Software-Assisted Manual Review of Clinical Next-Generation Sequencing Data: An Alternative to Routine Sanger Sequencing Confirmation with Equivalent Results in >15,000 Germline DNA Screens.

Clinical genomic tests increasingly use a next-generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In germline DNA screening, failure to correct such errors could have serious consequences for patients, who may follow an unwarranted screening or surgical management path. It has been suggested that routine orthogonal confirmation by Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction (<30% of alternate allele). We evaluated whether an alternative method of confirmation-software-assisted manual call review-performed comparably with Sanger confirmation in >15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch, sample, and variant levels, including raw NGS read pileups. Of ambiguous variant calls with <30% allele fraction (1707 total calls at 38 unique sites), manual call review classified >99% (n = 1701) as true positives (enriched for long insertions or deletions and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining <1% (n = 6) being mosaic. Critically, results from software-assisted manual review and retrospective Sanger sequencing were concordant for samples selected from all ambiguous sites. We conclude that the confirmation required for high confidence in NGS-based germline testing can manifest in different ways; a trained NGS expert operating platform-tailored review software achieves quality comparable with routine Sanger confirmation.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

A multicenter, cross-platform clinical validation study of cancer cytogenomic arrays.

Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.

Genomic imbalances in benign metastasizing leiomyoma: characterization by conventional karyotypic, fluorescence in situ hybridization, and whole genome SNP array analysis.

Benign metastasizing leiomyoma, a rare condition of controversial origin, is characterized by the occurrence of extrauterine smooth muscle tumors primarily affecting the lungs of women with a history of uterine leiomyomas. Numerous genetic studies of uterine leiomyoma with rearrangements of the HMGA2 and HMGA1 loci defined in prominent subgroups have been conducted. In contrast, cytogenetic and molecular descriptions of benign metastasizing leiomyoma are few, and, in particular, this entity has not been previously subjected to single nucleotide polymorphism (SNP) array analysis. In this study, conventional karyotypic, and/or molecular cytogenetic, and SNP array characterization of a pleuropulmonary benign mestasizing leiomyoma and a synchronous deep soft tissue leiomyoma of the thigh, which arose in a 56-year-old female with a remote history of uterine leiomyomata, revealed rearrangement of the HMGA1 (6p21) locus and nearly identical genomic profiles, including loss of chromosome 7 material in both lesions. These findings suggest that both the deep soft tissue and pleuropulmonary lesions were derived from the same abnormal clone and are genetically related to uterine leiomyomata.

A common 8q (MYC) amplification detected in a multifocal anaplastic astrocytoma by SNP array karyotyping.

The distinction of multifocal versus multicentric gliomas can conceivably have important therapeutic implications. We present a 27-year-old man with two radiologically distinct non-enhancing infiltrative masses in the anterior frontal lobe and the posterior temporoparietal region. No intervening disease was evident on MRI modalities; the lesions were stable over a period of many months. He underwent two separate resections a few months apart. Given the question of whether his tumors represented two de novo primary multicentric tumors or one multifocal tumor, single nucleotide polymorphism (SNP) array karyotyping and in situ hybridization studies were performed on both tumors. The two tumor profiles looked remarkably similar, histologically and genetically: both were anaplastic astrocytomas with a common 33Mb gain/ amplification of 8q23.3-q24.3, including MYC amplification, suggesting a monoclonal origin. The temporoparietal neoplasm showed several additional genetic alterations. This case illustrates that even with today's advanced neuroimaging modalities, extensive radiologically invisible tumor may be present between seemingly separate sites of glioma involvement. Thus modern global genomic studies of such tumors may help distinguish whether multiple tumors represent one extensive neoplasm with microscopically invasive disease or multiple genetically distinct tumors.

Reproducibility and performance of virtual karyotyping with SNP microarrays for the detection of chromosomal imbalances in formalin-fixed paraffin-embedded tissues.

BACKGROUND: Chromosomal imbalances are commonly seen in cancer and inherited genetic diseases. These imbalances may assist in the diagnosis, prognosis, and/or therapeutic management of certain neoplasms. Several methods for detecting chromosomal imbalances, such as, fluorescent in situ hybridization, array comparative genomic hybridization, and single nucleotide polymorphism (SNP) arrays have proven useful in formalin-fixed paraffin-embedded (FFPE) tissues. Here, we report the performance and reproducibility of virtual karyotyping of FFPE tissues with Affymetrix SNP arrays. METHODS: Virtual karyotypes from 442 FFPE tumor samples were generated using the Affymetrix GeneChip Mapping 10K Xba 2.0 and/or 250K Nsp SNP mapping arrays. Samples ranged from a few weeks to 17 years in archival storage. Virtual karyotypes were assessed for copy number changes, loss of heterozygosity, and acquired uniparental disomy. RESULTS: Overall, 75.3% of samples produced interpretable virtual karyotypes with the 10K arrays and 76.7% in the 250K arrays. Parameters for the selection of samples for hybridization were determined, which increased the success rate in both platforms to 81.3 and 92.6%, respectively. FFPE virtual karyotypes generated with both 10K Xba 2.0 and 250K Nsp arrays showed 100% concordance in intralaboratory and interlaboratory reproducibility studies. Samples older than 7 years showed decreased performance. CONCLUSIONS: SNP arrays are a reliable, reproducible, and robust platform for the virtual karyotyping of FFPE tumor tissues with performance characteristics adequate for clinical application. Parameters that most significantly affected sample performance were sample age and storage conditions.

