Author Proof. Genetic background of idiopathic pulmonary fibrosis. Review

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1 Genetic background of idiopathic pulmonary fibrosis Simona Santangelo*, Simone Scarlata, Anna Zito, Domenica Chiurco, Claudio Pedone and Raffaele Antonelli Incalzi Area of Geriatrics Unit of Respiratory Pathophysiology, Campus Biomedico University and Teaching Hospital, Rome, Italy *Author for correspondence: Tel.: Fax: s.santangelo@unicampus.it Expert Rev. Mol. Diagn. 13(4), (2013) Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by progressive fibrosing interstitial pneumonia. The histological pattern, which displays dense fibrosis with active areas of fibroblastic proliferation, suggests a pathogenetic role of aberrant response to healing of multiple microscopic, repeated alveolar epithelial injuries. Although the exact etiology of the disease is still under investigation, several studies suggest that a combination of genetic and environmental factors may play a causal role. The aim of this review is to describe the genetic background of IPF, reporting the latest advancements made possible by genomic techniques that allow a high-throughput analysis and the identification of target genes implicated in IPF. This information may help to clarify pivotal aspects on prognosis and diagnosis, and may help to identify potential targets for future therapies. Keywords: DNA methylation gene mutation idiopathic pulmonary fibrosis lung microarray mirna next-generation sequencing Idiopathic pulmonary fibrosis (IPF) is defined by the official American Thoracic Society/ European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association statement as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, occurring primarily in older adults and limited to the lungs. The diagnosis of IPF is descriptive, based on clinical, radiologic and histopathologic examination. Other possible causes of interstitial lung disease must be ruled out, and the presence of the usual interstitial pneumonia (UIP) pattern should also be ascertained by high-resolution computed tomography (HRCT) or, in patients subjected to surgical lung biopsy, by a combination of HRCT and histological pattern [1]. Genetic traits are of potential interest, but they lack diagnostic properties for clinical purposes [2]. IPF is characterized by progressively worsening dyspnea and lung function, and is usually associated with poor prognosis. Its incidence is estimated to be 10.7/100,000 person-years in men and 7.4/100,000 person-years in women. It has been shown that both prevalence and incidence increase after the sixth decade of life [3]. The median survival for patients affected by IPF varies from 2 to 5 years; patients exhibit variable disease courses and prognosis [4]. Although the initiating event of the disease is not completely understood, the histological pattern, characterized by dense fibrosis with active areas of fibroblastic proliferation, suggests a pathogenetic role of aberrant response to multiple microscopic, repeated alveolar epithelial injuries (both exogenous and endogenous) due to a dysregulation of the intricate network of local and systemic factors (e.g., fibroblasts, circulating fibrocytes, chemokines, growth factors and clotting factors), which eventually lead to pulmonary fibrosis. Alternatively, an excess of repeated injury into the lung may damage reparative mechanisms and lead to pulmonary fibrosis [5]. In the normal healing process, alveolar epithelial cells release inflammatory signals and fibroblasts are activated and differentiate into myofibroblasts that secrete collagen and other extracellular matrix (ECM) proteins. Furthermore, some epithelial cells may differentiate into myofibroblasts by epithelial mesenchymal transition (EMT). While in normal wound healing this mechanism is terminated by signals that stop fibroblasts accumulation and collagen deposition, it has been suggested that in IPF this regulatory mechanism fails, leading to fibrosis. The alveolar epithelial injury could be activated from the presence of persistent irritant (e.g., cigarette smoking) [6], prolonged exposure to occupational or environmental contaminants or dusts [7], viral or bacterial lung infections or medication [8,9]. Selected risk factors for IPF have been summarized in Box /ERM Expert Reviews Ltd ISSN

2 Santangelo, Scarlata, Zito, Chiurco, Pedone & Incalzi Other proposed pathogenetic mechanisms are genetic predisposition or disproportionate autoimmune pathway activation. Recent reports suggest that selected genetic polymorphisms may contribute to the development of lung fibrosis [10,11], and the presence of familial cases of pulmonary fibrosis (FPF) suggests a genetic basis for some forms of the disease. The same criteria are used to define IPF in familial and sporadic cases because the two forms are clinically and histologically indiscernible, although familial forms seem to have different patterns of gene transcription and their onset may be at an earlier age compared with the sporadic cases. The occurrence of a vertical transmission observed in familial IPF is supporting evidence for the existence of a genetic background of this disease. A case control study performed by García-Sancho et al. showed that the risk of IPF is strongly associated with a family history of pulmonary fibrosis, with an estimated IPF prevalence of 20% in patients with a parent and/or a sibling with pulmonary fibrosis [12]. On these bases, the authors reviewed the genetic background of IPF with special attention to the diagnostic and prognostic role of genetic biomarkers. The authors will summarize the current evidence in order to provide an overview on gene alleles that may confer the risk of developing pulmonary fibrosis or increase the progression rate of this disease (Table 1). All the genes subject to this review and their proposed involvement in the pathogenesis of IPF are summarized in Table 2 and described in the Figure 1. Genes for surfactant protein Surfactant protein C mutations Surfactant-associated protein C (SFTPC) genes have been associated with FPF. SP-C is a hydrophobic protein produced by type II pneumocytes whose physical and chemical properties reduce the surface tension of alveolar fluid [13], avoiding the collapse of alveolar units. The pulmonary SFTPC gene has been localized on the short arm of chromosome 8 [14]. It is a relatively small gene spanning 3.5 kilobases and composed of six exons [15]. Multiple SNPs have been identified in this gene, which cause type 2 pulmonary surfactant metabolism dysfunction, also called