ABSTRACT
Tumor purity, or the relative contribution of tumor cells out of all cells in a pathological specimen, influences mutation identification and clinical interpretation of cancer panel next generation sequencing results. Here, we describe a method of calculating tumor purity using pathologist-guided copy number analysis from sequencing data. Molecular calculation of tumor purity showed strong linear correlation with purity derived from driver KRAS or BRAF variant allele fractions in colorectal cancers (R2 = 0.79) compared to histological estimation in the same set of colorectal cancers (R2 = 0.01) and in a broader dataset of cancers with various diagnoses (R2 = 0.35). We used calculated tumor purity to quantitate ERBB2 copy number in breast carcinomas with equivocal immunohistochemical staining and demonstrated strong correlation with fluorescence in situ hybridization (R2 = 0.88). Finally, we used calculated tumor purity to infer the germline status of variants in breast and ovarian carcinomas with concurrent germline testing. Tumor-only next generation sequencing correctly predicted the somatic versus germline nature of 26 of 26 (100%) pathogenic TP53, BRCA1 and BRCA2 variants. In this article, we describe a framework for calculating tumor purity from cancer next generation sequencing data. Accurate tumor purity assessment can be assimilated into interpretation pipelines to derive clinically useful information from cancer genomic panels.
Subject(s)
Breast Neoplasms , Colorectal Neoplasms , Ovarian Neoplasms , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Colorectal Neoplasms/genetics , DNA Copy Number Variations , Female , Germ Cells/pathology , Germ-Line Mutation , High-Throughput Nucleotide Sequencing , Humans , In Situ Hybridization, Fluorescence , Mutation , Ovarian Neoplasms/pathology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Receptor, ErbB-2ABSTRACT
Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.
Subject(s)
Cell Cycle Proteins/metabolism , Cilia/pathology , Cyclin-Dependent Kinases/metabolism , DNA Replication/genetics , Polycystic Kidney Diseases/pathology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , S Phase/physiology , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Culture Techniques , Cell Cycle Checkpoints , Cell Cycle Proteins/genetics , Cilia/metabolism , Cyclin-Dependent Kinases/genetics , DNA Damage/genetics , Genomic Instability , Humans , Mice , Mutation/genetics , NIMA-Related Kinases , Phosphorylation , Polycystic Kidney Diseases/metabolism , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Stress, PhysiologicalABSTRACT
A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8(-/-) and Pkd2(-/-) embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.
Subject(s)
Body Patterning , Cilia/physiology , Polycystic Kidney Diseases/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Biomarkers/metabolism , Disease Models, Animal , Female , Heart Defects, Congenital/embryology , Heterozygote , Homozygote , Male , Mice , Mice, Inbred C57BL , NIMA-Related Kinases , Phenotype , Protein Serine-Threonine Kinases/metabolism , TRPP Cation Channels/metabolism , ZebrafishABSTRACT
BACKGROUND: Leptomeningeal metastases occur across multiple solid and lymphoid cancers, and patients typically undergo cytopathologic assessment of cerebrospinal fluid (CSF) in this setting. For patients diagnosed with metastatic cancer, the detection of actionable somatic mutations in CSF can provide clinically valuable information for treatment without the need for additional tissue collection. METHODS: The authors validated a targeted next-generation sequencing assay for the detection of somatic variants in cancer (OncoPanel) on cell-free DNA (cfDNA) isolated from archival CSF specimens in a cohort of 25 patients who had undergone molecular testing of a prior tumor specimen. RESULTS: CSF storage time and volume had no impact on cfDNA concentration or mean target coverage of the assay. Previously identified somatic variants in CSF cfDNA were detected in 88%, 50%, and 27% of specimens diagnosed cytologically as positive, suspicious/atypical, and negative for malignancy, respectively. Somatic variants were identified in 81% of CSF specimens from patients who had leptomeningeal enhancement on magnetic resonance imaging compared with 31% from patients without such enhancement. CONCLUSIONS: These data highlight the stability of cfDNA in CSF, which allows for cytopathologic evaluation before triage for next-generation sequencing assays. For a subset of cases in which clinical suspicion is high but cytologic or radiographic studies are inconclusive, the detection of pathogenic somatic variants in CSF cfDNA may aid in the diagnosis of leptomeningeal metastases.
