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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. S1-S81, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0071SU

Supplement

Functional Genomics of Lung Disease

Jeffrey A. Whitsett, Cindy J. Bachurski, Kathleen C. Barnes, Paul A. Bunn, Jr., Lisa M. Case, Donald N. Cook, Denise Crooks, Mark W. Duncan, Lori Dwyer-Nield, Robert C. Elston, Michael B. Fessler, Wilbur A. Franklin, Nir Friedman, Joe G. N. Garcia, Mark W. Geraci, Connie Glasgow, Stephan W. Glasser, William D. Hardie, Lisa M. Henning, Gary L. Johnson, Kamon Kawkitinarong, Robert L. Keith, Thomas R. Korfhagen, George D. Leikauf, Stephen B. Liggett, Kenneth C. Malcolm, Alvin M. Malkinson, Thomas J. Mariani, Susan A. McDowell, Dennis W. McGraw, Mario Medvedovic, Joel Moss, Lawrence M. Nogee, Stephanie Nonas, Gustavo Pacheco-Rodriguez, Lyle J. Palmer, David G. Peters, Daniel R. Prows, Susan Redline, Aviv Regev, Maureen A. Sartor, David A. Schwartz, Edwin K. Silverman, Wendy K. Steagall, Robert S. Stearman, Angelo Taveira-DaSilva, Jay W. Tichelaar, Craig R. Tomlinson, Katsuya Tsukada, Timothy E. Weaver, Susan E. Wert, Scott C. Wesselkamper, G. S. Worthen, Yan Xu, Laura Zerbe, Yi Zhang, Yingze Zhang, Augustine M. K. Choi and Naftali Kaminski

CONTENTS

Introduction S3

Surfactant Protein C: Regulation and Interstitial Lung Disease S4

Genes and Gene Polymorphisms Associated with Idiopathic Pulmonary Fibrosis S9

Progress in Chronic Obstructive Pulmonary Disease Genetic Epidemiology S13

Single Nucleotide Polymorphisms, Chips, and Functional Genomics in Acute Lung Injury S18

Resolving Gene–Environment Interactions in Complex Traits: Acute Lung Injury S23

Genetics of Obstructive Sleep Apnea and Related Phenotypes S35

Expression Analysis in Lung Cancer: The Search for Biomarkers S39

Genetics and Gene Expression in Lymphangioleiomyomatosis S45

Genetic Regulation of Innate Immunity: Lessons Learned from TLR4 S48

Regulation of Alveogenesis by Reciprocal Proximodistal Fibroblast Growth Factor and Retinoic Acid Signaling S52

Biochemical Remodeling of Airway Smooth Muscle Relaxation–Contraction by ß2-Adrenergic Receptor Crosstalk S58

New Experimental Approaches to the Signaling of Innate Immunity S62

Digital Gene Expression Profiling by Serial Analysis of Gene Expression S67

Identifying Regulatory Networks: Lessons from Yeast to Humans S72

The Second Pittsburgh Lung Conference: Are We Ready for Pulmonomics? S76

Author Affiliations, Acknowledgments, and Conflict of Interest Statements S79

 

Introduction

This 2004 August Supplement to the American Journal of Respiratory Cell and Molecular Biology highlights the Second Annual Pittsburgh International Lung Conference held on October 12–16, 2003 at the Nemacolin Woodlands Resort outside of Pittsburgh, Pennsylvania. The Second Annual Pittsburgh International Lung Conference focused on the evolving and expanding field of functional genomics in lung disease. A broad range of lung disorders was included for discussion and review, including asthma, chronic obstructive pulmonary disease, lung cancer, idiopathic pulmonary fibrosis, adult respiratory distress syndrome, obstructive sleep apnea, pulmonary hypertension, and lung transplantation. Leaders in the field of gene expression profiling, genetics, proteomics, and bioinformatics convened to discuss the current understanding of the functional genomics in lung diseases, and to prepare the pulmonary community for the expected and unexpected developments in genomics of lung diseases in the future.

From the opening plenary address by Dr. Jeffrey A. Whitsett highlighting the importance of genetics and expression of surfactant-B in lung disorders to Dr. Naftali Kaminski's conference summary, our pulmonary trainees and investigators were treated to an exciting venue. An internationally recognized faculty highlighted the breadth and complexity of this topic in the field of respiratory medicine. The conference also served to highlight future directions for investigators in the field.

Our goal with the Pittsburgh International Lung Conference is to establish a forum for junior and senior lung investigators to isolate themselves, focus on state-of-the-art investigation in a selected area of lung disease, and foster collaborative, collegial, and productive interactions. Akin to the successful First Pittsburgh International Lung Conference on Idiopathic Pulmonary Fibrosis, the Second Pittsburgh International Lung Conference on Functional Genomics of Lung Disease was a huge success! We will try our best once again to achieve this goal with the Third Annual Pittsburgh International Lung Conference on October 16–21, 2004. The focus for this conference will be Adult Respiratory Distress Syndrome/Acute Lung Injury. We welcome all of you to visit our website (http://paccm.upmc.edu) to learn more about the conference and register for attendance.

Augustine M. K. Choi, M.D.

Division of Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania

 

Surfactant Protein C

Regulation and Interstitial Lung Disease

Morphogenesis of the lung begins as an evagination of epithelial cells from the foregut endoderm at E9–9.5 in the mouse embryo. Undifferentiated respiratory epithelial cells proliferate, forming lung tubules that undergo branching morphogenesis to form the conducting airways and peripheral lung saccules. In the latter stages of lung morphogenesis, peripheral lung tubules dilate, forming saccules that further septate during the perinatal period to form the alveolar gas exchange surface of the lung. Lung morphogenesis is controlled by complex autocrine–paracrine interactions between epithelial and mesenchymal cells, which induce transcriptional pathways that direct differentiation and organization of the epithelium to form conducting and peripheral airways. Likewise, signals from the epithelium to the undifferentiated pulmonary mesenchyme direct differentiation of blood vessels, lymphatics, and other stromal components of the lung. Before birth, epithelial cells in the peripheral lung undergo dramatic changes in morphology and gene expression associated with the synthesis of pulmonary surfactant required for transition to air breathing at the time of birth. Identification of the surfactant proteins, their cDNAs and genes have provided molecular reagents and models that have been useful in further understanding of lung morphogenesis and function critical for perinatal survival.