pulmonary Box 1. Main identified factors associated with pulmonary fibrosis. alveolar proteinosis due to surfactant protein C deficiency. In children, pulmonary alveolar proteinosis has been associated with early onset nonspecific interstitial pneumonia (NSIP), desquamative interstitial pneumonitis (DIP) and pulmonary alveolar proteinosis. In adults, it has been associated with IPF and, to a minor degree, NSIP and DIPs. The first mutation identified in the SFTPC gene was a heterozygous guanine (G) to adenine (A) transition substitution (G>A) in the splice donor site of intron 4 [cdna base (c G>A)] in an infant affected by NSIP and in his mother affected by DIP. The nucleotide substitution resulted in the skipping of exon 4 and the deletion of 37 amino acids in the C-terminal domain of the precursor protein. Consequently the precursor protein, which lacked a cysteine residue important for disulphide-mediated protein folding, was not processed and secreted normally. Indeed, it was observed that the mature SP-C was absent in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore the identification of this mutation in only one allele for each patient is consistent with an autosomal dominant pattern [16]. Later, Thomas et al. [17] identified a heterozygous thymidine to adenine (T>A) transversion at genomic position exon T>A in the SFTPC gene by analyzing the DNA of 14 relatives affected by FPF. The identified mutation results in a leu188 to gln (L188Q) substitution in a highly conserved region of the C-terminal domain, crucial for the correct protein intracellular trafficking/folding. The showed family phenotype was found in adults with signs of UIP and children with signs of NSIP. Another heterozygous g+1286t>c transition in exon 3 of the SFTPC gene (resulting in an ile73 to thr, I73T substitution in the C-terminal propeptide) was identified by Brasch et al. [18] in a patient with combined histologic pattern of NSIP and pulmonary alveolar proteinosis. The mutation results in abnormal proprotein trafficking, with accumulation of aberrantly processed pro(sp)-c within alveoli. Finally, van Moorsel et al. identified two other mutations, c435+2 T>C (IVS4+2) and c.211 A>G (M71V), but the these mutations were found only in five out of 20 adult patients with FPF, and never in sporadic disease [19]. Risk factors for the development of pulmonary fibrosis: Cigarette smoking (particularly for individuals with a smoking history of more than 20 pack-years) Prolonged exposure to occupational or environmental contaminants or dusts: metal dusts (brass, lead and steel) and wood dust (pine), farming, raising birds, hair dressing, stone cutting/polishing and exposure to livestock and to vegetable dust/animal dust Viral or bacterial lung infections. Most research has been focused on Epstein Barr virus and hepatitis C. Both the protein and the DNA of Epstein Barr virus have been identified in lung tissue of patients with idiopatic pulmonary fibrosis, usually in the alveolar epithelial cells Medications: antibiotics (nitrofurantoin, sulfasalazine), antiarrythmics (amiodarone, propranolol), anticonvulsants (phenytoin), chemotherapeutic agents (methotrexate, bleomycin, oxaliplatin, erbital) and therapeutic radiation Acid reflux disease (gastroesophageal reflux disease) Diabetes mellitus Genetic factors These observations must be interpreted with great caution, since epidemiologic studies of environmental risk factors are subject to a variety of biases and limitations [1] 2 Expert Rev. Mol. Diagn. 13(4), (2013)

3 Genetics of idiopathic pulmonary fibrosis Review Table 1. Different classes of genes suggested to be involved in the pathogenesis of idiopatic pulmonary fibrosis. Gene ontology Surfactant proteins ABCA3 TERT/TERC MUC5B Study (year) Nogee et al. (2001) Thomas et al. (2002) Brasch et al. (2004) Official gene symbol SFTPC Name Chromosome Variation Association with IPF susceptibility Surfactant protein C Ref. 8p21 c.460+1g>a (I4) In FPF [16] +128T>A (E5) In FPF [17] +1286T>C (E3) In NSIP mixed PAP [18] Lawson (2004) +6108T>C UIP [20] Markart et al. (2007) van Moorsel et al. (2010) Selman et al. (2003) Wang et al. (2009) Selman et al. (2003) Shulenin et al. (2005) Young et al. (2008) Armanios et al. (2007) Tsakiri et al. (2007) Mushiroda et al. (2008) Seibold et al. (2011) SFTPA1 SFTPA2 SFTPB ABCA3 TERT/TERC Surfactant protein A1 Surfactant protein A2 Surfactant protein B ATP-binding cassette, subfamily A, member 3 (ABCA3) Telomerase reverse transcriptase/ telomerase RNA component c.413 C>A (E4); c.557 G>A (E5) c.211 A>G; c T>C 10q22.3 SP-A1_6A 4, AA219_T, AA50_C, AA62_G No [21] In FPF [19]. In NS only (OR = 3.67, p = 0.01; OR = 3.13, p = 0.02; OR = 6.68, p = <0.01; OR = 3.34, p = <0.01) 10q22.3 g.593t>c (E6) No [23] g.692g>t (E6) 2p12-p T>C 1580C in Sm only (OR = 7.63; p = 0.01) 16p13.3 NS and FM No [24] 5p15.33/3p26 c1 28A>G (E1); IVS9 + 11C>T (I9); c3765c>g TERT: CTG>CAG (E1); IVS1+1 G>A; cod112, delc (E2); IVS92 A>C (E10); ACG>ATG (E16). TERC: 98G>A No [22] [22] UIP [26] FPF [27] TERT: c.3346_3522del; c.2594g>a;; c.2240delt; c.1456c>t; c.97c>t; c.430g>a; c.2593c>t. TERC r.37a>g IS IPF [28] rs p = in J [30] MUC5B Mucin 5B 11p15 rs IPF (allelic association, p = ); FPF (allelic association p = Zhang et al. (2011) rs IPF (p = ) [34] E: Exon; FM: Frameshift mutation; FPF: Familial pulmonary fibrosis; HL: Heterozygous allele; I: Intron; I/D: Insertion/delection; IPF: Idiopatic pulmonary fibrosis; IS: Increase susceptibility; J: Japanese population; LL: Low expression allele polymorphisms; NS: Nonsense; NSIP: Nonspecific interstitial pneumonia; OR: Odds ratio; PAP: Pulmonary alveolar proteinosis; PG: Progressive group; PR: Promoter region; Sm: Smokers; S: Sarcoidosis; SS: Systemic sclerosis; UIP: Usual interstitial pneumonia. [33] 3

