY2 (HR: 1.25, 95% CI: 1.02 - 1.53) in liver, but lower expression in bone (HR: 0.95, 95% CI: 0.90 - 1.00) was associated with an increased risk of death. We found no association between ARNTL, NR1D1, NPAS2, PER3, or RORA and survival in any metastatic site. Conclusion: Our results show that circadian gene expression is altered in tissue from mCRPC patients, with substantial heterogeneity in circadian related expression patterns between metastatic tissue sites. These results support prior research on the role of circadian gene expression, particularly CRY1 and CLOCK, and outcomes in localized prostate cancer. Citation Format: Benjamin D. Booker, Konrad H. Stopsack, Travis A. Gerke, Kathryn Penny, Philip W. Kantoff, Lorelei A. Mucci, Sarah C. Markt, PCF/SU2C International Prostate Cancer Dream Team. Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 863.

Expression of Functional Human Proteinase Activated Receptor (PAR)-1 on Mouse Platelets

Thrombin activates platelets by specific cleavage of PARs leading to platelet aggregation. Human (h) platelets express PAR-1 and PAR-4, each with a different sensitivity to thrombin and specific kinetics of activation/deactivation influencing thrombogenesis and thrombus stability. PAR-1 and PAR-4 are candidates for the generation of anti-thrombotic drugs; indeed, a PAR-1 inhibitor (Vorapaxar) is already in clinical use. However, side effects such as bleeding warrant a continuous effort towards the generation of novel alternative inhibitors. In this regard, human PAR-4 may be a more selective antithrombotic target with a lesser impact on hemostasis. Mice are commonly used to test anti-thrombotic drugs and their mechanisms of action, but cannot be used in this instance because mouse (m) platelets express different PAR subtypes than human, i.e. PAR-3 and PAR-4. Moreover, mPAR-3 cannot be cleaved by thrombin and only acts as a co-receptor enhancing mPAR-4 response. In human platelets, PAR-1 and PAR-4 can act independently of one another. Yet, a different platelet membrane receptor, unrelated to PARs, which binds thrombin with high affinity - glycoprotein (GP) Ibα in the GPIb-IX-V complex - enhances PAR-1 response to low-dose thrombin through a mechanism only partially elucidated. Humanizing mouse platelets with respect to PAR expression would greatly facilitate explaining the functional interplay of GPIb with PAR-1 and PAR-4 and, notably, the role of these different PARs in hemostasis and thrombosis. Attempts to introduce hPAR-1 in mouse platelets have been so far unsuccessful (French et al. 2016, Arachiche et al. 2014) and claims have been made that obstacles to achieve this goal may be unsurpassable. Using a different approach, we introduced a floxed hPAR-1 transgene in the ROSA26 locus of C57BL6/J mice under the strong CAG promoter. The presence of an IRES sequence and a second transgene for eGFP allowed us to screen efficiently for platelets expressing hPAR1. Mice positive for the floxed-transgene were then bred with either a germ-line Cre recombinase-expressing strain (EIIa-Cre) or one in which Cre expression was linked to a platelet specific receptor (PF4-Cre). The percentage of platelets isolated from EIIa-Cre/PAR1+/- mice expressing eGFP was between 35 to 40% and breeding to homozygosity did not increase transgene expression. In contrast, eGFP expression was positive in nearly all PF4-Cre/PAR1+/-mice, suggesting that suppression of hPAR1 expression at the germ-line level could be advantageous for megakaryocyte development. As a functional test for transgene-expressed hPAR-1 we measured aggregation and intracellular calcium increase in washed platelets from PF4-Cre/PAR1+/+ mice stimulated by 5 μmol/liter of a specific PAR-1 activation peptide (TFLLRN, P1-AP). In both assays, the response of human or hPAR-1 expressing mouse platelets was indistinguishable; littermate control platelets lacking transgene expression (floxed hPAR1) did not respond to a 10 times higher concentration of peptide. Interestingly, when stimulated with a low dose of thrombin (0.25 nM) aggregation and intracellular calcium increase of PF4-Cre/PAR1+/+ platelets was similar to littermate controls. We reasoned that this could be a consequence of the presence of mPAR3, owning to its high affinity for thrombin. Thus we proceeded to cross PF4-Cre/PAR1+/+ with mPAR3-/- mice, so to obtain mice that express only mPAR4 and hPAR1. Aggregation induced with P1-AP (5 μM) was similar with platelets expressing PF4-Cre/PAR1+/+ and mPAR3-/-/PF4-Cre/PAR1+/+. As expected from the cofactor role of mPAR3, the minimal amount of thrombin necessary to aggregate fully platelets from mPAR3-/-/PF4-Cre/PAR1-/- was higher than in wild-type mice (2 nM vs 0.25 nM); however, 2 nM was also the minimum thrombin concentration necessary to activate mPAR3-/-/PF4-Cre/PAR1+/+ platelets. Therefore, the presence of a functional hPAR1 responsive to P1-AP does not influence the response of mouse platelets to low thrombin concentrations mediated by mPAR4, indicating that hPAR-1 and mPAR-4, like the human homologue, respond to thrombin independently of one another. In conclusion, to date we have shown that expression of a functional hPAR1 in mouse platelets is possible and should provide a versatile animal model to address several open questions on the role of platelet PAR-1 and GPIbα in hemostasis and thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Dual RNAseq analyses at soma and germline levels reveal evolutionary innovations in the elephantiasis-agent Brugia malayi, and adaptation of its Wolbachia endosymbionts.

