Publications by authors named "Eleni Giannoulatou"

74 Publications

Whole genome sequencing in transposition of the great arteries and associations with clinically relevant heart, brain and laterality genes.

Am Heart J 2022 Feb 17;244:1-13. Epub 2021 Oct 17.

Heart Centre for Children, The Children's Hospital at Westmead, Sydney, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia; Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia; St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, Australia; Cincinnati Children's Hospital Medical Center, Heart Institute, Cardiothoracic Surgery, Cincinnati, OH. Electronic address:

Background: The most common cyanotic congenital heart disease (CHD) requiring management as a neonate is transposition of great arteries (TGA). Clinically, up to 50% of TGA patients develop some form of neurodevelopmental disability (NDD), thought to have a significant genetic component. A "ciliopathy" and links with laterality disorders have been proposed. This first report of whole genome sequencing in TGA, sought to identify clinically relevant variants contributing to heart, brain and laterality defects.

Methods: Initial whole genome sequencing analyses on 100 TGA patients focussed on established disease genes related to CHD (n = 107), NDD (n = 659) and heterotaxy (n = 74). Single variant as well as copy number variant analyses were conducted. Variant pathogenicity was assessed using the American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines.

Results: Fifty-five putatively damaging variants were identified in established disease genes associated with CHD, NDD and heterotaxy; however, no clinically relevant variants could be attributed to disease. Notably, case-control analyses identified significantly more predicted-damaging, silent and total variants in TGA cases than healthy controls in established CHD genes (P < .001), NDD genes (P < .001) as well as across the three gene panels (P < .001).

Conclusion: We present compelling evidence that the majority of TGA is not caused by monogenic rare variants and is most likely oligogenic and/or polygenic in nature, highlighting the complex genetic architecture and multifactorial influences on this CHD sub-type and its long-term sequelae. Assessment of variant burden in key heart, brain and/or laterality genes may be required to unravel the genetic contributions to TGA and related disabilities.
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http://dx.doi.org/10.1016/j.ahj.2021.10.185DOI Listing
February 2022

Tumor Genotyping and Homologous Recombination Repair Gene Variants in Patients With Epithelial Ovarian Cancer: Is Pathogenic Enough?

Front Oncol 2021 1;11:683057. Epub 2021 Jun 1.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece.

Our hypothesis was that the predictive accuracy of pathogenic variants in genes participating in the homologous recombination repair (HRR) system in patients with epithelial ovarian cancer (EOC) could be improved by considering additional next-generation sequencing (NGS) metrics. NGS genotyping was performed in tumor tissue, retrospectively and prospectively collected from patients with EOC, diagnosed from 8/1998 to 10/2016. Variants were considered clonal when variant allele frequencies corresponded to >25%. The primary endpoint was overall survival (OS). This study included 501 patients with EOC, predominantly with high-grade serous (75.2%) and advanced stage tumors (81.7%); median age was 58 years (22-84). Pathogenic and clonal pathogenic variants in HRR and/or TP53 genes were identified in 72.8% and 66.5% tumors, respectively. With a median follow-up of 123.9 months, the presence of either pathogenic or clonal pathogenic HRR-only variants was associated with longer OS compared to HRR/ co-mutation (HR=0.54; 95% CI, 0.34-0.87, Wald's p=0.012 and HR=0.45; 95% CI, 0.27-0.78, Wald's p=0.004, respectively). However, only the presence of clonal HRR-only variants was independently associated with improved OS (HR=0.55; 95% CI, 0.32-0.94, p=0.030). Variant clonality and co-occuring TP53 variants affect the predictive value of HRR pathogenic variants for platinum agents in patients with EOC.

Clinical Trial Registration: [ClinicalTrials.gov], identifier [NCT04716374].
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http://dx.doi.org/10.3389/fonc.2021.683057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204021PMC
June 2021

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Nat Commun 2021 06 8;12(1):3447. Epub 2021 Jun 8.

Department of Physiology, Anatomy and Genetics, BHF Centre of Research Excellence, University of Oxford, Oxford, UK.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.
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http://dx.doi.org/10.1038/s41467-021-23660-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187484PMC
June 2021

Genotyping data of routinely processed matched primary/metastatic tumor samples.

Data Brief 2021 Feb 11;34:106646. Epub 2020 Dec 11.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece.