In the present work, we will review the structure, function, and regulation of surfactant protein C (SP-C). Recent studies, both in transgenic mice and in humans, demonstrate the importance of SP-C in surfactant function and alveolar homeostasis. Mutations in the gene encoding SP-C (SFTPC) cause acute and chronic lung disease in infants, children, and adults.

Structure and Function of SP-C

SP-C is an extremely hydrophobic 33– to 34–amino acid polypeptide isolated from surfactant phospholipids obtained by alveolar lavage (1, 2). SP-C mRNA is selectively expressed in type II alveolar epithelial cells in the lung, and depending upon alternative splicing at the beginning of exon 5 is translated to produce a 191– or 197–amino acid polypeptide that is palmitoylated and proteolytically processed to the active peptide found in the alveolus (Figure 1) (3, 4). Complete proteolytic processing of proSP-C requires the presence of SP-B (2, 5). The active SP-C peptide is trafficked to lamellar bodies and packaged with phospholipids before secretion (6). Thereafter, SP-B, SP-C, and surfactant phospholipids are secreted into the airspace to form tubular myelin and the surface active monolayers and bilayers that reduce surface tension at the air–liquid interface. SP-C perturbs phospholipid acyl packing, enhancing surface absorption and stability of surfactant lipids in the alveolus (7, 8). SP-C plays an important role in the recruitment and maintenance of the surfactant film that reduces surface tension throughout the respiratory cycle. Of clinical importance, SP-C is an active protein ingredient in surfactant preparations used for therapy of respiratory distress syndrome (RDS) in preterm infants (9). Recent studies demonstrate that SP-C may also play a role in innate host defense, binding LPS with high affinity (10).



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  		Figure 1. Gene regulation and proteolytic processing of proSP-C. The human SFTPC gene is located on chromosome 8. TTF-1 (thyroid transcription factor-1), NF-1 (nuclear factor-1), and GATA-6 are nuclear transcription factors that bind to cis-acting elements forming a transcription complex in the 5' region of the gene, activating expression of the SFTPC. SP-C mRNA is produced by transcription of exons 1–6 and is translated to produce a 191– or 197–amino acid precursor (proSP-C) that is palmitoylated in the endoplasmic reticulum and proteolytically processed during transport from multivesicular bodies to lamellar bodies in type II alveolar epithelial cells. The 34–amino acid active SP-C peptide (dark shading, 24–58) is secreted with surfactant lipids and SP-B into the alveolus.

 
Role of the SFTPC Gene In Vivo

Gene Targeting
In initial studies in Swiss black outbred mice, inactivation of the SFTPC locus modestly disturbed surfactant function, but did not alter perinatal survival (11). Surfactant isolated from the SP-C null mutant (SP-C–/–) mice was less stable than normal surfactant when studied by bubble surfactometry. In sharp contrast, when SP-C–/– mice were backcrossed into the 129/Sv strain, marked changes in lung function and histology were readily apparent in the postnatal period (12). Postnatal survival of adult SP-C–/– 129/Sv mice was decreased and associated with the development of emphysema, pulmonary inflammation, abnormal tissue lipid accumulations, and goblet cell hyperplasia. These differences demonstrated that SP-C is required for alveolar homeostasis in the postnatal period and that this requirement is strongly influenced by modifier genes, which differ in the 129/Sv compared with the Swiss black strain.

Mutation of the SP-C Proprotein Structure Disrupted Lung Morphogenesis
A truncated SP-C peptide, expressed under control of the mouse 13-kb SFTPC promoter that is selectively active in type II epithelial cells, disrupted lung morphogenesis, causing death at birth (13). Transgenic mice expressing a SFTPC mutation associated with interstitial lung disease in humans also died at birth with severe lung dysmorphogenesis (14). Both the truncated and mutant transgenes were expressed in the presence of two normal SFTPC alleles indicating that the mutant gene was dominant. Thus, either deletion or mutation of the SFTPC gene caused severe lung pathology.

Mutations in the Human SFTPC Gene Cause Familial and Sporadic Interstitial Lung Disease

Severe interstitial lung disease was associated with the absence of SP-C expression (15). Likewise, dominantly inherited mutations in the SFTPC gene were associated with both acute lung failure and chronic interstitial lung disease in human patients (16, 17). A number of distinct mutations in the SFTPC gene have been associated with interstitial lung disease. Lung pathology in these patients was classified as either chronic idiopathic pneumonitis of childhood (CIP), desquamating interstitial pneumonitis (DIP), usual interstitial pneumonitis (UIP), or nonspecific interstitial pneumonitis (NSIP). The histologic findings varied with age and severity of lung disease, perhaps indicating modification of the disease process by environmental and genetic factors. Thomas and coworkers reported a large kindred of individuals with dominantly inherited, interstitial lung disease, who exhibited variability in age of onset and severity of lung disease associated with a mutation in the SFTPC gene (17). Sporadic ILD associated with an apparent de novo SP-C gene mutation has also been recognized (18). Recently, a number of infants with SFTPC gene mutations were identified who suffered severe, and even lethal, neonatal respiratory distress (L. M. Nogee and colleagues, unpublished observations). Thus, mutations in the SFTPC gene cause diverse inherited forms of lung disease with remarkable differences in severity, age of onset and disease progression. Both acute respiratory distress syndrome and chronic interstitial lung disease with pulmonary fibrosis have been associated with SFTPC gene mutations. Therefore, pulmonary disorders associated with SFTPC gene mutations appear to be strongly influenced by modifier genes and/or environmental factors in both mice and humans.

