Genetics and Physiology of Surfactant Protein Deficiencies

Acknowledgments

The authors thank Jeffrey A. Whitsett, MD, Darrell Kotton, MD, Timothy Weaver, PhD, Aaron Hamvas, MD, F. Sessions Cole, MD, Frances V. White, MD, Lisa Young, MD, Robin Deterding, MD, Alicia Casey, MD, Martha Fishman, MD, Susan Guttentag, MD, Michael Beers, MD, Surafel Mulugeta, PhD, and Elizabeth Fiorino, MD, for their collaboration, along with Susan Wert, PhD, who also provided photographs of immunostained lung tissue. The authors also thank Dan Wegner, MS, Hillary B. Heins, BS, and Ping Yang, MS, at Washington University School of Medicine for their research contributions. This work was supported by grants from the National Institutes of Health, the American Thoracic Society/Children’s Interstitial Lung Disease Foundation, the Children’s Discovery Institute, and the Eudowood Foundation.

Introduction

The principal cause of respiratory distress syndrome (RDS) in prematurely born infants is an inability to produce sufficient amounts of pulmonary surfactant because of immaturity. Surfactant phospholipids in combination with specific proteins help reduce alveolar surface tension at the air-liquid interface and prevent end-expiratory collapse. DNA sequence variants in the genes encoding proteins important for surfactant function and metabolism may result in diffuse lung disease in term neonates as well as older infants and children. Loss-of-function DNA sequence variants in the genes encoding surfactant protein B (SP-B) and adenosine triphosphate (ATP)-binding cassette member A3 (ABCA3) may cause neonatal respiratory failure inherited in an autosomal recessive fashion. Pathogenic variants in the gene encoding surfactant protein C (SP-C) cause lung disease of more variable onset and severity, either inherited in an autosomal dominant pattern or causing sporadic disease from de novo pathogenic variants.

Pathogenic variants in the gene NKX2-1 , which encodes thyroid transcription factor 1 which is required for expression of surfactant-related and other genes, can also cause phenotypically similar lung disease. Pathogenic variants in NKX2-1 may also cause disease in other organ systems including the brain and thyroid, whereas diseases due to pathogenic variants in the genes encoding SP-B, SP-C, or ABCA3 are restricted to the lung. These conditions are rare but cause significant morbidity and mortality, and it is important that they be recognized in a timely fashion to appropriately counsel families of affected infants and children. Treatments are limited and nonspecific, and lung transplantation, with substantial associated mortality and morbidities, remains the definitive treatment for progressive respiratory failure. Genetic surfactant dysfunction disorders also provide insights into the roles of these proteins in normal lung function and surfactant homeostasis and demonstrate how genetic mechanisms may contribute to the development of more common forms of lung disease.

Pulmonary Surfactant Proteins and Their Function

SP-B and SP-C are extremely hydrophobic proteins that enhance the surface tension–lowering properties of surfactant phospholipids, and both are found in varying amounts in the mammalian-derived surfactant preparations used to treat premature infants with RDS. SP-B and SP-C are encoded by single genes on chromosomes 2 and 8, respectively, with the genetic loci referred to as SFTPB and SFTPC . The mature forms of SP-B and SP-C are proteolytically processed from precursor proteins (pro-SP-B, pro-SP-C), routed in alveolar epithelial type II cells (AEC2s) to lysosomally derived intracellular organelles called lamellar bodies, and secreted by exocytosis along with surfactant phospholipids. ABCA3 is a transmembrane protein located on the limiting membrane of lamellar bodies. The ATP-binding cassette proteins are a large family of transporter proteins that use the energy from the hydrolysis of ATP to move substances across biologic membranes. ABCA3 transports phospholipids (e.g., phosphatidylcholine and phosphatidylglycerol) essential for surfactant function from the cytoplasm into lamellar bodies.

Pulmonary surfactant also contains two larger glycoproteins, surfactant protein A (SP-A) and surfactant protein D (SP-D). SP-A and SP-D are members of the collectin family, having both collagenous and carbohydrate-binding or lectin-like domains. SP-A and SP-D are important components of the innate immune system in the lung. They bind to a wide array of microorganisms, facilitate their uptake and/or killing by alveolar macrophages, and also have immunomodulatory functions. ^, Genetically engineered SP-A- and SP-D-deficient mice do not develop neonatal respiratory disease but do appear to have an increased susceptibility to infection with a variety of organisms that are relevant to human neonates, including group B streptococcus and respiratory syncytial virus. With aging, SP-D-deficient mice develop emphysema and fibrosis, suggesting a role for SP-D and surfactant homeostasis in regulating chronic inflammation and lipoproteinosis. Two genes ( SFTPA1 , SFTPA2 ) and a pseudogene for SP-A (SFTPA3P) and a single gene for SP-D (SFTPD) are located on chromosome 10. Variants in SFTPA2 and rarely SFTPA1 have been associated with adult-onset pulmonary fibrosis and lung cancer. ^, Variants in SFTPD have not yet been associated with human lung disease.