4 Santangelo, Scarlata, Zito, Chiurco, Pedone & Incalzi Table 1. Different classes of genes suggested to be involved in the pathogenesis of idiopatic pulmonary fibrosis (cont.). Gene ontology MHC TGF-β1 ACE Study (year) Falfán-Valencia et al. (2005) Aquino-Galvez A Official gene symbol Name Chromosome Variation Association with IPF susceptibility MHC MHC 6p21.31 (HLA alleles) HLA-B*15 DRB1*0101 DQB1*0501 Ref. OR=10.72, P=0.011 [39] MICA *001/*00201 OR = 4.72, p = 0.01 [40] Xue et al. (2011) HLA-DRB1*1501 OR = 2.0, p = [41] Xaubet et al. (2003) Fatini et al. (1990) Interleukins du Bois et al. (2002) CR1 FcgRIIa MMP1 PAI-1 Whyte et al. (2000) Pantelidis et al. (2001) Whittington et al. (2003) Vasakova et al. (2011) TGF-β1 ACE Transforming growth factor-β1 Angiotensin I converting enzyme 19q T>C (E1) No [47] +915G>C (E1) 17q23.3 I/D (I16) In SS [57] IL-1α IL-1α 2q14-889C>T No [62] IL-1β IL-1β 2q14-511C>T No IL1RN IL-1 receptor antagonist +3953C>T 2q C>T OR = 3.8 [60] IL-6 IL-6 7p21 A>G (I4) No [63] TNF-RII Tumor necrosis factor-receptor 2 1p C>T IL-10 IL-10 1q31-q32 +43G>A (PR) No [64] IL-4 IL-4 5q C>T; -33C>T p < , p < Ahn et al. (2011) IL-8 IL-8 4q13-q21 rs4073t>a (PR) OR = 0.46, p = [74] Zorzetto et al. (2002) Yuan et al. (2011) Bournazos et al. (2009) Checa et al. (2008) Kim et al. (2003) CR1 CR1 FcgRIIa Complement component (3b/4b) receptor 1 Complement component (3b/4b) receptor 1 Fc fragment of IgG, low affinity IIa, receptor No No rs t>g (i) OR = 0.48, p = q C>G (E33) p = (in S) [75] MMP1 PAI-1 Matrix metalloproteinase Plasminogen activator inhibitor type 1 1q A>G HL and LL genotype (p < 0.05 on average) 1q23 rs p = (PG) [78] 11q I/D (PR) p < 0.008; OR = 1.7 [82] -755 T>T p = 0.03 in Sm IPF vs Sm controls 7q21.3 q22 4G/5G (PR) IS in NSIP not in UIP [97] E: Exon; FM: Frameshift mutation; FPF: Familial pulmonary fibrosis; HL: Heterozygous allele; I: Intron; I/D: Insertion/delection; IPF: Idiopatic pulmonary fibrosis; IS: Increase susceptibility; J: Japanese population; LL: Low expression allele polymorphisms; NS: Nonsense; NSIP: Nonspecific interstitial pneumonia; OR: Odds ratio; PAP: Pulmonary alveolar proteinosis; PG: Progressive group; PR: Promoter region; Sm: Smokers; S: Sarcoidosis; SS: Systemic sclerosis; UIP: Usual interstitial pneumonia. [65] [77] 4 Expert Rev. Mol. Diagn. 13(4), (2013)

5 Genetics of idiopathic pulmonary fibrosis Review Table 2. Gene functions and their likely role in pathogenesis. Genes Function Potential pathogenetic mechanism SFTPC ABCA3 TERT, TERC ELMOD2 MUC5B HLA TGF-β 1 ACE2 IL-1Rα IL-6 Gene that encodes for the pulmonary-associated surfactant protein C, a surfactant protein that promotes alveolar stability essential for lung function and homeostasis. Pulmonary surfactant is a lipoprotein complex composed of 90% lipids and 10% proteins as plasma proteins and apolipoproteins SP-A, SP-B, SP-C and SP-D Gene that encodes for a protein with the function of the maintenance of pulmonary surfactant phospholipid homeostasis Telomerase reverse transcriptase encodes the catalytic component of telomerase. The telomerase RNA component provides a template for the repeat sequence added in tandem to the ends of chromosomes. Telomerase expression has a role in cellular senescence Gene that encodes for one of six engulfment and motility domain-containing proteins. This gene is thought to play a role in antiviral responses Gene that encodes for the major gel-forming mucin in mucus, that conferring the viscoelastic and lubrification properties of saliva, normal lung mucus and cervical mucus Present peptides derived from the endoplasmic reticulum lumen This gene encodes for a member of the transforming growth factor-β family of cytokines, that regulate proliferation, differentiation, adhesion, migration and other functions in many cell types ACE-2 enzyme that degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation The protein encoded by this gene is a member of the IL-1 cytokine family. This cytokine is a pleiotropic cytokine involved in various immune responses, inflammatory processes and hematopoiesis Gene that encodes for a cytokine related to inflammation and B-cell maturation ANG: Angiotensin; IPF: Idiopathic pulmonary fibrosis. The presence of missense or short deletion mutations might cause abnormal pro-protein trafficking, production and function of the protein. The abnormalities in the protein may cause type-ii epithelial cells injury. The protein is also involved in host defense and tissue repair [16 18] ABCA3 mutations were associated with an abnormal ABCA3 intracellular trafficking and/or function that can cause a loss of protein function critical for surfactant metabolism and responsible of interstitial lung disease [24 26] The association of mutant telomerase and FPF can be explained with the hypothesis that cell death or cell cycle arrest can occur in response to short dysfunctional telomeres that activate a DNA damage response. The loss of alveolar cells provoke the fibrotic lesion in patients with short telomeres. The process of telomere shortening still contributes to the pathogenesis: conditions that increase cell turnover (e.g., smoking or aging) might lead to fibrosis [27] Participates in antiviral responses and is expressed in the relevant cell types in the lung, in agreement with the hypothesis of virus-induced epithelial injury as one of the triggers in IPF. Also provides a specific molecular mechanistic model linking genetic susceptibility to disease pathogenesis [31,32] The presence of the rs MUC5B promoter SNP can increase the expression of the gene and decrease the clearance of the protein. MUC5B excess leads to a reduction in lung clearance of inhaled particles and microorganisms with consequent lung injury [33,34] Because the HLA haplotype inheritance regulates the antigen repertoire that stimulates the T-cell response, it is possible that immunoregulatory elements within the HLA Class II complex are involved in the pathogenesis of IPF: immunogenetic processes can lead to a susceptibility to IPF or influence its manifestation [41]. TGF-β 1 is considered one of the critical mediators for its profibrogenic effects in the development of IPF [43,44]. Polymorphism +869T>C at signal sequence region can influence protein secretion, might regulate its expression and influence intracellular trafficking or export efficiency of the protein [45 47] ACE-2 may protect the lung against fibrogenesis through limiting local accumulation and degradation of ANG II produced in response to bleomycin: downregulation of ACE-2 may be a critical profibrotic event in IPF [59] The role of IL-1Rα in preventing fibrosis suggests that insufficiently expressed IL-1Rα can promote fibrogenesis and predispose to IPF [68] The genotype is associated with rapidity of disease progression as measured by impairment of carbon monoxide transfer [63] 5

