A Bayesian framework for efficient and accurate variant prediction.

There is a growing need to develop variant prediction tools capable of assessing a wide spectrum of evidence. We present a Bayesian framework that involves aggregating pathogenicity data across multiple in silico scores on a gene-by-gene basis and multiple evidence statistics in both quantitative and qualitative forms, and performs 5-tiered variant classification based on the resulting probability credible interval. When evaluated in 1,161 missense variants, our gene-specific in silico model-based meta-predictor yielded an area under the curve (AUC) of 96.0% and outperformed all other in silico predictors. Multifactorial model analysis incorporating all available evidence yielded 99.7% AUC, with 22.8% predicted as variants of uncertain significance (VUS). Use of only 3 auto-computed evidence statistics yielded 98.6% AUC with 56.0% predicted as VUS, which represented sufficient accuracy to rapidly assign a significant portion of VUS to clinically meaningful classifications. Collectively, our findings support the use of this framework to conduct large-scale variant prioritization using in silico predictors followed by variant prediction and classification with a high degree of predictive accuracy.

TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome.

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2, MSH6, MLH1, PMS2 and EPCAM. Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

Sanger Confirmation Is Required to Achieve Optimal Sensitivity and Specificity in Next-Generation Sequencing Panel Testing.

Next-generation sequencing (NGS) has rapidly replaced Sanger sequencing as the method of choice for diagnostic gene-panel testing. For hereditary-cancer testing, the technical sensitivity and specificity of the assay are paramount as clinicians use results to make important clinical management and treatment decisions. There is significant debate within the diagnostics community regarding the necessity of confirming NGS variant calls by Sanger sequencing, considering that numerous laboratories report having 100% specificity from the NGS data alone. Here we report our results from 20,000 hereditary-cancer NGS panels spanning 47 genes, in which all 7845 nonpolymorphic variants were Sanger- sequenced. Of these, 98.7% were concordant between NGS and Sanger sequencing and 1.3% were identified as NGS false-positives, located mainly in complex genomic regions (A/T-rich regions, G/C-rich regions, homopolymer stretches, and pseudogene regions). Simulating a false-positive rate of zero by adjusting the variant-calling quality-score thresholds decreased the sensitivity of the assay from 100% to 97.8%, resulting in the missed detection of 176 Sanger-confirmed variants, the majority in complex genomic regions (n = 114) and mosaic mutations (n = 7). The data illustrate the importance of setting quality thresholds for panel testing only after thousands of samples have been processed and the necessity of Sanger confirmation of NGS variants to maintain the highest possible sensitivity.

Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection.

The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants.

Mutations in RASA1 and GDF2 identified in patients with clinical features of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder caused by mutations in ENG, ACVRL1 and SMAD4, which function in regulating the transforming growth factor beta and bone morphogenetic protein signaling pathways. Symptoms of HHT can be present in individuals who test negative for mutations in these three genes indicating other genes may be involved. In this study, we tested for mutations in two genes, RASA1 and GDF2, which were recently reported to be involved in vascular disorders. To determine whether RASA1 and GDF2 have phenotypic overlap with HHT and should be included in diagnostic testing, we developed a next-generation sequencing assay to detect mutations in 93 unrelated individuals who previously tested negative for mutations in ENG, ACVRL1 and SMAD4, but were clinically suspected to have HHT. Pathogenic mutations in RASA1 were identified in two samples (2.15%) and a variant of unknown significance in GDF2 was detected in one sample. All three individuals experienced epistaxis with dermal lesions described in medical records as telangiectases. These results indicate that the inclusion of RASA1 and GDF2 screening in individuals suspected to have HHT will increase the detection rate and aid clinicians in making an accurate diagnosis.

The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay.

Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS) has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH) to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.

High-Resolution Genomic Profiling of Chromosomal Abnormalities in Human Stem Cells Using the 135 K StemArray.

Culturing stem cells for an extended period of time can lead to acquired chromosomal aberrations. Determining the copy number variant (CNV) profile of stem cell lines is critical since CNVs can have dramatic effects on gene expression and tumorigenic potential. Here, we describe an improved version of our StemArray, a stem-cell-focused comparative genomic hybridization (aCGH) microarray, which contains 135,000 probes and covers over 270 stem cell and cancer related genes at the exon level. We have dramatically increased the median probe spacing throughout the genome in order to obtain a higher resolution genetic profile of the cell lines. To illustrate the importance of using the StemArray, we describe a karyotypically normal iPSC line in which we detected acquired chromosomal variations that could affect the cellular phenotype of the cells. Identifying adaptive chromosomal aberrations in stem cell lines is essential if they are to be used in regenerative medicine.

Rapid detection of the ACMG/ACOG-recommended 23 CFTR disease-causing mutations using ion torrent semiconductor sequencing.

Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49-98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput general-population carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.

Array-comparative genomic hybridization characterization of human pluripotent stem cells.