Genotypic and phenotypic comparisons of tumors in multiple tissue samples from the same patient are important for understanding disease evolution and treatment possibilities. Panel NGS genotyping is currently widely used in this context, whereby NGS variant filtering and final evaluation constitute the basis for meaningful comparisons. Here, we present the genotype data used for genotype / phenotype comparisons between matched primary / metastatic colorectal tumors in the work by Chatzopoulos et al (doi: 10.1016/j.humpath.2020.10.009), as well as the process followed for obtaining these data. We describe key issues while processing routinely formalin-fixed paraffin-embedded (FFPE) tumors for genotyping, NGS application (Ion Torrent), a stringent variant filtering algorithm for genotype analyses in FFPE tissues and particularly in matched tumor samples, and provide the respective datasets. Apart from research, tumor NGS genotyping is currently applied for clinical diagnostic purposes in Oncology. The datasets and method description provided herein (a) are important for comprehending the peculiarities of FFPE tumor genotyping, which is still mostly based on principles of germline DNA genotyping; (b) can be used in pooled analyses, e.g., of primary / metastatic tumors for the investigation of tumor evolution.
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http://dx.doi.org/10.1016/j.dib.2020.106646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749371PMC
February 2021

Dynamics of Transforming Growth Factor (TGF)-β Superfamily Cytokine Induction During HIV-1 Infection Are Distinct From Other Innate Cytokines.

Front Immunol 2020 24;11:596841. Epub 2020 Nov 24.

Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.

Human immunodeficiency virus type 1 (HIV-1) infection triggers rapid induction of multiple innate cytokines including type I interferons, which play important roles in viral control and disease pathogenesis. The transforming growth factor (TGF)-β superfamily is a pleiotropic innate cytokine family, some members of which (activins and bone morphogenetic proteins (BMPs)) were recently demonstrated to exert antiviral activity against Zika and hepatitis B and C viruses but are poorly studied in HIV-1 infection. Here, we show that TGF-β is systemically induced with very rapid kinetics (as early as 1-4 days after viremic spread begins) in acute HIV-1 infection, likely due to release from platelets, and remains upregulated throughout infection. Contrastingly, no substantial systemic upregulation of activins A and B or BMP-2 was observed during acute infection, although plasma activin levels trended to be elevated during chronic infection. HIV-1 triggered production of type I interferons but not TGF-β superfamily cytokines from plasmacytoid dendritic cells (DCs) , putatively explaining their differing induction; whilst lipopolysaccharide (but not HIV-1) elicited activin A production from myeloid DCs. These findings underscore the need for better definition of the protective and pathogenic capacity of TGF-β superfamily cytokines, to enable appropriate modulation for therapeutic purposes.
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http://dx.doi.org/10.3389/fimmu.2020.596841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732468PMC
June 2021

Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.

Hum Mol Genet 2020 12;29(22):3662-3678

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON M5G1X3, Canada.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.
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http://dx.doi.org/10.1093/hmg/ddaa258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823106PMC
December 2020

Genotype-phenotype associations in colorectal adenocarcinomas and their matched metastases.

Hum Pathol 2021 01 5;107:104-116. Epub 2020 Nov 5.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece; Aristotle University of Thessaloniki, Thessaloniki, Greece; German Oncology Center, Limassol, Cyprus.

Although primary colorectal carcinomas (CRCs) frequently share genetic alterations with their metastases, morphologic surrogates reflecting the genotype contexture of metastases remain largely unknown. We investigated phenotype/genotype associations in paired primary and metastatic colorectal adenocarcinomas from 75 patients. Thirty-three (44%) metastatic lesions were synchronous and 42 (56%) were metachronous. Tumor budding, micronecrosis, and tumor-infiltrating lymphocyte (TIL) density were compared with matched next-generation sequencing genotypes. Micronecrosis in the primary were significantly associated with nodal status (P = 0.0054) and with micronecrosis in metastatic sites (P = 0.0216), particularly in metachronous metastases (P = 0.0033). With a 57-gene panel, one or more mutations were identified in 64 (85.3%) cases. In metastases, high (brisk) TILs were associated with overall mutational burden (P = 0.0058) and with mutations in EGF (P = 0.0325), RAS genes (P = 0.0043), and MMR genes (P = 0.0069), whereas high-level micronecrosis correlated with mutations in APC (P = 0.0004) and MSH6 (P = 0.0385) genes. Genomic alterations were shared in 90.1% of primary/metastatic pairs, but clonality of the same mutation was shared in only 57.1% of paired lesions. Compared with synchronous, metachronous metastases had more private clonal alterations (P = 0.0291); in this group, clonal alterations coincided with brisk TILs (P = 0.0334) and high micronecrosis (P = 0.0133). High TILs in metastatic lesions were predictive of favorable overall survival (log-rank P = 0.044). The observed phenotype/genotype associations favor the clonal evolution model in CRC metastases that seems accompanied by intense host immune response. If the role of micronecrosis and brisk TILs in metachronous metastases is validated in larger studies, these histologic parameters will be worth adding in the armamentarium for the evaluation of metastatic CRC.
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http://dx.doi.org/10.1016/j.humpath.2020.10.009DOI Listing
January 2021

Spontaneous Coronary Artery Dissection: Insights on Rare Genetic Variation From Genome Sequencing.