6 Santangelo, Scarlata, Zito, Chiurco, Pedone & Incalzi Table 2. Gene functions and their likely role in pathogenesis (cont.). Genes Function Potential pathogenetic mechanism IL-10 The protein encoded by this gene is a cytokine that downregulates the expression of Th1 cytokines, MHC class II receptors and costimulatory molecules on macrophages. It improves B-cell survival, proliferation and antibody production, and is able to block B-cell NF-kB activity. It is also involved in the regulation of the JAK-STAT signalling pathway The presence of the IL G>A substitution influences the efficiency of protein translocation and signal peptide cleavage, and could lead to lower levels of IL-10 protein secretion [64] IL-4 IL-8 CR1 IgG receptor FcgRIIa MMPs The protein encoded by this gene is a pleiotropic cytokine produced by activated T cells. This cytokine is a ligand for the IL-4 receptor. The IL-4 receptor also binds to IL-13, which may contribute to many overlapping functions of this cytokine and IL-13. STAT6, a signal transducer and activator of transcription, has been shown to play a central role in mediating the immune regulatory signal of this cytokine The protein encoded is a chemokine secreted by several cell types and functions as chemoattractant and angiogenic factor Gene that encodes a monomeric single-pass type I membrane protein. This protein regulates the cellular binding to immune complexes FcgRIIa R131H polymorphism determines receptor affinity for IgG subclasses. Since FcgRIIa is expressed by diverse leukocyte types, including macrophages and neutrophils Family of zinc-dependent endopeptidases that are capable of cleaving all extracellular matrix substrates, playing a relevant role in diverse physiological and pathological processes. MMP-1 has a prominent role in initial cleavage of the extracellular matrix PAI-1 Gene that encodes for a member of the serine proteinase inhibitor (serpin) superfamily. This member is the principal inhibitor of tissue plasminogen activator and urokinase, and hence is an inhibitor of fibrinolysis and impairs the dissolution of clots ANG: Angiotensin; IPF: Idiopathic pulmonary fibrosis. Gene polymorphisms are likely to play a pathogenic role in IPF and in modification of its clinical presentation and severity. It is unknown whether these polymorphisms can influence the production of IL-4, or how can influence its affinity to the IL-4 receptors on lung fibroblasts [65,66] A SNP in the promoter of this gene (rs4073t>a) causes an upregulation of IL-8 protein synthesis leading to an increased susceptibility of developing IPF [74]. The role of IL-8 in pulmonary fibrosis is confirmed by an animal study demonstrating that neutralization of IL-8 attenuates the bleomycin induced lung fibrosis [70] An impaired clearance of immune complexes containing viral particles and/or complement opsonised viruses could be related to a CR1 polymorphisms that implies a lower CR1 expression. The consequence are repeated episodes of lung injury and potentially aberrant wound healing [75,77] FcgRIIa R131H polymorphism seems to influence IgG mediated effector responses since the engagement of FcgRIIa with IgG-containing immune complexes initiates a number of leukocyte effector responses. Damage to the alveolar walls and pulmonary interstitium could lead to fibroblast activation and aberrant deposition of fibrotic tissue which is characteristic of IPF [79] Members of the MMP family might be involved in the deregulation of the synthesis and degradation of extracellular matrix proteins a process that leads to the enlarged extracellular matrix deposition in the tissue phenotype of fibrosis [84] Fibrin deposits observed in fibrotic lung disease might persist because the normal fibrinolytic activity within the alveolar space is suppressed by increase in proteolytic inhibitors such as PAI-1 [92] The fact that sequence variation in SFPTC is rarely found in the DNA of subjects with sporadic IPF indicates that SFTPC mutations do not contribute to the pathogenesis of sporadic IPF cases. Indeed, Lawson and colleagues studying the SFTPC alterations in UIP, demonstrated that only 13 patients out of 135 had SFTPC genetic sequence variations, and only one had a sequence variation (+6108T>C) causing an aminoacid change (I73T) [20]. The authors concluded that, despite the findings from familial SFTPC cases, genetic mutations in SFTPC are not common in sporadic cases of IPF. Also in the study by Markart and colleagues on 35 adult patients (25 IPF + 10 NSIP), only two nonsynonymous variants of the SFTPC gene, T138N (c.413 C>A) and S186N (c.557 G>A), were identified, with similar allele frequency in cases and controls [21]. They concluded that in both sporadic cases of IPF and NSIP, mutations in the SFPTC gene are not common and not pathogenetic. While SFTPC may not be the primary gene of interest in sporadic IPF, the study of SFTPC mutations may nonetheless help us to understand the pathogenesis of this disease. The presence of identified missense or short deletion mutations can influence 6 Expert Rev. Mol. Diagn. 13(4), (2013)

7 Genetics of idiopathic pulmonary fibrosis Review ELMOD2 (antiviral responce) HLA (immunogenetic processes) Figure 1. Proposed genes role in the development of pulmonary fibrosis. the processing, production and function of the protein. These abnormalities may cause type II epithelial cell injury and the initiation and propagation of disease. Surfactant protein A mutations The identification of SFTPC mutations led to the hypothesis that mutations in the genes for other surfactant proteins might be found in IPF. By studying 84 unrelated patients with IPF, Selman et al. found that, compared with healthy controls, three (AA50_C, AA62_G, AA219_T ) of five SNPs that characterizes the SP-A1 6A 4 allele, and one SNP of the surfactant protein B allele (SP-B) (1580 T>C) were more frequent in the nonsmoker and smoker subgroups, respectively (p 0.01) [22]. Wang and colleagues described two more mutations in this gene: heterozygosity for a g.692g>t transversion in exon 6 of the SFTPA2 gene and heterozygosity for a g.593t>c transition in exon 6 of the SFTPA2 gene. The first mutation was identified in affected members of a four-generation family with IPF (some of whom had lung cancer) and results in a gly231 to val (G231V) substitution at a highly conserved residue in the carbohydrate recognition domain. The second mutation was observed in a patient with IPF and lung cancer and caused a phe198 to ser (F198S) substitution at a highly Alveolar epithelial injuries TERC and TERT (short dysfunctional telomerase) MUCB5 (impairs mucosal host defense, results in excessive lung injury from inhaled substances) SP-C (role in host defence against pathogens) ABCA3 (critical function for surfactant metabolism) Fibroblast activation/myofibroblastic differentiation/emt TGF -β1 (differentiates fibroblast to myofibroblast) ACE (activates fibroblast and macrophages) Disordered coagulation pathway/fails of regulatory mechanism PAI1 (suppresses fibrinolysis) MMPs and TIMPS (role in initial cleavage of extracellular matrix substrates) Inflammation CR1 (clearance of