Nick Fishbane

Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

Background The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. Results We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. Conclusions Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

Molecular mechanisms underlying variations in lung function: a systems genetics analysis

BACKGROUND Lung function measures reflect the physiological state of the lung, and are essential to the diagnosis of chronic obstructive pulmonary disease (COPD). The SpiroMeta-CHARGE consortium undertook the largest genome-wide association study (GWAS) so far (n=48,201) for forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FEV1/FVC) in the general population. The lung expression quantitative trait loci (eQTLs) study mapped the genetic architecture of gene expression in lung tissue from 1111 individuals. We used a systems genetics approach to identify single nucleotide polymorphisms (SNPs) associated with lung function that act as eQTLs and change the level of expression of their target genes in lung tissue; termed eSNPs. METHODS The SpiroMeta-CHARGE GWAS results were integrated with lung eQTLs to map eSNPs and the genes and pathways underlying the associations in lung tissue. For comparison, a similar analysis was done in peripheral blood. The lung mRNA expression levels of the eSNP-regulated genes were tested for associations with lung function measures in 727 individuals. Additional analyses identified the pleiotropic effects of eSNPs from the published GWAS catalogue, and mapped enrichment in regulatory regions from the ENCODE project. Finally, the Connectivity Map database was used to identify potential therapeutics in silico that could reverse the COPD lung tissue gene signature. FINDINGS SNPs associated with lung function measures were more likely to be eQTLs and vice versa. The integration mapped the specific genes underlying the GWAS signals in lung tissue. The eSNP-regulated genes were enriched for developmental and inflammatory pathways; by comparison, SNPs associated with lung function that were eQTLs in blood, but not in lung, were only involved in inflammatory pathways. Lung function eSNPs were enriched for regulatory elements and were over-represented among genes showing differential expression during fetal lung development. An mRNA gene expression signature for COPD was identified in lung tissue and compared with the Connectivity Map. This in-silico drug repurposing approach suggested several compounds that reverse the COPD gene expression signature, including a nicotine receptor antagonist. These findings represent novel therapeutic pathways for COPD. INTERPRETATION The system genetics approach identified lung tissue genes driving the variation in lung function and susceptibility to COPD. The identification of these genes and the pathways in which they are enriched is essential to understand the pathophysiology of airway obstruction and to identify novel therapeutic targets and biomarkers for COPD, including drugs that reverse the COPD gene signature in silico. FUNDING The research reported in this article was not specifically funded by any agency. See Acknowledgments for a full list of funders of the lung eQTL study and the Spiro-Meta CHARGE GWAS.

Serum IgG and risk of exacerbations and hospitalizations in chronic obstructive pulmonary disease

Fernando Sergio Leitao Filho, MD, PhD, Seung Won Ra, MD, PhD, Andre Mattman, MD, Robert S. Schellenberg, MD, Nick Fishbane, MSc, Gerard J. Criner, MD, Prescott G. Woodruff, MD, MPH, Stephen C. Lazarus, MD, Richard Albert, MD, John E. Connett, PhD, Meilan K. Han, MD, MSc, Fernando J. Martinez, MD, MSc, Janice M. Leung, MD, S.F. Paul Man, MD, Shawn D. Aaron, MD, Robert M. Reed, MD, Don D. Sin, MD, MPH

Nondestructive cryomicro-CT imaging enables structural and molecular analysis of human lung tissue

Micro-computed tomography (CT) enables three-dimensional (3D) imaging of complex soft tissue structures, but current protocols used to achieve this goal preclude cellular and molecular phenotyping of the tissue. Here we describe a radiolucent cryostage that permits micro-CT imaging of unfixed frozen human lung samples at an isotropic voxel size of (11 µm)3 under conditions where the sample is maintained frozen at -30°C during imaging. The cryostage was tested for thermal stability to maintain samples frozen up to 8 h. This report describes the methods used to choose the materials required for cryostage construction and demonstrates that whole genome mRNA integrity and expression are not compromised by exposure to micro-CT radiation and that the tissue can be used for immunohistochemistry. The new cryostage provides a novel method enabling integration of 3D tissue structure with cellular and molecular analysis to facilitate the identification of molecular determinants of disease. NEW & NOTEWORTHY The described micro-CT cryostage provides a novel way to study the three-dimensional lung structure preserved without the effects of fixatives while enabling subsequent studies of the cellular matrix composition and gene expression. This approach will, for the first time, enable researchers to study structural changes of lung tissues that occur with disease and correlate them with changes in gene or protein signatures.