Circ Genom Precis Med 2020 12 30;13(6):e003030. Epub 2020 Oct 30.

Department of Cardiovascular Sciences and NIHR Leicester Biomedical Research Centre, University of Leicester, United Kingdom (A.A.B., T.R.W., S.E.H., D.P., A.A.-H., A.W., D.K., N.J.S., D.A.).

Background: Spontaneous coronary artery dissection (SCAD) occurs when an epicardial coronary artery is narrowed or occluded by an intramural hematoma. SCAD mainly affects women and is associated with pregnancy and systemic arteriopathies, particularly fibromuscular dysplasia. Variants in several genes, such as those causing connective tissue disorders, have been implicated; however, the genetic architecture is poorly understood. Here, we aim to better understand the diagnostic yield of rare variant genetic testing among a cohort of SCAD survivors and to identify genes or gene sets that have a significant enrichment of rare variants.

Methods: We sequenced a cohort of 384 SCAD survivors from the United Kingdom, alongside 13 722 UK Biobank controls and a validation cohort of 92 SCAD survivors. We performed a research diagnostic screen for pathogenic variants and exome-wide and gene-set rare variant collapsing analyses.

Results: The majority of patients within both cohorts are female, 29% of the study cohort and 14% validation cohort have a remote arteriopathy. Four cases across the 2 cohorts had a diagnosed connective tissue disorder. We identified pathogenic or likely pathogenic variants in 7 genes (, , , , , , and ) in 14/384 cases in the study cohort and in 1/92 cases in the validation cohort. In our rare variant collapsing analysis, was the highest-ranked gene, and several functionally plausible genes were enriched for rare variants, although no gene achieved study-wide statistical significance. Gene-set enrichment analysis suggested a role for additional genes involved in renal function.

Conclusions: By studying the largest sequenced cohort of SCAD survivors, we demonstrate that, based on current knowledge, only a small proportion have a pathogenic variant that could explain their disease. Our findings strengthen the overlap between SCAD and renal and connective tissue disorders, and we highlight several new genes for future validation.
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http://dx.doi.org/10.1161/CIRCGEN.120.003030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7748045PMC
December 2020

Transposon clusters as substrates for aberrant splice-site activation.

RNA Biol 2021 03 23;18(3):354-367. Epub 2020 Sep 23.

School of Medicine, University of Southampton, Southampton, UK.

Transposed elements (TEs) have dramatically shaped evolution of the exon-intron structure and significantly contributed to morbidity, but how recent TE invasions into older TEs cooperate in generating new coding sequences is poorly understood. Employing an updated repository of new exon-intron boundaries induced by pathogenic mutations, termed DBASS, here we identify novel TE clusters that facilitated exon selection. To explore the extent to which such TE exons maintain RNA secondary structure of their progenitors, we carried out structural studies with a composite exon that was derived from a long terminal repeat (LTR78) and J and was activated by a C > T mutation optimizing the 5' splice site. Using a combination of SHAPE, DMS and enzymatic probing, we show that the disease-causing mutation disrupted a conserved J stem that evolved from helix 3.3 (or 5b) of 7SL RNA, liberating a primordial GC 5' splice site from the paired conformation for interactions with the spliceosome. The mutation also reduced flexibility of conserved residues in adjacent exon-derived loops of the central hairpin, revealing a cross-talk between traditional and auxilliary splicing motifs that evolved from opposite termini of 7SL RNA and were approximated by Watson-Crick base-pairing already in organisms without spliceosomal introns. We also identify existing exons activated by the same RNA rearrangement. Collectively, these results provide valuable TE exon models for studying formation and kinetics of pre-mRNA building blocks required for splice-site selection and will be useful for fine-tuning auxilliary splicing motifs and exon and intron size constraints that govern aberrant splice-site activation.
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http://dx.doi.org/10.1080/15476286.2020.1805909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951965PMC
March 2021

Spontaneous Coronary Artery Dissection and Fibromuscular Dysplasia: Vasculopathies With a Predilection for Women.