Proposed Mechanisms Underlying the Pathogenesis of Pulmonary Disease in SFTPC Mutations

Findings in both mice and humans support the hypothesis that deficiency of SP-C underlies abnormalities in lung function and remodeling, leading to acute and chronic lung disease. This concept is supported by the finding of patients lacking SP-C expression and null mice lacking expression of SP-C or proSP-C who develop interstitial lung disease (Figure 2) (12, 15). Expression of mutant proSP-C peptides is also associated with intracellular accumulation of proSP-C, raising the possibility that misfolded protein, as well as SP-C deficiency, may contribute to disease pathogenesis. Mutations in one SFTPC allele, which lead to misfolding of the encoded SP-C proprotein, result in retention and degradation of the mutant protein via the endoplasmic reticulum–associated degradation (ERAD) pathway. Because proSP-C can form homodimers (19), the product of the wild-type SFTPC allele may also be trapped in the ER and degraded. The net effect of the mutation is deficiency of SP-C in the airspaces. Under certain circumstances, degradation of misfolded proSP-C may be compromised leading to accumulation of a cytotoxic form of the proprotein and inappropriate cell death. In transgenic mice expressing mutant SP-C proprotein, accumulation of misfolded protein likely occurred because the high concentration of the transgenic protein saturated the degradation pathway (14). In human patients bearing SFTPC gene mutations, accumulation of misfolded proprotein may occur when additional stress, e.g., viral infection, is superimposed on a clearance pathway that is already operating at or near capacity. Thus, both intracellular accumulation of a misfolded, cytotoxic SP-C proprotein and deficiency of the mature peptide in the airspaces may contribute to the pathogenesis of lung disease (Figure 3).



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  		Figure 2. Pulmonary histopathology in SP-C–/– null mutant mice and in a patient with an SFTPC mutation. (A) Normal pulmonary alveoli are observed in lungs from adult wild-type mice. (B) Interstitial lymphocytic pneumonitis with mild alveolar septal wall thickening and macrophage accumulations are observed in the lungs of SP-C null mutant mice. (C) Alveolar septal wall thickening, accumulation of alveolar macrophages and amorphous, eosinophilic material (lipoproteinosis) in the alveolar spaces are found in the lungs of a 7-mo-old child diagnosed with childhood interstitial pneumonitis caused by a mutation in exon 4 of the SFTPC gene. (D) Accumulation of abnormal proSP-C in alveolar type II cells is detected in the lungs of this child using an antibody generated against proSP-C. A–C: Hematoxylin and eosin stains. D: Immunoperoxidase staining for proSP-C. Bar = 100 µm for all panels.

 


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  		Figure 3. Pathogenesis of pulmonary disease associated with mutation in SFTPC. A mutation in one allele of the SFTPC gene produces a mutant proSP-C protein that is misfolded. The mutant proSP-C interacts with wild-type SP-C proproteins in the endoplasmic reticulum, inhibiting processing of normal proSP-C and blocking production of the active peptide. Both proproteins are degraded leading to SP-C deficiency in the airspaces. Lung injury may be mediated by the lack of SP-C and the accumulation of abnormal proSP-C/mutant proSP-C proteins. ProSP-C may accumulate intracellularly resulting in cytotoxicity and lung pathology.

 
The SFTPC Gene Provides the Molecular Entrée to Elucidation of Genetic Programs Regulating Lung Function and Formation

With the recognition that SP-C was expressed in a highly lung epithelial–specific pattern and was induced in the perinatal period, studies were undertaken to identify the mechanisms controlling its expression. Bohinski and coworkers identified the critical role of thyroid transcription factor-1 (TTF-1) in the activation of promoter elements in the SFTPC gene, as well as other lung epithelial–selective genes (20). The SFTPC promoter has been highly useful in the introduction of genes into the lungs of transgenic mice, for gene addition, mutation, or deletion. Relatively small regions of the promoter are adequate to direct lung epithelial cell–specific expression (21, 22). The SFTPC promoter has been useful in studies to discern the roles of genes influencing lung formation and function. For example, SFTPC promoter elements have been used for constitutive expression of genes in the respiratory epithelium of transgenic mice and for conditional expression or deletion of genes under control of the SP-C-rtTA (reverse tetracycline transactivator protein) transgene (23). Conditional expression of Cre-recombinase with this system has been used for recombination of floxed genes in vivo, which has been useful for cell lineage analysis, as well as for gene addition and deletion, to study lung function and formation in vivo (24–26).

Genetic Elements Controlling Lung Epithelial Cell Gene Expression: TTF-1

Bohinski and colleagues identified a critical role for TTF-1 in the regulation of a number of genes selectively expressed in respiratory epithelial cells, including SP-A, SP-B, SP-C, and the Clara cell secretory protein (CCSP) (20). TTF-1 binds and activates cis-elements (TTF-1 response elements [TRE]) located in the 5' regions of these target genes. Mutations of TREs markedly or completely abrogated gene expression normally induced by the interaction of TTF-1 with the promoter elements in these target genes (Figure 1). TTF-1 is expressed in the forebrain, thyroid, and lung, and is a member of the Nkx family of homeodomain-containing nuclear transcription factors (27). Deletion of TTF-1 per se causes severe brain abnormalities, as well as thyroid and lung hypoplasia, demonstrating its critical role in brain, thyroid, and lung morphogenesis (28). Haploinsufficiency of TTF-1 gene (TITF-1) in humans resulting from loss-of-function mutations on one allele is associated with hypothyroidism, choreoathetosis, and chronic lung disease (29). In the fetal lung, TTF-1 is detected in respiratory epithelial cells lining both conducting and peripheral lung tubules. In the postnatal lung, TTF-1 is expressed in subsets of bronchiolar cells and type II epithelial cells in the alveolus, but not in type I epithelial cells (30, 31). Deletion of the TITF-1 gene causes tracheoesophageal fistula and marked lung hypoplasia associated with the absence of SP-A, -B, and -C (28, 32). In contrast, ciliated bronchiolar epithelial cells and associated markers, including FOXJ1 and ß-tubulin IV, are expressed in TTF-1–/– mice, demonstrating that TTF-1 selectively influences peripheral lung formation and gene expression (33).