During culture adaptation, human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) tend to acquire chromosomal aberrations. Generally, stem cell lines are screened for large-scale chromosomal changes using low resolution karyotype analysis. Recent studies characterizing human stem cells using array-comparative genomic hybridization (aCGH) suggests most abnormalities acquired during culture are under the resolution of karyotype analysis and therefore are routinely missed. Here, we describe a custom-designed stem cell focused microarray utilizing 44K probes, with increased resolution in relevant stem cell-associated and cancer-related genes.

High resolution array-CGH characterization of human stem cells using a stem cell focused microarray.

Human embryonic and induced pluripotent stem cells (ESCs, iPSCs) that are cultured for an extended period of time are susceptible to genomic instability. Chromosomal aberrations decrease the reliability and reproducibility of experiments and could deem the cells unusable for therapeutic purposes. The genetic stability of human ESCs and iPSCs is commonly monitored by karyotype analysis. However, this low-resolution technique can only identify large aneuploidies. A reliable, high-resolution technique to detect genomic aberrations at a cost comparable to karyotyping is needed to better characterize stem cell lines. We have designed a stem cell focused array-comparative genomic hybridization microarray that covers the entire genome at high resolution with increased probe coverage in over 60 stem cell associated genes and more than 195 cancer related genes. Several iPSC lines were analyzed using the focused microarray and compared with either karyotyping or a standard Agilent 44K microarray. In addition to the abnormalities detected by these platforms, the custom microarray identified several small duplications spanning stem cell and/or cancer related genes. Scientists using a stem cell focused microarray to characterize their stem cells will be aware of the structural variants present in their cells and be more confident in their experimental results.

ABCB8 mediates doxorubicin resistance in melanoma cells by protecting the mitochondrial genome.

Despite their initial effectiveness in the treatment of melanoma, chemotherapeutic agents are ultimately futile against this most aggressive form of skin cancer, and patients inevitably succumb to the disease. One of the mechanisms by which residual melanoma cells become chemoresistant is via the decreased efficiency of chemotherapeutics through the action of ATP-binding cassette (ABC) proteins that are variably expressed by the tumor cells. The clinical relevance of the ABC transporters in the context of cancer is paramount. Inhibitors of these transporters have been shown to increase the efficacy of standard therapy in experimental systems. Their clinical application requires better understanding of the role individual transporters play in the mechanism and the development of more specific inhibitors with minimal off target effects. ABC transporters in tumor cells have been shown to confer multidrug resistance in many solid tumors. However, their role in melanomas is far from clear. Here, we prospectively identify ABCB8 as a specific and major player in the chemoresistance of several melanoma cell lines. ABCB8 knockdown with shRNA reduced doxorubicin resistance approximately 3- to 4-fold in these cells. Furthermore, we show that this reversal is specific to doxorubicin and not to other commonly used chemotherapeutics. Our results also provide evidence that ABCB8 conferred resistance through the protection of mitochondrial DNA from doxorubicin-induced DNA damage.

Identifying genes differentially expressed between PGCs and ES cells reveals a role for CREB-binding protein in germ cell survival.

Primordial germ cells (PGCs) are the embryonic precursors of the adult gametes. Although restricted in developmental potency, PGCs express many of the same molecular markers as pluripotent embryonic stem (ES) cells and can give rise to embryonal carcinoma (EC) cells, the stem cells of testicular tumors, in vivo. Likewise, when exposed to specific growth factors in vitro PGCs can be converted into pluripotent embryonic germ (EG) cells. Here, we propose that genes differentially expressed between PGCs and ES cells are good candidates for regulating germline development. To identify genes important in regulating germ cell development and mammalian fertility, we performed suppression subtraction hybridization (SSH) between PGCs and ES cells whole gene set. Using this method, we identified the transcriptional coactivator/histone acetyltransferase CREB-binding protein (CBP) as being highly expressed in PGCs compared to ES cells. To elucidate the function of CBP in PGCs, we generated mice with a PGC-specific deletion of CBP. Loss of CBP in PGCs leads to increased apoptosis and subsequent reduction in PGC numbers. These data indicate an essential role for CBP in maintaining normal germ cell development.

Embryonic germ cells: when germ cells become stem cells.

Embryonic germ cells (EGCs) are pluripotent stem cells derived from primordial germ cells (PGCs). PGCs are progenitors of adult gametes, which diverge from the somatic lineage between late embryonic to early fetal development. First derived in the mouse, EGCs have also been derived from human, chicken, and pig. As pluripotent stem cells, EGCs demonstrate long-term self-renewal via clonal expansion in an undifferentiated state, and differentiate in vitro to form embryoid bodies containing cells that represent all three germ layers as well as mixed cell populations of less differentiated progenitors and precursors. This is also demonstrated in vivo by their formation into experimentally induced teratocarcinomas following transplantation. Furthermore, mice, pig, and chicken EGCs have also been shown to contribute to experimentally produced chimeric animals, including germline transmission. Importantly, EGCs demonstrate normal and stable karyotypes as well as normal patterns of genomic imprinting, including X-inactivation. Transplantation studies have begun in a variety of models in hopes of defining their potential use to treat a wide variety of human conditions, including diabetes and urological and neurological disorders.