Whole-Genome Sequencing of a Family with Hereditary Pulmonary Alveolar Proteinosis Identifies a Rare Structural Variant Involving CSF2RA/CRLF2/IL3RA Gene Disruption

Abstract

Pulmonary alveolar proteinosis (PAP) is a rare pulmonary disease in which the abnormalities in alveolar surfactant accumulation are caused by impairments of GM-CSF pathway attributing to defects in a variety of genes. However, hereditary PAP is extremely uncommon and a detailed understanding in the genetic inheritance of PAP in a family may provide timely diagnosis, treatment and proper intervention including genetic consultation. Here, we described a comprehensive analysis of genome and gene expression for a family containing one affected child with a diagnosis of PAP and two other healthy siblings. Family-based whole-genome analysis revealed a homozygous deletion that disrupts CSF2RA, CRLF2, and IL3RA gene in the pseudoautosomal region of the X chromosome in the affected child and one of asymptomatic siblings. Further functional pathway analysis of differentially expressed genes in IL-1β-treated peripheral blood mononuclear cells highlighted the insufficiency of immune response in the child with PAP, especially the protection against bacterial infection. Collectively, our results reveal a novel allele as the genetic determinant of a family with PAP and provide insights into variable expressivity and incomplete penetrance of this rare disease, which will be helpful for proper genetic consultation and prompt treatment to avoid mortality and morbidity.

Figure 1. Genetic pedigrees, chest radiograph, high-resolution computed tomography (HRCT) scan of the chest, and lung histopathology in the child with pulmonary alveolar proteinosis.

(A) Pedigree of family with cases of pulmonary alveolar proteinosis. (B) Chest radiograph at presentation showing bilateral diffuse alveolar infiltration with an air bronchogram. (C) Chest HRCT showing areas of ground-glass opacification with thickened inter- and intralobular septae, alternated with areas of normal lung. (D) Histological examination showing alveoli filled with granular lipoproteinaceous material, staining pink with periodic acid-Schiff (PAS) stain. (E) Protein expression analysis of GM-CSF receptors showing the absence of GM-CSF α chain in NGS1 and NGS2. Cropped blots were shown for clarity. Full-length blots/gels are presented in Supplementary Fig. S1A.

Figure 2. Identification of a structural variant in a family with PAP by whole-genome sequencing.

(A) Alignment of a subregion of the autosomal region of X chromosome (marked in red) by IGV (Intergative Genomics Viewer) illustrates a deletion of 0.425 Mb (XpΔ0.425) that disrupts the first two exons of the IL3RA gene and entire CRLF2 and CSF2RA gene. (B–C) Magnified images of the regions surrounding both breakpoints demonstrate the genotype of each family member. (D) Pedigree of the patient with PAP in this study. Filled symbol represents the proband, and the partially filled symbol indicates the asymptomatic, homozygous carrier.

Figure 3. Quantitative real-time PCR analysis, flow cytometry and signaling pathway analysis of CSF2RA, CRLF2, and IL3RA gene expression.

(A) Quantitative RT-PCR revealed a decrease in expression of these genes in NGS4 and NGS5 (parents) compared to NGS3 (healthy girl). (B) Histograms showed the shift in the fluorescence intensity that was seen for the monocytes (PE-labeled anti-human CSF2RA antibody) and for the plasmacytoid dendritic cells (PE-labeled anti-human IL3RA antibody) in NGS3, NGS4 and NGS5. (C) PBMCs stimulated with recombinant human GM-CSF and IL-3 but not TSLP revealed the phosphorylation of STAT5 in NGS3 and NGS4. Extremely low STAT5 phosphorylation was detected in PBMCs with GM-CSF, TSLP and IL-3 stimulation in NGS1 and NGS2. Negative control was cell incubated with PBS without stimulant individually. Cropped blots were shown for clarity. Full-length blots/gels are presented in Supplementary Fig. S1B.

Figure 4. Analysis of gene expression in PMBCs stimulated with IL-1β (10 ng/ml) using RNA sequencing.

(A) Venn diagram showed the distribution of the genes identified in NGS1, NGS2 and NGS3 (fold change ≥ 2, P value < 0.05). (B) A heat map of Hierarchical Clustering of 21 genes identified in common in all three subjects. (C) A heat map of Hierarchical Clustering of 63 genes identified just in common in NGS2 and NGS3 rather than NGS1. Red or green colours indicate higher and lower expression levels respectively of the genes correlating into a smaller number of uncorrelated variables called principal components.