Heart Lung Circ 2021 Jan 6;30(1):27-35. Epub 2020 Jul 6.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia; St Vincent's Clinical School, St Vincent's Hospital, Sydney, NSW, Australia; Faculty of Medicine, University of NSW, Sydney, NSW, Australia. Electronic address:

The burden of cardiovascular disease in women is being increasingly appreciated. Nevertheless, both clinicians and the general public are largely unaware that cardiovascular disease is the leading cause of death worldwide in women in all countries and that outcomes after a heart attack are worse for women than men. Of note, certain types of cardiovascular disease have a predilection for women, including spontaneous coronary artery dissection (SCAD) and fibromuscular dysplasia (FMD). Although uncommon, SCAD is being increasingly recognised as the cause of an acute coronary syndrome (ACS) and can recur. It is a potentially fatal, under-diagnosed condition that affects relatively young women, who often have few traditional risk factors, and is the commonest cause of a myocardial infarction associated with pregnancy. In contrast, FMD often remains silent but when manifested can also cause major sequelae, including renal infarction, stroke, cervical artery dissection and gut infarction. Here we provide an update on the diagnosis, aetiology and management of these important disorders that overwhelmingly affect women.
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http://dx.doi.org/10.1016/j.hlc.2020.05.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710561PMC
January 2021

Prognostic Biomarkers in Early-stage Gastric Adenocarcinoma Treated With Adjuvant Chemoradiotherapy.

Cancer Genomics Proteomics 2020 May-Jun;17(3):277-290

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece.

Background/aim: Early-stage gastric cancer has a high risk of recurrence, despite trimodality therapy with surgery, chemotherapy and radiation. To improve patient selection for adjuvant chemoradiotherapy, we evaluated the prognostic significance of immunohistochemical and genetic biomarkers in patients with resected gastric adenocarcinoma.

Patients And Methods: Tumors from 119 patients were subjected to immunohistochemistry for 12 protein biomarkers, as well as next-generation sequencing. Clinical and biomarker data were available for 91 patients.

Results: EBV-positive tumors and tumors with mutations had higher intratumoral CD8 tumor-infiltrating lymphocyte density (p=0.009 and p=0.017, respectively). PIK3CA mutations were correlated with VEGFA overexpression (p=0.042), while KRAS mutations and HER2 expression were mutually exclusive (p=0.036). PTEN expression univariately confirmed longer overall survival (HR=0.27; p=0.046), while there was a trend between the presence of KRAS mutations and inferior disease-free and overall survival.

Conclusion: PTEN protein expression and KRAS mutations may predict disease outcome in early-stage gastric cancer. These results need to be further validated in larger cohorts.
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http://dx.doi.org/10.21873/cgp.20188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259883PMC
November 2020

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer.

BMC Microbiol 2020 04 23;20(Suppl 1):83. Epub 2020 Apr 23.

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

Background: The human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking.

Results: We found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation.

Conclusions: Our study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.
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http://dx.doi.org/10.1186/s12866-020-01762-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178946PMC
April 2020

dv-trio: a family-based variant calling pipeline using DeepVariant.

Bioinformatics 2020 06;36(11):3549-3551

Victor Chang Cardiac Research Institute, Sydney, Australia.

Motivation: In 2018, Google published an innovative variant caller, DeepVariant, which converts pileups of sequence reads into images and uses a deep neural network to identify single-nucleotide variants and small insertion/deletions from next-generation sequencing data. This approach outperforms existing state-of-the-art tools. However, DeepVariant was designed to call variants within a single sample. In disease sequencing studies, the ability to examine a family trio (father-mother-affected child) provides greater power for disease mutation discovery.

Results: To further improve DeepVariant's variant calling accuracy in family-based sequencing studies, we have developed a family-based variant calling pipeline, dv-trio, which incorporates the trio information from the Mendelian genetic model into variant calling based on DeepVariant.

Availability And Implementation: dv-trio is available via an open source BSD3 license at GitHub (https://github.com/VCCRI/dv-trio/).

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa116DOI Listing
June 2020

Tumor Mutational Patterns and Infiltrating Lymphocyte Density in Young and Elderly Patients With Breast Cancer.

Cancer Genomics Proteomics 2020 Mar-Apr;17(2):181-193

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece.

Background/aim: Age may pertain to different tumor genotype characteristics which may interfere with treatment efficacy and prognosis. We investigated the distribution and prognostic effect of mutations and tumor infiltrating lymphocyte (stromal TIL density) in young (≤35 years) and elderly (>65 years) early breast cancer patients.

Materials And Methods: Paraffin tumor genotypes of all clinical subtypes from 345 patients were examined.

Results: A total of 638 mutations were detected in 221 patients (64.1%). Compared to young, elderly patients presented with lower TIL density (p<0.001) but more TILs in TP53 mutated tumors (p=0.042). Mutation in one, rather than in 2 or more genes, conferred better outcome (DFS: HR=0.51, p=0.016; OS: HR=0.47, p=0.015) but the effect was age-independent.

