GEnomes Management Application (GEM.app): A New Software Tool for Large‐Scale Collaborative Genome Analysis

Novel genes are now identified at a rapid pace for many Mendelian disorders, and increasingly, for genetically complex phenotypes. However, new challenges have also become evident: (1) effectively managing larger exome and/or genome datasets, especially for smaller labs; (2) direct hands‐on analysis and contextual interpretation of variant data in large genomic datasets; and (3) many small and medium‐sized clinical and research‐based investigative teams around the world are generating data that, if combined and shared, will significantly increase the opportunities for the entire community to identify new genes. To address these challenges, we have developed GEnomes Management Application (GEM.app), a software tool to annotate, manage, visualize, and analyze large genomic datasets ( https://genomics.med.miami.edu/">https://genomics.med.miami.edu/">https://genomics.med.miami.edu/ ). GEM.app currently contains ～1,600 whole exomes from 50 different phenotypes studied by 40 principal investigators from 15 different countries. The focus of GEM.app is on user‐friendly analysis for nonbioinformaticians to make next‐generation sequencing data directly accessible. Yet, GEM.app provides powerful and flexible filter options, including single family filtering, across family/phenotype queries, nested filtering, and evaluation of segregation in families. In addition, the system is fast, obtaining results within 4 sec across ～1,200 exomes. We believe that this system will further enhance identification of genetic causes of human disease.     

The role of tandem duplicator phenotype in tumour evolution in high-grade serous ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is characterized by genomic instability, ubiquitous TP53 loss, and frequent development of platinum resistance. Loss of homologous recombination (HR) is a mutator phenotype present in 50% of HGSOCs and confers hypersensitivity to platinum treatment. We asked which other mutator phenotypes are present in HGSOC and how they drive the emergence of platinum resistance. We performed whole-genome paired-end sequencing on a model of two HGSOC cases, each consisting of a pair of cell lines established before and after clinical resistance emerged, to describe their structural variants (SVs) and to infer their ancestral genomes as the SVs present within each pair. The first case (PEO1/PEO4), with HR deficiency, acquired translocations and small deletions through its early evolution, but a revertant BRCA2 mutation restoring HR function in the resistant lineage re-stabilized its genome and reduced platinum sensitivity. The second case (PEO14/PEO23) had 216 tandem duplications and did not show evidence of HR or mismatch repair deficiency. By comparing the cell lines to the tissues from which they originated, we showed that the tandem duplicator mutator phenotype arose early in progression in vivo and persisted throughout evolution in vivo and in vitro, which may have enabled continual evolution. From the analysis of SNP array data from 454 HGSOC cases in The Cancer Genome Atlas series, we estimate that 12.8% of cases show patterns of aberrations similar to the tandem duplicator, and this phenotype is mutually exclusive with BRCA1/2 carrier mutations.     

Use of panel tests in place of single gene tests in the cancer genetics clinic

Improved technology has made it possible to test for mutations within multiple genes simultaneously. It is not clear when these gene ‘panels’ should be used in the hereditary cancer setting. These analyses were intended to guide panel testing criteria. Offering hereditary panel testing as a first and final, ‘single‐tier’, option was explored. A ‘two‐tiered’ approach, in which panel testing is offered reflexively following stricter criteria, was then applied to the same data. Within our cohort of 105 patients, the single‐tier approach was associated with a higher mutation detection rate (6.7% vs 3.8%) and variant of uncertain significance (VUS) rate (0.94 vs 0.23 average per person) compared to a two‐tiered approach. Of the VUSs also identified in other patients by another lab, 53% were classified differently between laboratories. Individuals reporting African American race had more VUSs compared to other ancestry groups (p = 0.001). The test cost for a single‐tier test was 21% more than a two‐tiered approach. Single‐tier panel testing was associated with higher mutation and VUS rates, and there is inconsistent classification of the VUS/low penetrant genes between laboratories.     

Integrating next‐generation sequencing into the diagnostic testing of inherited cancer predisposition

The clinical application of next‐generation sequencing (NGS) as a diagnostic tool has become increasingly evident. The coupling of NGS technologies with new genomic sequence enrichment methods has made the sequencing of panels of target genes technically feasible, at the same time as making such an approach cost‐effective for diagnostic applications. In this article, we discuss recent studies that have applied NGS in the diagnostic setting in relation to hereditary cancer.     

Cancer genomics and pathology: All Together Now

Cancer develops from a single cell with stepwise accumulation of genomic alterations. Recent innovative sequencing technologies have made it possible to sequence the full cancer genome. Cancer genome sequencing has been productive and helpful in the discovery of novel cancer genes. It also has revealed previously unknown but intriguing features of the cancer genome such as chromothripsis and kataegis. However, careful comparison of these studies has suggested that analyses of most tumors still seem to be incomplete, and histopathological diagnosis/classification will be essential for refining these data. Based on the improvement of technology and the completion of the cancer gene catalog, genetic diagnosis, such as examination of all potentially druggable mutations, of individual cancers will be performed routinely together with histological diagnosis. Pathologists will play a central role in both interpreting these patho‐molecular diagnoses for oncologists, and the process of decision‐making necessary for individualized medicine.     

Next-generation sequencing in molecular diagnosis: NUBPL mutations highlight the challenges of variant detection and interpretation

Next-generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an individual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild-type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch-site mutation in addition to the missense mutation. We demonstrate that the branch-site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus-specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow-up to confirm mutation pathogenicity.