Abbreviations

Introduction

A 48-year-old Italian woman was seen in our outpatient clinic in April 2010. She reported a 2-year history of progressive dyspnea and reduced exercise tolerance. She had sought medical attention when her cough, which her doctor attributed to an upper respiratory tract infection, did not go away with broadspectrum antibiotics. She had smoked 20 cigarettes daily for 25 years and was on no regular medication. She also reported a history of menorrhagia, decreased visual acuity with horizontal nystagmus, and easy bruising. She had no joint involvement or other signs or symptoms to suggest an underlying connective tissue disease. She denied signi cant environmental or occupational exposures and no precipitins to alveolitic antigens were detected. Family history revealed that one of the patient’s siblings had albinism. On examination, she had fair skin and digital clubbing; widespread velcro-type end-inspira- tory crackles could be heard on lung auscultation. Oxygen

P. Spagnolo (*) · N. Bernardinello

Respiratory Disease Unit, Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova, Padova, Italy

e-mail: paolo.spagnolo@unipd.it

saturation at rest on room air was 94% but rapidly dropped to 85% during a 6-min walk test. Chest radiograph showed bilateral reticular opacities. A CT thorax con rmed the presence of diffuse interstitial brosis with extensive subpleural honeycombing particularly in the upper zones (Fig. 28.1). Lung function tests showed a restrictive ventilatory defect (forced vital capacity 60% of predicted) with a severe reduction in gas transfer (27% of predicted). Cardiac ultrasonography showed an estimated right ventricular systolic pressure of 60 mmHg. The right ventricle was dilated and mildly hypokinetic, with no hypertrophy. She was treated with supportive care only. The patient rapidly deteriorated and died 11 months later while listed for lung transplantation.

Pulmonary brosis may occur in the context of several genetic disorders, the most common being dyskeratosis congenita and Hermansky-Pudlak syndrome. Disorders caused by the inheritance of a single defective gene are referred to as monogenic or single gene disorders. They may be either “recessive” (i.e., they produce the diseased phenotype only if a copy of the abnormal gene is transmitted by both parents) or “dominant” (i.e., the transmission of a single copy of the abnormal gene is suf cient for the disease to occur), and are generally due to rare genetic variants (i.e., mutations) often leading to a single amino acid change in the encoded protein. Conversely, complex diseases result from genetic variations relatively common in the general population (termed “polymorphisms”) and, importantly, involving multiple genes, each contributing an effect of varying magnitude. In addition, gene-gene and gene-environment interactions are believed to contribute signi cantly to disease pathogenesis and clinical manifestations.

The genetics of diffuse parenchymal lung disease is complex. For example, idiopathic interstitial types of pneumonia display considerable genetic heterogeneity and carriage of both rare (i.e., within surfactant protein C, surfactant protein A2, telomerase reverse transcriptase and telomerase RNA component genes, among others [1–7]) and common variants (i.e., within MUC5B gene) [8] increases the risk of developing the disease. In familial forms of pulmonary

brosis, many of the pedigrees show vertical transmission consistent with an autosomal dominant pattern of inheritance, though with variable penetrance. Indeed, as many as 45% of the pedigrees display phenotypic heterogeneity, suggesting that the underlying genetic factors may lead to an increased “generic” predisposition to pulmonary brosis with additional (largely unidenti ed) factors acting as disease modi ers [9].

Hermansky-Pudlak Syndrome

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculo-cutaneous albinism, bleeding diathesis, and accumulation of ceroid lipofuscin, an amorphous lipid-protein material, in the reticuloendothelial system of various tissues [10, 11]. Seemingly disparate, these abnormalities are believed to be related to defective formation, intracellular traf cking, or function of lysosomes (ceroid lipofuscin deposition), or lysosome-­related organelles such as melanosomes (oculo-­ cutaneous albinism) and platelet dense bodies (bleeding dysfunction) [12]. Speci cally, hypopigmentation is secondary to impaired melanosome formation, traf cking, or transfer to keratinocytes, while the bleeding diathesis is caused by the absence of platelet dense bodies, which are involved in secondary platelet aggregation.