Conclusion: There are fewer TILs and different mutations patterns in tumors from elderly patients compared to young. Age and TIL-independent gene agnostic co-mutations affect patient outcome.
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http://dx.doi.org/10.21873/cgp.20179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078837PMC
September 2020

Pathogenic mutations and overall survival in 3,084 patients with cancer: the Hellenic Cooperative Oncology Group Precision Medicine Initiative.

Oncotarget 2020 Jan 7;11(1):1-14. Epub 2020 Jan 7.

The University of Texas MD Anderson Cancer Center, Department of Investigational Cancer Therapeutics, Houston, TX, USA.

We evaluated the association between pathogenic mutations and overall survival (OS) in patients with cancer referred to Hellenic Cooperative Oncology Group-affiliated Departments. Patients referred from 12/1980 to 1/2017 had molecular testing (for research) of archival tumor tissue collected at the time of first diagnosis (non-metastatic, 81%; metastatic, 19%). Tumor-specific gene panels (16-101 genes) were used to identify pathogenic mutations in clinically relevant genes. NGS genotyping was performed at the Laboratory of Molecular Oncology, Aristotle University of Thessaloniki. Annotation of mutations was performed at MD Anderson Cancer Center. We analyzed 3,084 patients (median age, 57 years; men, 22%) with sequencing data. Overall, 1,775 (58% of 3,084) patients had pathogenic mutations. The median follow-up was 7.52 years (95% CI, 7.39-7.61). In patients with non-metastatic tumors, after stratification by tumor type, increasing age, higher grade, and histology other than adenocarcinoma were associated with shorter OS. OS was also shorter in patients with pathogenic (HR=1.36; p<0.001), (HR=1.64; p=0.005), and (HR=1.46; p=0.047) mutations compared to wild-type genes. In multivariate analyses, independent prognostic factors predicting shorter OS were pathogenic mutations in (HR=1.37, p=0.002) and (HR=1.50, p=0.027); increasing age (HR=1.02, p<0.001); and increasing grade (HR=1.46, p<0.001). In patients with metastatic cancer, older age and higher grade were associated with shorter OS and maintained their independent prognostic significance (increasing age, HR=1.03, p<0.001 and higher grade, HR=1.73, p<0.001). Analysis of molecular data reveals prognostic biomarkers, regardless of tissue or organ of origin to improve patient management.
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http://dx.doi.org/10.18632/oncotarget.27338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6967777PMC
January 2020

Is There an Independent Role of TERT and NF1 in High Grade Gliomas?

Transl Oncol 2020 Feb 28;13(2):346-354. Epub 2019 Dec 28.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, University Campus, Building 17B, 54006 Thessaloniki, Greece; Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; German Oncology Center, Avenue Nikis 1, 4108 Agios Athanasios, Limassol, Cyprus.

Background: High grade glioma molecular profiling is of particular interest in neurooncology. The role of telomerase reverse transcriptase (TERT) varies dependent upon other molecular parameters. We explored the role of TERT in 101 high-grade gliomas.

Methods: A total of 101 patients (pts) with grade III-IV gliomas treated with standard of care and informative tumor genotypes were included in the present study. Of 55 genes targeted with the next-generation sequencing panel, mutations (muts) were found in 37; these were included in the analysis. TERT mut were tested with Sanger sequencing. MGMT promoter methylation status was determined by methylation specific PCR.

Results: 270 mut were detected in 92/101 tumors (91.1%). TERT was the most frequently mutated gene (74.3%). IDH1/2 mut were mutually exclusive with mut in the neurofibromin 1 (NF1) gene. Mutated TERT was associated with wild-type (wt) IDH1/2 (p = 0.025). The 12-month overall survival (OS) rate was 74.3% (median OS: 22 months). Pts with TERT and NF1 wt had a median OS of 40.8 months, while among pts with NF1 wt/TERT mutant, the median OS was 18.5 months. NF1 and TERT mut univariately conferred shorter OS (HR = 3.19; p = 0.004 and HR = 2.28; p = 0.002). Upon multivariate analysis, mutated TERT showed marginal unfavorable prognostic significance for OS (p = 0.049), while NF1 lost its unfavorable significance (p = 0.151).

Conclusions: TERT is herein proven to confer poor prognosis in high grade gliomas, independent of IDH and MGMT. NF1 seems to also confer poor prognosis although our small numbers do not allow for firm conclusions.
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http://dx.doi.org/10.1016/j.tranon.2019.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940679PMC
February 2020

Comparison of somatic variant detection algorithms using Ion Torrent targeted deep sequencing data.

BMC Med Genomics 2019 12 24;12(Suppl 9):181. Epub 2019 Dec 24.

