Hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy

Abstract

Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor function, and increased GM-CSF.

Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction.

Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls.

Measurement and main results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor β chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy.

Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.

Figure 1.

Serum granulocyte-macrophage colony–stimulating factor (GM-CSF) concentrations in children diagnosed with hereditary pulmonary alveolar proteinosis (hPAP), members of their immediate family who were healthy (family members), or healthy controls. The lower limit of quantification (LLOQ) of the assay was 7.8 pg/ml (dashed line). The serum levels of GM-CSF in children with hPAP (52 pg/ml [28–101 pg/ml]) were increased compared with healthy family members (0.0 pg/ml [0.0–3 pg/ml]) and unrelated healthy controls (0.0 pg/ml [0.0–1.9 pg/ml]) (n = 8, 11, 30, respectively; P < 0.001; Kruskal-Wallis analysis of variance on ranks with comparisons using Dunn's method).

Figure 2.

Segregation of CSF2RA mutations in six families with hereditary pulmonary alveolar proteinosis (PAP). (A) Pedigree of patients A–C. (B) Pedigree of patient D. (C) Pedigree of patient E. (D) Pedigree of patients F and G. (E) Pedigree of patient H. Squares represent male family members and circles represent female members. Filled symbols represent persons with hereditary PAP. Allelic variants segregating within a family are shown above the pedigree. The status of both alleles is indicated under the symbols according to the following key referring to CSF2RA sequences: N/N = two normal alleles; changed nucleotide (or amino acid)/N = one mutated and one normal allele; changed nucleotide (or amino acid)/changed nucleotide (or amino acid) = two abnormal alleles; NA = not available for testing. The colloquial nomenclature for each mutation is shown here and the formal genetic names are given below (Table 3). The CSF2RA gene abnormalities caused by chromosomal deletions at Xp33.22 are indicated by the size of the deletion in megabases (mb): XpΔ1.6 = Xp33.22del1.6mb; XpΔ0.41 = Xp33.22del0.41 mb; Xq^Houston = Xq chromosome in patient H identified in Houston (28). Patient H had Turner syndrome (46 Xi(Xq). The CSF2RA XpΔ0.41 mutation originated on the paternal X chromosome and was transmitted to the normal length isochromosome X of patient H; her Xq CSF2RA allele was absent because of the deletion of Xp. The proband in each family is indicated (arrows). A question mark indicates the status of the second CSF2RA allele, if present, is unknown.

Figure 3.

High-resolution computed tomography scans of the chest in patients with hereditary pulmonary alveolar proteinosis obtained at the time of diagnosis. (A) Patient A at age 5 years and asymptomatic on room air. A small region of ground glass opacification is indicated (arrow). (B) Patient B at age 3 years and dyspneic on oxygen via nasal canula at 2 L per minute. (C) Patient C at age 4 years and dyspneic on oxygen via nasal canula at 2 L per minute. (D) Patient D at age 11 years and dyspneic on room air. (E) Patient E at age 11 years and dyspneic on 100% oxygen via facemask. All scans show findings at the level of the main carina.

Figure 4.

BAL cell cytology, alveolar macrophage ultrastructure, and lung histopathology in patients with hereditary pulmonary alveolar proteinosis. (A–D) Cytology and ultrastructure of bronchoalveolar lavage cells obtained from patient A at the time of diagnosis (5 yr). (A) Diff-Quick staining showing numerous enlarged, foamy alveolar macrophages (original magnification ×20, bar = 20 μm; inset original magnification ×100, bar = 2 μm). (B) Periodic acid–Schiff (PAS) staining showing glycoprotein accumulation in alveolar macrophages (original magnification ×20, bar = 20 μm; inset original magnification ×100, bar = 2 μm). (C) Oil red O staining and hematoxylin counterstaining showing accumulation of neutral lipids in alveolar macrophages (original magnification ×20, bar = 20 μm; inset original magnification ×100, bar = 2 μm). (D) Ultrastructural analysis demonstrating that the foamy appearance of alveolar macrophages is caused by the accumulation of lamellar body (open arrows) and lipid droplet (closed arrows) inclusions. The extracellular space had an abnormal abundance of surfactant (asterisk) (original magnification ×6,000, bar = 2 μm). (E–H) Histopathologic appearance of the lung biopsy obtained from patient D at the time of diagnosis (11 yr) after PAS staining (original magnification ×20). (E) Alveoli in some areas are completely filled with granular, PAS-positive material. (F) Region showing the sharp interface between alveoli filled with PAS-positive material and empty, normal alveoli. (G) Region showing well-preserved alveolar wall structure without abnormalities. (H) Region showing the accumulation of foamy alveolar macrophages (arrows) and small amounts of extracellular, intraalveolar PAS-positive material. Note the well-preserved alveolar walls.

Figure 5.

Radiographic appearance of the lungs in patients with hereditary pulmonary alveolar proteinosis before and after whole-lung lavage. Posteroanterior chest radiographs of patient E are shown (A) before and (B) 1 week after the second of two bilateral whole-lung lavage procedures (13 L saline each) separated by 3 months. The oxyhemoglobin saturation was 90% on 100% oxygen before the first and 97% on room air after the second procedure. Note the marked clearing of the infiltrate in both lungs and improved visualization of the cardiac border after lavage. High-resolution computed tomography scans of the chest of patient C are shown (C) before and (D) 8 weeks after a right whole single-lung lavage with 5.35 L and 12 weeks after left whole single-lung lavage with 3.78 L of saline. Note the marked clearing of the infiltrates in both lungs after lavage.

