background Pulmonary surfactant forms a lipid-rich monolayer that coats the airways of the lung and is essential for proper inflation and function of the lung. Surfactant is produced by alveolar type II cells, stored intracellularly in organelles known as lamellar bodies, and secreted by exocytosis. The gene for ATP-binding cassette transporter A3 (ABCA3) is expressed in alveolar type II cells, and the protein is localized to lamellar bodies, suggesting that it has an important role in surfactant metabolism. methods We sequenced each of the coding exons of the ABCA3 gene in blood DNA from 21 racially and ethnically diverse infants with severe neonatal surfactant deficiency for which the etiologic process was unknown. Lung tissue from four patients was examined by high-resolution light and electron microscopy. results Nonsense and frameshift mutations, as well as mutations in highly conserved residues and in splice sites of the ABCA3 gene were identified in 16 of the 21 patients (76 percent). In five consanguineous families with mutations, each pair of siblings was homozygous for the same mutation and each mutation was found in only one family. Markedly abnormal lamellar bodies were observed by ultrastructural examination of lung tissue from four patients with different ABCA3 mutations, including nonsense, splice-site, and missense mutations. conclusions Mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. ABCA3 is critical for the proper formation of lamellar bodies and surfactant function and may also be important for lung function in other pulmonary diseases. Since it is closely related to ABCA1 and ABCA4, proteins that transport phospholipids in macrophages and photoreceptor cells, it may have a role in surfactant phospholipid metabolism.

https://www.nejm.org/doi/pdf/10.1056/NEJMoa032178

ABCA3 gene mutations in newborns with fatal surfactant deficiency.

Mutations in the genes encoding the surfactant proteins B and C (SP-B and SP-C) and the phospholipid transporter, ABCA3, are associated with respiratory distress and interstitial lung disease in the pediatric population. Expression of these proteins is regulated developmentally, increasing with gestational age, and is critical for pulmonary surfactant function at birth. Pulmonary surfactant is a unique mixture of lipids and proteins that reduces surface tension at the air-liquid interface, preventing collapse of the lung at the end of expiration. SP-B and ABCA3 are required for the normal organization and packaging of surfactant phospholipids into specialized secretory organelles, known as lamellar bodies, while both SP-B and SP-C are important for adsorption of secreted surfactant phospholipids to the alveolar surface. In general, mutations in the SP-B gene SFTPB are associated with fatal respiratory distress in the neonatal period, and mutations in the SP-C gene SFTPC are more commonly associated with interstitial lung disease in older infants, children, and adults. Mutations in the ABCA3 gene are associated with both phenotypes. Despite this general classification, there is considerable overlap in the clinical and histologic characteristics of these genetic disorders. In this review, similarities and differences in the presentation of these disorders with an emphasis on their histochemical and ultrastructural features will be described, along with a brief discussion of surfactant metabolism. Mechanisms involved in the pathogenesis of lung disease caused by mutations in these genes will also be discussed.

Genetic disorders of surfactant dysfunction

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of alveolar macrophages. PAP results in progressive dyspnoea of insidious onset, hypoxaemic respiratory failure, secondary infections and pulmonary fibrosis. PAP can be classified into different types on the basis of the pathogenetic mechanism: primary PAP is characterized by the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling and can be autoimmune (caused by elevated levels of GM-CSF autoantibodies) or hereditary (due to mutations in CSF2RA or CSF2RB, encoding GM-CSF receptor subunits); secondary PAP results from various underlying conditions; and congenital PAP is caused by mutations in genes involved in surfactant production. In most patients, pathogenesis is driven by reduced GM-CSF-dependent cholesterol clearance in alveolar macrophages, which impairs alveolar surfactant clearance. PAP has a prevalence of at least 7 cases per million individuals in large population studies and affects men, women and children of all ages, ethnicities and geographical locations irrespective of socioeconomic status, although it is more-prevalent in smokers. Autoimmune PAP accounts for >90% of all cases. Management aims at improving symptoms and quality of life; whole-lung lavage effectively removes excessive surfactant. Novel pathogenesis-based therapies are in development, targeting GM-CSF signalling, immune modulation and cholesterol homeostasis.