immune-complexes) IgG receptor FcgRIIa (initiates a number of leukocyte effector responses as release of proinflammatory cytokines) ILs (cytokine and chemokine) Pulmonary fibrosis conserved residue in the carbohydrate recognition domain. These two mutations were not found in population-based controls [23]. ABCA3: maintenance of pulmonary surfactant phospholipid homeostasis The processing and secretion of SP-C are dependent on ABCA3, a lamellar body membrane protein expressed exclusively in the type II cells producing surfactant protein A and fundamental for the homeostasis of pulmonary surfactant phospholipids. ABCA3 mutations have been associated with an abnormal ABCA3 intracellular trafficking and/or function that can cause a loss of protein function critical for surfactant metabolism and responsible for interstitial lung disease. In 2004, Shulenin et al. identified in 16 racially and ethnically diverse infants with severe neonatal surfactant metabolism dysfunction, 12 different homozygous or compound heterozygous nonsense and frameshift mutations in highly conserved residues and splice site of the ABCA3 gene [24]. Subsequently, it was showed by Bullard et al. that the heterozygosity for ABCA3 mutations modifies the severity of lung disease associated with a SFTPC mutation in pediatric patients [25], but a later study by van Moorsel and colleagues did not find 7

8 Santangelo, Scarlata, Zito, Chiurco, Pedone & Incalzi an association between ABCA3 variation and SFTPC mutations [19]. Interestingly, in 2008 a case of UIP in an adolescent with three novel ABCA3 heterozygous variants (c1-28a>g, IVS9+11C>T intron 9 and c.3765c>g) was reported. In this case the altered ABCA3 protein function was considered to be the etiology of the histopathologic manifestations of UIP [26]. TERT, TERC Several reports have shown an association between mutant telomerase, FPF and sporadic IPF. Telomerase is a polymerase that adds telomere repeats (a repeat that comprises the six TTAGGG nucleotides complementary to the template telomerase reverse transcriptase, TERT) at the end of chromosome and is formed by the following two components such as TERT, which encodes the catalytic component of telomerase and the telomerase RNA component (TERC), that provides a template for the repeat sequence added in tandem to the ends of chromosome. Telomeres seem to act as a counter of cell duplications as they shorten at each cell cycle, and conditions that increase cell turnover (e.g., smoking or aging) are associated with short telomeres [27]. In 2007, Armanios et al. studied the involvement of the pathways leading to telomere shortening in IPF in six families with the presence of two or more cases of IPF [27]. They found that germline mutations in the genes TERT and TERC cause autosomal dominant dyskeratosis congenita, an hereditary disorder associated to bone marrow failure due to aplastic anemia, and pulmonary fibrosis. They identified five heterozygous mutations in TERT (two missense, two splice junction and one frameshift) and one mutation in TERC; none of these was found in controls. The association of the mutant telomerase with FPF may be explained by the fact that short dysfunctional telomeres activate a DNA damage response that eventually leads to cell death or cell cycle arrest. These processes lead to a depletion of local progenitor reserve that is essential for the turnover of the bronchoalveolar epithelium, with eventual loss of alveolar cells and development of fibrosis. The loss of alveolar cells, in turn, causes the fibrotic lesion, similar to the one observed in autoimmune disease associated with lung fibrosis. The presence of pulmonary fibrosis can be explained by the fact that the broncoalveolar epithelium is continuously replaced and relies to the local progenitor reserve limited by short telomerase. Conditions that increase cell turnover (e.g., smoking or aging), are associated with short telomeres. Although the mutations in TERT and TERC do not seem to be present in the majority of FPF cases, the process of telomere shortening may nonetheless contribute to the pathogenesis of this disease. These findings were replicated by Tsakiri et al. who, using a genetic linkage approach, identified two frameshift deletion and five missense mutations in TERT, and one heterozygous mutation in TERC in patients with IPF [28]. Furthermore, Alder et al. studied patients without family history of idiopathic interstitial pneumonia (IIP) to explore if short telomeres might contribute to the development of sporadic IIP. Leukocytes from IIP patients had shorter telomeres compared to leukocytes from age-matched controls (p < ). Furthermore, compared with healthy controls, IPF patients had shorter telomeres in alveolar epithelial cells (p < ), similar to familial cases with mutations. Although mutations in TERC were revealed in only one patient out of a series of 100 cases, the same group identified a cluster of individuals (3%) affected by both IPF and cryptogenic liver cirrhosis, suggesting a role of telomere shortening in both lung and liver fibrosis [29]. Finally, after genotyping 159 Japanese IPF patients and 934 controls, Mushiroda et al. found a SNP in intron 2 of the telomerase reverse transcriptase gene that was significantly associated with IPF [30]. Alveolar epithelial injuries ELMOD2 In 2006, Hodgson et al., through a genome-wide association study performed on six Finnish families with FPF identified five loci of interest. In particular, two functionally uncharacterized genes, ELMOD2 and LOC152586, were significantly more frequent among patients compared with population-based controls. In particular, ELMOD2 was identified as a possible candidate gene for susceptibility to familial IPF, as it is expressed in the lung and has a significantly decreased mrna expression in lungs of IPF patients compared with that of healthy controls (p = 0.05) [31]. A recent study confirmed the role of ELMOD2 as susceptibility gene for IPF [32]. Taking into account that the virus-induced epithelial injury is one of the hypothesized causes and risk factors for development of IPF, it was demonstrated that ELMOD2 regulates IFN-related antiviral responses and that its expression is decreased in response to viral infections. Furthermore, it was expressed in a relevant manner in lung epithelial cells and alveolar macrophages (AMs), the fundamental cell for response to respiratory viruses infections [32]. MUC5B An association has been found between a common variant in the putative promoter of the MUC5B gene and the development of both FPF and sporadic IPF. Using a