Using clinical chest CT scans, we have previously identified emphysema-predominant probands in the Boston Early-Onset COPD Study (8
). Without CT scans on all family members, we could not perform genome-wide linkage analysis of CT emphysema; stratified linkage analysis of airflow obstruction phenotypes in families of emphysema-predominant probands served as a surrogate phenotype. We identified a region on chromosome 1p with significantly increased evidence for linkage in the subset of families of the emphysema-predominant probands. This linkage peak from the stratified analysis pointed to a chromosomal region that may be important in emphysema, in addition to the linkage peaks for spirometric traits previously identified in all subjects (16
). In a previous publication, we found no association with GSTM1, a potential candidate gene (4
). In the current study, we have tested two additional candidate genes for emphysema located in the linked region, finding SNP-level replication of the association between two intronic SNPs in TGFBR3 (also known as betaglycan) and COPD phenotypes in three study samples (Boston Early-Onset COPD Study, NETT-NAS and ICGN), with gene-level replication in a fourth study (LHS). A coding SNP (Ser15Phe), or proxy SNPs for this coding SNP, was associated in three of the four populations. In the Boston Early-Onset COPD Study, the ICGN, and the LHS, the main associated phenotype was FEV1
; subjects in NETT-NAS were ascertained based on FEV1
levels, among other criteria. The two intronic SNPs also showed a trend toward association with quantitative emphysema measurements in the subset of ICGN participants who had chest CT scans. In NETT, all subjects had emphysema on chest CT, and in the Boston Early-Onset COPD Study, the association remained significant in the families of emphysema-predominant probands, also pointing to the potential relevance of TGFBR3 as a candidate gene for the emphysema component of COPD. Chest CT scans were not available in the Lung Health Study.
The only reported genome-wide linkage analyses for COPD-related traits have been performed in the Boston Early-Onset COPD Study (16
). However, a genome-wide linkage analysis in 144 families from the Collaborative Study for the Genetics of Asthma found evidence of linkage to asthma on chromosome 1p only in subjects exposed to environmental tobacco smoke (42
), pointing to the potential relevance of genes on chromosome 1p in smoking-related lung diseases. Our study is the first published report of an association between polymorphisms in TGFBR3 and COPD.
Other human and murine genetics studies demonstrate the potential importance of TGFBR3 in COPD and emphysema. In a microarray study of human lung tissue, Golpon and colleagues found reduced expression of TGFBR3 in lungs from five patients with emphysema compared with five normal lungs (43
); this difference was validated by RT-PCR. In a genome-wide linkage analysis in mice, Reinhard and coworkers found significant linkage to lung function on murine chromosome 5, where the orthologous murine gene tgfbr3
is located (44
). Sequencing of the tgfbr3
gene found three nonsynonymous coding variants between the two parental strains. However, Reinhard and colleagues did not describe a SNP corresponding to the human Ser15Phe variant, and a similar mouse variant is not found in the dbSNP database.
The functional effects of the two associated intronic SNPs are unknown. These SNPs may have functional consequences, or more likely, they are in LD with the causal variant(s). The Ser15Phe variant is located in the signal peptide of TGFBR3; the functional consequence of this amino acid change is not known, but it is predicted to be possibly damaging, according to PolyPhen analysis (45
). We demonstrated associations with several SNPs in this large gene (223.5 kb), suggesting that multiple TGFBR3 variants may affect emphysema susceptibility. It also possible that the causal variant or variants are not located in the TGFBR3 gene, and that the associations we found are due to LD with other chromosome 1 genes. However, we did not find associations with two other COPD candidate genes in the region, and TGFBR3 is known to be expressed in human emphysematous lung (43
). Several other lines of evidence have pointed to the importance of the TGF-β pathway in COPD (40
The linkage and association analyses in the Boston Early-Onset COPD Study are limited by the availability of chest CT scans only on the probands; since CT scans were performed for clinical indications at the discretion of the local physician, and were not part of the study protocol, CTs were not available in the family members. Linkage and association analyses for qualitative or quantitative CT traits could not be performed, though we have previously demonstrated that emphysema predominance on CT can be used to define clinically relevant subgroups (8
). We did not find association with emphysema measurements in NETT, though the sample size in NETT with quantitative CT data was limited. In the larger sample from the ICGN study, we were able to find trends for association with quantitative emphysema measurements.
Spurious association due to multiple testing is a concern in genetic association studies (6
). To guard against multiple testing, we used a staged genotyping and replication approach (). First, we screened a limited number of SNPs in TGFBR3 in the Boston Early-Onset COPD Study families and in the NETT-NAS case-control study. Only after identifying a replicated association did we test additional SNPs in that gene. The screening set of ht-SNPs plus additional significant SNPs were then tested in the large ICGN family study. We then compared the results to independent genotyping performed in a separate cohort study of patients with COPD from the LHS. Consistent replication of the results across multiple patient samples, using different study designs, offers strong protection against false positive results (5
). The use of family-based study designs in the Boston Early-Onset COPD Study and the ICGN and the testing of a panel of unlinked SNPs in the NETT-NAS case-control study protect against spurious association due to population stratification.
To refine the heterogeneous syndrome of COPD, we used chest CT scans to define a set of early-onset COPD probands with emphysema-predominant COPD. Genome-wide linkage analysis followed by association testing identified variants in TGFBR3, a biologically plausible candidate gene, to be associated with COPD and with FEV1, an important intermediate phenotype. SNP-level replication was confirmed in follow-up studies comparing emphysema cases from NETT with control smokers and in a large family-based study from the ICGN. The same SNPs were not tested in the LHS, but gene-level replication was found for TGFBR3. COPD is, by definition, a complex disease. There are likely to be some genes relevant for COPD in general and other genes relevant only for COPD subtypes, such as emphysema or airway disease. Genetic studies in patients with COPD defined only on the basis of airflow obstruction may be sufficient to identify the former set of genes. However, more precise characterization of subjects with COPD, using chest CT scans, will be required to find distinct genes for emphysema and airway disease.