Victor Chang Cardiac Research Institute, Darlinghurst, Australia.

Background: The application of next-generation sequencing in cancer has revealed the genomic landscape of many tumour types and is nowadays routinely used in research and clinical settings. Multiple algorithms have been developed to detect somatic variation from sequencing data using either paired tumour-blood or tumour-only samples. Most of these methods have been developed and evaluated for the identification of somatic variation using Illumina sequencing datasets of moderate coverage. However, a comprehensive evaluation of somatic variant detection algorithms on Ion Torrent targeted deep sequencing data has not been performed.

Methods: We have applied three somatic detection algorithms, Torrent Variant Caller, MuTect2 and VarScan2, on a large cohort of ovarian cancer patients comprising of 208 paired tumour-blood samples and 253 tumour-only samples sequenced deeply on Ion Torrent Proton platform across 330 amplicons. Subsequently, the concordance and performance of the three somatic variant callers were assessed.

Results: We have observed low concordance across the algorithms with only 0.5% of SNV and 0.02% of INDEL calls in common across all three methods. The intersection of all methods showed better performance when assessed using correlation with known mutational signatures, overlap with COSMIC variation and by examining the variant characteristics. The Torrent Variant Caller also performed well with the advantage of not eliminating a high number of variants that could lead to high type II error.

Conclusions: Our results suggest that caution should be taken when applying state-of-the-art somatic variant algorithms to Ion Torrent targeted deep sequencing data. Better quality control procedures and strategies that combine results from multiple methods should ensure that higher accuracy is achieved. This is essential to ensure that results from bioinformatics pipelines using Ion Torrent deep sequencing can be robustly applied in cancer research and in the clinic.
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http://dx.doi.org/10.1186/s12920-019-0636-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929331PMC
December 2019

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Hum Mol Genet 2020 03;29(4):566-579

Victor Chang Cardiac Research Institute, Sydney, NSW, 2010, Australia.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.
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http://dx.doi.org/10.1093/hmg/ddz270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068028PMC
March 2020

VPOT: A Customizable Variant Prioritization Ordering Tool for Annotated Variants.

Genomics Proteomics Bioinformatics 2019 10 22;17(5):540-545. Epub 2019 Nov 22.

Victor Chang Cardiac Research Institute, Sydney 2010, Australia; St Vincent's Clinical School, University of New South Wales, Sydney 2052, Australia. Electronic address:

Next-generation sequencing (NGS) technologies generate thousands to millions of genetic variants per sample. Identification of potential disease-causal variants is labor intensive as it relies on filtering using various annotation metrics and consideration of multiple pathogenicity prediction scores. We have developed VPOT (variant prioritization ordering tool), a python-based command line tool that allows researchers to create a single fully customizable pathogenicity ranking score from any number of annotation values, each with a user-defined weighting. The use of VPOT can be informative when analyzing entire cohorts, as variants in a cohort can be prioritized. VPOT also provides additional functions to allow variant filtering based on a candidate gene list or by affected status in a family pedigree. VPOT outperforms similar tools in terms of efficacy, flexibility, scalability, and computational performance. VPOT is freely available for public use at GitHub (https://github.com/VCCRI/VPOT/). Documentation for installation along with a user tutorial, a default parameter file, and test data are provided.
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http://dx.doi.org/10.1016/j.gpb.2019.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056850PMC
October 2019

Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.

Hum Mol Genet 2020 05;29(7):1068-1082

Victor Chang Cardiac Research Institute, Department of Embryology, New South Wales, 2010 Sydney, Australia.

Pre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice.
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http://dx.doi.org/10.1093/hmg/ddz231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206850PMC
May 2020

Opposite Prognostic Impact of Single PTEN-loss and Mutations in Early High-risk Breast Cancer.

Cancer Genomics Proteomics 2019 May-Jun;16(3):195-206

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki, Thessaloniki, Greece.

Background/aim: PTEN-loss and PIK3CA mutations have been addressed as markers of PI3K activation in breast cancer. We evaluated these markers in early high-risk breast cancer (EBC) focusing on PTEN immunohistochemistry (IHC) issues, particularly in HER2-positive disease.

Materials And Methods: We examined PTEN-loss and PIK3CA mutations in 1265 EBC patients treated with adjuvant chemotherapy within two clinical trials. Two different methods for the evaluation of PTEN IHC were used, one upfront binary (loss; no-loss) and the other initially multi-scale allowing for the classification of "grey zone" tumors with low and very low PTEN protein expression.