Gene Substitution: TTF-1 Phosphorylation Mutant

The severe lung hypoplasia characteristic of TTF-1–/– mice limited their utility for identification of the molecular targets and functions of TTF-1 later in gestation (28). Because phosphorylation of TTF-1 influences its activity in the transcriptional activation of target genes, the endogenous TITF-1 allele was replaced with a mutant allele in which serine phosphorylation sites were mutated to alanine (TTF-1[PM]) (34). Lung formation was substantially rescued in the TTF-1(PM) mice compared with the TTF-1–/– mice. TTF-1(PM) mice died of respiratory failure at the time of birth, indicating that the mutant represented a hypomorphic allele. To identify potential transcriptional targets of TTF-1 before birth, RNA microarray analysis was performed on lungs at E18.5. A number of genes whose expression was influenced by TTF-1(PM) were identified, including genes regulating surfactant homeostasis, host defense, fluid and electrolyte transport, gene transcription, differentiation, vasculogenesis, and alveologenesis (see Figures 4 and 5). Promoter elements from a number of these genes contained TREs located in the regulatory regions, indicating the likelihood that these genes represent direct transcriptional targets of TTF-1 in respiratory epithelial cells. A number of known molecular targets of TTF-1, including SP-A, -B, -C, and CCSP, as well as previously unknown targets (tcf7, Lef1, ß-catenin, Sox17, and Mdk) were identified. Subsets of genes expressed primarily in nonepithelial cells are likely to be indirect targets of TTF-1, their expression being dependent upon paracrine or cell–cell signals derived from the respiratory epithelium, which, in turn, influence expression and formation of the underlying stromal elements that form the alveolar walls, as well as the vascular and lymphatic systems of the lung.



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  		Figure 4. Heat map of hierarchical clustering of lung mRNAs influenced by the TTF-1(PM). Microarray analysis was performed on lung mRNA at E18.5 in TTF-1(PM) mutant mice (Mut) and control littermates (Con). Red represents mRNAs that were significantly increased, whereas green represents those decreased in TTF-1(PM) lung tissue. The heat map provides a representative list of altered genes. A complete list of these genes is provided in deFelice and coworkers (34).

 


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  		Figure 5. Schemata of classes of mRNAs influenced by the TTF-1(PM) mutation. TTF-1 influences the expression of gene transcription, surfactant homeostasis, ion and water transport, signaling, vasculogenesis, cell differentiation, and host defense. The sites of serine to alanine substitutions (A) made in the TTF-1(PM) phosphorylation mutant gene are shown as described in deFelice and coworkers (34). HD is the homeodomain region of TTF-1 that binds to cis-acting elements in TTF-1 target genes.

 
Summary

Study of the SP-C protein and SFTPC gene have provided unexpected insights into the molecular pathways influencing the pathogenesis of interstitial lung disease and the regulation of gene expression in the peripheral lung. The molecular tools and models derived from these studies have been useful in identifying genes and processes that are likely to mediate adaptation to air breathing at birth. It is hoped that future studies of the pathways mediating lung formation, as well as those linking SP-C to acute and chronic lung disease, will provide new insights into the pathogenesis of pulmonary disease, which will be useful in identifying novel therapies for both RDS and interstitial lung disease.

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Genes and Gene Polymorphisms Associated with Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by extracellular matrix deposition in the interstitial spaces of the lung and severe distortion of the pulmonary architecture (1). Spatial and temporal appearances of fibroblast foci and honeycomb in affected lungs are unique histologic features of IPF (2). The incidence of IPF is 7–10 per million with an average onset between 50 and 70 yr of age. The prevalence and death rate of IPF increase with the aging process. In general, the 5-yr mortality rate is 68% and the overall median survival rate is 2.8 yr (3).

Historically, the lung fibrosis observed in patients with IPF was considered to be a deteriorating consequence of persistent lung inflammation associated with continuous cellular injury from unknown insults (4). However, growing evidence in recent years suggests that the inflammatory response may not precede the fibrosis nor play a major role in the IPF development. This is supported by the lack of responsiveness of patients with IPF to anti-inflammatory treatments. Instead, IPF may be a disease of impaired wound healing involving the epithelial/fibroblastic pathway (5, 6). The triggering event in IPF may be multiple and continuous microscopic insults to the alveolar epithelial cells. Aberrant wound healing subsequently leads to fibroblast proliferation, transformation into myofibroblasts, formation of fibroblastic foci, deposition of extracellular matrix, and ultimately fibrosis.

IPF is a complex disease influenced by multiple genetic predisposition factors and environmental exposure. Gene expression analysis of lung tissues from patients with IPF has yielded insights into the molecular pathways that may be important in IPF development (7). However, genetic studies of IPF have been hindered by limited familial cases, late disease onsets, phenotypic heterogeneity, and the high mortality rate associated with this disease. Identification of genetic factors that provide susceptibility to IPF and/or modify disease progression will be vital in understanding the pathogenesis of IPF.