Subject(s)
Cell-Free Nucleic Acids , Neoplasms , Humans , Cell-Free Nucleic Acids/genetics , Mutation , Neoplasms/diagnostic imaging , Neoplasms/genetics , High-Throughput Nucleotide Sequencing/methodsABSTRACT
Standard methods of variant assessment in hereditary cancer susceptibility genes are limited by the lack of availability of key supporting evidence. In cancer, information derived from tumors can serve as a useful source in delineating the tumor behavior and the role of germline variants in tumor progression. We have previously demonstrated the value of integrating tumor and germline findings to comprehensively assess germline variants in hereditary cancer syndromes. Building on this work, herein, we present the development and application of the INT2GRATE|HPPGL platform. INT2GRATE (INTegrated INTerpretation of GeRmline And Tumor gEnomes) is a multi-institution oncology consortium that aims to advance the integrated application of constitutional and tumor data and share the integrated variant information in publicly accessible repositories. The INT2GRATE|HPPGL platform enables automated parsing and integrated assessment of germline, tumor, and genetic findings in hereditary paraganglioma-pheochromocytoma syndromes (HPPGLs). Using INT2GRATE|HPPGL, we analyzed 8600 variants in succinate dehydrogenase (SDHx) genes and their associated clinical evidence. The integrated evidence includes germline variants in SDHx genes; clinical genetics evidence: personal and family history of HPPGL-related tumors; tumor-derived evidence: somatic inactivation of SDHx alleles, KIT and PDGFRA status in gastrointestinal stromal tumors (GISTs), multifocal or extra-adrenal tumors, and metastasis status; and immunohistochemistry staining status for SDHA and SDHB genes. After processing, 8600 variants were submitted programmatically from the INT2GRATE|HPPGL platform to ClinVar via a custom-made INT2GRATE|HPPGL variant submission schema and an application programming interface (API). This novel integrated variant assessment and data sharing in hereditary cancers aims to improve the clinical assessment of genomic variants and advance precision oncology.
ABSTRACT
BACKGROUND: Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease. METHODS: We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation. RESULTS: Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied. CONCLUSIONS: GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.
Subject(s)
Chondrocytes/cytology , Codon, Nonsense , Nuclear Proteins/genetics , Osteochondrodysplasias/genetics , Animals , Cell Differentiation , Cell Proliferation , Cytoskeletal Proteins , Endoplasmic Reticulum/ultrastructure , Genes, Recessive , Glycosylation , Golgi Apparatus/ultrastructure , Humans , Mice , Mice, Mutant Strains , Nuclear Proteins/deficiency , Phenotype , Polymorphism, Single Nucleotide , Protein Processing, Post-Translational/physiology , Sequence Analysis, DNAABSTRACT
The presence of variants of uncertain significance (VUS) in DNA mismatch repair (MMR) genes leads to uncertainty in the clinical management of patients being evaluated for Lynch syndrome (LS). Currently, there is no platform to systematically use tumor-derived evidence alongside germline data for the assessment of VUS in relation to LS. We developed INT2GRATE (INTegrated INTerpretation of GeRmline And Tumor gEnomes) to leverage information from the tumor genome to inform the potential role of constitutional VUS in MMR genes. INT2GRATE platform has two components: a comprehensive evidence-based decision tree that integrates well-established clinico-genomic data from both the tumor and constitutional genomes to help inform the potential relevance of germline VUS in LS; and a web-based user interface (UI). With the INT2GRATE decision tree operating in the backend, INT2GRATE UI enables the front-end collection of comprehensive clinical genetics and tumor-derived evidence for each VUS to facilitate INT2GRATE assessment and data sharing in the publicly accessible ClinVar database. The performance of the INT2GRATE decision tree was assessed by qualitative retrospective analysis of genomic data from 5057 cancer patients with MMR alterations which included 52 positive control cases. Of 52 positive control cases with LS and pathogenic MMR alterations, 23 had all the testing parameters for the evaluation by INT2GRATE. All these variants were correctly categorized as INT2GRATE POSITIVE. The stringent INT2GRATE decision tree flagged 29 of positive cases by identifying the absence or unusual presentation of specific evidence, highlighting the conservative INT2GRATE logic in favor of a higher degree of confidence in the results. The remaining 99% of cases were correctly categorized as INCONCLUSIVE due to the absence of LS criteria and ≥1 tumor parameters. INT2GRATE is an effective platform for clinical and genetics professionals to collect and assess clinical genetics and complimentary tumor-derived information for each germline VUS in suspected LS patients. Furthermore, INT2GRATE enables the collation of integrated tumor-derived evidence relevant to germline VUS in LS, and sharing them with a large community, a practice that is needed in precision oncology.