Results: PTEN-loss (33.4% and 22.1%, depending on the IHC method) and PIK3CA mutations (29.6%) were associated with ER/PgR/HER2-negative and ER/PgR-positive disease, respectively. Concordance of the two IHC methods was moderate (Cohen's kappa 0.624). PTEN-loss discrepancy and intra-tumor heterogeneity concerned "grey zone" tumors that were prevalent among HER2-positive cancers. PTEN-loss independently conferred higher risk for relapse and death. Compared to single PIK3CA mutations,single PTEN-loss was independently associated with increased risk for relapse and death. Depending on the evaluation method, in HER2-positive cancer, PTEN-loss was without- or of marginal unfavorable prognostic significance.

Conclusion: In EBC, PTEN-loss is an independent predictor of poor outcome. When occurring singly, PTEN-loss and PIK3CA mutations have opposite prognostic impact. In HER2-positive disease, assessment of PTEN-loss by IHC appears unreliable and the marker is without clear prognostic significance.
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http://dx.doi.org/10.21873/cgp.20125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542648PMC
September 2019

Spliceogen: an integrative, scalable tool for the discovery of splice-altering variants.

Bioinformatics 2019 11;35(21):4405-4407

Victor Chang Cardiac Research Institute, Sydney, Australia.

Motivation: In silico prediction tools are essential for identifying variants which create or disrupt cis-splicing motifs. However, there are limited options for genome-scale discovery of splice-altering variants.

Results: We have developed Spliceogen, a highly scalable pipeline integrating predictions from some of the individually best performing models for splice motif prediction: MaxEntScan, GeneSplicer, ESRseq and Branchpointer.

Availability And Implementation: Spliceogen is available as a command line tool which accepts VCF/BED inputs and handles both single nucleotide variants (SNVs) and indels (https://github.com/VCCRI/Spliceogen). SNV databases with prediction scores are also available, covering all possible SNVs at all genomic positions within all Gencode-annotated multi-exon transcripts.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz263DOI Listing
November 2019

Big data: the elements of good questions, open data, and powerful software.

Biophys Rev 2019 Feb 25;11(1):1-3. Epub 2019 Jan 25.

Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010, Australia.

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http://dx.doi.org/10.1007/s12551-019-00500-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381355PMC
February 2019

Host and microbiome multi-omics integration: applications and methodologies.

Biophys Rev 2019 Feb 9;11(1):55-65. Epub 2019 Jan 9.

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, No.79 Qingchun Road, Hangzhou, 310003, Zhejiang, China.

The study of the microbial community-the microbiome-associated with a human host is a maturing research field. It is increasingly clear that the composition of the human's microbiome is associated with various diseases such as gastrointestinal diseases, liver diseases and metabolic diseases. Using high-throughput technologies such as next-generation sequencing and mass spectrometry-based metabolomics, we are able to comprehensively sequence the microbiome-the metagenome-and associate these data with the genomic, epigenomics, transcriptomic and metabolic profile of the host. Our review summarises the application of integrating host omics with microbiome as well as the analytical methods and related tools applied in these studies. In addition, potential future directions are discussed.
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http://dx.doi.org/10.1007/s12551-018-0491-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381360PMC
February 2019

Association of the PHACTR1/EDN1 Genetic Locus With Spontaneous Coronary Artery Dissection.

J Am Coll Cardiol 2019 01;73(1):58-66

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia; St. Vincent's Clinical School, University of New South Wales, Kensington, New South Wales, Australia.

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene.

Objectives: This study sought to test the association between the rs9349379 genotype and SCAD.

Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD.

Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence.

Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD.
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http://dx.doi.org/10.1016/j.jacc.2018.09.085DOI Listing
January 2019

Relapsed and De Novo Metastatic HER2-positive Breast Cancer Treated With Trastuzumab: Tumor Genotypes and Clinical Measures Associated With Patient Outcome.

Clin Breast Cancer 2019 04 5;19(2):113-125.e4. Epub 2018 Nov 5.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research/Aristotle University of Thessaloniki, Thessaloniki, Greece; Aristotle University of Thessaloniki, Thessaloniki, Greece.

Background: We examined tumor genotype characteristics of human epidermal growth factor receptor 2 (HER2)-positive relapsed (R-) and de novo (dn-) metastatic breast cancer (MBC) in trastuzumab-treated patients who were previously not exposed to this agent.

Materials And Methods: We analyzed genotypes obtained upon deep sequencing from 113 HER2-positive primary tumors from 69 patients with R-MBC and 44 patients with dn-MBC.