Surfactant Protein B Deficiency

SP-B deficiency was the first recognized inborn error of surfactant metabolism. Affected infants are usually term and present with clinical and radiographic features of RDS or hyaline membrane disease as observed in premature infants. Although some affected infants may have relatively milder initial respiratory symptoms, most have severe disease requiring positive pressure support. A need for support with high-frequency ventilation or even extracorporeal membrane oxygenation is not uncommon. Lung disease in SP-B deficiency is invariably progressive, with escalating need for respiratory support and persistent and worsening alveolar and interstitial infiltrates seen on chest radiographs. An initial positive response to surfactant replacement therapy may be observed but with diminished response to subsequent doses. Glucocorticoids may improve the lung disease in some affected infants, perhaps because they have variants that allow some SP-B production. The disease is generally rapidly fatal, with the majority of affected infants dying within the first 3 to 4 months without lung transplantation. ^,

Molecular Genetics and Epidemiology

The SP-B gene (SFTPB) contains 12 exons (initially thought to be 11), the first and last of which are untranslated, and is transcribed into a 2-kilobase messenger RNA, which directs the synthesis of a 393–amino acid pre-proprotein, with the signal peptide removed co-translationally. Mature SP-B corresponds to codons 213 (encoding phenylalanine) to 291 (encoding methionine) of the messenger RNA and is encoded in exons 7 and 8 of the gene. Pro-SP-B contains 3 tandem domains with structural homology to the saposins, lysosomal proteins that bind lipids and activate lysosomal hydrolases, with the 79–amino acid mature SP-B corresponding to the middle domain of the proprotein.

The first and most frequently identified SFTPB pathogenic variant is a frameshift resulting from a substitution of three bases (GAA) for one base (C) located upstream of the sequence encoding mature SP-B. This variant introduces a premature codon for the termination of translation, resulting in a transcript that is unstable owing to nonsense-mediated decay and a lack of detectable SP-B messenger RNA. The frameshift upstream of the mature SP-B encoding sequence and premature termination codon would also preclude production of mature SP-B from the mutant sequence. This variant is thus a complete null allele, consistent with a loss-of-function mechanism. The variant was originally termed 121ins2 for the net 2-base insertion into codon 121 of the SFTPB transcript. On the basis of homology studies, the reference SFTPB transcript is now believed to contain an additional 12 upstream codons and the current nomenclature for this pathogenic variant is c.397delCinsGAA (or p.Pro133Gln fs Ter95 for the predicted change in the protein). Pathogenic variants on both SFTPB alleles in affected infants are consistent with an autosomal recessive inheritance pattern.

Multiple other pathogenic variants and a sizable deletion in SFTPB have been identified. Some pathogenic variants allow production of pro-SP-B but processing of pro-SP-B to mature SP-B is impaired, resulting in the lack of mature SP-B in lung tissue and tracheal secretions of affected infants. Pathogenic variants in the mature SP-B domain could theoretically result in the production of a SP-B with abnormal surface properties. However, a synthetic peptide containing one such variant (previously p.Arg236Cys, p.Arg248Cys in the current nomenclature) was able to augment surface tension lowering normally in an in vitro system, suggesting that the disease in infants with this variant also resulted from impaired processing of pro-SP-B containing the variant. SP-B forms higher-order oligomers dependent on sulfhydryl-bond formation and ionic interactions. Variants that prevent oligomerization of SP-B could also in theory result in SP-B with decreased activity, and potentially act in a dominant negative fashion to interfere with SP-B from a normal allele, but such a mechanism for SP-B deficiency has not been reported.

The p.Pro133GlnfsTer95 variant has been identified mostly among individuals of European descent and likely results from a common ancestral origin (founder effect). Two other SFTPB pathogenic variants have been identified within five nucleotides of the site of this variant, and a single nucleotide polymorphism is located within 30 base pairs of the site of the p.Pro133GlnfsTer95 variant, suggesting that this region of SFTPB could also be a “hot spot” for variation. Other pathogenic variants have been observed in more than one unrelated individual from specific ethnic backgrounds. Rarely, children with biallelic SFTPB variants and relatively milder phenotypes have been identified. ^, ^, ^, The variants in these children may allow the production of some SP-B and thus result in partial deficiency. Whether the small amount of mature SP-B (approximately 8% to 10% of control levels) or some retained function of pro-SP-B accounts for the milder phenotype is unclear. These observations suggest that there is a critical level of SP-B needed for normal lung function. This hypothesis is supported by studies in which mice genetically engineered so that their production of SP-B could be experimentally reduced developed respiratory failure when their SP-B levels fell to 20% to 30% of those observed in control animals.