genome-wide linkage scan study approach, Seibold et al. detected a linkage between IIP and a 3.4-Mb region of chromosome 11p15 in 82 families. They found an association of the minor allele (T) of a SNP in the promoter of MUC5B gene, rs , with IPF (allelic association, p = ) and with FPF (allelic association p = ) [33]. In a study of 341 cases with IPF from the University of Pittsburgh (PA, USA) and the University of Chicago (IL, USA) and 802 controls from the same 2 centers, Zhang et al. confirmed the results of Seibold et al., finding a strong association of the minor allele at rs with IPF (p = ) [34]. The presence of too much MUC5B (due to a promoter SNP that may cause an upregulation of the gene or influence the clearance of the protein) could impair mucosal host defense with the results of an excessive lung injury from inhaled substances and development of IIP. Furthermore, it is also possible that an excess of MUCB5 could interfere and prevent the alveolar repair mechanism through the interaction between the type II alveolar epithelial cells and the underlying matrix or with the surface-tension properties of surfactant. If the process of re-epithelialization of 8 Expert Rev. Mol. Diagn. 13(4), (2013)

9 Genetics of idiopathic pulmonary fibrosis Review the basal lamina of the alveolus fails or surfactant activity is to some extent defective, ongoing alveolar collapse and fibrosis of adjacent bronchoalveolar units may be enhanced. HLA The human MHC complex on chromosome 6p21.31 is characterized by the presence of numerous, extraordinarily polymorphic HLA alleles [35 38]. Studying polymorphisms of the MHC, locus HLA-B, DRB1 and DQB1 in a cohort of 75 IPF patients and 95 controls by using PCR and hybridization, Falfán-Valencia et al. demonstrated the association of the following three haplotypes with IPF: HLA-B*15 DRB1*0101 DQB1*0501 (odds ratio: 10.72; CI: ); HLA-B*52 DRB1*1402 DQB1*0301 (odds ratio: 4.42; CI: ) and HLA-B*35 DRB1*0407 DQB1*0302 (odds ratio: 4.73; CI: ) [39]. Another study in a cohort of 80 sporadic IPF patients compared to 201 controls, found a significant association between MHC class I chain-related gene A allele 1 (MICA *001) and IPF (odds ratio: 2.91; p = 0.03). A stronger association was found with the MICA *001/*00201 genotype (odds ratio= 4.72; p < 0.01). Furthermore, a strong immunoreactive MICA staining was found in alveolar epithelial cells and fibroblasts from IPF lungs, but not in control lungs. Soluble MICA was also detected in 35% of IPF patients compared with 12% of control subjects of (p = ), and a significantly decreased MICA receptor NKG2D expression was found in γ/δ-t cells and NK cells obtained from IPF lungs. The proposed conclusion was that the presence of MICA polymorphisms and an abnormal expression of NKG2D might confer a susceptibility to IPF [40]. The HLA class I and class II were further investigated by Xue et al. in 2011, who found an increased prevalence of DRB1*1501 in IPF patients compared with normal subjects [41]. Since the HLA haplotype inheritance regulates the antigens repertoire that stimulates the T-cell response, it is possible that immunoregulatory elements within the HLA Class II complex are involved in the pathogenesis of IPF: immunogenetic processes can lead to a susceptibility to IPF or influence its manifestation. Thus, the HLA-DR locus could represent a specific chromosomal area for genomic studies aimed at the identification of the various clinical IPF phenotypes [41]. Fibroblast activation/myofibroblastic differentiation/emt Profibrotic molecules TGF-β 1 TGF-β 1 is a growth factor produced by several cell types that is chemotactic for fibroblasts, induces the synthesis of matrix proteins and glycoprotein and inhibits collagen degradation by induction of protease inhibitors. It has been also reported that TGF-β 1 is implicated in tissue remodeling and promotes the differentiation of fibroblasts into myofibroblasts [42]. Furthermore, TGF-β 1 inhibits alveolar epithelial cells type II proliferation [43] and induces EMT in cultured alveolar epithelial cells [44]. Due to all this evidence, and because of its profibrogenic effects, TGF-β 1 is considered one of the critical mediators in the development of IPF; and increased expression of TGF-β 1 has been found in lung tissue from patients with this disease and in animal models of pulmonary fibrosis [45,46]. The human gene encoding TGF-β 1 is located on chromosome 10q13 and seven polymorphisms in this gene have been identified. Xaubert et al. assessed polymorphisms in TGF-β 1 in white IPF patients from Spain. Studying two exon 1 polymorphisms in codons 10 and 25 (+869T>C and +915G>C), both resulting in amino acid substitutions, and found that these polymorphisms do not predispose to the development of IPF, but may affect IPF progression increasing TGF-β 1 production [47]. Accordingly, the inhibition of TGF-β 1 activity could be considered a potential therapeutic strategy in IPF. A recent investigation in the Han population found a significant difference in +869T>C genotype distribution of TGF-β 1 between IPF cases and controls, a significant negative association between T>C genotype and the development of IPF, and a positive association between C>C genotype and the development of IPF [48]. Enzymes ACE ACE is a dipeptidyl carboxypeptidase that hydrolyzes angiotensin 1 (ANG I) into angiotensin 2 (ANG II), a potent vasopressor and aldosterone-stimulating peptide. It is encoded by a gene localized on chromosome 17q23 [49]. It has been reported that ANG II promotes the proliferation and collagen synthesis of cardiac fibroblasts, and that this phenomenon is inhibited by ANG II receptor type 1 (AGTR1) antagonists [50 52]. These findings indicate that ANG II activates fibroblasts and macrophages by signaling via AGTR1, resulting in clinical findings of heart injury and fibrosis. Thus, it is conceivable that the local renin angiotensin aldosterone system in the lung might play a role in lung fibrogenesis. Using a rat bleomycin-induced model of pulmonary fibrosis, Otsuka et al. demonstrated that the AGTR1 expression is upregulated in fibrotic lungs and that the inhibition of AGTR1 reduces bleomycin-induced lung fibrosis in rats [53]. In a retrospective study of 478 patients with IPF, however, Nadrous and colleagues concluded that there were no differences in mortality between the groups receiving ACE inhibitors and/or statins versus neither and that ACE inhibitors and/or statins are not associated with improved survival in IPF [54]. At any rate, a functional insertion/ deletion polymorphism has been found in intron 16 of the ACE gene responsible for almost half of the variance in serum ACE levels [55,56] and the deletion allele has been shown to be associate with systemic sclerosis, a disease showing a high propensity for lung fibrosis [57]. In line with these findings, Morrison et al. detected an increased frequency of allele deletion polymorphism of the ACE gene in a small cohort of mixed IPF cases (69 vs 54% in controls) [58]. In support of a role of the renin angiotensin aldosterone system in the lung fibrogenesis, Li et al. reported a marked reduction of ACE-2 mrna protein (that degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation), and its enzymatic activity in both human and experimental lung fibrosis induced by bleomycin. ACE-2 might protect the lung against fibrogenesis through limiting local 9