Figure 6.

Analysis of granulocyte-macrophage colony–stimulating factor (GM-CSF) receptors in patients with hereditary pulmonary alveolar proteinosis. Fresh heparinized blood was obtained from patient B, her unaffected parents, and an unrelated healthy control and blood leukocytes were evaluated for GM-CSF signaling (A) or GM-CSF receptor α chains on the cell surface (B) or in cell lysates (C). (A) Blood was incubated without (-) or with (+) GM-CSF (10 ng/ml, 15 minutes) followed by Western blotting to detect phosphorylated STAT5 (pSTAT5), total STAT5 (STAT5), or actin (to ensure equal loading of cell lysates). As a positive control for STAT5 phosphorylation in patient leukocytes, cells were incubated with IL-2, which resulted in readily detectable STAT5 phosphorylation as expected (42) (not shown). (B) Blood was immunostained and flow cytometry was used to detect GM-CSF receptor α protein on the cell surface of blood leukocytes from patient B, her father and mother, or an unrelated healthy control (C). In each histogram, the y axis scale represents 0–40 cells per histogram cell and the x axis scale represents 10–1,000 fluorescence intensity units. Note the absence of GM-CSF receptor α for patient B and its presence for family members and control. All patients, family members, and controls had readily detected GM-CSF receptor β on blood leukocytes (not shown). (C) Blood was used to prepare leukocyte lysates, which were fractionated on polyacrylamide gradient gels and subjected to Western analysis to detect and determine the molecular mass of GM-CSF receptor α. Western analysis of parallel samples for actin ensured equal loading of cell lysates. The band corresponding to GM-CSF receptor α glycoprotein is broad because of glycosylation at multiple sites (43). Note the absence of GM-CSF receptor α in patient B but not her parents or control.

Figure 7.

Analysis of CSF2RA mutations in patients with hereditary pulmonary alveolar proteinosis. (A) Schematic of CSF2RA and the polymerase chain reaction (PCR) amplification and strategy used for sequencing. The locations of Alu sequence high copy number genomic repeat sequence motifs are indicated (hatched boxes). (B) Partial nucleotide sequence of CSF2RA exon 8 in genomic DNA from patient B and a healthy control. Numbering is according to the CSF2RA reference sequence in Genbank (accession No. NM_006140.3). (C) Partial nucleotide sequence CSF2RA exon 10 in mRNA from patient D and a healthy control. (D) PCR amplification of the CSF2RA coding sequence from mRNA from patient E, her father (F), mother (M), brother (B), or a healthy individual (C). (E) PCR amplification of CSF2RA exons 1–13 in genomic DNA from patient E and family members (indicated as in D). (F) PCR amplification of genomic DNA from patient E and family members (indicated as in D) in the region surrounding the Alu family repeats flanking CSF2RA exon 7 as shown in the schematic in A. Note the small band in the patient, small and large bands in each parent, and only the large band in the brother. (G) Partial nucleotide sequence of CSF2RA intron 6 (black arrows) and exon 7 (red arrow) in genomic DNA from patient E and a healthy control. (H) Partial nucleotide sequence of CSF2RA exons 6–10 in mRNA from patient E and a healthy control showing a single mRNA transcript sequence in the healthy control and two overlapping transcript sequences beginning at Lys158 in the patient. (I) Proposed model for the generation of the CSF2RA mutation present in patient E (homozygous) and her parents (heterozygous). See text for details. The locations of the two Alu family repeats flanking Exon 7 are indicated (hatched regions). These regions are 90% homologous at the DNA level, and contain a 10-nucleotide central region of 100% homology (black region within the hatched region).

Figure 8.

CSF2RA mutations and granulocyte-macrophage colony–stimulating factor (GM-CSF) receptor α abnormalities associated with the development of hereditary pulmonary alveolar proteinosis. (Top) Schematic of the normal GM-CSF receptor α protein showing regions corresponding to the signal peptide, extracellular GM-CSF binding domain, transmembrane spanning domain, and short intracellular, cytoplasmic domain, along with the 11 N-glycosylation sites (43). The asterisk indicates the glycosylation site disrupted by the G196R mutation. (Middle) Schematic of the CSF2RA mRNA showing exons 1–13. The start codon (ATG) and stop codon (TGA) are indicated. (Bottom) Schematic representations of the effects of each CSF2RA mutation (indicated at left) on the GM-CSF receptor α protein structure are shown. The colloquial nomenclature is used for each CSF2RA abnormality. In each, stippled bars represent the signal peptide, black bars represent the extracellular domain, gray bars represent the transmembrane domain, white bars represent the cytoplasmic domain, hatched bars represent regions in which the reading frame is shifted, and dotted bars represent regions that are missing in the predicted protein product. Bars indicate the size of a peptide 50 amino acids in length (50 aa) or a nucleotide sequence 100 nucleotides in length (100 nt).