Results: Mutations were observed in 90 (79.6%) tumors, 56 R-MBC and 34 dn-MBC (median number per tumor: 2; mean: 11.2; range: 0-150). The top mutated gene was TP53 (63.7%) followed by PIK3CA (24.8%) and others that were mostly co-mutated with TP53 (eg, 22 of 28 PIK3CA mutated tumors were co-mutated in TP53, 17 of these were R-MBC [P = .041]). dn-MBC had higher CEN17 average copies (P = .048). Tumor mutational burden inversely correlated with average HER2 copies (rho -0.32; P < .001). In all patients, PIK3CA mutations and higher proliferation rate were independent unfavorable prognosticators. In R-MBC, longer disease-free interval between initial diagnosis and relapse conferred lower risk for time-to-progression (P < .001) and death (P = .009); PIK3CA mutations conferred higher risk for death (P = .035). In dn-MBC, surgical removal of the primary tumor before any other therapy was favorable for time-to-progression (P = .002); higher tumor mutational burden was unfavorable for survival (P = .026).

Conclusions: Except for the overall unfavorable prognostic effect of PIK3CA mutations in trastuzumab-treated MBC, our exploratory findings indicate that the outcome of patients with R-MBC is related to patient benefit from the preceding adjuvant chemotherapy and provide initial evidence that tumor mutational burden may be related to prognosis in dn-MBC, which is of potential clinical relevance and merits further investigation.
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http://dx.doi.org/10.1016/j.clbc.2018.10.014DOI Listing
April 2019

Antiviral activity of bone morphogenetic proteins and activins.

Nat Microbiol 2019 02 3;4(2):339-351. Epub 2018 Dec 3.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.
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http://dx.doi.org/10.1038/s41564-018-0301-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590058PMC
February 2019

Pathogenic BRCA1 mutations may be necessary but not sufficient for tissue genomic heterogeneity: Deep sequencing data from ovarian cancer patients.

Gynecol Oncol 2019 02 14;152(2):375-386. Epub 2018 Nov 14.

Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki, Thessaloniki, Greece; Aristotle University of Thessaloniki, Thessaloniki, Greece. Electronic address:

Background: Tissue genomic heterogeneity (t-HET) in patients with epithelial ovarian cancer (OVCA) is related to tissue plasticity, i.e., flexibility to adapt to adverse molecular environments. Here, we interrogated the presence and clinical relevance of OVCA t-HET.

Methods: We applied high-depth (>2000×) sequencing on 297 paraffin tissue samples (fallopian tubes, ovaries, intra-abdominal metastases) from 71 treatment-naïve patients who subsequently received first-line platinum-based chemotherapy. Based on tissue mutation patterns, we distinguished tissue genotypes into: no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed t-HET in 69 patients. Predicted pathogenic mutations refer to germline and/or tissues.

Results: Among all 71 patients, 46 (64.8%) had pathogenic BRCA1 mutations and 15 (21.7%) had BRCA1/2 disruption (i.e., pathogenic mutations with position-LOH). We classified 29 patients with t-HET (42%), all with pathogenic BRCA1; t-HET was observed in 64% with such mutations (p < 0.001). As opposed to non-t-HET, matched tissues in t-HET shared pathogenic BRCA1 (p < 0.001) but not BRCA2 and TP53. Germline BRCA1 mutations in tissues exhibited position-LOH; heterozygous status; or, partial loss of the inherited allele accompanied by additional clonal mutations. Patients with t-HET had worse outcome (log-rank p = 0.048 [progression-free]; p = 0.037 [overall survival]), including 12/15 patients with disrupted BRCA1/2 and 3 BRCA1 carriers with partial germline loss in tissues.

Conclusions: Pathogenic BRCA1 mutations appear necessary but may not be sufficient for the establishment of t-HET. t-HET may be associated with worse outcome, including in patients with disrupted BRCA1/2, which is usually considered as a favourable marker. OVCA t-HET may need to be addressed for treatment decisions.
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http://dx.doi.org/10.1016/j.ygyno.2018.11.016DOI Listing
February 2019

Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes.

Genome Res 2018 12 24;28(12):1779-1790. Epub 2018 Oct 24.

Clinical Genetics Group, MRC-Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (, , , , , and ). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (, , , , , and ), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.
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http://dx.doi.org/10.1101/gr.239186.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280762PMC
December 2018

Identification of clinically actionable variants from genome sequencing of families with congenital heart disease.

Genet Med 2019 05 8;21(5):1111-1120. Epub 2018 Oct 8.

Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia.

Purpose: Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of genome sequencing (GS) of a heterogeneous cohort of CHD patients.

Methods: Ninety-seven families with probands born with CHD requiring surgical correction were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. GS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines.

Results: Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families.

Conclusions: Interrogating GS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation, will increase diagnostic yield during future reassessment of our GS data.
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http://dx.doi.org/10.1038/s41436-018-0296-xDOI Listing
May 2019
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