Extracutaneous manifestations in phacomatosis cesioflammea and cesiomarmorata: Case series and literature review.

Phacomatosis pigmentovascularis (PPV) comprises a family of rare conditions that feature vascular abnormalities and melanocytic lesions that can be solely cutaneous or multisystem in nature. Recently published work has demonstrated that both vascular and melanocytic abnormalities in PPV of the cesioflammea and cesiomarmorata subtypes can result from identical somatic mosaic activating mutations in the genes GNAQ and GNA11. Here, we present three new cases of PPV with features of the cesioflammea and/or cesiomarmorata subtypes and mosaic mutations in GNAQ or GNA11. To better understand the risk of potentially occult complications faced by such patients we additionally reviewed 176 cases published in the literature. We report the frequency of clinical findings, their patterns of co-occurrence as well as published recommendations for surveillance after diagnosis. Features assessed include: capillary malformation; dermal and ocular melanocytosis; glaucoma; limb asymmetry; venous malformations; and central nervous system (CNS) anomalies, such as ventriculomegaly and calcifications. We found that ocular findings are common in patients with phacomatosis cesioflammea and cesiomarmorata. Facial vascular involvement correlates with a higher risk of seizures (p = .0066). Our genetic results confirm the role of mosaic somatic mutations in GNAQ and GNA11 in phacomatosis cesioflammea and cesiomarmorata. Their clinical and molecular findings place these conditions on a clinical spectrum encompassing other GNAQ and GNA11 related disorders and inform recommendations for their management.

Long-read genome sequencing identifies causal structural variation in a Mendelian disease.

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

A genome-wide association study of non-HPV-related head and neck squamous cell carcinoma identifies prognostic genetic sequence variants in the MAP-kinase and hormone pathways.

BACKGROUND: Carcinomas of the oral cavity, pharynx and larynx are referred to as head and neck cancers (HNC); together they account for 2-3% of all newly diagnosed cancers in North America. Between 40-50% of HNC are early diagnosed at stages I-II. The 5-year and 10-year relative survival rates are 61% and 50%, respectively. Germline genetic sequence variants (GSV) have become increasingly found to have prognostic implications in a variety of cancers. Identifying these variants may have important clinical and biological implications. METHODS: We conducted a genome-wide association study (GWAS) in 531 Stage I-II radiation-treated HNC patients (originally recruited for α-tocopherol/ß-carotene placebo-controlled secondary prevention study) and used a replication cohort of 566 HNC patients of all stages, of mostly non-HPV-related cancers. Survival rates were estimated by the Kaplan-Meier method. Cox proportional hazards models adjusted for potential clinical factors and principal components were used to test for associations between the GSV and overall survival (OS) in these tumors. RESULTS: The median follow-up time for OS was 9.21 years (GWAS cohort) and 2.37 years (replication cohort). In both cohorts, CACNA2D1:rs2299187, ESRRG:rs946465 and ESRRG:rs1416612 were each individually significantly associated with survival. In silico analysis of ESRRG:rs946465 identifies that it produces a splice variant in ESRRG. Variant alleles of CACNA2D1:rs2299187 and ESRRG:rs946465 were associated with higher expression of the corresponding protein. CONCLUSIONS: Putatively functional polymorphisms in the MAP-Kinase and estrogen pathways, identified through GWAS and replicated in an independent dataset were associated with the survival of HNC patients.

Medical implications of technical accuracy in genome sequencing.

BACKGROUND: As whole exome sequencing (WES) and whole genome sequencing (WGS) transition from research tools to clinical diagnostic tests, it is increasingly critical for sequencing methods and analysis pipelines to be technically accurate. The Genome in a Bottle Consortium has recently published a set of benchmark SNV, indel, and homozygous reference genotypes for the pilot whole genome NIST Reference Material based on the NA12878 genome. METHODS: We examine the relationship between human genome complexity and genes/variants reported to be associated with human disease. Specifically, we map regions of medical relevance to benchmark regions of high or low confidence. We use benchmark data to assess the sensitivity and positive predictive value of two representative sequencing pipelines for specific classes of variation. RESULTS: We observe that the accuracy of a variant call depends on the genomic region, variant type, and read depth, and varies by analytical pipeline. We find that most false negative WGS calls result from filtering while most false negative WES variants relate to poor coverage. We find that only 74.6% of the exonic bases in ClinVar and OMIM genes and 82.1% of the exonic bases in ACMG-reportable genes are found in high-confidence regions. Only 990 genes in the genome are found entirely within high-confidence regions while 593 of 3,300 ClinVar/OMIM genes have less than 50% of their total exonic base pairs in high-confidence regions. We find greater than 77 % of the pathogenic or likely pathogenic SNVs currently in ClinVar fall within high-confidence regions. We identify sites that are prone to sequencing errors, including thousands present in publicly available variant databases. Finally, we examine the clinical impact of mandatory reporting of secondary findings, highlighting a false positive variant found in BRCA2. CONCLUSIONS: Together, these data illustrate the importance of appropriate use and continued improvement of technical benchmarks to ensure accurate and judicious interpretation of next-generation DNA sequencing results in the clinical setting.

Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data.

High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework.

Spatial genomic heterogeneity within localized, multifocal prostate cancer.

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Combining tumor genome simulation with crowdsourcing to benchmark somatic single-nucleotide-variant detection.

The detection of somatic mutations from cancer genome sequences is key to understanding the genetic basis of disease progression, patient survival and response to therapy. Benchmarking is needed for tool assessment and improvement but is complicated by a lack of gold standards, by extensive resource requirements and by difficulties in sharing personal genomic information. To resolve these issues, we launched the ICGC-TCGA DREAM Somatic Mutation Calling Challenge, a crowdsourced benchmark of somatic mutation detection algorithms. Here we report the BAMSurgeon tool for simulating cancer genomes and the results of 248 analyses of three in silico tumors created with it. Different algorithms exhibit characteristic error profiles, and, intriguingly, false positives show a trinucleotide profile very similar to one found in human tumors. Although the three simulated tumors differ in sequence contamination (deviation from normal cell sequence) and in subclonality, an ensemble of pipelines outperforms the best individual pipeline in all cases. BAMSurgeon is available at https://github.com/adamewing/bamsurgeon/.

Developing a prognostic micro-RNA signature for human cervical carcinoma.

Cervical cancer remains the third most frequently diagnosed and fourth leading cause of cancer death in women worldwide. We sought to develop a micro-RNA signature that was prognostic for disease-free survival, which could potentially allow tailoring of treatment for cervical cancer patients. A candidate prognostic 9-micro-RNA signature set was identified in the training set of 79 frozen specimens. However, three different approaches to validate this signature in an independent cohort of 87 patients with formalin-fixed paraffin-embedded (FFPE) specimens, were unsuccessful. There are several challenges and considerations associated with developing a prognostic micro-RNA signature for cervical cancer, namely: tumour heterogeneity, lack of concordance between frozen and FFPE specimens, and platform selection for global micro-RNA expression profiling in this disease. Our observations provide an important cautionary tale for future miRNA signature studies for cervical cancer, which can also be potentially applicable to miRNA profiling studies involving other types of human malignancies.

Identification of a microRNA signature associated with risk of distant metastasis in nasopharyngeal carcinoma.

PURPOSE: Despite significant improvement in locoregional control in the contemporary era of nasopharyngeal carcinoma (NPC) treatment, patients still suffer from a significant risk of distant metastasis (DM). Identifying those patients at risk of DM would aid in personalized treatment in the future. MicroRNAs (miRNAs) play many important roles in human cancers; hence, we proceeded to address the primary hypothesis that there is a miRNA expression signature capable of predicting DM for NPC patients. METHODS AND RESULTS: The expression of 734 miRNAs was measured in 125 (Training) and 121 (Validation) clinically annotated NPC diagnostic biopsy samples. A 4-miRNA expression signature associated with risk of developing DM was identified by fitting a penalized Cox Proportion Hazard regression model to the Training data set (HR 8.25; p < 0.001), and subsequently validated in an independent Validation set (HR 3.2; p = 0.01). Pathway enrichment analysis indicated that the targets of miRNAs associated with DM appear to be converging on cell-cycle pathways. CONCLUSIONS: This 4-miRNA signature adds to the prognostic value of the current "gold standard" of TNM staging. In-depth interrogation of these 4-miRNAs will provide important biological insights that could facilitate the discovery and development of novel molecularly targeted therapies to improve outcome for future NPC patients.

Integrated omic analysis of oropharyngeal carcinomas reveals human papillomavirus (HPV)-dependent regulation of the activator protein 1 (AP-1) pathway.