Surfactant Protein C Gene Mutation in Familial IPF

Based on a clinical and epidemiologic study conducted in the United Kingdom, the incidence of familial IPF was estimated to be ~ 2% of all diagnosed IPF cases (8). However, according to a recent report, as many as 10% of all patients with IPF may have IPF history in their families (9). In comparison with sporadic cases, the patients with familial IPF have earlier disease onset and nearly 20% mortality before age 50 (9). Using a family affected by IPF, consisting of five generations and eleven affected individuals, Thomas and coworkers have identified a heterozygous exon 5 +128 T to A transversion in the surfactant protein C (SP-C) gene from all affected individuals (10). SP-C is a member of the surfactant-associated protein family that affects lung mechanics, gas exchange, and host defense (11). The exon 5 +128 T to A mutation results in a glutamine for leucine substitution at a highly conserved codon within the carboxyl-terminal domain of the SP-C protein precursor. Aberrant cellular distribution of the SP-C protein was observed in lung tissues of affected individuals from this family. The exon 5 +128 T to A mutation may produce a misfolded protein with abnormal cellular trafficking. Given the important roles of SP-C in the structural integrity of alveolar type II cells, alterations in the cellular distribution of SP-C protein may provide susceptibility to injury for the alveolar type II cells. Missense and splicing site mutations of the SP-C gene were also reported in sporadic and familial cases of other interstitial lung diseases (12, 13).

Gene Polymorphisms Associated with Sporadic IPF

Although SP-C gene mutations form a genetic basis for some familial cases of IPF, mutations in SP-C are not associated with all of the families with IPF. In addition, the majority of patients with IPF lack a family history suggesting a complex genetics of this disease. The development of IPF is likely to be determined by multiple genetic factors that each contribute a modest effect to the predisposition of this disease. In combination with appropriate environmental or cellular triggers, individuals who possess these predisposition factors may develop IPF. The lack of genome-wide linkage analyses and chromosomal loci for IPF make the positional cloning of these genes impossible and, therefore, population-based association is the method of choice for the genetic study of IPF.

To date, limited gene and gene polymorphisms have been evaluated in IPF cohorts and even fewer of them have demonstrated confirmed associations (Table 1). Coinciding with the traditional paradigm for the pathogenesis of IPF, candidate genes that have been analyzed for this disease are primarily involved in inflammatory and immune responses. Given the intrinsic pitfalls of population stratification, misclassification of phenotypes, and insufficient study subjects associated with genetic case-control studies, cautious interpretation of the outcomes of these studies is important.


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  		TABLE 1. Gene and gene polymorphisms evaluated in sporadic idiopathic pulmonary fibrosis

 
Surfactant Protein Gene Family
Association of the SP-C gene mutation with familial IPF suggests that genes involved in maintaining the alveolar structure and function are important for the development of IPF. In addition to the SP-C gene, SP-A1, SP-A2, SP-B, and SP-D are also members of the surfactant-associated protein family (11). Using a Mexican IPF cohort, Selman and colleagues evaluated gene polymorphisms in the SP-A1, SP-A2, SP-B, SP-C, and SP-D genes (14). Stratification of the study cohort based on their smoking habit has demonstrated associations for the 6A4 haplotype of SP-A1 gene with nonsmoking patients with IPF and the 1580C allele of SP-B gene with patients with IPF who were also smokers. Functional analyses of the SP-A proteins with different SP-A1 6A4 haplotypes demonstrated allele-specific aggregation of the SP-A proteins. However, associations with IPF were not detected for multiple polymorphisms in the SP-C and SP-D genes (14).

Transforming Growth Factor-ß Gene
Transforming growth factor-ß (TGF-ß) is a central player in pulmonary fibrosis, and its expression is elevated in the lung tissues of patients with interstitial lung diseases (15, 16). Overexpression of TGF-ß in the lung tissues induced prolonged pulmonary fibrosis in an animal model (17). Using a cohort consisting of 128 subjects with IPF and 140 ethnicity-matched healthy control subjects, Xaubet and associates have demonstrated no association for two nonsynonymous SNPs, 869T>C/L10P and 915G>C/R25P, of the TGF-ß gene with the IPF phenotype (18). However, the 10P allele was associated with an increased deterioration of the disease as measured by gas exchange (18). As noted by the authors, this allele was not correlated with other functional parameters of IPF, and the levels of TGF-ß in both cases and control subjects were not determined. Therefore, further evaluation using independent IPF cohorts is necessary to determine the true roles of the 10P allele as well as other polymorphisms within the TGF-ß gene in IPF.

Interleukin-1 Gene Cluster
Interleukin-1 (IL-1) is a proinflammatory and fibrogenic cytokine. IL-1{alpha}, IL-1ß, and their naturally occurring inhibitor IL-1 receptor antagonist (IL-1RN) are localized in a cluster on chromosome 2q13 (19). Using an IPF cohort from the United Kingdom and Italy, Whyte and coworkers demonstrated an association between the rare C allele of the +2018C>T SNP in the IL-1RN gene for both populations (20). However, this association was not confirmed with cohorts from the United States and Europe (21, 22). The –889T allele in the IL-1{alpha} gene was also associated with severity of gas transfer deficits in patients with IPF. No association was identified for multiple polymorphisms in IL-1{alpha}, IL-1ß, and IL-1RN genes using an IPF cohort from the Czech Republic (22).

Tumor Necrosis Factor-{alpha}, IL-6, and Related Genes
Expression analysis of the tumor necrosis factor-{alpha} (TNF-{alpha}) in lung tissues of patients with IPF has shown an elevated level in the alveolar type II epithelial cells (23, 24). Using two well-defined IPF cohorts, Whyte and colleagues first reported an association with the –308A allele of TNF-A gene (20). However, a subsequent study using a third study cohort did not detect the same association between IPF and the –308A allele of TNF-A gene (25). In addition, association was not detected for multiple polymorphisms in the TNF-A, TNF-receptor II (TNF-RII) and lymphotoxin {alpha} (LT-{alpha}) genes using the third IPF cohort (25). Interestingly, the G allele of the IL-6 intron 4 A>G SNP was associated with rapid disease progression of the patients with IPF in this cohort (25). Co-carriage of the TNF-RII 1690C allele and the IL-6 intron 4G allele were also strongly associated with the IPF phenotype, suggesting a combinatory effect of these two genes in the IPF development (25).