Ten types of HPS associated with mutations in 10 different genes have been described. HPS types 1 and 4 are the most severe forms of the disease and are associated with pulmonary brosis, hemorrhage, and granulomatous colitis while types 3, 5, and 6 diseases are associated with a milder phenotype. Prevalence is estimated at 1–2 per million population worldwide but is as high as 1 in 1800 in northwestern Puerto Rico, making it the most common single-gene disorder in this population and accounting for approximately 50% of all cases globally [13]. Notably, most Puerto Rican individuals affected by HPS type 1 carry the same 16-base-pair duplication in exon 15 of HPS1 gene (10q23.1), suggesting a founder effect [14]. Additional mutations associated with HPS are located within AP3B1 (5q14.1; HPS-2), HPS3 (3q24), HPS4 (22q11.2-q12.2), HPS5 (11p15-p13), HPS6 (10q24.32), DTNBP1 (6p22.3; HPS-7), BLOC1S3 (19q13; HPS-8), PLDN (15q21.1; HPS9), and AP3D1 (19p13.3; HPS10), which encode components in one of four protein complexes (i.e., adapter protein 3 [AP-3] and biogenesis of lysosomerelated organelles complex 1, 2, and 3 [BLOC-1, BLOC-2, BLOC-3]) that support intracellular biogenesis and traf cking of lysosome and lysosome-related organelles [11].

The diagnosis of HPS can be suspected in patients with skin and hair color lighter than the other family members and with a history of excessive bleeding and bruising, early-onset pulmonary brosis, or granulomatous colitis, but is generally established by “whole mount” electron microscopy of plate-

lets showing the absence of dense bodies [15, 16]. Conversely, bleeding time assessment is not reliable and is not recommended in the diagnostic work-up of HPS [17]. Genetic testing con rms the diagnosis of HPS and determines the disease subtype. All HPS subtypes are associated with oculo-­ cutaneous albinism, although with variable degrees of hypopigmentation. Bleeding diathesis is similarly present in all HPS patients with severity ranging from easy bruising and epistaxis to postpartum hemorrhage and serious bleeding during surgery [18].

Progressive pulmonary brosis, the most serious complication, usually presents in the third or fourth decade— although has been reported as early as in adolescence—and occurs only in patients with HPS types 1 (100% of cases), 2, and 4 [19, 20]. While the pathogenic mechanisms of HPS-­ related pulmonary brosis remain largely unknown, dysfunction of lamellar bodies in type II pneumocytes, which synthesize, store, and release surfactant, is probably a contributing factor in causing deposition of ceroid and degeneration and death of type II cells [21]. However, the early development of pulmonary brosis in HPS suggests that environmental or external insults, acting either alone or coupled with abnormal repair mechanisms, may represent contribute to disease pathogenesis. Chest radiograph and high-resolu- tion computed tomography (HRCT) show nonspeci c patterns of diffuse, peripheral reticulation, traction bronchiectasis and bronchiolectasis, subpleural cysts, and ground glass opacities [22]. Honeycombing, when presents, prevail in the lower lobes but is not predominantly subpleural as in the usual interstitial pneumonia (UIP) pattern of pulmonarybrosis seen in idiopathic pulmonary brosis (IPF). Similarly, the active fbroblastic foci characteristic of UIP are rarely present in HPS [23]. HRCT ndings tend to differ somehow as the disease progresses. In milder cases, the most common abnormalities include thickened interlobular septa, reticular changes, and ground glass opacities [22]. Histologic examination of the lung, in addition to the extensive brosis of the alveolar walls, reveals the presence of macrophages lled with ceroid throughout the interstitium and alveolar spaces. Lung disease is the leading cause of death in HPS, followed by gastrointestinal and hemorrhagic complications. There are currently no effective treatments for HPS-related pulmonarybrosis and the average life expectancy of individuals affected is 40–50 years. Therefore, lung transplantation remains the only potentially life-extending therapy for progressive lung disease, though only in a selected minority of patients [24]. An initial trial of 21 Puerto Rican patients with HPS type 1 suggested that pirfenidone, a drug approved for the treatment of IPF, might delay disease progression in individuals with forced vital capacity (FVC) >50% predicted [25]. However, a subsequent study in patients with HPS-­related pulmonarybrosis and preserved lung function showed no bene t from pirfenidone and was prematurely discontinued [26].