10 Santangelo, Scarlata, Zito, Chiurco, Pedone & Incalzi accumulation and degradation of ANG II produced in response to bleomycin: downregulation of ACE-2 may be a critical profibrotic event in IPF [59]. Inflammation Genes encoding for cytokines and chemokines IL-1 comprises two structurally distinct forms, IL1-α and IL1-β, both potent proinflammatory cytokines with fibrogenic properties encoded together with their inhibitor IL-1 receptor antagonist (IL1RN), by a cluster genes localized on chromosome 2q14. In 2000, Whyte et al. were the first to report an association between the increased risk of developing fibrosing alveolitis and the single base variation of IL1RN (+2018C>T) allele 2 and of TNF-α (-308G>A) allele 2 [60]. The IL1RN association with susceptibility to IPF was not confirmed in patients studied by Hutyrová et al. [61], but an association between the -889C>T allele of IL1-α and severity of gas transfer deficits in patients with IPF has been reported by du Bois [62]. In addition, although Pantelidis et al. did not detect an association between the TNF -380G>A allele and IPF in UK, an increased frequency of cocarriage of the IL-6 intron 4 A>G and the TNF-RII 1690C>T alleles was found despite the different chromosomes location. This genotype seems to be associated with rapidity of disease progression as measured by impairment of carbon monoxide transfer [63]. IL-10 has an anti-inflammatory effect and a regulatory role affecting TNF-α production. In 2003 Whittington et al. demonstrated changes in IL-10 protein production by AMs from patients with IPF. They speculated that a +43G>A substitution in the start codon of the IL-10 gene could affect the efficiency of protein translocation and signal peptide cleavage resulting in lower levels of IL-10 protein secretion, but no difference in allele frequency between patients and controls was observed [64]. Moreover, Vasakova et al. speculated on the pathogenic role in IPF of two polymorphisms: the -590C>T genotype of IL-4 promoter and the -33C>T genotype were more frequent in the IPF group (p < ) compared with healthy controls [65]. The same authors found a correlation between respiratory function parameters, BALF cell counts, and HRCT alveolar and interstitial scores with the polymorphisms of promoter regions of IL1-α at -889C>T position and IL-4 at -1098T>G, -590C>T and -33C>T position gene polymorphisms. They conclude that these gene polymorphisms are likely to play a pathogenic role in IPF and in modification of its clinical presentation and severity but it is not known; however, whether these polymorphisms can influence the production of IL-4, or how they can influence its affinity to the IL-4 receptors on lung fibroblasts [66]. In a recent article, Barlo et al. demonstrate a significant decrease of IL-1 receptor antagonist (IL-1Rα)/IL-1β ratio both in serum and BALF in 77 IPF patients compared with 349 healthy controls. One SNP of the IL1RN gene was associated with both the susceptibility to IPF and reduced IL-1Rα/IL-1β ratio in BALF. These results indicate that the genetic variability in the IL1RN gene may play a role more important than it has been until recently thought in the pathogenesis of IPF [67]. Finally, in a Caucasian population, Korthagen et al. performed a linkage analysis of five case control studies, finding a close linkage disequilibrium between the variable number tandem repeat (VNTR), minor alleles rs and rs419598, allowing the polymorphisms to be combined into a VNTR*2 haploblock [68]. They concluded that polymorphisms associated with the IL1RN VNTR is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Rα mrna expression. The role of IL-1Rα in preventing fibrosis suggests that insufficiently expressed IL-1Rα can promote fibrogenesis and predispose to IPF [68]. IL-8 IL-8 concentration in the BALF and IL-8 protein mrna expression in AMs is increased in patients with IPF [69]. This may be related to neutrophil accumulation in the lower respiratory tract, which is a typical finding in IPF; recruitment and activation of these cells is believed to play a fundamental role in the development of lung injury that precedes normal repair [69]. The role of IL-8 has been demonstrated in an animal study showing an attenuation of bleomycin-induced lung fibrosis by the neutralization of IL-8 [70]. The gene encoding IL-8,located on chromosome 4q12-q21, is composed of four exons and three introns [71]. The SNPs within IL-8 gene are candidates for cystic fibrosis and a neutrophil-dominant inflammatory lung disease like IPF [72]. While in a comparison of 237 subjects with IPF and 456 normal controls, association has been found between IPF risk and these SNPs [73], by the genotyping of one promoter (rs4073t>a) and two intronic SNPs (rs t>g and rs c>t). Ahn et al. demonstrated that the IL-8 rs4073 T allele is significantly associated with an increased risk of IPF in the Korean population, maybe due to an upregulation of IL-8 protein synthesis in the lung [74]. Immunomodulatory genes CR1 CR1 complement component (3b/4b) receptor 1 is located in the long arm of chromosome 1 at band 32 (1q32). The monomeric single-pass type I membrane glycoprotein encoded by this gene (expressed on erythrocytes, leukocytes, glomerular podocytes and splenic follicular dendritic cells) allows the cellular binding to particles and immune complexes. The decreased expression of this protein and/or mutations in its gene has been correlated to gall bladder carcinomas, mesangiocapillary glomerulonephritis, systemic lupus erythematosus and sarcoidosis. In 2002 Zorzetto et al. demonstrated a significant association between the +5507C>G polymorphism, that is thought to result in low expression of CR1 on erythrocytes, a marker of progression of sarcoidosis to IPF. These CR1 polymorphisms related to a low CR1/erythrocyte ratio might contribute to impaired clearance of immune complexes containing viral particles and/or complement-opsonised viruses, and in association to environmental and genetically factors, could lead to an aberrant wound healing after repeated episodes of lung injury [75]. To replicate this finding, Kubistova et al. compared 53 Czech IPF patients with 203 Czech healthy controls and 70 English 10 Expert Rev. Mol. Diagn. 13(4), (2013)