Angiotensin-Converting Enzyme Gene
A functional insertion/deletion polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene has been associated with plasma levels of ACE and angiotensin (26). The D allele correlating with high plasma ACE level has been shown to have association with systemic sclerosis, a disease with high propensity to interstitial lung diseases (27). Using a very small North American IPF cohort, Morrison and colleagues have detected an increased D allele frequency for IPF cases in comparison with matched control subjects (42% versus 31%) (28). Because this study population consists of both subjects with IPF and subjects with NSIP (nonspecific interstitial pneumonia), no reliable conclusion should be made from this small study cohort.

Complement Receptor 1 Gene
Recently, Zorzetto and colleagues analyzed polymorphisms of the complement receptor 1 (CR1) gene using an Italian IPF cohort (29). CR1 is important in the clearance of immune complexes. The polymorphism C5570G in exon 33 has been correlated with the levels of CR1 expressed on erythrocytes that may directly correlate with the clearance of the immune complex (30). Using the RFLP method, they demonstrated significant association for the GG genotype of the C5570G polymorphisms with IPF (29). The GG genotype was also associated with sarcoidosis in the same population (31). Because sarcoidosis and IPF are two clinically distinct identities of interstitial lung diseases, the likelihood of sharing the same genetic predisposition factors are low and, therefore, confirmation of the CR1 association in additional IPF study cohorts are necessary.

In contrast to the genes and gene polymorphisms that have been shown to be associated with IPF, lack of association with IPF development has been documented for polymorphisms in plasminogen activator inhibitor-1 and genes involved in the Th1/Th2 response (32–34). Systematic analyses of these genes using functional genomic tools may be necessary to determine their roles in the development of IPF.

Future Genetic Polymorphism Studies in IPF

With limited familial cases and late disease onset associated with IPF, identification of genetic predisposition factors will mainly rely on population-based association studies. One of the major potential pitfalls of case-control genetic association studies is population stratification (35). Population stratification arises as a result of admixture of two distinct populations that differ in allele frequencies at both the marker and trait loci. This allele frequency difference can mimic allelic association (even when the trait and marker loci are unlinked) that can be replicated in subsequent sampling from the same admixed population. Therefore, every effort should be made in selecting ethnicity-matched control subjects to minimize population stratification. The population stratification may also be controlled experimentally. One of the approaches that have been used to control this potential problem is the Genomic Control (GC) method (36, 37). GC automatically adjusts test statistics of association for the presence of population stratification by evaluating other markers. Although the impact of population stratification on well-selected association studies is unclear, GC is straightforward to implement (36, 37). Devlin and Roeder present GC methods that can be implemented in the simplest situations in case-control studies (36). GC methods provide the advantage of robustness against spurious association due to population stratification while eliminating the need to collect a significantly greater number of samples for the case-control study (37). Alternatively, as introduced by Pritchard and Rosenberg, the population stratification can also be controlled by simply analyzing 15–20 unlinked microsatellite markers of the study population (38).

Another critical component for conducting a case-control study is the accurate characterization of clinical phenotypes. According to the American Thoracic Society/European Respiratory Society (ATS/ERS), IPF is a "Specific form of chronic fibrosing interstitial pneumonia limited to the lung and associated with the histologic appearance of usual interstitial pneumonia (UIP)." It is characterized by progressive dyspnea, worsening pulmonary function, and a typical radiographic pattern (39). According to Dacic and Yousem, diagnosis of IPF should be based on (i) histological findings consistent with UIP from multiple lung biopsies, (ii) clinical findings excluding known underlying diseases such as connective tissue and autoimmune diseases, and (iii) radiographic presentations demonstrating typical features of pulmonary fibrosis with little active inflammation (2). Misdiagnosis of cases in an IPF study cohort may mask the genetic differences between the case and control groups and prevent the identification of IPF-associated genes.

Unlike the rare gene mutations associated with Mendelian genetic diseases, genetic polymorphisms associated with complex traits may be common in the population. The presence of one disease-associated allele may not be sufficient to cause disease. Therefore, carriage of a disease-associated polymorphism does not translate into a clinical diagnosis. It is the specific combination of multiple disease-associated loci (gene–gene interaction) and the presence of environmental triggers (gene–environmental interactions) that lead to the development of specific complex phenotypes. The disease-associated alleles may either provide susceptibility to initial disease triggers or modify the disease development after initial pathogenic changes.

Integration of gene expression/proteomic studies of affected tissues may permit the identification of cellular and molecular pathways important in the disease pathogenesis. Identification of these pathways is crucial for guiding the selection of candidate genes in association studies. As demonstrated in autoimmune diseases, different genes or gene polymorphisms that are involved in the same pathway may independently contribute to the development of same or related clinical phenotypes. Recently, three distinct regulatory polymorphisms have been independently associated with systemic lupus erythematosus, rheumatoid arthritis, and psoriasis (40–42). Although these three polymorphisms are localized in different genomic locations and may regulate four candidate genes for autoimmunity, the genetic variations in these polymorphisms alter the binding sites for a common transcription factor, runt-related transcription factor 1 (RUNX1). Therefore, systemic analysis of crucial genes involved in important pathways of IPF pathogenesis will be vital in determining their combinatory roles in the IPF development.