Telomerase-Associated Pulmonary Fibrosis

Telomeres are regions of repetitive nucleotide sequences (TTAGGG) that protect the ends of the chromosomes from degradation. Indeed, with repeated cell division, telomeres tend to shorten and the chromosomes may become unstable, fused, or lost, leading to cell apoptosis or senescence. In addition to age, environmental factors, such as smoking, affect telomere function by reducing their length [27]. A complex of proteins and RNA called telomerase is essential in extending telomeres, by adding TTAGGG repeats to the chromosome end, thus preventing its shortening. The reverse transcriptase component TERT and the RNA template component TERC are key components of the telomerase complex, whereas several small nucleolar ribonucleoproteins are responsible for the assembly and stability of the telomerase [28]. Mutations in the genes encoding any of the factors implicated in telomere function lead to abnormally short telomeres.

Dyskeratosis congenita  Dyskeratosis congenita (DC) is a rare systemic disorder with an overall incidence of 1 in 1,000,000 persons that generally manifests in the rst or second decade with bone marrow failure, the leading cause of death, and the classic triad of abnormal skin pigmentation dystrophic nails, and oral leukoplakia [29]. DC is considered a syndrome of premature aging as suggested by features, such as premature graying of the hair, pulmonarybrosis, dental abnormalities, arteriovenous malformations, testicular atrophy, cryptogenic cirrhosis, osteoporosis, and increased risk of several malignancies (i.e., myelodysplastic syndrome, leukemia, and solid tumors). Further corroborating this hypothesis, DC has been the rst disease recognized to result from impaired telomere maintenance [30]. The majority of DC patients carry a pathogenic mutation in one of the following genes encoding components or regulators of the telomerase complex: ACD (adrenocortico dysplasia homolog), CTC1 (conserved telomere maintenance component 1), DKC1 (dyskerin 1), NAF1 (NEF-associated factor 1), NHP2 (non-histone protein 2), NOP10 (nucleolar protein 10), PARN (polyadenyl- ate-speci c ribonuclease), RTEL1 (regulator of telomere elongation helicase 1), STN1 (subunit of CST complex), TERC (telomerase RNA component); TERT (telomerase reverse transcriptase), TINF2 (TRF1-interating nuclear factor 2), WRAP53 (WD repeat-containing protein antisense to TP53). The mode of inheritance may be autosomal dominant, autosomal recessive, or X-linked, depending on the affected gene. Speci cally, the autosomal dominant disease has been observed with mutations in, among others, TERC, TERT, PARN, TINF2, and RTEL1; autosomal recessive disease has been observed with mutations in, among others, TERT, PARN, and RTEL1; whereas X-linked DC results

from mutations in DKC1. Accordingly, there is a wide variation in severity and spectrum of manifestations, although the more clinically severe forms of DC are associated with the greatest reduction in telomere length. For instance, patients carrying mutations in TINF2 have extremely short telomeres and tend to present with bone marrow failure before 5 years of age [31]. Affected cases may also demonstrate genetic anticipation, the occurrence of more severe and earlier onset disease in later generations secondary to the progressive shortening of telomeres [32]. While telomere length is uniformly reduced in DC, thus indicating a shared mechanism, as many as 30% of patients do not have an identi able pathogenic mutation.

The range of pulmonary phenotypes associated with mutations in telomere-related genes is wide, ranging from pulmonary brosis to emphysema and pulmonary vascular disease [33]. Pulmonary brosis develops in approximately 20% of cases and tends to present in adulthood (fourth orfth decade). The histology most often demonstrates UIP, although other patterns such as bronchiolocentric infammation and nonspeci c interstitial pneumonia (NSIP) have also been described. The prognosis of DC patients after the development of pulmonary brosis is poor, with death occurring 12–40 months after the onset of dyspnea [34]. Pulmonary complications may also arise after stem cell transplantation is used to treat bone marrow failure [30].