ABSTRACT
CONTEXT.: The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature. OBJECTIVE.: To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens. DESIGN.: We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens. RESULTS.: Identification of specimen contamination was validated via in silico and in vitro mixtures, and reference range and reproducibility were established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively applied this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) were predicted to be affected by greater than 5% contamination. CONCLUSIONS.: Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.
Subject(s)
High-Throughput Nucleotide Sequencing , Neoplasms , High-Throughput Nucleotide Sequencing/methods , Humans , Neoplasms/diagnosis , Neoplasms/genetics , Pathology, Molecular , Polymorphism, Single Nucleotide , Reproducibility of ResultsABSTRACT
The interpretation of hereditary genetic sequencing variants is often limited due to the absence of functional data and other key evidence to assess the role of variants in disease. Cancer genetics is unique, as two sets of genomic information are often available from a cancer patient: somatic and germline. Despite the progress made in the integrated analysis of somatic and germline findings, the assessment of pathogenicity of germline variants in high penetrance genes remains grossly underutilized. Indeed, standard ACMG/AMP guidelines for interpreting germline sequence variants do not address the evidence derived from tumor data in cancer. Previously, we have demonstrated the utility of somatic tumor data as supporting evidence to elucidate the role of germline variants in patients suspected with VHL syndrome and other cancers. We have leveraged the key elements of cancer genetics in these cases: genes with expected high disease penetrance and those with a known biallelic mechanism of tumorigenicity. Here we provide our optimized protocol for evaluating the pathogenicity of germline VHL variants using informative somatic profiling data. This protocol provides details of case selection, assessment of personal and family evidence, somatic tumor profiles, and loss of heterozygosity (LOH) as supporting evidence for the re-evaluation of germline variants.
ABSTRACT
Genomic profiles of tumors are often unique and represent characteristic mutational signatures defined by DNA damage or DNA repair response processes. The tumor-derived somatic information has been widely used in therapeutic applications, but it is grossly underutilized in the assessment of germline genetic variants. Here, we present a comprehensive approach for evaluating the pathogenicity of germline variants in cancer using an integrated interpretation of somatic and germline genomic data. We have previously demonstrated the utility of this integrated approach in the reassessment of pathogenic germline variants in selected cancer patients with unexpected or non-syndromic phenotypes. The application of this approach is presented in the assessment of rare variants of uncertain significance (VUS) in Lynch-related colon cancer, hereditary paraganglioma-pheochromocytoma syndrome, and Li-Fraumeni syndrome. Using this integrated method, germline VUS in PMS2, MSH6, SDHC, SHDA, and TP53 were assessed in 16 cancer patients after genetic evaluation. Comprehensive clinical criteria, somatic signature profiles, and tumor immunohistochemistry were used to re-classify VUS by upgrading or downgrading the variants to likely or unlikely actionable categories, respectively. Going forward, collation of such germline variants and creation of cross-institutional knowledgebase datasets that include integrated somatic and germline data will be crucial for the assessment of these variants in a larger cancer cohort.
ABSTRACT
Von Hippel-Lindau (VHL) syndrome is a hereditary cancer genetic condition associated with inactivating pathogenic alterations in the VHL tumor suppressor gene located at 3p (short arm of chromosome 3). Classic features of VHL include clear cell renal cell carcinoma, hemangioblastomas of the brain, spinal cord, and retina, pheochromocytoma, pancreatic cysts, and neuroendocrine tumors. Two sets of genomic information may be available from patients with VHL: the germline data showing the constitutional genetic profile and somatic profile obtained from patient tumor(s). Here we present both somatic and germline dataset from heterozygous carriers of germline VHL variants who exhibit non-syndromic VHL phenotypes. This data description article accompanies the paper "Pathogenicity of VHL variants in families with non-syndromic von Hippel-Lindau phenotypes: an integrated evaluation of germline and somatic genomic results'' by Huma Q. Rana, Diane R. Koeller, Alison Schwartz, Danielle K. Manning, Katherine A. Schneider, Katherine M. Krajewski, Toni K. Choueiri, Neal I. Lindeman, Judy E. Garber, Arezou A. Ghazani. We provide next generation sequencing (NGS) data obtained from DNA from tumors (renal cancer, bladder cancer, and cerebral hemangioblastoma) of three VHL carriers. The somatic dataset was analyzed for single nucleotide variants (SNVs) and copy number variants (CNVs) in 447 cancer genes, and structural variation (SVs) in 191 regions across 60 genes for rearrangements. We also present germline raw NGS data and analyzed SNV and CNV data in exonic regions of 133 hereditary cancer genes obtained from the peripheral blood of two VHL carriers.
