—  SYMPOSIUM #31  —

Children's Interstitial Lung Disease Network:
A Cooperative Approach to the Classification of Pediatric Diffuse Lung Diseases
Moderator: Dr. Claire Langston

Section 2 - Surfactant Disorders: Clinical and Pathologic Spectrum
(Surfactant Metabolism and Deficiencies in Surfactant Proteins)

Susan E. Wert
Cincinnati Children's Hospital Medical Center
Cincinnati , OH


Introduction:
Genetic disorders of surfactant metabolism have been recognized as underlying causes of acute respiratory distress and failure in full term neonates, as well as of interstitial lung disease (ILD) in older infants, children, and adults. The genes involved in these disorders are the surfactant protein B gene (SFTPB), the surfactant protein C gene (SFTPC) and the ABCA3 gene. Surfactant protein B (SP-B) and C (SP-C) are small hydrophobic proteins that are synthesized in alveolar type 2 cells and secreted into the alveolar lumen, where they function to stabilize and enhance spreading of surfactant phospholipids along the alveolar surface. This mixture of proteins and lipids reduces surface tension at the air-liquid interface, preventing collapse of the lung at the end of expiration. ABCA3 is an integral membrane protein that has been localized to the limiting (outer) membrane of the lamellar body, which is the intracellular storage organelle for surfactant lipids and proteins in alveolar type 2 cells. ABCA3 is a member of a large family of ATP-binding cassette proteins that transport a variety of substances across biological membranes, including lipids.

Surfactant Metabolism:
Surfactant lipids are composed primarily of phosphatidylcholine (PC or lecithin) and, in particular, disaturated or dipalmitoylated phosphatidylcholine (DSPC or DPPC), with lesser amounts of phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and cholesterol. SP-B is encoded by a single gene on human chromosome 2, while SP-C is located on human chromosome 8. Both proteins are synthesized as large precursor proteins (proSP-B and proSP-C), which are then processed by several proteolytic enzymes (Napsin A, Cathepsin H and Pepsinogen C) to yield the smaller, hydrophobic, active airway peptides. The final processing steps for both SP-B and SP-C take place in the lamellar body, where these proteins associate with surfactant phospholipids and are then secreted together with the lipids into the airway. Both surfactant phospholipids and proteins are recycled by the type 2 cell or catabolized by alveolar macrophages.

SFTPB Mutations :
Hereditary SP-B deficiency has been reported in over 75 individual children from unrelated families and is inherited as an autosomal recessive disease. Over 40 distinct mutations in the SFTPB gene have been identified and characterized to date by Dr. Lawrence Nogee, Johns Hopkins University , Baltimore , MD. Two-thirds of the mutant alleles are accounted for by a common mutation, 121ins2, in exon 4. The gene frequency is one mutation per 1000-3000 individuals. Mutations in SFTPB cause acute respiratory distress at birth, which is progressive and usually fatal by 3 months of age. Lung disease is caused by the complete absence of SP-B (loss-of function), and clinical findings are consistent with those seen in preterm infants with RDS due to insufficient surfactant stores. In general, SP-B deficiency is associated with histopathologic diagnoses of congenital pulmonary alveolar proteinosis (PAP) and desquamative interstitial pneumonits (DIP). Histopathologic features of genetic SP-B deficiency include: 1) a severe alveolar proteinosis admixed with desquamated type II cells and alveolar macrophages, 2) intact, hyperplastic, alveolar epithelia with prominent alveolar Type II cells, and 3) interstitial thickening. Immunohistochemical features include 1) absence of immunostaining for proSP-B and the mature SP-B peptide and (2) increased amounts of SP-A and proSP-C immunopositive material in the airspaces. The presence of increased amounts of partially processed proSP-C in the airspace suggests that SP-B is required for normal processing or trafficking of SP-C. Aberrant or incompletely processed forms of proSP-B may or may not be detected depending on the location of the mutation in the gene. Phospholipid profiles may be abnormal, resembling that of an immature lung; and lamellar body formation is perturbed. No mature lamellar bodies are found, although many large multivesicular bodies are detected by electron microscopy.

