Using a dense set of SNPs to fine map a region on chromosome 12p linked to COPD, we identified SOX5 as a candidate gene for COPD. The association was discovered in the NETT-NAS case-control study and replicated in the family-based BEOCOPD study. This combined analysis remained significant with a strict multiple testing correction. The SNP association could not be confirmed in a third study, the ICGN. However, there was a trend for association with earlier-onset COPD in the ICGN. SOX5 gene expression was reduced, and significantly correlated with lung function, in the lung tissue of some subjects with COPD. A Sox5 null mouse model demonstrated abnormalities in embryonic lung development and reduced expression of fibronectin, an extracellular matrix component previously shown to be critical for lung morphogenesis. Lungs of Sox5+/− heterozygous mice showed less severe developmental abnormalities, which may be more comparable to lung disease in humans carrying SOX5 variants than is the extreme phenotype of Sox5−/− mice.
There are no previous animal or human reports on the role of SOX5 in lung disease. There are two major isoforms of SOX5 (24
). The short isoform is expressed primarily in testis, whereas the long isoform is expressed in many tissues, including lung (24
). In mouse models, Sox5 has been primarily studied in relation to cartilage (19
) and nervous system development (26
). Smits and colleagues observed that Sox5−/−
mice died at birth from respiratory distress, with a cleft palate and abnormalities of the bony thoracic cage (19
). Developmental abnormalities in the lungs of Sox5−/−
mice had not been previously described; thus, it was unknown if lethality was due to defects in lung development. The lungs of newborn Sox5−/−
mice appeared severely affected, demonstrating a premature (canalicular rather than saccular) structure (data not shown). To determine if these abnormalities contributed to neonatal death, we studied embryonic lung development in these mice. We observed a profound and consistent deficiency in lung development in embryonic Sox5−/−
mutant lungs. It is possible that abnormalities in rib cage and/or palate formation contributed to the developmental lung defects in these mice. Prior animal models have demonstrated associations between cartilage/bone formation and lung development (27
We studied molecular changes in the lungs of Sox5−/− mice, but found no significant differences in the expression of markers for cell proliferation or cell lineage at the steady-state mRNA level. Sox5 is known to regulate the expression of many extracellular matrix genes. Such a process could contribute, at least in part, to the defects in lung development observed in mutant mice. We found a significant reduction in the expression of fibronectin, but not other extracellular matrix molecules. This is of particular interest as fibronectin is specifically necessary for branching morphogenesis. Interestingly, fibronectin expression was intermediately affected in Sox5+/− heterozygous lungs consistent with the mild to moderate histological abnormalities in the developing lungs of these mice. Further in vivo and in vitro studies will be required to define whether changes in fibronectin expression explain the phenotype in these mice and to more completely determine the specific contributions of Sox5 to mammalian lung development.
The animal model demonstrated the importance of Sox5 in normal mouse lung development. It is plausible that events in early human lung development may eventually predispose to COPD (29
). Abnormalities in lung development may reduce the maximally attained level of lung function, predisposing to reduced lung function in adulthood, according to the classic Fletcher and Peto diagram (31
) and supported by more recent longitudinal studies (32
). Additionally, lungs with subtle developmental abnormalities, which may not be clinically relevant in childhood, may have an increased susceptibility to the damaging effects of exposures such as cigarette smoke (30
). Similarly, functional variants of developmentally important genes may be less capable of contributing to injury-repair processes necessary for lung maintenance. Several genes have been identified that may have effects on lung development as well as COPD susceptibility, including members of the TGF-β signaling pathway (34
). For example, mice genetically deficient in latent TGF-β binding protein-4 (ltpb4) have emphysematous changes present at birth (38
); our group has shown that genetic variants in LTBP4 may influence COPD-related phenotypes (39
Recently, variants in SOX5 have been associated with a variety of human traits through GWAS. None of these studies has focused on musculoskeletal or lung diseases. In GWAS, SNPs in SOX5 have been associated with rapid progression of AIDS (40
), electrocardiographic P-R interval (41
), triglyceride levels in subjects taking statins (42
), and metabolic side effects of antipsychotic drugs, specifically high-density lipoprotein levels in patients taking perphenazine (43
). Most of these associations have been confined to the 5′ end of the gene, whereas the SNP we identified to be associated with COPD was located 3′ to the SOX5 transcript. SOX5 SNPs have also been associated with pharmacogenetic effects of bupropion for smoking cessation, a potentially relevant phenotype for COPD (44
). However, the specific associated SNPs were not detailed.
In the present study, the most strongly associated SOX5 SNP in NETT-NAS (rs11046966) was also strongly associated with COPD in the BEOCOPD study. We were unable to replicate the associations in a third population, the ICGN. The genetic associations in the first two populations and the animal model both point to the potential importance of SOX5 in COPD, yet rs11046966 may not be the functional variant. SNP rs11046966 is located more than 7 kb downstream from the 3′ UTR of SOX5, so it is unlikely to affect an miRNA binding site. Despite sequencing the exons of SOX5 and highly conserved regions in the 3′ end of the gene, we were unable to identify a putative functional variant. SNP rs11046966 is located in a conserved region that extends from the 3′ end of SOX5. According to MAPPER analysis (45
), rs11046966 is located within a predicted binding sequence for interferon response factor. Based on SNP data in whites from phase 3 of the International HapMap project (46
), rs11046966 is in moderate linkage disequilibrium (r2
> 0.66) with additional SNPs 3′ to the transcript and with intronic SNPs (Table E3), but the roles of these SNPs in COPD are unknown as well. However, these SNPs do point to SOX5 as the relevant gene in the linkage region, justifying the studies in the Sox5 null mouse model. Subtle differences in the linkage disequilibrium patterns between rs11046966 and the functional variant comparing the two U.S. populations (NETT-NAS and BEOCOPD) to the combined North American and European subjects in the ICGN may account for the lack of association in the latter study. Differences in COPD severity across the study populations may also explain the nonreplication in ICGN (Figure E1).
In summary, using a systematic fine mapping approach, we identified genetic associations between a variant near SOX5 and COPD in two of the three populations tested. SOX5 gene expression was reduced in lung tissue from patients with COPD in one of two populations tested. A Sox5 null mouse demonstrated abnormal lung development, and heterozygous animals showed an intermediate severity phenotype. However, the specific role of SOX5 as it relates to human COPD is not clear. For example, we do not know whether its role in human COPD is confined to lung development or whether it plays a role in lung repair. Future research in the mouse model, such as determining the effects of cigarette smoke exposure in the heterozygous model, and future studies in emphysematous human lung tissue will be required to answer these questions about the role of SOX5 in COPD.