Carriage of mutations in genes involved in the telomere biology and short telomere length is also well described in familial and sporadic pulmonary brosis cases without features of DC. Approximately one-quarter of familial cases and 10% of sporadic cases of idiopathic pulmonary brosis are associated with mutations within telomere-related genes [33], and individuals carrying such mutations invariably have telomeres shorter than age-matched controls [35]. However, short telomeres are also found in 25% of sporadic and 37% of familial cases of pulmonary brosis that do not carry pathogenic mutations within telomere-related genes [4].

In a large series of patients with mutations in TERT (n = 75), TERC (n = 7), RTEL1 (n = 14) and PARN (n = 19), 46% had IPF, 20% unclassi able lung brosis, 12% chronic hypersensitivity pneumonitis, 10% pleuroparenchymalbroelastosis (10%), 7% interstitial pneumonia with autoimmune features, 4% idiopathic interstitial pneumonia (other than IPF) and 4% connective tissue disease-related interstitial brosis [36]. Notably, mutations in these genes were associated with progressive disease irrespective of the underlying speci c diagnosis. Moreover, there is evidence that short telomere length may be a risk factor for the development of diseases outside the lung, such as liver cirrhosis, cancer, diabetes, sepsis, and stroke [37–40].

Lysosomal Storage Diseases

Lysosomes are cytoplasmic membrane-bound organelles involved in the breakdown and recycling of macromolecules such as lipids, proteins, and carbohydrates. Lysosomal storage diseases (LSDs) are a highly heterogeneous group of inherited disorders of lysosomal catabolism, with an estimated incidence ranging from 1 in 50,000 to 1 in 250,000 live births [41]. LSDs are characterized by speci c enzyme de ciencies resulting in endolysosomal accumulation of non-degraded or partially degraded substrates and impaired lysosomal function. LSDs typically manifest in infancy and childhood with a broad spectrum of clinical phenotypes depending on the substrate involved, residual enzyme activity, and site of accumulation. Because LSDs are systemic disorders, virtually all organs may be involved, including the respiratory system, either at presentation or as a late-onset complication.

Gaucher’s Disease  Gaucher’s disease (GD)—the most common LSD—is an autosomal recessive disorder caused by mutations in glucocerebrosidase 1 (GBA1) gene, leading to defective glucocerebrosidase activity and endolysosomal accumulation of insoluble glucocerebroside (glucosylceramide) in monocytes and macrophages [42]. GD has been identi ed throughout the world and has an estimated prevalence of 0.7–1.75 per 100,000 [43]; however, the disease is more common among Jewish [44]. The pathologic hallmark of GD is the accumulation of lipid-laden and distended macrophages (Gaucher cells) in the macrophage-monocyte system, particularly in the liver, spleen, and bone marrow. Gaucher cells, which are 20–100 μm in diameter, have a characteristic wrinkled-paper appearance and stain positively with PAS, can also in ltrate the lung interstitium, alveolar spaces, or pulmonary vasculature, although the precise mechanism by which organ damage develops remains unclear [45].

Patients with GD may display a number of clinical phenotypes ranging from subclinical adults to children with devastating neurological diseases. Indeed, GD is classi ed into three broad phenotypes based on the presence or absence of neurological involvement: type 1 (non-neuronopathic), the most common, type 2 (acute neuronopathic), and type 3 (subacute neuronopathic) [42].

GBA1 gene is located at 1q21 and comprises 11 exons. Nearly 300 mutations within GBA1 have been identi ed in GD, including frame-shift mutations, point mutations, deletions, insertions, splice site mutations, and recombinant alleles [46]. Based on the level of glucosylceramidase production, these mutations are commonly classi ed as null, severe, or mild. In the presence of null mutations, such as

