Curating Clinically Relevant Transcripts for the Interpretation of Sequence Variants.

Variant interpretation depends on accurate annotations using biologically relevant transcripts. We have developed a systematic strategy for designating primary transcripts and have applied it to 109 hearing loss-associated genes that were divided into three categories. Category 1 genes (n = 38) had a single transcript; category 2 genes (n = 33) had multiple transcripts, but a single transcript was sufficient to represent all exons; and category 3 genes (n = 38) had multiple transcripts with unique exons. Transcripts were curated with respect to gene expression reported in the literature and the Genotype-Tissue Expression Project. In addition, high-frequency loss-of-function variants in the Genome Aggregation Database and disease-causing variants in ClinVar and the Human Gene Mutation Database across the 109 genes were queried. These data were used to classify exons as clinically significant, insignificant, or of uncertain significance. Interestingly, 6% of all exons, containing 124 reportedly disease-causing variants, were of uncertain significance. Finally, we used exon-level next-generation sequencing quality metrics generated at two clinical laboratories and identified a total of 43 technically challenging exons in 20 different genes that had inadequate coverage and/or homology issues that might lead to false-variant calls. We have demonstrated that transcript analysis plays a critical role in accurate clinical variant interpretation.

Next generation sequencing-based copy number analysis reveals low prevalence of deletions and duplications in 46 genes associated with genetic cardiomyopathies.

BACKGROUND: Diagnostic testing for genetic cardiomyopathies has undergone dramatic changes in the last decade with next generation sequencing (NGS) expanding the number of genes that can be interrogated simultaneously. Exon resolution copy number analysis is increasingly incorporated into routine diagnostic testing via cytogenomic arrays and more recently via NGS. While NGS is an attractive option for laboratories that have no access to array platforms, its higher false positive rate requires weighing the added cost incurred by orthogonal confirmation against the magnitude of the increase in diagnostic yield. Although copy number variants (CNVs) have been reported in various cardiomyopathy genes, their contribution has not been systematically studied. METHODS: We performed single exon resolution NGS-based deletion/duplication analysis for up to 46 cardiomyopathy genes in >1400 individuals with cardiomyopathies including HCM, DCM, ARVC, RCM, and LVNC. RESULTS AND CONCLUSION: Clinically significant deletions and duplications were identified in only 9 of 1425 (0.63%) individuals. The majority of those (6/9) represented intragenic events. We conclude that the added benefit of exon level deletion/duplication analysis is low for currently known cardiomyopathy genes and may not outweigh the increased cost and complexity of incorporating it into routine diagnostic testing for these disorders.

Improving hearing loss gene testing: a systematic review of gene evidence toward more efficient next-generation sequencing-based diagnostic testing and interpretation.

PURPOSE: With next generation sequencing technology improvement and cost reductions, it has become technically feasible to sequence a large number of genes in one diagnostic test. This is especially relevant for diseases with large genetic and/or phenotypic heterogeneity, such as hearing loss. However, variant interpretation remains the major bottleneck. This is further exacerbated by the lack in the clinical genetics community of consensus criteria for defining the evidence necessary to include genes on targeted disease panels or in genomic reports, and the consequent risk of reporting variants in genes with no relevance to disease. METHODS: We describe a systematic evidence-based approach for assessing gene-disease associations and for curating relevant genes for different disease aspects, including mode of inheritance, phenotypic severity, and mutation spectrum. RESULTS: By applying this approach to clinically available hearing loss gene panels with a total of 163 genes, we show that a significant number (45%) of genes lack sufficient evidence of association with disease and thus are expected to increase uncertainty and patient anxiety, in addition to intensifying the interpretation burden. Information about all curated genes is summarized. Our retrospective analysis of 539 hearing loss cases tested by our previous OtoGenomeV2 panel demonstrates the impact of including genes with weak disease association in laboratory wet-bench and interpretation processes. CONCLUSION: Our study is, to our knowledge, the first to highlight the urgent need for defining the clinical validity of gene-disease relationships for more efficient and accurate clinical testing and reporting.Genet Med 18 6, 545-553.

VisCap: inference and visualization of germ-line copy-number variants from targeted clinical sequencing data.

PURPOSE: To develop and validate VisCap, a software program targeted to clinical laboratories for inference and visualization of germ-line copy-number variants (CNVs) from targeted next-generation sequencing data. METHODS: VisCap calculates the fraction of overall sequence coverage assigned to genomic intervals and computes log2 ratios of these values to the median of reference samples profiled using the same test configuration. Candidate CNVs are called when log2 ratios exceed user-defined thresholds. RESULTS: We optimized VisCap using 14 cases with known CNVs, followed by prospective analysis of 1,104 cases referred for diagnostic DNA sequencing. To verify calls in the prospective cohort, we used droplet digital polymerase chain reaction (PCR) to confirm 10/27 candidate CNVs and 72/72 copy-neutral genomic regions scored by VisCap. We also used a genome-wide bead array to confirm the absence of CNV calls across panels applied to 10 cases. To improve specificity, we instituted a visual scoring system that enabled experienced reviewers to differentiate true-positive from false-positive calls with minimal impact on laboratory workflow. CONCLUSIONS: VisCap is a sensitive method for inferring CNVs from targeted sequence data from targeted gene panels. Visual scoring of data underlying CNV calls is a critical step to reduce false-positive calls for follow-up testing.Genet Med 18 7, 712-719.Genetics in Medicine (2016); 18 7, 712-719. doi:10.1038/gim.2015.156.