Brugia malayi is a human filarial nematode responsible for elephantiasis, a debilitating condition that is part of a broader spectrum of diseases called filariasis, including lymphatic filariasis and river blindness. Almost all filarial nematode species infecting humans live in mutualism with Wolbachia endosymbionts, present in somatic hypodermal tissues but also in the female germline which ensures their vertical transmission to the nematode progeny. These α-proteobacteria potentially provision their host with essential metabolites and protect the parasite against the vertebrate immune response. In the absence of Wolbachia wBm, B. malayi females become sterile, and the filarial nematode lifespan is greatly reduced. In order to better comprehend this symbiosis, we investigated the adaptation of wBm to the host nematode soma and germline, and we characterized these cellular environments to highlight their specificities. Dual RNAseq experiments were performed at the tissue-specific and ovarian developmental stage levels, reaching the resolution of the germline mitotic proliferation and meiotic differentiation stages. We found that most wBm genes, including putative effectors, are not differentially regulated between infected tissues. However, two wBm genes involved in stress responses are upregulated in the hypodermal chords compared to the germline, indicating that this somatic tissue represents a harsh environment to which wBm have adapted. A comparison of the B. malayi and C. elegans germline transcriptomes reveals a poor conservation of genes involved in the production of oocytes, with the filarial germline proliferative zone relying on a majority of genes absent from C. elegans. The first orthology map of the B. malayi genome presented here, together with tissue-specific expression enrichment analyses, indicate that the early steps of oogenesis are a developmental process involving genes specific to filarial nematodes, that likely result from evolutionary innovations supporting the filarial parasitic lifestyle.

Homologous recombination deficiency in breast cancer: Implications for risk, cancer development, and therapy.

An underlying cause of breast cancers has been largely attributed to defects in the DNA damage response (DDR) pathway. In particular, the homologous recombination (HR) pathway repairs double-stranded breaks (DSBs) in DNA, ultimately protecting the cell from genomic instability and thus preventing the accumulation of transforming mutations. In line with this, mutations in a number of genes encoding HR proteins are a well-studied cause of HR deficiency (HRD), and, at the germline level, can confer risk to breast cancer but also occur somatically, contributing to sporadic breast cancer development, progression and response to therapy. Our understanding of the biological processes involved in HR and how these become compromised during breast cancer development has led to a better understanding of how HRD cells can be targeted with specific DNA damaging agents and/or with synthetic lethal targeting approaches such as PARP inhibition. Additionally, in vitro and preclinical modeling has supported the development of clinical trials to assess targeted therapies such as PARP inhibitors (PARPis), ultimately leading to development of therapies with greater clinical benefit. A number of challenges have been encountered, including resistance to therapy; however, addressing these challenges head-on and continually driving scientific research and clinical trials with innovative therapies will contribute to our ability to target HRD in breast cancers. Ongoing research efforts into HRD in breast cancer development are therefore essential, even in the era of targeted therapies, to provide innovative strategies for improved tumor responses.