Integrative Genomics of Emphysema-Associated Genes Reveals Potential Disease Biomarkers

Abstract Chronic obstructive pulmonary disease is the third leading cause of death worldwide. Gene expression profiling across multiple regions of the same lung identified genes significantly related to emphysema. We sought to determine whether the lung and epithelial expression of 127 emphysema‐related genes was also related to lung function in independent cohorts, and whether any of these genes could be used as biomarkers in the peripheral blood of patients with chronic obstructive pulmonary disease. To that end, we examined whether the expression levels of these genes were under genetic control in lung tissue (n = 1,111). We then determined whether the mRNA levels of these genes in lung tissue (n = 727), small airway epithelial cells (n = 238), and peripheral blood (n = 620) were significantly related to lung function measurements. The expression of 63 of the 127 genes (50%) was under genetic control in lung tissue. The lung and epithelial mRNA expression of a subset of the emphysema‐associated genes, including ASRGL1, LPHN2, and EDNRB, was strongly associated with lung function. In peripheral blood, the expression of 40 genes was significantly associated with lung function. Twenty‐nine of these genes (73%) were also associated with lung function in lung tissue, but with the opposite direction of effect for 24 of the 29 genes, including those involved in hypoxia and B cell‐related responses. The integrative genomics approach uncovered a significant overlap of emphysema genes associations with lung function between lung and blood with opposite directions between the two. These results support the use of peripheral blood to detect disease biomarkers.

Surfactant protein D is a causal risk factor for COPD: results of Mendelian randomisation

Surfactant protein D (SP-D) is produced primarily in the lung and is involved in regulating pulmonary surfactants, lipid homeostasis and innate immunity. Circulating SP-D levels in blood are associated with chronic obstructive pulmonary disease (COPD), although causality remains elusive. In 4061 subjects with COPD, we identified genetic variants associated with serum SP-D levels. We then determined whether these variants affected lung tissue gene expression in 1037 individuals. A Mendelian randomisation framework was then applied, whereby serum SP-D-associated variants were tested for association with COPD risk in 11 157 cases and 36 699 controls and with 11 years decline of lung function in the 4061 individuals. Three regions on chromosomes 6 (human leukocyte antigen region), 10 (SFTPD gene) and 16 (ATP2C2 gene) were associated with serum SP-D levels at genome-wide significance. In Mendelian randomisation analyses, variants associated with increased serum SP-D levels decreased the risk of COPD (estimate −0.19, p=6.46×10−03) and slowed the lung function decline (estimate=0.0038, p=7.68×10−3). Leveraging genetic variation effect on protein, lung gene expression and disease phenotypes provided novel insights into SP-D biology and established a causal link between increased SP-D levels and protection against COPD risk and progression. SP-D represents a very promising biomarker and therapeutic target for COPD. Surfactant protein D is a causal risk factor for COPD http://ow.ly/n1OG30eUQlf

The Effect of Statins on Blood Gene Expression in COPD

Background COPD is currently the fourth leading cause of death worldwide. Statins are lipid lowering agents with documented cardiovascular benefits. Observational studies have shown that statins may have a beneficial role in COPD. The impact of statins on blood gene expression from COPD patients is largely unknown. Objective Identify blood gene signature associated with statin use in COPD patients, and the pathways underpinning this signature that could explain any potential benefits in COPD. Methods Whole blood gene expression was measured on 168 statin users and 451 non-users from the ECLIPSE study using the Affymetrix Human Gene 1.1 ST microarray chips. Factor Analysis for Robust Microarray Summarization (FARMS) was used to process the expression data. Differential gene expression analysis was undertaken using the Linear Models for Microarray data (Limma) package adjusting for propensity score and surrogate variables. Similarity of the expression signal with published gene expression profiles was performed in ProfileChaser. Results 25 genes were differentially expressed between statin users and non-users at an FDR of 10%, including LDLR, CXCR2, SC4MOL, FAM108A1, IFI35, FRYL, ABCG1, MYLIP, and DHCR24. The 25 genes were significantly enriched in cholesterol homeostasis and metabolism pathways. The resulting gene signature showed correlation with Huntington’s disease, Parkinson’s disease and acute myeloid leukemia gene signatures. Conclusion The blood gene signature of statins’ use in COPD patients was enriched in cholesterol homeostasis pathways. Further studies are needed to delineate the role of these pathways in lung biology.

