Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA

Abstract

Primary pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by accumulation of surfactant in the lungs that is presumed to be mediated by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling based on studies in genetically modified mice. The effects of GM-CSF are mediated by heterologous receptors composed of GM-CSF binding (GM-CSF-Ralpha) and nonbinding affinity-enhancing (GM-CSF-Rbeta) subunits. We describe PAP, failure to thrive, and increased GM-CSF levels in two sisters aged 6 and 8 yr with abnormalities of both GM-CSF-Ralpha-encoding alleles (CSF2RA). One was a 1.6-Mb deletion in the pseudoautosomal region of one maternal X chromosome encompassing CSF2RA. The other, a point mutation in the paternal X chromosome allele encoding a G174R substitution, altered an N-linked glycosylation site within the cytokine binding domain and glycosylation of GM-CSF-Ralpha, severely reducing GM-CSF binding, receptor signaling, and GM-CSF-dependent functions in primary myeloid cells. Transfection of cloned cDNAs faithfully reproduced the signaling defect at physiological GM-CSF concentrations. Interestingly, at high GM-CSF concentrations similar to those observed in the index patient, signaling was partially rescued, thereby providing a molecular explanation for the slow progression of disease in these children. These results establish that GM-CSF signaling is critical for surfactant homeostasis in humans and demonstrate that mutations in CSF2RA cause familial PAP.

Figure 1.

Phenotypic characterization of patients with familial PAP. (A) Chest radiograph (top) and high-resolution computed tomogram of the chest (bottom) of the index patient at presentation. (B) Histopathologic appearance of the open lung biopsy from the index patient after staining with hematoxylin and eosin (H&E) or immunostaining for SP-A, mature SP-B, Pro–SP-C, SP-D, and ABCA3. Note the lymphocytosis in the low-power hematoxylin and eosin–stained section and the intact alveolar wall in the high-powered hematoxylin and eosin–stained section. Images were obtained at a total magnification of 50× (top) or 400× (all others). Bars: (top) 100 μm; (all others) 10 μm. (C) Chest radiograph of the index patient 4 mo after presentation immediately before (top) and 5 d after (bottom) whole lung lavage therapy. (D) Serum SP-D levels in affected siblings, parents, and a control. The mean ± SD serum SP-D concentration in 67 healthy controls was 63.5 ± 39 ng/ml (hatched region) and in 12 patients with autoimmune PAP was 174 ± 83 ng/ml (not indicated). (E) High-resolution computed tomogram showing diffuse patchy ground glass opacities representing mild PAP in the affected sister.

Figure 2.

Structural and functional analysis of GM-CSF receptor defects. (A) Flow cytometry demonstrating GM-CSF-Rα and GM-CSF-Rβ on the cell surface of peripheral blood leukocytes in all family members. (B) Western blots of PBMC lysates using antibodies to detect GM-CSF-Rα or actin as indicated. (C) GM-CSF clearance assay. Blood leukocytes from the patient were unable to remove exogenous GM-CSF added to culture medium at time zero in contrast to leukocytes from two controls that rapidly bound and cleared GM-CSF. Error bars show the means ± SE. (D and E) Measurement of GM-CSF concentration by ELISA. GM-CSF was readily detected in lavage from the patient but was not detected (ND) in lung lavage from nine healthy controls (D). GM-CSF was detected in the serum of affected siblings but not their parents or a control (E). (F) Blood leukocytes isolated from the indicated family members were incubated for 15 min in the absence (−) or presence (+) of 10 ng/ml GM-CSF, followed by Western blotting of cell lysates to detect total STAT5 (STAT5), phosphorylated STAT5 (pSTAT5), or actin. (G) Measurement of the GM-CSF–stimulated increase in CD11b levels on leukocytes in whole blood. The CD11b stimulation index (24) was calculated as the mean fluorescence of CD11b on leukocytes incubated with GM-CSF minus that of unstimulated cells divided by that of unstimulated cells and multiplied by 100. (H) Similar to F except that a higher GM-CSF concentration (1,000 ng/ml) was used and cell lysates were immunoprecipitated with anti-STAT5 antibody before Western blotting to detect total STAT5 (STAT5) or phosphorylated STAT5 (pSTAT5).

Figure 3.