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the ß1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.

Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands.

Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.

Novel PRKD gene rearrangements and variant fusions in cribriform adenocarcinoma of salivary gland origin.

Polymorphous low-grade adenocarcinoma (PLGA) and cribriform adenocarcinoma of minor salivary gland (CAMSG) are low-grade carcinomas arising most often in oral cavity and oropharynx, respectively. Controversy exists as to whether these tumors represent separate entities or variants of one spectrum, as they appear to have significant overlap, but also clinicopathologic differences. As many salivary carcinomas harbor recurrent translocations, paired-end RNA sequencing and FusionSeq data analysis was applied for novel fusion discovery on two CAMSGs and two PLGAs. Validated rearrangements were then screened by fluorescence in situ hybridization (FISH) in 60 cases. Histologic classification was performed without knowledge of fusion status and included: 21 CAMSG, 18 classic PLGA, and 21 with "mixed/indeterminate" features. The RNAseq of 2 CAMSGs showed ARID1A-PRKD1 and DDX3X-PRKD1 fusions, respectively, while no fusion candidates were identified in two PLGAs. FISH for PRKD1 rearrangements identified 11 additional cases (22%), two more showing ARID1A-PRKD1 fusions. As PRKD2 and PRKD3 share similar functions with PRKD1 in the diacylglycerol and protein kinase C signal transduction pathway, we expanded the investigation for these genes by FISH. Six additional cases each showed PRKD2 and PRKD3 rearrangements. Of the 26 (43%) fusion-positive tumors, there were 16 (80%) CAMSGs and 9 (45%) indeterminate cases. A PRKD2 rearrangement was detected in one PLGA (6%). We describe novel and recurrent gene rearrangements in PRKD1-3 primarily in CAMSG, suggesting a possible pathogenetic dichotomy from "classic" PLGA. However, the presence of similar genetic findings in half of the indeterminate cases and a single PLGA suggests a possible shared pathogenesis for these tumor types.

A case report and genetic characterization of a massive acinic cell carcinoma of the parotid with delayed distant metastases.

We describe the presentation, management, and clinical outcome of a massive acinic cell carcinoma of the parotid gland. The primary tumor and blood underwent exome sequencing which revealed deletions in CDKN2A as well as PPP1R13B, which induces p53. A damaging nonsynonymous mutation was noted in EP300, a histone acetylase which plays a role in cellular proliferation. This study provides the first insights into the genetic underpinnings of this cancer. Future large-scale efforts will be necessary to define the mutational landscape of salivary gland malignancies to identify therapeutic targets and biomarkers of treatment failure.

Genome-wide association scan for diabetic nephropathy susceptibility genes in type 1 diabetes.

OBJECTIVE: Despite extensive evidence for genetic susceptibility to diabetic nephropathy, the identification of susceptibility genes and their variants has had limited success. To search for genes that contribute to diabetic nephropathy, a genome-wide association scan was implemented on the Genetics of Kidneys in Diabetes collection. RESEARCH DESIGN AND METHODS: We genotyped approximately 360,000 single nucleotide polymorphisms (SNPs) in 820 case subjects (284 with proteinuria and 536 with end-stage renal disease) and 885 control subjects with type 1 diabetes. Confirmation of implicated SNPs was sought in 1,304 participants of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, a long-term, prospective investigation of the development of diabetes-associated complications. RESULTS: A total of 13 SNPs located in four genomic loci were associated with diabetic nephropathy with P < 1 x 10(-5). The strongest association was at the FRMD3 (4.1 protein ezrin, radixin, moesin [FERM] domain containing 3) locus (odds ratio [OR] = 1.45, P = 5.0 x 10(-7)). A strong association was also identified at the CARS (cysteinyl-tRNA synthetase) locus (OR = 1.36, P = 3.1 x 10(-6)). Associations between both loci and time to onset of diabetic nephropathy were supported in the DCCT/EDIC study (hazard ratio [HR] = 1.33, P = 0.02, and HR = 1.32, P = 0.01, respectively). We demonstratedexpression of both FRMD3 and CARS in human kidney. CONCLUSIONS: We identified genetic associations for susceptibility to diabetic nephropathy at two novel candidate loci near the FRMD3 and CARS genes. Their identification implicates previously unsuspected pathways in the pathogenesis of this important late complication of type 1 diabetes.