Small airways disease in mild and moderate chronic obstructive pulmonary disease: a cross-sectional study

BACKGROUND The concept that small conducting airways less than 2 mm in diameter become the major site of airflow obstruction in chronic obstructive pulmonary disease (COPD) is well established in the scientific literature, and the last generation of small conducting airways, terminal bronchioles, are known to be destroyed in patients with very severe COPD. We aimed to determine whether destruction of the terminal and transitional bronchioles (the first generation of respiratory airways) occurs before, or in parallel with, emphysematous tissue destruction. METHODS In this cross-sectional analysis, we applied a novel multiresolution CT imaging protocol to tissue samples obtained using a systematic uniform sampling method to obtain representative unbiased samples of the whole lung or lobe of smokers with normal lung function (controls) and patients with mild COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1), moderate COPD (GOLD 2), or very severe COPD (GOLD 4). Patients with GOLD 1 or GOLD 2 COPD and smokers with normal lung function had undergone lobectomy and pneumonectomy, and patients with GOLD 4 COPD had undergone lung transplantation. Lung tissue samples were used for stereological assessment of the number and morphology of terminal and transitional bronchioles, airspace size (mean linear intercept), and alveolar surface area. FINDINGS Of the 34 patients included in this study, ten were controls (smokers with normal lung function), ten patients had GOLD 1 COPD, eight had GOLD 2 COPD, and six had GOLD 4 COPD with centrilobular emphysema. The 34 lung specimens provided 262 lung samples. Compared with control smokers, the number of terminal bronchioles decreased by 40% in patients with GOLD 1 COPD (p=0·014) and 43% in patients with GOLD 2 COPD (p=0·036), the number of transitional bronchioles decreased by 56% in patients with GOLD 1 COPD (p=0·0001) and 59% in patients with GOLD 2 COPD (p=0·0001), and alveolar surface area decreased by 33% in patients with GOLD 1 COPD (p=0·019) and 45% in patients with GOLD 2 COPD (p=0·0021). These pathological changes were found to correlate with lung function decline. We also showed significant loss of terminal and transitional bronchioles in lung samples from patients with GOLD 1 or GOLD 2 COPD that had a normal alveolar surface area. Remaining small airways were found to have thickened walls and narrowed lumens, which become more obstructed with increasing COPD GOLD stage. INTERPRETATION These data show that small airways disease is a pathological feature in mild and moderate COPD. Importantly, this study emphasises that early intervention for disease modification might be required by patients with mild or moderate COPD. FUNDING Canadian Institutes of Health Research.

Longitudinal study of surrogate aging measures during human immunodeficiency virus seroconversion

Persons living with human immunodeficiency virus (HIV) harbor an increased risk of age-related conditions. We measured changes in telomere length and DNA methylation in the peripheral blood of 31 intravenous drug users, who were followed longitudinally with blood samples pre-HIV (T1), immediately post-HIV (T2; 1.9±1 year from T1), and at a later follow-up time (T3; 2.2±1 year from T2). Absolute telomere length measurements were performed using polymerase chain reaction methods. Methylation profiles were obtained using the Illumina Human Methylation450 platform. Methylation aging was assessed using the Horvath method. Telomere length significantly decreased between T1 and T2 (227±46 at T1 vs. 201±48 kbp/genome at T2, p=0.045), while no differences were observed between T2 and T3 (201±48 at T2 vs. 186±27 kbp/genome at T3, p=0.244). Methylation aging as measured by the age acceleration residual increased over the time course of HIV infection (p=0.035). CpG sites corresponding to PCBP2 and CSRNP1 were differentially methylated between T1 and T2 at a q-value <0.05. Telomere shortening and methylation changes can therefore be observed in the short-term period immediately following HIV seroconversion. Further studies to confirm these results in larger sample sizes and to compare these results to non-HIV and non-injection drug users are warranted.

Relationship of Absolute Telomere Length With Quality of Life, Exacerbations, and Mortality in COPD

Background COPD is an age‐related disease. The role of cellular senescence in COPD has not been fully elucidated. This study examined the relationship between telomere length of peripheral blood leukocytes and clinical outcomes, including health status, rate of exacerbations, and risk of mortality in individuals with COPD. Methods Using quantitative polymerase chain reaction, we measured the absolute telomere length (aTL) of DNA extracted from blood samples of 576 participants with moderate‐to‐severe COPD treated with either azithromycin or placebo for 12 months in the Macrolide Azithromycin for Prevention of Exacerbations of COPD (MACRO) study. All participants were followed for approximately 13 months, during which time health status and exacerbations were carefully ascertained, and an additional 29 months for mortality. The rates of exacerbation and mortality were determined by dividing the aTL into two groups using the median value as the cutoff. Results Participants with shorter telomere length had worse health status defined by higher St. George’s Respiratory Questionnaire scores (&bgr; = −0.09, P = .034). In the placebo arm of the study, the rate of exacerbation (rate ratio, 1.50; 95% CI, 1.16‐1.95; P = .002) and the risk of mortality (hazard ratio, 9.45; 95% CI, 2.85‐31.36; P = .015) were significantly higher in the shorter telomere group than in the longer telomere group; these differences were not observed in the azithromycin arm (interaction P = .008 for exacerbation and interaction P = .017 for mortality) Conclusions These data suggest that replicative senescence may help to predict poor outcomes in COPD. Shorter leukocyte telomere lengths may represent a clinically translatable biomarker for identifying individuals at increased risk of poor clinical outcomes in COPD.