ABSTRACT
Von Hippel-Lindau (VHL) syndrome is a hereditary tumor syndrome associated with germline loss-of-function pathogenic variants (PVs) in the VHL gene. VHL is classically associated with a high penetrance for many different tumor types. The same tumors may be sporadic in the setting of somatic VHL PVs. With more large-scale genome sequencing, variants with low penetrance or variable expressivity are identified. This has introduced challenges in patient management and the clinical interpretation of germline VHL variants identified in non-classic families. Herein, we report individuals from 3 non-classic families with VHL variants who presented with unexpected or non-syndromic phenotypes, but often with a VHL component tumor. In family 1, two siblings, age 61, with pathogenic VHL p.Leu188Val presented with clear cell renal cell carcinoma and lobular breast cancer. In family 2, the proband, age 82, was found to have pathogenic germline VHL p.Tyr98His on testing for metastatic bladder cancer. In family 3, four members carried germline VHL p.Pro81Ser (variant of uncertain significance), after the proband, age 40, presented with cerebellar hemangioblastoma. None of the individuals in the above three families met clinical criteria of classic VHL, suggesting germline VHL p.Leu188Val, p.Y98H, and p.Tyr98His may be low penetrant variants. Large studies are needed to evaluate penetrance and possible effect of genetic and non-genetic modifiers. Somatic sequencing performed on their respective tumors could help discern the etiology of the component tumors, highlighting the role of somatic evaluation in these cases. Paired examination of somatic and germline findings provided a more complete landscape of genome alterations in cancer development.
Subject(s)
Genetic Predisposition to Disease/genetics , Germ-Line Mutation/genetics , von Hippel-Lindau Disease/genetics , Adult , Aged, 80 and over , Humans , Male , Middle Aged , Pedigree , PhenotypeABSTRACT
PURPOSE: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.
Subject(s)
Neoplasms/genetics , Whole Genome Sequencing/standards , Adolescent , Adult , Child , Child, Preschool , Female , Genetic Predisposition to Disease/genetics , Humans , Infant , Male , Precision Medicine/methods , Precision Medicine/standards , Precision Medicine/trends , Whole Genome Sequencing/methods , Whole Genome Sequencing/statistics & numerical dataABSTRACT
BACKGROUND The diagnoses of adenomatous polyposis coli (APC)-associated polyposis conditions are typically based on suggestive personal features and/or family history, and the identification of a pathogenic variant in the APC gene. However, with large-scale genome sequencing, it is now possible to identify pathogenic variants before or even without the presentation of the expected clinical features. This case describes a novel pathogenic APC variant. CASE REPORT We report the unexpected identification of a rare, pathogenic germline APC variant, p.S2627Gfs*12 in an 80-year-old man with a diagnosis of renal cell carcinoma, without any family history of APC-associated polyposis or personal history of colorectal cancer. After the identification of the APC variant, a review of the patient's medical records showed a personal history of 15 adenomatous polyps over a decade ago, with no follow-up genetic testing at the time. CONCLUSIONS This novel APC variant has not been characterized to date. The presence of the APC-p.S2627Gfs*12 variant in this patient led to the recommendation of additional cascade genetic testing and surveillance measures for any family members who tested positive for this variant. This report highlights the broad spectrum of the APC-associated polyposis features, and a mild phenotype associated with the pathogenic APC p.S2627Gfs*12 variant.
Subject(s)
Adenomatous Polyposis Coli , Genes, APC , Adenomatous Polyposis Coli/genetics , Adenomatous Polyposis Coli Protein/genetics , Aged, 80 and over , Germ Cells , Germ-Line Mutation , Humans , Male , PhenotypeABSTRACT
Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platforms-anchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capture-were analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE, with linear regression slopes near 1 for ITDs not duplicating primers, and systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.
Subject(s)
Algorithms , Diagnosis, Computer-Assisted/methods , High-Throughput Nucleotide Sequencing/methods , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Medical Informatics/methods , Tandem Repeat Sequences/genetics , fms-Like Tyrosine Kinase 3/genetics , Alleles , Cohort Studies , Exons , Gene Frequency , Humans , Multiplex Polymerase Chain Reaction/methods , Mutation , Sensitivity and SpecificityABSTRACT
Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.