c.84dupG (84 GG), there is no enzyme production, while severe mutations, such as c.1448 T > C (L444P), though leading to enzyme production, are usually associated with Type 2 or 3 diseases when inherited with a null or another severe mutation. Conversely, mild mutations, such as c.1226A > G (N370S), are only associated with type 1 disease [47]. Type 1 GD is often referred to as adult-type GD, but the majority of symptomatic patients are diagnosed with the disease before reaching adulthood [45]. Hepatosplenomegaly, and hematological and bone abnormalities are the predominant manifestations of the disease [48], whereas pulmonary involvement is a rare nding (<5% of cases) [49]. However, pulmonary function or radiological abnormalities have been observed in as many as 68% and 17%, respectively, of (mostly asymptomatic) patients [50]. Type 2 disease manifests before the rst year of life with a rapidly progressive course leading to death in the early years of life whereas type 3 GD patients tend to experience a slowly progressive disease course [51]. The distinction between type 2 and type 3 diseases is often dif cult, and some authors believe they may represent a spectrum of disease manifestations. The diagnosis of GD requires the detection of de cient glucocerebrosidase activity in peripheral blood leucocytes; genetic testing can detect pathogenic variants within GBA1 thus con rming the diagnosis.

Four patterns of pulmonary involvement may be observed, namely intracapillary, patchy interstitial in ltrates in a lymphatic distribution, massive interstitial thickening of alveolar septa and intra-alveolar in ltrates, which result from in ltration of alveolar, interstitial, perivascular, and peribronchial spaces by Gaucher cells. Accordingly, chest X-ray and HRCT show bilateral interstitial in ltration, in the form of either a predominant ground glass pattern with superimposed thickening of interlobular septa (“crazy paving”) or a diffuse reticular in ltration [52]. L444P homozygotes appear at major risk for developing the pulmonary disease, even at an early age [52]. On the other hand, pulmonary hypertension, strongly associated with splenectomy and female gender, may occur in subjects with non-N370S mutation, positive family history, and angiotensin-converting enzyme I gene polymorphism [53]. Despite signi cant advances in our knowledge of the spectrum of GBA1 mutations, the possibility to make prognostic predictions from genotype data remains limited. Indeed, while it is possible to enumerate individual mutant alleles encountered in patients with type 1, 2, and 3 GD, the clinical phenotype results from the combination of mutations on both alleles. In addition, similar phenotypes may result from different genotypes, but, at the same time, individuals sharing the same genotype can exhibit different disease manifestations, clinical courses, and responses to therapy [46].

Gaucher disease is the rst lysosomal lipid storage diseases to be successfully treated by enzyme replacement ther-

apy (ERT) [54]. However, ERT, which consists of intravenous infusions of recombinantly produced glucocerebrosidase, is a costly and lifelong treatment. In addition, in contrast to the remarkable effect of ERT on hepatosplenomegaly and hematological abnormalities, the response to pulmonary disease is variable [55]. Similarly, ERT does not prevent or halt neurologic involvement, as it does not cross the blood–brain barrier. Plasma chitotriosidase, a biomarker of macrophage activation may be useful for monitoring disease severity and the effectiveness of therapy. Indeed, a chitotriosidase level usually decreases and remains stable with adequate ERT or substrate reduction therapy [56]. Successful bilateral lung transplantation has been described in a patient with pulmonary hypertension [57] but this remains a viable therapeutic option for only a selected minority of patients.

Niemann-Pick Type Disease  The eponym “Niemann-Pick disease” (NPD) refers to a heterogeneous group of autosomal recessive disorders of lysosomal lipid storage characterized by sphingomyelin and cholesterol accumulation in reticuloendothelial and parenchymal tissues, with or without neurological involvement [58]. NPD is commonly classi ed into three major subgroups, which, despite a common name, differ in disease mechanisms, pathogenesis, and clinical manifestations. NPD type A is characterized by severe and early central nervous system deterioration and massive visceral and cerebral sphingomyelin storage leading to death in the rst years of life; type B has a chronic course with marked visceral involvement but a sparing of the nervous system; type C is characterized by a subacute nervous system involvement with a slower course and milder visceral storage. NPD types A and B are caused by de cient lysosomal acid sphingomyelinase activity secondary to mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1). Most of the SMPD1 mutations that cause types A and B NPD are “private,” having been described only in one or a few families. Frameshift mutations, small and large insertions, and deletions, and splicing defects typically result in little or no residual acid sphingomyelinase activity and are called type A alleles. Conversely, mutations that retain signi cant residual activity (>5% of in vitro-expressed wild-type activity) are neuroprotective and are called type B alleles [59]. Inheritance of two type A alleles predicts a type A phenotype with neurodegenerative disease. In contrast, the inheritance of only one type B allele is neuroprotective and predictive for a type B phenotype, even if the other allele has a type A abnormality. The overall prevalence of type A/B NPD is estimated at around 1:250,000 [60] and the diagnosis requires the observation of elevated plasma levels of oxysterols, particularly lyso-sphingomyelin, along with the identi cation of pathogenic SMPD1 variants. Type C NPD is caused by nonfunctional NPC1 (95% of patients) or NPC2 genes that alter lipid processing and transport result-