1897P Germline and somatic mutational landscape in a cohort of malignant pleural mesothelioma patients

Germline mutations in BAP1 and in other genes, such as BRCA2, CHEK2, CDKN2A or ATM, have been associated to hereditary predisposition to malignant pleural mesothelioma (MPM). We aim to characterize the genomic landscape in MPM patients at germline and somatic level.

Abstract 5446: Genetic bases of testicular seminoma and non-seminoma tumors

Abstract Testicular Germ Cell Tumor (TGCT) is the most common cancer type in men between 15-45 years old (1 per 250 men). Around 1-2% of all cases are familial (with at least two affected individuals within the same family), between 3-5% are bilateral (they develop two tumors, one in each testicle), and the rest are sporadic patients. There are two main types of histologies, seminomas and non-seminomas, and there are also tumors that contain both seminoma and non-seminoma cells, and are called mixed tumors. No great genetic differences have been found so far between them. In order to identify susceptibility genes that could explain these histological differences, we performed Whole Exome Sequencing (WES) in peripheral blood of 61 members from 17 families with at least two affected members with TGCT. Twenty one members had seminoma, seven members had non-seminomas, and nine had mixed tumor. We did not find significant genetic differences between the two histological types at the germline level. Therefore, an evaluation at a somatic level was carried out to determine if the differences between the tumor types are in the tumor. To this end, WES of blood and tumor tissue was performed in three bilateral cases with different histologies (a seminoma in one testicle and a non-seminoma in the other). The comparison between pairs of tumors from each of the patients showed that they shared in average a 30% of the somatic mutations between both the histologies. Even more, 27 genes were shared among the two tumors in the three patients. We next performed independency tests, comparing the somatic similarities and differences between the two tumors from each patient (intrapatient) and the two histologies (interpatients), which suggest a common genetic background that could predispose to testicular cancer development. We are currently performing gene ontology analysis with the genes that were shared among the pairs of tumors from each patient. Partially supported by CIBERER and FIS 16/00440 Citation Format: Paloma Martin-Gimeno, Beatriz Paumard-Hernandez, Oriol Calvete, Javier Benitez. Genetic bases of testicular seminoma and non-seminoma tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5446.

MET alterations in biphasic squamoid alveolar papillary renal cell carcinomas and clinicopathological features.

Biphasic squamoid alveolar papillary renal cell carcinoma (BSA-PRCC) is a recently studied lesion considered a morphologic variant of papillary renal cell carcinoma (RCC), more closely related to type 1. Considering the role of proto-oncogene MET in both sporadic type 1 papillary RCC and hereditary papillary RCC, we aimed to explore the role of MET activation in the oncogenesis of BSA-PRCC. We identified 17 patients with either unique (n = 14) or multiple (n = 3) BSA-PRCC, all localized, and performed an integrative analysis of MET status in 18 formalin-fixed paraffin-embedded tumors combining next-generation sequencing analysis, fluorescent in situ hybridization and immunohistochemistry. Trisomy 7 was found in 86% of tumors (14/16) without MET amplification at 7q31 (15/15). A pathogenic MET genetic variant was identified in 60% (9/15) of cases, at the germline level in 57% (4/7) of tested patients or at the somatic level (5/11). MET expression was observed in all tumors with a higher value of combined score in large cells (mean 97%, range 80-100%) than in small cells (mean 74%, range 10-100%) and was lower in two cases without MET copy number gain. In conclusion, our study provides additional evidence to consider biphasic squamoid alveolar papillary RCC as a morphological variant of type 1 papillary renal RCC. Our data strongly suggest that MET represents a major oncogenic driver gene in BSA-PRCC, harboring a higher frequency of MET mutation that encourages to further explore the benefice of anti-MET targeted therapies for aggressive BSA-PRCC.