11 Genetics of idiopathic pulmonary fibrosis Review IPF patients with 149 English controls, finding no significant differences in the distribution of CR C>G variants [76]. Subsequently, Yuan et al. exploring by PCR-restriction fragment length polymorphism (PCR-RFLP), the association between the erythrocyte CR1 genomic density and +3650A>G site polymorphism and IPF, found an association between the HL and LL genotypes of CR1 gene with IPF. The final conclusion was been that individuals carrying the L allele might be susceptible to develop IPF [77]. IgG receptor FcgRIIa In many chronic inflammatory diseases there is a persistently high concentration of antibody antigen complexes, both circulating and as tissue deposits, that in a normal immune response are removed by tissue-resident phagocytes. The ensuing activation of complement and engagement of leukocyte Fc receptors can lead to tissue injury [5,6]. A pivotal role in this pathogenetic process is played by Fcγ receptors, that are expressed principally by leukocytes and are responsible for the recognition of IgG containing immune complexes. Genetic variation (in the form of SNPs) of the gene encoding for this receptor have been reported to be related to some chronic inflammatory and autoimmune diseases [78]. In 2010, Bournazos et al. found a strong relationship between IgG receptor FcγRIIa rs (R131H) variant substitution and severity and progression IPF. This variant interacts with the Fc region of IgG determining the affinity of FcgRIIa for human IgG. Since the H131, but not the R131 variant, can interact with IgG2 binding and mediate phagocytosis of IgG2-coated particles, the FcgRIIa R131H polymorphism influence the IgGmediated effector responses. The engagement of FcgRIIa with IgG-containing immune complexes initiates a number of leukocyte effector responses as antibody-dependent cellular cytotoxicity and phagocytosis, that might contribute to the damage to the alveolar walls. It was hypothesized that the consequent fibroblast activation and aberrant deposition of fibrotic tissue could lead to IPF. These findings support the role of immunological mechanisms and the pathogenic potential of immune complexes in the development of IPF [79]. Disordered coagulation pathway/fails of regulatory mechanism Extracellular matrix MMPs & TIMPs The inflammatory process in IPF is characterized by infiltration of various inflammatory cell types, release of cytokines and chemokines and secretion of matrix remodeling proteinases like the matrix metalloproteinases (MMPs) [80]. MMPs, a family of zinc (Zn)-dependent endopeptidases, virtually cleave all ECM substrates and their catalytic activity is accomplished by their inhibitors named tissue inhibitors of MMPs (TIMPs). The imbalance between MMPs and TIMPs may play an important role in fibrogenesis. There are structurally and functionally diverse types of MMPs. The most ubiquitously expressed interstitial collagenase is MMP-1, which has the role of initiating the cleavage of the ECM. The identified insertion/deletion of G at position of the MMP-1 gene promoter creates an allele having a single guanine (1G) and another having two guanines (2G) [81]. The presence of the 2G allele in polymorphism of MMP-1 might increase the level of protein expression, leading to a more intense degradation of ECM, an important process in tissue remodeling and repair during development and inflammation. In a case control study of 130 IPF patients and 305 healthy controls, Checa et al. studied the 2G polymorphism at using PCR-RFLP analysis technique [82]. This polymorphism has been previously shown to generate the core of an AP-1 binding site, and its transcriptional activity is correlated with risk of developing IPF. Indeed, a higher frequency of the 2G/2G genotype was found in IPF than in controls (63 vs 49%; odds ratio=1.7, p = 0.008). In contrast, chromatin immunoprecipitation assay performed on IPF fibroblasts with either -755 genotype [TT(-755) and GT(-755) genotypes] did not reveal a significant difference between IPF and healthy controls in the frequency of the SNP -755T>G, previously identified by sequencing the MMP-1 promoter. After stratification for smoking status, however, a significant increase in the -755T>T genotype frequency was observed in smokers compared with nonsmoking controls (45 vs 26%; odds ratio=2.3, p = 0.03). In conclusion, polymorphisms of the MMP-1 promoter may increase risk of IPF and it is possible a gene environment interaction occurs with smoke. The first study using a gene expression profiling approach, published in 2002 by Zuo et al. [83], found an upregulation of MMPs, predominantly MMP-1 and MMP-7 (matrilysin) in the lungs of patients with UIP. In their data set, they found significantly higher levels of MMP-1, MMP-2 and MMP-9 in UIP lungs: these members of the MMP family might be involved in the deregulation of the synthesis and degradation of ECM proteins a process that leads to enlarged ECM deposition in the tissue phenotype of fibrosis [84]. Furthermore, studies conducted in human pulmonary fibrosis and animal models of pulmonary fibrosis, showed the upregulation of MMP-1 (collagenase-1 that degrades fibrillar collagens) as well as MMP-2 and MMP-9 (gelatinases A and B, which have a broad range of substrates including type IV collagens from basement membranes [85 87]). Others studies suggested an increased production of TIMP-1, -2, -3 and -4 in IPF [85,88]. Supporting the hypothesis that MMPs and their inhibitors are involved in pulmonary fibrosis, it has been shown that atorvastatin, an inhibitor of the hydroxy-methyl-glutaryl-coa reductase with anti-inflammatory effects and capability to downregulate MMPs, inhibits the synthesis and release of MMP-9 and TIMP-1 in the BALF of rats with bleomycin-induced pulmonary fibrosis. Atorvastatin, however, has no significant effect on circulating MMP-9 and TIMP-1 [89]. Coagulation pathway gene PAI-1 PAI-1, encoded by the SERPINE1 gene, is a member of the serine protease inhibitors. It inhibits PLAT and PLAU that proteolytically activate plasminogen into plasmin, which breaks down fibrin 11