Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the U.S, is strongly influenced by cigarette smoking and genetic predisposition. The primary objectives of this proposal are to identify functional genetic variants in two COPD genome-wide association (GWAS) loci, HHIP and FAM13A regions, and to define the molecular mechanisms by which these functional variants alter gene expression and cellular phenotypes relevant to COPD. We hypothesize that functional variants in these two loci are regulatory variants that can be measured by quantifying enhancer activity in human bronchial epithelial cells. Furthermore, we hypothesize that functional variants, through differential bindin to specific transcription factors, regulate HHIP or FAM13A gene expression, thus affecting cellular sensitivity to smoke-induced cell injury. To test these hypotheses, in the R21 phase of the project, we will apply recently developed massively parallel reporter assays (MPRA) to identify functional variants that show allele-specific enhancer activity in Beas-2B cells exposed to either room air or cigarette smoke (CS), followed by validation in reporter assays. We will also confirm allele- specific open chromatin in primary human bronchial epithelial cells (HBE) using FAIRE, and we will assess long-range interactions of identified regulatory elements with the relevant promoters using chromosome conformation capture assays. In the R33 phase of the project, we will determine the role of the functional variants in smoke-induced cell death in both Beas-2B cells and HBE. Subsequently, we will apply an siRNAs library targeting 1529 known human transcription factors (TFs) to identify TFs that bind to the identified enhancer elements with differential affinity to genetic variants (by EMSA and ChIP), alter enhancer activity (using co-transfection and reporter assays), and regulate HHIP or FAM13A expression (by RT-PCR following gene silencing) in Beas-2B cells. To demonstrate the clinical relevance of these mechanistic studies, we will perform in vivo validation in HBE cells from smokers with established COPD vs. smoking controls. Ultimately, these functional characterizations will identify biological pathways with potential therapeutic implications.

Public Health Relevance

Chronic obstructive pulmonary disease (COPD) is a major public health problem which is strongly influenced by cigarette smoking and genetic predisposition. The primary objectives of this proposal are to identify functional genetic variants in two COPD genome-wide association (GWAS) loci, HHIP and FAM13A, and to define the molecular mechanisms by which these functional variants regulate gene expression and predefine cellular sensitivity to smoke-induced injury. Identification and investigation of these genetic determinants may lead to new insights into the biological mechanisms causing COPD and suggest new pathways for treatment. (End of Abstract)

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33HL120794-04
Application #
9230434
Study Section
Special Emphasis Panel (ZHL1-CSR-H (S1))
Program Officer
Gan, Weiniu
Project Start
2014-03-01
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
4
Fiscal Year
2017
Total Cost
$529,538
Indirect Cost
$231,207
Name
Brigham and Women's Hospital
Department
Type
Independent Hospitals
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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