Papillary renal cell-like carcinoma in a retroperitoneal teratoma.

We report a case of somatic type malignancy with papillary renal cell carcinoma differentiation arising in a retroperitoneal mixed germ cell tumor. The patient was a 36-year-old man with a synchronous mediastinal teratoma. The somatic type malignancy in the retroperitoneal tumor was composed of papillary structures covered by atypical epithelial cells with eosinophilic cytoplasm, prominent nucleoli and pseudostratified nuclei. Papillary cores contained numerous aggregates of foamy macrophages, typical of type I papillary renal cell carcinomas. The immunohistochemical profile was consistent with papillary renal cell carcinoma, including positive reactions for cytokeratin 7 and alpha-methyl acyl CoA racemase. There was no somatic type malignancy component in the synchronous mediastinal teratoma. Both the retroperitoneal and the mediastinal tumor showed gains of 12p and chromosome 17 material. There was no c-MET mutation in the somatic type malignancy. To our knowledge, this is the first report of a somatic type malignancy with features of papillary renal cell carcinoma arising in a germ cell tumor. It is important not to confuse such a retroperitoneal tumor with a conventional papillary renal cell carcinoma, because presence of other malignant histologies within the germ cell tumor may warrant different treatment. In such cases, the presence of isochromosome 12p can be helpful to the diagnosis.

Array-based karyotyping for prognostic assessment in chronic lymphocytic leukemia: performance comparison of Affymetrix 10K2.0, 250K Nsp, and SNP6.0 arrays.

Specific chromosomal alterations are recognized as important prognostic factors in chronic lymphocytic leukemia (CLL). Array-based karyotyping is gaining acceptance as an alternative to the standard fluorescence in situ hybridization (FISH) panel for detecting these aberrations. This study explores the optimum single nucleotide polymorphism (SNP) array probe density for routine clinical use, presents clinical validation results for the 250K Nsp Affymetrix SNP array, and highlights clinically actionable genetic lesions missed by FISH and conventional cytogenetics. CLL samples were processed on low (10K2.0), medium (250K Nsp), and high (SNP6.0) probe density Affymetrix SNP arrays. Break point definition and detection rates for clinically relevant genetic lesions were compared. The 250K Nsp array was subsequently validated for routine clinical use and demonstrated 98.5% concordance with the standard CLL FISH panel. SNP array karyotyping detected genomic complexity and/or acquired uniparental disomy not detected by the FISH panel. In particular, a region of acquired uniparental disomy on 17p was shown to harbor two mutated copies of TP53 that would have gone undetected by FISH, conventional cytogenetics, or array comparative genomic hybridization. SNP array karyotyping allows genome-wide, high resolution detection of copy number and uniparental disomy at genomic regions with established prognostic significance in CLL, detects lesions missed by FISH, and provides insight into gene dosage at these loci.

Role of GPR81 in lactate-mediated reduction of adipose lipolysis.

Heavy exercise or oxygen deficit often links with higher levels of arterial lactate and lower levels of plasma free fatty acids (FFA). Treatment with lactate reduces circulating levels of FFA in vivo and lipolysis in adipose tissues in vitro. However, the underlying mechanism has remained unclear. Here we employ pharmacological and genetic approaches to show that GPR81, an orphan G-protein-coupled receptor with relatively restricted expression in the adipose tissues, functions as a receptor for lactate and can mediate an anti-lipolytic effect of lactate. GPR81 may thus function as a sensor of lactate that can modulate the FFA pool under exercise or conditions of oxygen deficit.

Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion.

Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.

Deficiency of adiponectin receptor 2 reduces diet-induced insulin resistance but promotes type 2 diabetes.

Adiponectin/adiponectin receptors (AdipoR) are involved in energy homeostasis and inflammatory pathways. To investigate the role of AdipoR2 in metabolic control, we studied the lipid and glucose metabolic phenotypes in AdipoR2-deficient mice. AdipoR2 deletion diminished high-fat diet-induced dyslipidemia and insulin resistance yet deteriorated glucose homeostasis as high-fat feeding continued, which resulted from the failure of pancreatic beta-cells to adequately compensate for the moderate insulin resistance. A defect in the AdipoR2 gene may represent a mechanism underlying the etiology of certain subgroups of type 2 diabetic patients who eventually develop overt diabetes, whereas other obese patients do not.

An integrative genomics approach to infer causal associations between gene expression and disease.

A key goal of biomedical research is to elucidate the complex network of gene interactions underlying complex traits such as common human diseases. Here we detail a multistep procedure for identifying potential key drivers of complex traits that integrates DNA-variation and gene-expression data with other complex trait data in segregating mouse populations. Ordering gene expression traits relative to one another and relative to other complex traits is achieved by systematically testing whether variations in DNA that lead to variations in relative transcript abundances statistically support an independent, causative or reactive function relative to the complex traits under consideration. We show that this approach can predict transcriptional responses to single gene-perturbation experiments using gene-expression data in the context of a segregating mouse population. We also demonstrate the utility of this approach by identifying and experimentally validating the involvement of three new genes in susceptibility to obesity.

Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior.

The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels (NaV1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes NaV1.9. Our results confirm that NaV1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of NaV1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.