Pulmonary alveolar proteinosis.

Pulmonary surfactant is required for adaptation to air breathing after birth, reducing surface tension at the air-liquid interface in the alveolus to maintain lung volumes during the respiratory cycle. Disorders of pulmonary surfactant homeostasis are associated with acute and chronic respiratory disease in infants and adults. Mutations in the surfactant protein genes encoding SP-B and SP-C cause severe respiratory failure in infancy and chronic interstitial lung disease in older individuals.

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Diseases of Pulmonary Surfactant Homeostasis

Mutations in the surfactant protein C gene (SFTPC) were recently reported in patients with interstitial lung disease. In a 13-month-old infant with severe respiratory insufficiency, a lung biopsy elicited combined histological patterns of nonspecific interstitial pneumonia and pulmonary alveolar proteinosis. Immunohistochemical and biochemical analyses showed an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, mature SP-B, aberrantly processed proSP-C, as well as mono- and dimeric SP-C. Sequencing of genomic DNA detected a de novo heterozygous missense mutation of the SFTPC gene (g.1286T>C) resulting in a substitution of threonine for isoleucine (173T) in the C-terminal propeptide. At the ultrastructural level, abnormal transport vesicles were detected in type-II pneumocytes. Fusion proteins, consisting of enhanced green fluorescent protein and wild-type or mutant proSP-C, were used to evaluate protein trafficking in vitro. In contrast to wild-type proSP-C, mutant proSP-C was routed to early endosomes when transfected into A549 epithelial cells. In contrast to previously reported mutations, the 173T represents a new class of surfactant protein C gene mutations, which is marked by a distinct trafficking, processing, palmitoylation, and secretion of the mutant and wild-type surfactant protein C. This report heralds the emerging diversity of phenotypes associated with the expression of mutant surfactant C proteins.

https://erj.ersjournals.com/content/erj/24/1/30.full.pdf

Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene

Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Reunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Reunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities.

Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island

Fe ´deration de Genetique et INSERM U458, Hoˆpital Robert-Debre (AP-HP), Paris, FranceWehaveanalyzedsurfactantproteinB(SP-B)and its encoding gene (SFTPB, MIM 178640)in 40 unrelated pediatric patients with un-explained respiratory distress (URD). Therewas high consanguinity (eight kindreds)and an underlying autosomal recessive traitcould be inferred in most cases, with overallhigh sex ratio (32/17) suggesting proband’sgendertoimpactonpenetrance.Theclinical/biological presentations ﬁtted into threemajor nosologic frameworks. I: SP-B de-ﬁciency (nine probands), complete or in-complete, with homozygous/compoundlyheterozygous mutations identiﬁed (six pro-bands), including one from the populationisolate of Re´union Island (496delG). In ad-dition, there was a consanguineous kindredin which incomplete deﬁciency was unam-biguously unlinked to SFTPB. II: pulmonaryalveolar proteinosis (PAP, 19 probands),withtypicalstorageofPAS-positivematerialwithin the alveoli with foamy macrophagesandvariableinterstitialreaction,whichwasdiagnosed in most patients from Re´unionIsland. In contrast to previously publishedﬁndings, mutation and/or segregation ana-lyses excluded SFTPB as a disease locus,although slight metabolic derangementrelated to SP-B and/or mild SFTPB changescould somehow contribute to disease. III:URD without evidence for SP-B deﬁciencyor PAP (12 probands), equally unlinked toSFTPB, although a single patient had apossibly causal, maternally-derived, hetero-zygous genetic change (G4521A). The popu-lation frequency of ﬁve known and fournovel SNPs was studied, providing as manypotential markers for pulmonary diseaserelated to SFTPB. Overall, URD was foundto be heterogeneous, both phenotypicallyand genetically, even in population isolateswhere a founder effect might have beenexpected. When disease loci are identiﬁed,patient genotyping will be crucial as a diag-nostic aid, for devising proper treatment,and as a basis for genetic counseling.

François Cartault

Papers

Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island

Analysis of 40 sporadic or familial neonatal and pediatric cases with severe unexplained respiratory distress: relationship to SFTPB.