In addition to the genetic predisposition, environmental exposure may be equally important in the development of IPF. One of the well-known risk factors for IPF is smoking. Stratification of the study populations based on their environmental exposure in genetic association studies is essential to determine gene–environment interactions in IPF. Once a gene or gene polymorphism is identified, it is important to verify the functional relevance in IPF using molecular and functional genomic approaches as well as animal models. Identification of genetic factors that either offer susceptibility or modify the disease development of IPF will ultimately provide genetic profiles that may predict disease onsets, disease progression, and treatment outcomes.

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Progress in Chronic Obstructive Pulmonary Disease Genetic Epidemiology

Chronic obstructive pulmonary disease (COPD) has been defined as "a disease state characterized by airflow limitation that is not fully reversible" (1). Cigarette smoking is a proven and critical environmental risk factor for the development of COPD. However, considerable variability in the development of airflow obstruction in response to smoking has been observed (2, 3). For example, Burrows and colleagues found that only approximately 15% of the variability in FEV1 was explained by pack-years of cigarette smoking. Cigarette smoking is usually required to develop severe chronic airflow obstruction, but the FEV1 values of many heavy cigarette smokers remain within the normal range (2). Thus, in most cases, cigarette smoking is likely a necessary but not sufficient exposure to lead to COPD. The low percentage of pulmonary function variation explained by smoking suggests that differences in genetic susceptibility to the effects of smoking may be present. However, there appears to be a gradient in susceptibility to the effects of smoking, which may be determined by multiple genetic variants; some individuals develop COPD after minimal or no smoking exposure, other individuals develop COPD after heavy smoking exposure, and still other individuals are resistant to the development of COPD despite heavy smoking exposure.

In this review, we will briefly discuss {alpha}1-antitrypsin (AAT) deficiency as a proven genetic determinant of COPD, we will consider the evidence for familial aggregation of COPD unrelated to AAT deficiency, and we will focus on the recent genetic linkage analysis studies of COPD.

Severe AAT Deficiency

Approximately 1–2% of patients with COPD inherit severe AAT deficiency (typically phenotype PI Z) (4). AAT is encoded by the PI (protease inhibitor) gene, which is located on chromosome 14. Individuals with phenotype PI Z (corresponding to genotypes ZZ or Znull) have severely reduced serum AAT levels and are clearly at increased risk for the development of COPD—especially if they smoke (5–7).

Because most subjects with phenotype PI Z are identified because they already have COPD, ascertainment bias has had a significant impact on the perceived natural history of lung disease in severe AAT deficiency. In a study of 52 subjects with phenotype PI Z and their families at Washington University in St. Louis, nonindex subjects with phenotype PI Z, who were not ascertained due to COPD, often had normal pulmonary function (8). Not surprisingly, index subjects with phenotype PI Z, who were ascertained with COPD, all had significant airflow obstruction. Seersholm and colleagues also found significantly higher baseline FEV1 values in nonindex subjects with phenotype PI Z compared to index subjects with phenotype PI Z despite similar ages and smoking histories in the index and nonindex subjects (9).

Cigarette smoking is clearly a major determinant of pulmonary function variability among individuals with phenotype PI Z. However, some smokers with phenotype PI Z maintain normal FEV1 values at least into middle age; by this age, some nonsmokers with phenotype PI Z have already developed significant airflow obstruction. Results of genetic modeling from the St. Louis AAT Study suggested that additional genetic factors influence the development of airflow obstruction in AAT deficiency (10). By comparing the slopes of the relationship between FEV1 and pack-years of smoking (known as the norms of reaction) in subjects with phenotypes PI Z, PI MZ, and PI M, significant genotype-by-environment interaction between PI type and pack-years of smoking was demonstrated in the St. Louis AAT Study (11).

Only a few other studies have considered whether factors other than cigarette smoking influence the development of lung disease in subjects with phenotype PI Z. For example, Black and Kueppers found significant variability in pulmonary function and clinical symptoms among nonsmoking individuals with phenotype PI Z (12). In a study of more than 200 nonsmokers with phenotype PI Z, Piitulainen and colleagues showed that, as expected, increasing age was associated with reduced pulmonary function (13). Among nonsmokers with phenotype PI Z older than 50 years, male sex, wheezing symptoms, and occupational exposures to respiratory irritants were also associated with reduced pulmonary function. Ongoing studies in AAT-deficient families hold promise to identify genetic modifiers of the development of lung disease in individuals with phenotype PI Z.

Familial Aggregation of Spirometry and COPD

Studies of spirometric measurements performed in families and in twins from the general population have suggested that genetic factors influence pulmonary function variation (14, 15). Studies in relatives of individuals with COPD have also suggested a role for genetic factors in COPD susceptibility by demonstrating higher rates of airflow obstruction in first-degree relatives of patients with COPD compared with control subjects (16, 17).

In the Boston Early-Onset COPD Study, our research group has focused on severe, early-onset COPD probands, because the study of early-onset cases in other complex disorders has successfully led to the identification of susceptibility genes (e.g., breast cancer). Severe early-onset COPD probands in this study met the following criteria: Age < 53 yr; FEV1 < 40% predicted; physician-diagnosed COPD; absence of severe AAT deficiency; and no other lung disease to account for chronic airflow obstruction. The majority of these severe, early-onset COPD probands have been identified from lung transplantation and lung volume reduction surgery programs.

To maximize the genetic information from these rare severe, early-onset COPD probands, all available first-degree relatives (siblings, parents, and children) and older second-degree relatives (half-siblings, aunts, uncles, and grandparents) were invited to participate in the study. In addition, other relatives with diagnosed COPD were included, and all first-degree relatives of any family members with moderate airflow obstruction (FEV1 < 60% predicted, FEV1/FVC < 90% predicted) were also invited to participate.