The paternally imprinted DLK1-GTL2 locus is differentially methylated in embryonal and alveolar rhabdomyosarcomas.

Parental imprinting of differentially methylated regions (DMRs) contributes to appropriate expression of several developmentally important genes from paternally or maternally derived chromosomes. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is associated with altered expression of certain parentally imprinted genes. As previously reported, RMS cells display loss of imprinting (LOI) of the DMR at the IGF2-H19 locus, resulting in insulin-like growth factor 2 (IGF2) transcription from both paternally and maternally inherited chromosomes, and overall IGF2 overexpression. As the DLK1-GTL2 locus is structurally similar to the IGF2-H19 locus, the status of parental imprinting of the DLK1-GTL2 locus was studied in RMS. We observed that while both embryonal and alveolar rhabdomyosarcomas (ERMS and ARMS, respectively) show LOI of the DMR at the IGF2-H19 locus, imprinting of the DMR at the DLK1-GTL2 locus varies in association with the histological subtype of RMS. We found that, while ERMS tumors consistently show LOI of the DMR at the DLK1-GTL2 locus, ARMS tumors have erasure of imprinting (EOI) at this locus. These changes in imprinting status of the DLK1-GTL2 locus result in a higher GTL2/DLK1 mRNA ratio in ARMS as compared to ERMS. This difference in imprinting elucidates a novel genetic difference between these two RMS subtypes and may provide a potential diagnostic tool to distinguish between these subtypes.

IMP-1 displays cross-talk with K-Ras and modulates colon cancer cell survival through the novel proapoptotic protein CYFIP2.

Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expression and translation. In contrast to normal adult tissue, IMP-1 is reexpressed and/or overexpressed in human cancers. We show that knockdown of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further show that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase- and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3'UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible proapoptotic protein CYFIP2 is upregulated in IMP-1 knockdown SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3- and PARP-mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knockdown. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (P ≤ 0.0001) and correlates with K-Ras expression (r = 0.35, P ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-Myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis.

Parvin-beta inhibits breast cancer tumorigenicity and promotes CDK9-mediated peroxisome proliferator-activated receptor gamma 1 phosphorylation.

Parvin-beta is a focal adhesion protein downregulated in human breast cancer cells. Loss of Parvin-beta contributes to increased integrin-linked kinase activity, cell-matrix adhesion, and invasion through the extracellular matrix in vitro. The effect of ectopic Parvin-beta expression on the transcriptional profile of MDA-MB-231 breast cancer cells, which normally do not express Parvin-beta, was evaluated. Particular emphasis was placed upon propagating MDA-MB-231 breast cancer cells in three-dimensional culture matrices. Interestingly, Parvin-beta reexpression in MDA-MB-231 cells increased the mRNA expression, serine 82 phosphorylation (mediated by CDK9), and activity of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), and there was a concomitant increase in lipogenic gene expression as a downstream effector of PPARgamma. Importantly, Parvin-beta suppressed breast cancer growth in vivo, with associated decreased proliferation. These data suggest that Parvin-beta might influence breast cancer progression.

N-cadherin and keratinocyte growth factor receptor mediate the functional interplay between Ki-RASG12V and p53V143A in promoting pancreatic cell migration, invasion, and tissue architecture disruption.

The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.

PRR5 encodes a conserved proline-rich protein predominant in kidney: analysis of genomic organization, expression, and mutation status in breast and colorectal carcinomas.

Loss of heterozygosity on chromosome 22q13.31 is a frequent event during human breast and colorectal carcinogenesis. Herein we characterize a novel gene at chromosome 22q13.31 designated PRR5. Alternative promoter usage and splicing converge to generate five PRR5 transcript variants with maximum mRNA expression in kidney. In vitro transcription/translation demonstrated that the five variants generate three protein isoforms differing in their N-terminal length. Mutational analysis of PRR5 in human breast and colorectal tumors did not reveal somatic mutations. However, mRNA expression analyses revealed PRR5 overexpression in a majority of colorectal tumors but substantial downregulation of PRR5 expression in a subset of breast tumors and reduced expression in two breast cancer cell lines. Treatment with trichostatin A increased PRR5 mRNA levels in BT549 and MDA-MB-231 cells, whereas 5'-aza-2'-deoxycytidine induced expression in MDA-MB-231 cells only. Thus, PRR5 may represent a potential candidate tumor suppressor gene in breast cancer.