Subject(s)
Genome, Human , Genomics , High-Throughput Nucleotide Sequencing , Neoplasms/diagnosis , Neoplasms/etiology , Tumor Virus Infections/complications , Tumor Virus Infections/virology , Cell Transformation, Viral , Computational Biology/methods , Genome, Viral , Genomics/methods , Genomics/standards , Genotype , High-Throughput Nucleotide Sequencing/methods , Humans , Phylogeny , Polymorphism, Single Nucleotide , Precision Medicine/methods , Sensitivity and Specificity , Tumor Virus Infections/diagnosis , Virus IntegrationABSTRACT
Nek8 is a serine/threonine kinase that is mutated in the jck (juvenile cystic kidneys) mouse, a model of autosomal recessive juvenile polycystic kidney disease, but its function is poorly understood. We used the jck mouse to study the functional relationship between Nek8 and other proteins that have been implicated in polycystic kidney diseases. In the collecting tubules and collecting ducts of wild-type mice, we found that Nek8 was localized to the proximal portion of primary cilia and was weakly detected in the cytosol. In the jck mutant, however, Nek8 was found along the entire length of cilia. Coimmunoprecipitation experiments demonstrated that Nek8 interacted with polycystin-2, but not with polycystin-1, and that the jck mutation did not affect this interaction. Western blot analysis and real-time reverse transcriptase PCR revealed that the protein and mRNA expression of polycystin-1 (PC1) and polycystin-2 (PC2) were increased in jck mouse kidneys. The jck mutation also led to abnormal phosphorylatin of PC2, and this was associated with longer cilia and ciliary accumulation of PC1 and PC2. Our data suggests that Nek8 interacts with the signal transduction pathways of the polycystins and may control the targeting of these ciliary proteins. Dysfunction Nek8 may lead to cystogenesis by altering the structure and function of cilia in the distal nephron.
Subject(s)
Cilia/metabolism , Kidney Tubules/metabolism , Protein Kinases/metabolism , TRPP Cation Channels/metabolism , Animals , Cilia/pathology , Kidney Tubules/pathology , Mice , Mutation , NIMA-Related Kinases , Phosphorylation , Protein Serine-Threonine Kinases , Up-RegulationABSTRACT
The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.
Subject(s)
Body Patterning/physiology , Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , Body Patterning/genetics , Carrier Proteins/genetics , Cell Line , Female , Humans , Kinesins/genetics , Kinesins/metabolism , Male , Mice , Morphogenesis/genetics , Morphogenesis/physiology , Mutation , NIMA-Related Kinases , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Rats , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.
Subject(s)
Axons/physiology , Cerebral Cortex , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Mutation/genetics , Phenotype , Thalamus , Transcription Factors/genetics , Alkylating Agents/pharmacology , Animals , Body Patterning/drug effects , Body Patterning/genetics , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , Embryo, Mammalian , Ethylnitrosourea/pharmacology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Gene Expression Regulation, Developmental/drug effects , Genetic Testing/methods , Lac Operon/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Pathways/abnormalities , Neural Pathways/embryology , Neural Pathways/growth & development , Reelin Protein , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Thalamus/embryology , Thalamus/growth & development , Thalamus/metabolism , beta-Galactosidase/metabolismABSTRACT
A primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. It is structurally divided along its vertical axis into sub-compartments that include the ciliary tip, the shaft, the ciliary necklace segment, the transitional zone and the basal body. We recently discovered that the shaft of the primary cilia has a distinct molecular compartment, termed the "Inv compartment", which is characterized by the accumulation of Inv at the base of primary cilia. Inv was discovered as a causative gene in inv mutant mice. It was later found to be responsible for the infantile type of nephronophthisis (NPHP2). Nephronophthisis (NPHP) is an autosomal recessive kidney disease. Nine causative genes have been identified, with all examined products thought to function in cilia, basal body and/or centrioles. However, their exact intra-ciliary localization and relationship have not been clear. Here, we report that products of Nphp3 and Nek8 (the mouse orthologs of the causative genes for NPHP3 and NPHP9, respectively) localize to the Inv compartment. We also show that Inv is essential for the compartmental localization of Nphp3 and Nek8, whereas localization of Inv does not require Nphp3 or Nek8. Nphp1 and Nphp4 also localize at the proximal region of the cilium, but not in Inv compartment. Our results indicate that Inv acts as an anchor for Nphp3 and Nek8 in the Inv compartment, and suggest that Inv compartment is a candidate site for intra-ciliary interaction of Inv, Nphp3 and Nek8.