SFTPC Mutations:
Over 40 distinct mutations in the SFTPC gene have been identified and characterized to date (personal communication, Dr. Lawrence Nogee), with almost all mapping to the carboxy-terminal domain of proSP-C. This causes misfolding of proSP-C, resulting in incomplete processing of the propeptide. A common mutation, I73T, is found in about 25% of these cases. SFTPC mutations are associated with chronic interstitial lung disease (ILD) in both children and adults. Lung disease due to SFTPC mutations is inherited as an autosomal dominant trait with variable penetrance and severity (45%), or as sporadic disease due to a de novo mutation (51%). Approximately 44% of patients with a SFTPC mutation developed respiratory symptoms within the first month of life (as neonates), while another 40% developed symptoms between 1 and 6 months of life (as infants). Onset of respiratory symptoms has been reported, however, in both older children and adults; and at least 6 asymptomatic individuals with SP-C mutations have been identified (personal communication, Dr. Lawrence Nogee). Although the incidence of lung disease due to SFTPC mutations is currently unknown, recent studies indicate that SFTPC mutations are a rare cause of adult ILD. The population frequency of disease causing SFTPC variants is, however, currently unknown. In general, SP-C deficiency is associated with histopathologic diagnoses of DIP, PAP, chronic pneumonitis of infancy (CPI), and non-specific interstitial pneumonitis (NSIP), although the most common histopathologic diagnosis in infants is CPI. Immunohistochemical analysis reveals robust staining for all of the surfactant proteins including proSP-C, which is restricted to alveolar type 2 cells. Two different patterns of proSP-C staining have been observed: 1) diffuse staining of the cytoplasm and 2) accumulation of immunoreactive material in a perinuclear compartment. Accumulation of incompletely processed proSP-C in alveolar type 2 cells may be toxic, resulting in chronic lung injury and disease, although decreased levels of mature SP-C are also found in these patients, suggesting that routing and processing of protein from the normal allele is perturbed.

ABCA3 Mutations:
Over 100 distinct mutations have been identified in the ABCA3 gene (personal communication, Dr. Lawrence Nogee), making this the largest class of genetic mutations that cause surfactant deficiency. Population frequencies for these mutations are, however, currently unknown. Genetic ABCA3 deficiency is inherited as an autosomal recessive disease and is associated with surfactant deficiency (loss-of-function) and fatal respiratory distress in the neonatal period. In general, ABCA3 deficiency is associated with histopathologic diagnoses of DPI and PAP, as well as with aberrant lamellar body formation. Therefore, the clinical presentation, mortality rate, and histopathology for ABCA3 deficiency are similar to those for genetic SP-B deficiency for the majority of neonates with ABCA3 mutations on both alleles. Recently, a common mutation, E292V, has been identified in 10 older children who were diagnosed with chronic ILD. Milder neonatal disease with presentation of non-specific symptoms and findings, including cough, tachypnea, hypoxemia in room air, clubbing, and failure to thrive, were reported in some of these children, while other children in the study did not have any respiratory symptoms or findings until later in childhood. In general, immunostaining for all of the surfactant proteins, as well as ABCA3, is robust, although a subset of cases exhibits poor immmunostaining for the mature SP-B peptide. Unlike SP-B deficiency, proSP-C staining is restricted to the cytoplasm of alveolar type 2 cells and is not found in the airspaces. Multiple, small, lamellar bodies with densely packed phospholipid membranes and eccentrically placed electron-dense cores are detected in type 2 cells, while phospholipid analyses in a subset of patients revealed abnormal phospholipid profiles with decreased proportions of phosphatidylcholine. Funding: NIH HL-57403 and HL-56387.

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