ing in multiorgan (mainly brain, liver, and spleen) accumulation of low-density lipoprotein cholesterol [61]. The disease, which has an estimated prevalence of about 1 in 150,000 [61], is transmitted in an autosomal recessive manner with nonsense or frameshift mutations within NPC1 being associated with the most severe neurological involvement. Abnormal NPC1 and NPC2 proteins are believed to function in a coordinate fashion in the post-lysosomal/late endosomal transport of cholesterol and other molecules, although their precise function is unknown [62, 63].

Clinical presentation of NPC disease is extremely heterogeneous with the age of onset ranging from the perinatal period to adulthood. Similarly, the lifespan of the affected individuals varies widely, although the majority of patients experience progressive deterioration and die within the second decade of life. Apart from a subset of patients who die at birth or within therst 6 months of life from hepatic or respiratory failure, all patients will ultimately develop progressive neuropathy, which manifests with cerebellar ataxia, dysarthria, dysphagia, and progressive dementia. The majority of cases show also a characteristic vertical supranuclear gaze palsy [64].

The prevalence of lung involvement in NPD is unknown. In a retrospective study of 13 patients, with type A (n = 1), type B (n = 10), and type C (n = 2) disease aged 2 months to 9 years at diagnosis, respiratory symptoms were present at diagnosis in 10 patients and developed during follow-up in the remaining 3 patients. In addition, all patients showed signs of interstitial lung disease either on chest X-ray or CT scan. Bronchoalveolar lavage fuid analysis was performed in seven patients and revealed a marked accumulation of foamy macrophages (Niemann-Pick cells) in all patients. At follow up, one patient died of respiratory failure, ve required

long-term oxygen therapy, six patients manifested chronic obstructive pulmonary disease and one complained of chronic cough [65]. In a study of 53 patients with type B disease, interstitial abnormalities on HRCT were present in 51 of them (98%) with upper lobe predominant ground glass opacity and basilar predominant interlobular septal thickening representing the most common features (Figs. 28.2 and 28.3) [66]. When abnormal, pulmonary function tests generally reveal a restrictive ventilatory defect with or without the reduced diffusing capacity of the lung for carbon monoxide (DLCO) [66]. ILD may be the presenting feature of type C disease; indeed, a subset of infants carrying loss-of-function NPC2 mutations may develop pulmonary alveolar proteinosis (PAP) and respiratory failure secondary to the endoalveolar accumulation of cholesterol-rich surfactant [67]. Pulmonary disease may also manifest as endogenous lipoid pneumonia, which is characterized by the accumulation of sphingomyelin-laden and foamy-appearing macrophages that stain deep blue with May-Grunwald-Giemsa (“sea-blue histiocytes” or Niemann-Pick cells; Figs. 28.4 and 28.5) within bronchial walls, alveolar spaces and alveolar septa [66, 68]. Improvement of endogenous lipid pneumonia has been reported with whole lung lavage [68]; however, progressive respiratory failure leading to death following whole-­ lung lavage has also been reported [69]. Lung transplantation has also been successfully performed in type B disease, although this represents a realistic therapeutic option for only a selected minority of patients [70]. No speci c therapies for NPD are available, but the search for novel treatments is very active.

Fabry Disease  Fabry disease (FD), also known as Anderson-­ Fabry disease, is a rare X-linked disorder, resulting from

Figs. 28.2 and 28.3  Type B Niemann-Pick disease. A transverse CT scan of middle lung zones in a 33-year-old woman shows severe interstitial changes. Note the presence of ground-glass opacities and the

intermixed thickened interlobular septa and intralobular lines in some areas; these ndings are suggestive of the “crazy paving” sign