Extreme Trait Whole-Genome Sequencing Identifies PTPRO as a Novel Candidate Gene in Emphysema with Severe Airflow Obstruction

Abstract

Rationale: Genetic association studies in chronic obstructive pulmonary disease have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. Because rare variation is also likely to contribute to susceptibility, we used whole-genome sequencing of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to chronic obstructive pulmonary disease susceptibility.

Objectives: To identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction.

Methods: We identified heavy smokers that were resistant (n = 65) or susceptible (n = 64) to emphysema with severe airflow obstruction in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We filtered whole-genome sequencing results to include only rare variants and conducted single variant tests, region-based tests across the genome, gene-based tests, and exome-wide tests.

Measurements and main results: We identified several suggestive associations with emphysema with severe airflow obstruction, including a suggestive association of all rare variation in a region within the gene ZNF816 (19q13.41; P = 4.5 × 10^-6), and a suggestive association of nonsynonymous coding rare variation in the gene PTPRO (P = 4.0 × 10^-5). Association of rs61754411, a rare nonsynonymous variant in PTPRO, with emphysema and obstruction was demonstrated in all non-Hispanic white individuals in the Pittsburgh Specialized Center of Clinically Oriented Research cohort. We found that cells containing this variant have decreased signaling in cellular pathways necessary for survival and proliferation.

Conclusions: PTPRO is a novel candidate gene in emphysema with severe airflow obstruction, and rs61754411 is a previously unreported rare variant contributing to emphysema susceptibility. Other suggestive candidate genes, such as ZNF816, are of interest for future studies.

Keywords: chronic obstructive pulmonary disease; emphysema; genetic association studies; whole-genome sequencing.

Figure 1.

Extreme trait whole-genome sequencing study design. (A) Patient selection and quality control resulted in (B) final analysis using three different group-wise tests comparing rare variants or rare nonsynonymous variants in 65 individuals resistant to emphysema and 64 individuals susceptible to emphysema. F-950 = Hounsfield units value less than −950; MAF = minor allele frequency; Pred = predicted; SCCOR = Specialized Center of Clinically Oriented Research; SNP = single-nucleotide polymorphisms.

Figure 2.

Analyzed subcohorts represent clinical extremes in terms of obstruction as measured by (A) percent predicted FEV₁ and (B) F-950 despite having very similar (D) smoking histories (pack-years) at a similar (C) age. Dashed lines represent subcohort means. F-950 = Hounsfield units value less than −950.

Figure 3.

Manhattan plot of 30-kb regions spanning the whole genome. Each dot represents a single 30-kb region and the strength of its association with emphysema is plotted on the y-axis. The top association at 19q31 is labeled. Other top associations are reported in Table 2. The top threshold represents genome-wide significant association (P ≤ 5.4 × 10⁻⁷), and the bottom threshold represents suggestive association (P ≤ 1.1 × 10⁻⁵) after Bonferroni correction.

Figure 4.

Nonsynonymous variants in PTPRO are associated with emphysema. (A) Manhattan plot of exome-wide test taking into consideration only nonsynonymous variation. The top association is the gene PTPRO on chromosome 12, and other top associations are reported in Table 4. The top threshold represents genome-wide significant association, and the bottom threshold represents genome-wide suggestive association after Bonferroni correction. The most prevalent nonsynonymous single-nucleotide polymorphism in this gene was significantly associated with (B) % predicted FEV₁ and (C) F-950 in the entire Pittsburgh Specialized Center of Clinically Oriented Research cohort. F-950 = Hounsfield units value less than −950; PTPRO = protein tyrosine phosphatase, type O.

Figure 5.

PTPRO rs61754411 genotype-specific intracellular signaling necessary for cell survival and proliferation after CSE and EGF treatments of human primary human bronchial epithelial cells. Human bronchial epithelial cells from individuals with no lung diseases bearing the PTPRO rs61754411-CG or -CC genotype (n = 2 of each) were cultured in air–liquid interface and treated with CSE for the times indicated (A–E). (A) Whole-cell lysates from treated cells were blotted for the EGFR subunits ErbB1 and ErbB2 and for the downstream signaling molecules Erk and STAT3. Blots above the blue line are representative of one unique individual, and blots below are representative of the other unique individual. As controls, PTPRO and β-actin were also analyzed. (B–E) Densitometry analysis (ratio of p-Akt to total Akt) was performed on these results. Data are presented as mean ± SEM. These same cell lines were cultured in monolayer and treated with recombinant human EGF for the times indicated (F–I). (F) Whole-cell lysates from treated cells were blotted for the EGFR subunit ErbB1 and for the downstream signaling molecules Erk and STAT3. Similarly, PTPRO and GAPDH were also analyzed as controls. (G–I) Densitometry analysis was performed on the blots shown in F for two unique individuals with PTPRO rs61754411-CC and -CG genotypes. Values are presented as a relative ratio of p-Akt to total Akt band intensity compared with those observed in basal, untreated conditions. *P < 0.05. For both treatments, the cells were starved with growth media without growth factor supplements for 4 hours before the treatment in growth media with reduced growth factor supplements (10% of normal culture conditions). Akt = protein kinase B; CSE = cigarette smoke extract; EGF = epidermal growth factor; EGFR = EGF receptor; ErbB1/2 = EGFR/human epidermal growth factor receptor (HER2); Erk = extracellular signal regulated kinase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; p-Akt = phosphorylated Akt; p-Erk = phosphorylated Erk; p-STAT3 = phosphorylated STAT3; PTPRO = protein tyrosine phosphatase, type O; STAT3 = signal transducer and activator of transcription 3.

Figure 5.

PTPRO rs61754411 genotype-specific intracellular signaling necessary for cell survival and proliferation after CSE and EGF treatments of human primary human bronchial epithelial cells. Human bronchial epithelial cells from individuals with no lung diseases bearing the PTPRO rs61754411-CG or -CC genotype (n = 2 of each) were cultured in air–liquid interface and treated with CSE for the times indicated (A–E). (A) Whole-cell lysates from treated cells were blotted for the EGFR subunits ErbB1 and ErbB2 and for the downstream signaling molecules Erk and STAT3. Blots above the blue line are representative of one unique individual, and blots below are representative of the other unique individual. As controls, PTPRO and β-actin were also analyzed. (B–E) Densitometry analysis (ratio of p-Akt to total Akt) was performed on these results. Data are presented as mean ± SEM. These same cell lines were cultured in monolayer and treated with recombinant human EGF for the times indicated (F–I). (F) Whole-cell lysates from treated cells were blotted for the EGFR subunit ErbB1 and for the downstream signaling molecules Erk and STAT3. Similarly, PTPRO and GAPDH were also analyzed as controls. (G–I) Densitometry analysis was performed on the blots shown in F for two unique individuals with PTPRO rs61754411-CC and -CG genotypes. Values are presented as a relative ratio of p-Akt to total Akt band intensity compared with those observed in basal, untreated conditions. *P < 0.05. For both treatments, the cells were starved with growth media without growth factor supplements for 4 hours before the treatment in growth media with reduced growth factor supplements (10% of normal culture conditions). Akt = protein kinase B; CSE = cigarette smoke extract; EGF = epidermal growth factor; EGFR = EGF receptor; ErbB1/2 = EGFR/human epidermal growth factor receptor (HER2); Erk = extracellular signal regulated kinase; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; p-Akt = phosphorylated Akt; p-Erk = phosphorylated Erk; p-STAT3 = phosphorylated STAT3; PTPRO = protein tyrosine phosphatase, type O; STAT3 = signal transducer and activator of transcription 3.