RNAseq analysis of bronchial epithelial cells to identify COPD-associated genes and SNPs.

BACKGROUND: There is a need for more powerful methods to identify low-effect SNPs that contribute to hereditary COPD pathogenesis. We hypothesized that SNPs contributing to COPD risk through cis-regulatory effects are enriched in genes comprised by bronchial epithelial cell (BEC) expression patterns associated with COPD. METHODS: To test this hypothesis, normal BEC specimens were obtained by bronchoscopy from 60 subjects: 30 subjects with COPD defined by spirometry (FEV1/FVC < 0.7, FEV1% < 80%), and 30 non-COPD controls. Targeted next generation sequencing was used to measure total and allele-specific expression of 35 genes in genome maintenance (GM) genes pathways linked to COPD pathogenesis, including seven TP53 and CEBP transcription factor family members. Shrinkage linear discriminant analysis (SLDA) was used to identify COPD-classification models. COPD GWAS were queried for putative cis-regulatory SNPs in the targeted genes. RESULTS: On a network basis, TP53 and CEBP transcription factor pathway gene pair network connections, including key DNA repair gene ERCC5, were significantly different in COPD subjects (e.g., Wilcoxon rank sum test for closeness, p-value = 5.0E-11). ERCC5 SNP rs4150275 association with chronic bronchitis was identified in a set of Lung Health Study (LHS) COPD GWAS SNPs restricted to those in putative regulatory regions within the targeted genes, and this association was validated in the COPDgene non-hispanic white (NHW) GWAS. ERCC5 SNP rs4150275 is linked (D' = 1) to ERCC5 SNP rs17655 which displayed differential allelic expression (DAE) in BEC and is an expression quantitative trait locus (eQTL) in lung tissue (p = 3.2E-7). SNPs in linkage (D' = 1) with rs17655 were predicted to alter miRNA binding (rs873601). A classifier model that comprised gene features CAT, CEBPG, GPX1, KEAP1, TP73, and XPA had pooled 10-fold cross-validation receiver operator characteristic area under the curve of 75.4% (95% CI: 66.3%-89.3%). The prevalence of DAE was higher than expected (p = 0.0023) in the classifier genes. CONCLUSIONS: GM genes comprised by COPD-associated BEC expression patterns were enriched for SNPs with cis-regulatory function, including a putative cis-rSNP in ERCC5 that was associated with COPD risk. These findings support additional total and allele-specific expression analysis of gene pathways with high prior likelihood for involvement in COPD pathogenesis.

Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples.

Reliable breakpoint cluster region (BCR)--Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and ß-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 10(3) GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 10(3) GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.