Mutation Spectrum of EGFR From 21,324 Chinese Patients With Non-Small Cell Lung Cancer (NSCLC) Successfully Tested by Multiple Methods in a CAP-Accredited Laboratory.

Genotyping epidermal growth factor receptor (EGFR) gene in patients with advanced non-small cell lung cancers (NSCLC) is essential for identifying those patients who may benefit from targeted therapies. Systemically evaluating EGFR mutation detection rates of different methods currently used in clinical setting will provide valuable information to clinicians and laboratory scientists who take care of NSCLC patients. This study retrospectively reviewed the EGFR data obtained in our laboratory in last 10 years. A total of 21,324 NSCLC cases successfully underwent EGFR genotyping for clinical therapeutic purpose, including 5,244 cases tested by Sanger sequencing, 13,329 cases tested by real-time PCR, and 2,751 tested by next-generation sequencing (NGS). The average EGFR mutation rate was 45.1%, with 40.3% identified by Sanger sequencing, 46.5% by real-time PCR and 47.5% by NGS. Of these cases with EGFR mutations identified, 93.3% of them harbored a single EGFR mutation (92.1% with 19del or L858R, and 7.9% with uncommon mutations) and 6.7% harbored complex EGFR mutations. Of the 72 distinct EGFR variants identified in this study, 15 of them (single or complex EGFR mutations) were newly identified in NSCLC. For these cases with EGFR mutations tested by NGS, 65.3% of them also carried tumor-related variants in some non-EGFR genes and about one third of them were considered candidates of targeted drugs. NGS method showed advantages over Sanger sequencing and real-time PCR not only by providing the highest mutation detection rate of EGFR but also by identifying actionable non-EGFR mutations with targeted drugs in clinical setting.

Mechanism of REST/NRSF regulation of clustered protocadherin α genes.

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

Regulation of the protocadherin Celsr3 gene and its role in globus pallidus development and connectivity.

The globus pallidus (GP) is a central component of basal ganglia whose malfunctions cause a variety of neuropsychiatric disorders as well as cognitive impairments in neurodegenerative diseases such as Parkinson's disease. Here we report that the protocadherin gene Celsr3 is regulated by the insulator CCCTC-binding factor (CTCF) and the repressor neuron-restrictive silencer factor (NRSF, also known as REST) and is required for the development and connectivity of GP. Specifically, CTCF/cohesin and NRSF inhibit the expression of Celsr3 through specific binding to its promoter. In addition, we found that the Celsr3 promoter interacts with CTCF/cohesin-occupied neighboring promoters. In Celsr3 knockout mice, we found that the ventral GP is occupied by aberrant calbindin-positive cholinergic neurons ectopic from the nucleus basalis of Meynert. Furthermore, the guidepost cells for thalamocortical axonal development are missing in the caudal GP. Finally, axonal connections of GP with striatum, subthalamic nucleus, substantia nigra, and raphe are compromised. These data reveal the essential role of Celsr3 in GP development in the basal forebrain and shed light on the mechanisms of the axonal defects caused by the Celsr3 deletion.