The first phase of the Boston Early-Onset COPD Study included 44 severe, early-onset COPD probands with 204 first-degree relatives (18). To confirm that the Boston Early-Onset COPD Study probands were correctly diagnosed with COPD, reports of previously performed chest CT scans and lung pathological samples were reviewed. Chest CT scan reports were available for 36 subjects; 34/36 chest CT reports noted emphysema, and for one of the subjects without gross emphysema, attenuated pulmonary vasculature consistent with emphysema was noted. Reports regarding lung pathological specimens were available for 17 subjects, and all of these specimens showed emphysema. Thus, the Boston Early-Onset COPD Study probands had severe airflow obstruction with emphysema, and the diagnosis of COPD among these COPD probands appears to be quite accurate.

The 44 early-onset COPD probands were selected to have severe airflow obstruction at an early age, and a profound degree of airflow obstruction in these subjects (mean FEV1 16.9% predicted) was observed. Only 2 of the first 44 early-onset COPD probands were lifelong nonsmokers.

A high prevalence of females (> 70%) was found among the Boston Early-Onset COPD Study probands; subsequent studies also demonstrated an increased risk to smoking female first-degree relatives of early-onset COPD probands for severe airflow obstruction compared with smoking male first-degree relatives (19). The etiology of the female predominance in the Boston Early-Onset COPD Study compared with the male predominance in most previous studies of severe COPD remains to be defined. However, our sample included, on average, younger subjects with lower FEV1 values than previous studies.

The key phenotypes selected for familial aggregation analysis in the Boston Early-Onset COPD Study were spirometric measures of airflow obstruction, including FEV1 and FEV1/FVC. Because reduced FEV1 can result from restrictive lung diseases (e.g. interstitial lung diseases, neuromuscular diseases), analysis of the FEV1/FVC ratio selects for effects of airflow obstruction. In addition, chronic bronchitis, a clinically defined condition of cough productive of phlegm for at least 3 mo/yr for at least 2 yr, is a COPD-related phenotype that can be assessed reasonably well by questionnaire.

The 204 first-degree relatives of severe, early-onset COPD probands were compared to 83 control subjects that participated in previous population-based studies at the Channing Laboratory and agreed to participate in this study. First-degree relatives of early-onset COPD probands had significantly lower mean values for FEV1 (percent predicted) and FEV1/FVC (percent predicted) than control subjects (P < 0.01), despite similar values for age and pack-years of smoking. Although the difference in mean FEV1 (% predicted) between all first-degree relatives of early-onset COPD probands and all control subjects was modest, compared with current or ex-smoking control subjects, current or ex-smoking first-degree relatives of early-onset COPD probands had substantially lower FEV1 (76.1% predicted versus 89.2% predicted) and FEV1/FVC (83.5% predicted versus 94.3% predicted) (P < 0.01 for each comparison). The distribution of FEV1 values in smoking first-degree relatives of early-onset COPD probands compared with smoking control subjects is shown in Figure 1. On the other hand, the FEV1 and FEV1/FVC values in lifelong nonsmoking first-degree relatives and nonsmoking control subjects were quite similar.



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  		Figure 1. Significantly lower FEV1 (% predicted) values were found in smoking first-degree relatives of severe, early-onset COPD probands (filled bars) compared to smoking control subjects (open bars) in the Boston Early-Onset COPD Study (from Ref. 18).

 
To determine whether relatives of the Boston Early-Onset COPD Study probands have increased risk for reduced FEV1 after adjustment for the effects of age, smoking, and the inclusion of multiple study participants from each family, odds ratios for various levels of reduction in FEV1 were calculated with generalized estimating equations. Comparing all first-degree relatives to all control subjects, increased risk of FEV1 < 80% predicted (odds ratio 3.4 with 95% C.I. 1.5–7.8) was found; a trend toward increased risk of FEV1 < 60% predicted (odds ratio 2.5 with 95% C.I. 0.8–7.7) was also observed. Further analysis demonstrated that these risks were significantly increased only in cigarette smokers. Among current or ex-smokers, the odds ratio was 4.5 (95% C.I. 1.8–11.5) for FEV1 < 80% predicted and 3.5 (95% C.I. 1.0–12.9) for FEV1 < 60% predicted. First-degree relatives that were lifelong nonsmokers did not have an increased risk for reduced FEV1. A similar pattern of smoking-related susceptibility was observed for chronic bronchitis. More recent results from the Boston Early-Onset COPD Study have shown that nonsmoking first-degree relatives of early-onset COPD probands do have reduced levels of more sensitive measurements of airway dysfunction, including FEF25-75 and FEF25-75/FVC, compared with nonsmoking control subjects (20). Thus, there may be some mild baseline airway abnormalities in first-degree relatives of early-onset COPD probands, which can lead to significant airflow obstruction if such individuals smoke cigarettes.

Very similar results in terms of familial aggregation of airflow obstruction were also found by McCloskey, Lomas, and colleagues in a sample of families from the United Kingdom (21). Significantly increased odds for airflow obstruction were found in smoking first-degree relatives of COPD probands compared to smoking control subjects, but no increased risk for airflow obstruction was found in nonsmoking first-degree relatives of COPD probands.

The results of the Boston Early-Onset COPD Study and the McCloskey/Lomas study suggest that the increased risk for airflow obstruction found only in current or ex-smoking first-degree relatives of COPD probands may relate to genotype-by-environment interactions between cigarette smoking and one or more unidentified genetic factors.

Linkage Analysis of Severe, Early-Onset COPD

After the analysis of familial aggregation of COPD-related phenotypes in the Boston Early-Onset COPD Study pedigrees, additional pedigrees were recruited, and 607 members of 72 early-onset COPD pedigrees were included in a genome scan linkage analysis. After excluding 22 subjects due to pedigree inconsistencies (likely related to nonpaternity), a 9-cM genome scan of short tandem repeat (STR) markers, performed by the NHLBI Mammalian Genotyping Service, was analyzed for linkage to COPD-related phenotypes. Because the optimal COPD-related phenotypes for