Genetic analysis of CSF2RA gene defects. (A) Nucleotide sequence of CSF2RA in genomic DNA from each family member, including nt 580–591 of the coding sequence (numbered relative to the initiation codon; from GenBank/EMBL/DDBJ under accession no. NM_006140.3). The index patient and her sister exhibited only a G→A point mutation at nt 586. The father was heterozygous for this substitution and the mother exhibited only the normal sequence. (B) FISH analysis to detect CSF2RA sequences in genomic DNA from the father and the patient. The probe (CTD-3047L21), which maps to the pseudoautosomal region (Xp22.33 and Yp11.32), hybridized to both X and Y chromosomes in the father (white arrows) and to one (white arrow), but not the other (yellow arrow), X chromosome in the patient. 8–10 metaphase cells and 25 interphase cells were evaluated for each individual. Similar FISH analyses are shown for each family member in the supplemental material (available at http://www.jem.org/cgi/content/full/jem.20080990/DC1). Images were obtained at a total magnification of 1000×. (C) CGH analysis for the patient in the region of Xp22.33. The relative fluorescence of fluorescently labeled DNA from the patient (open circles) compared with a same-sex reference DNA (filled circles) after hybridization to various BAC clones on the SignatureSelect V2 chip representing the Xp22.33 region is shown. Reduced hybridization to several BAC clones (clones B and C) is indicated by the lower fluorescence of the patient's DNA compared with the reference DNA for these BAC clones. The telomeric breakpoint is mapped to between clones A and B and the centromeric breakpoint is mapped to between clones C and D. These data indicate an interstitial deletion of ∼1.264 Mb at Xp22.33 encompassing ∼1,610,183–2,873,864 bp. The dashed line represents a relative fluorescence of zero. (D) High-resolution SNP mapping of the Xp22 region for paternal and maternal X chromosomes. A schematic shows the locations of the point mutation (CSF2RA^G174R) in the paternal X chromosome and the 1.6-Mb deletion at Xp22.33 in the maternal X chromosome and the genes encompassed. The base 2 ratio of normalized hybridization intensities for patient and reference samples (log R ratio) is shown. Similar SNP analyses for each family member are shown in the supplemental material (available at http://www.jem.org/cgi/content/full/jem.20080990/DC1). (E) Map showing a portion of the X chromosome summarizing the genetic analysis used to identify the small maternal X chromosomal deletion at Xp22.33 encompassing CSF2RA. The probe used for FISH analysis (hatched bar) is the same as clone B on the CGH microarray chip. The locations of selected CGH microarray probes in the region of the CSF2RA gene are shown. Those CGH probes showing balanced hybridization to patient and control DNA are shown as clear boxes (A and D), whereas those showing an unbalanced hybridization representing sequences deleted in the patient are shown in black. High-resolution SNP analysis revealed that the deletion (red bar) extended from 1,308,324 to 2,881,011 bp. Sequence analysis (A) and PCR quantification of CSF2RA exon 7 (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20080990/DC1) demonstrated that the deletion included CSF2RA exon 7. The chromosomal location of the CSF2RA gene (1,347,701–1,388,827 bp) is indicated. (F) Pedigree of the family deduced from sequencing and CSF2RA allelic copy number determination experiments. The index case is indicated (arrow).

Figure 4.

Reproduction of the CSF2RA^G174R gene defect. (A) Western blots of nontransfected 293 cell lysates (control) or lysates of cells transfected with plasmids expressing CSF2RB and either CSF2RA^WT (WT) or CSF2RA^G174R (G174R), followed by incubation with (+) or without (−) Peptide: N-Glycosidase F (PNGase F) and detection with antibodies against GM-CSF-Rα or actin. (B) Evaluation of cell-mediated binding and removal of exogenous GM-CSF from the culture media. Asterisks indicate significant differences in levels of GM-CSF in media from cells expressing CSF2RA^WT/CSF2RB compared with cells expressing CSF2RA^G174R/CSF2RB, CSF2RB alone or media without cells. Error bars show the means ± SE. (C) Evaluation of GM-CSF–stimulated phosphorylation of STAT5 in 293 cells transfected as described in A and incubated for 15 min in the absence (−) or presence (+) of 10 ng/ml GM-CSF. Lysates were evaluated by Western blotting (WB) to detect total STAT5 (STAT5) or were immunoprecipitated with anti-STAT5 antibody and then evaluated by Western blotting (IP + WB) to detect total STAT5 or phosphorylated STAT5 (pSTAT5). (D) Similar to C, except that increased concentrations of GM-CSF were used for evaluate GM-CSF receptor function.