Pulmonary alveolar proteinosis caused by deletion of the GM-CSFRalpha gene in the X chromosome pseudoautosomal region 1

Abstract

Pulmonary alveolar proteinosis (PAP) is a rare lung disorder in which surfactant-derived lipoproteins accumulate excessively within pulmonary alveoli, causing severe respiratory distress. The importance of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of PAP has been confirmed in humans and mice, wherein GM-CSF signaling is required for pulmonary alveolar macrophage catabolism of surfactant. PAP is caused by disruption of GM-CSF signaling in these cells, and is usually caused by neutralizing autoantibodies to GM-CSF or is secondary to other underlying diseases. Rarely, genetic defects in surfactant proteins or the common beta chain for the GM-CSF receptor (GM-CSFR) are causal. Using a combination of cellular, molecular, and genomic approaches, we provide the first evidence that PAP can result from a genetic deficiency of the GM-CSFR alpha chain, encoded in the X-chromosome pseudoautosomal region 1.

Figure 1.

Absence of GM-CSFRα expression and responsiveness by the PAP patient's peripheral blood granulocytes. (A) CD14⁺ peripheral blood monocytes from the patient and family members were analyzed for the cell-surface expression of GM-CSFRα by flow cytometry. The dashed line is the isotype-negative control antibody. The patient does not express GM-CSFRα (green). The mother's monocytes give a single peak for GM-CSFRα expression (yellow). The father's and sister's monocytes give a bimodal pattern of staining for GM-CSFRα expression (blue and red). (B) Up-regulation of CD11b expression by stimulation of the patient's and mother's peripheral blood granulocytes with GM-CSF was measured by flow cytometry. The histograms demonstrate constitutive expression of CD11b by granulocytes from both the mother and the patient (shaded). Stimulation with 100 ng/ml GM-CSF resulted in up-regulation of CD11b on the mother's granulocytes, but not on the patient's granulocytes (solid line). The dashed lines are the isotype-matched controls. The MFI from the histograms from the mother's and patient's granulocytes before and after stimulation with 50 and 100 ng/ml GM-CSF were used to calculate the SI, as previously described (reference 25). The data were reproducible in duplicate samples and in two separate experiments.

Figure 2.

Molecular analysis of GM-CSFRα. (A, left) Karyotype analysis of the PAP patient demonstrating one grossly intact X chromosome (Xi) and one short X chromosome that is truncated in the P arm (Xq), characteristic of Turner syndrome. (A, right) FISH analysis of the PAP patient's chromosomes using a probe specific for the X centromere (teal) and a probe specific for the proximal PAR1 region of the X chromosome (red probe). The PAR1 probe hybridizes to the Xi chromosome but not the Xq chromosome. (B, left) Illustration of a subregion of the XPAR1, band p22.33. Highlighted in red is the GM-CSFRα gene, which is located between the genes for the TSLPR and the IL-3Rα, which is followed by the gene encoding ASMTL. (B, right) RT-PCR analysis of the genes examined in PAR1 region, as well as genes encoding IL-5Rα (Ch. 3p24-p26) and βc (Ch. 22q12.2-13.1). The data were reproducible in three out of three experiments.

Figure 3.

Genomic analysis of GM-CSFRα. (left) Genomic structure of the GM-CSFRα gene locus depicting the 11 coding exons (black boxes) and intronic regions (black line). (right) PCR amplification of the 11 exons (exons 3–13) encoding the GM-CSFRα gene using genomic DNA isolated from the PAP patient's leukocytes. Each GM-CSFRα primer set was designed to generate amplicons of 200–350 bp (bottom band), whereas the internal standard (SMG1, Ch. 16) was 627 bp (top band present in all lanes). The first lane in each group of amplicons is the PAP patient's DNA sample (P); the remaining three lanes are positive controls. Exons 3 and 4 are present, but exons 5–13 are missing from the patient's genomic DNA. The data were reproducible in three out of three experiments.