An estimate of the incidence of SP-B deficiency may be derived from the frequencies of known or likely pathogenic variants in SFTPB in selected populations or from publicly available databases of genetic variants identified by whole-exome or whole-genome sequencing. The carrier frequency for the p.Pro133Gln fs Ter95 variant is approximately 1 in 2300 individuals in the gnomAD database of over 140,000 adults ( https://gnomad.broadinstitute.org/ ) with a greater carrier frequency among European-descent individuals (approximately 1 of 1200 individuals). ^, This variant has accounted for approximately two thirds of the variant SFTPB alleles identified among infants with progressive respiratory failure to date. SP-B deficiency is an autosomal recessive disorder with a 25% chance of an affected child when both parents carry a pathogenic variant. Using the frequencies of null (e.g., nonsense and frameshift) variants reported in gnomAD (97 null alleles identified in approximately 140,000 individuals) the predicted incidence of disease is approximately 1 in 5 to 10 million births, indicating that SP-B deficiency is a very rare disease. However, this is most certainly an underestimate as missense variants, whose effects on protein function are more difficult to predict, also contribute to disease incidence.

SP-B production increases with advancing gestational age and is down-regulated with inflammation. Lung disease could develop in individuals with loss-of-function variants on one SFTPB allele (haploinsufficiency) if other factors, such as prematurity or infection, further delay or reduce SP-B expression below a critical level. Genetically engineered mice heterozygous for a null Sftpb allele have half normal levels of SP-B and were more susceptible to pulmonary oxygen toxicity than their wild-type littermates but did not develop neonatal respiratory disease. ^, In a large study from Denmark, pulmonary function tests were performed in adult carriers of the SFTPB p.Pro133Gln fs Ter95 pathogenic variant. Abnormalities with reductions in the forced expiatory volume in the first second of expiration and the ratio of this to the forced vital capacity were observed in individuals who also smoked, suggesting that haploinsufficiency for SP-B in conjunction with an environmental insult could predispose to chronic obstructive lung disease.

Pathophysiology of Surfactant Protein B Deficiency

Surfactant isolated from bronchoalveolar lavage fluid (BALF) of SP-B-deficient infants does not lower surface tension effectively, an unsurprising finding given the importance of SP-B in surfactant function. Tubular myelin, the lattice-like extracellular form of surfactant, the formation of which requires SP-B (as well as SP-A and calcium), is also not found in lung tissue of affected infants. However, additional disturbances in surfactant metabolism also contribute to the pathophysiology of the lung disease in SP-B-deficient infants. These include abnormal surfactant phospholipid composition, particularly a marked reduction in phosphatidylglycerol content, abnormal lamellar body formation, and an apparent block in posttranslational processing of pro-SP-C to mature SP-C. ^, Abnormal lamellar body formation and impaired pro-SP-C processing have also been observed in the lungs of genetically engineered mice unable to produce SP-B and that died within minutes of birth because of respiratory failure. These observations indicate a fundamental intracellular role for SP-B (or pro-SP-B) in surfactant metabolism in addition to its extracellular role in lowering surface tension.

The block in the final processing steps of pro-SP-C would result in a relative deficiency of mature SP-C, further contributing to surfactant dysfunction. The abnormal pro-SP-C peptides found in BALF from affected infants contain relatively hydrophilic amino-terminal epitopes, are not very surface-active, and are likely to further inhibit surfactant function. The precise mechanisms underlying this block in pro-SP-C processing are unknown. Ultrastructural examination of the lungs of SP-B-deficient infants reveals a lack of normally formed lamellar bodies, and instead AEC2s have poorly organized lamellar bodies with abnormal-appearing lipid vesicles. ^, The final steps in processing of pro-SP-C to mature SP-C occur late in the secretory pathway, either in lamellar bodies or in their immediate precursors. Thus, the inability to form these organelles because of the lack of SP-B results in incompletely processed pro-SP-C, as well as inadequate packaging of surfactant components for secretion. SP-B facilitates membrane fusion, and an explanation for these observations is that without SP-B, transport vesicles containing incompletely processed pro-SP-C are unable to fuse with developing lamellar bodies, thus exposing the amino-terminal epitopes of pro-SP-C to the necessary processing enzymes.

Treatments for infants with respiratory failure due to SP-B deficiency remain limited. However, recent applications of lentiviral-mediated gene correction and CRISPR-Cas9 gene editing to induced pluripotent stem cells (iPSC) from infants with SP-B deficiency have yielded promising results. Lentivirus carrying the wild-type SFTPB gene was used to restore SP-B protein production, lamellar body phenotype, and surfactant secretion in iPSC-derived lung organoids from an infant homozygous for p.Pro133Gln fs Ter95. CRISPR-Cas9 gene editing resulted in in vitro correction of both mutant SFTPB alleles and restoration of SP-B and SP-C processing and lamellar body ultrastructure of AEC2s generated from iPSCs from an affected infant. While significant challenges remain before these technologies can be applied clinically, these results offer hope for infants affected by this devastating disorder.

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