Mosaic structural variation in children with developmental disorders

Delineating the genetic causes of developmental disorders is an area of active investigation. Mosaic structural abnormalities, defined as copy number or loss of heterozygosity events that are large and present in only a subset of cells, have been detected in 0.2–1.0% of children ascertained for clinical genetic testing. However, the frequency among healthy children in the community is not well characterized, which, if known, could inform better interpretation of the pathogenic burden of this mutational category in children with developmental disorders. In a case–control analysis, we compared the rate of large-scale mosaicism between 1303 children with developmental disorders and 5094 children lacking developmental disorders, using an analytical pipeline we developed, and identified a substantial enrichment in cases (odds ratio = 39.4, P-value 1.073e − 6). A meta-analysis that included frequency estimates among an additional 7000 children with congenital diseases yielded an even stronger statistical enrichment (P-value 1.784e − 11). In addition, to maximize the detection of low-clonality events in probands, we applied a trio-based mosaic detection algorithm, which detected two additional events in probands, including an individual with genome-wide suspected chimerism. In total, we detected 12 structural mosaic abnormalities among 1303 children (0.9%). Given the burden of mosaicism detected in cases, we suspected that many of the events detected in probands were pathogenic. Scrutiny of the genotypic–phenotypic relationship of each detected variant assessed that the majority of events are very likely pathogenic. This work quantifies the burden of structural mosaicism as a cause of developmental disorders

Rational design of novel transcriptional regulators

The lac repressor is a transcriptional regulator found in E.coli that monitors the levels of available lactose and adjusts expression of the genes involved in lactose utilization accordingly. Since its discovery, the lac operon has served as a model system for exploring the molecular mechanisms of gene regulation, protein-DNA recognition and allosteric signaling. In recent years, the molecular switch has become a valuable tool for regulating expression in a number of bacterial and eukaryotic systems. With such widespread application of the lac repressor, we saw the need and opportunity to design the next generation of transcriptional regulators. In this dissertation, we have used directed evolution to create repressors which improve upon the wild type system by reducing the leakiness of the switch. A series of novel repressors have also been developed which are capable of repressing non-classical operator sites. These repressors heterodimerize and allow two different DNA binding domains to function together in recognizing asymmetric operators. Finally, a series of repressors have been created that contain altered effector specificity. Several of these repressors induce with a previously neutral effector, ONPG, while other repressors function in the reverse direction by a mechanism of co-repression. In an effort to produce novel switches, we have also discovered facets of the lac operon that are responsible for optimal functionality. We demonstrate that the operator is not a passive component of the molecular switch: it is responsible for establishing binding affinity, specificity, and translational efficiency of the resulting transcript. Using the heterodimeric construct we were also able to determine that binding of two inducers is required for full induction. Finally, determination of crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. These structures suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. This hydrogen bonding can account in part for the different structural conformations of the repressor and is vital for the allosteric transition

Rational design of novel transcriptional regulators

The lac repressor is a transcriptional regulator found in E.coli that monitors the levels of available lactose and adjusts expression of the genes involved in lactose utilization accordingly. Since its discovery, the lac operon has served as a model system for exploring the molecular mechanisms of gene regulation, protein-DNA recognition and allosteric signaling. In recent years, the molecular switch has become a valuable tool for regulating expression in a number of bacterial and eukaryotic systems. With such widespread application of the lac repressor, we saw the need and opportunity to design the next generation of transcriptional regulators. In this dissertation, we have used directed evolution to create repressors which improve upon the wild type system by reducing the leakiness of the switch. A series of novel repressors have also been developed which are capable of repressing non-classical operator sites. These repressors heterodimerize and allow two different DNA binding domains to function together in recognizing asymmetric operators. Finally, a series of repressors have been created that contain altered effector specificity. Several of these repressors induce with a previously neutral effector, ONPG, while other repressors function in the reverse direction by a mechanism of co-repression. In an effort to produce novel switches, we have also discovered facets of the lac operon that are responsible for optimal functionality. We demonstrate that the operator is not a passive component of the molecular switch: it is responsible for establishing binding affinity, specificity, and translational efficiency of the resulting transcript. Using the heterodimeric construct we were also able to determine that binding of two inducers is required for full induction. Finally, determination of crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. These structures suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. This hydrogen bonding can account in part for the different structural conformations of the repressor and is vital for the allosteric transition

Abstract 3428: Validation and utilization of next generation sequencing in the clinical assessment of gliomas

Abstract Background. Identifying prognostic and potentially therapeutic genetic alterations in gliomas is crucial to improve clinical outcome and guide future targeted therapies in this devastating disease. Here, we establish the feasibility of next generation sequencing (NGS) profiling of glioma samples in a clinical setting. Methods. We optimized and validated a 47 gene panel for use on both fresh and Formalin Fixed Paraffin embedded (FFPE) tissue specimens in a CLIA-certified laboratory. Validation was performed in 27 samples by comparing mutation detection on two different NGS platforms, and we have since incorporated routine molecular tumor profiling into the standard workup of glioma patients seen at our institution. Results. From analysis of 120 clinical samples, we found disease-associated changes in 90 patients (75%). Overall 184 changes including disease-associated point mutations and variants of unclear significance were identified across 98 samples, resulting in an average of 1.85 mutations per sample and a range of 1- 6 mutations. This included 49 amplifications across several genes (EGFR, PDGFRA and KIT). Conclusions. The final validated assay allows for a cost effective and efficient analysis of a spectrum of clinically relevant and actionable biomarkers including point mutations, insertions, deletions and gene amplifications. Given the high fraction of tumors presenting with known disease associated changes, routine genomic profiling has the promise of improving patient outcomes and allow for access to targeted therapies. Citation Format: MacLean P. Nasrallah, Maria Martinez-Lage, Alan Fox, Shrey Sukhadia, Arati Desai, Donald M. O'Rourke, Steven Brem, David Roth, Jennifer J.D. Morrissette, Robert D. Daber. Validation and utilization of next generation sequencing in the clinical assessment of gliomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2014-3428</jats:p

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing

<div><p>Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (<i>EGFR</i>, <i>KRAS</i>, <i>BRAF</i>, <i>NPM1</i>, <i>FLT3</i>, and <i>JAK2</i>). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two <i>FLT3</i> internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.</p></div

Next-generation sequencing data analysis pipeline.

<p>Data analysis occurs in three sequential stages, pre-processing of NGS reads, variant calling, and variant annotation. Of note, large indels are detected by an examination of reads that failed to map to target regions of the reference genome and are recovered from a pool of rejected reads (“Trash”). SNVs, single nucleotide variants. CNVs, copy number variation.</p

Tumor Percentage of Specimens Analyzed by Targeted <i>EGFR</i> Tests.

<p>Cases were placed into 5 bins (<5%, 5–10%, 11–25%, 26–50%, >50% tumor cells in specimen).</p

Proposed Workflow for NGS and Single-Gene Assays.

<p>Three main decision points are highlighted. Specimens requiring an urgent turnaround time are routed directly for single-gene testing (possibly followed by NGS). Additionally, single-gene testing is performed on samples with less than 10% tumor or DNA inadequate for NGS (i.e., degraded or low quantity). In samples not meeting any of the above criteria, NGS is performed instead of single-gene testing. NGS results do not require confirmation by single-gene testing.</p