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.
Granulocyte-macrophage colony–stimulating factor (GM-CSF) plays a critical role in alveolar macrophage function and pulmonary surfactant homeostasis. Disruption of GM-CSF signaling by GM-CSF receptor mutations in mice or by GM-CSF autoantibodies in humans results in pulmonary alveolar proteinosis (PAP).
Results identify various CSF2RA mutations causing hereditary PAP in humans. Patients with this disease presented with progressive dyspnea of insidious onset between the ages of 1.5 and 9 years; some were asymptomatic. They were identified and distinguished from autoimmune PAP by serum biomarkers and responded well to whole-lung lavage therapy.
GM-CSF is a hematopoietic cytokine that signals via heterodimeric cell–surface receptors comprised of ligand-binding α (CD116) and affinity-enhancing β (CD131) chains, controlling myeloid cell survival, proliferation, differentiation, and functional activation (9). Ligand binding causes the formation of active dodecameric signaling complexes comprised of α and β chains and β chain–associated Janus kinase-2 subunits that transphosphorylate the receptor and the signal transducer and activator of transcription 5 (STAT5), activating multiple signaling pathways (10). The β chain is also common to receptors for IL-3 and IL-5 (11). GM-CSF is required for the terminal differentiation of alveolar macrophages in mice (4) and likely in humans. GM-CSF, via the transcription factor PU.1, regulates numerous functions of alveolar macrophages including the ability to catabolize surfactant lipids and proteins (3, 5, 12, 13). GM-CSF also promotes survival and regulates multiple functions in circulating neutrophils (14).
Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by the accumulation of surfactant in alveolar macrophages and alveoli resulting in respiratory insufficiency and, in severe cases, respiratory failure (7). Although PAP comprises a heterogeneous group of diseases, autoimmune PAP represents approximately 90% of cases and occurs in men, women, and children with an overall prevalence of approximately 6 to 7 per million (15). It is caused by high levels of neutralizing GM-CSF autoantibodies that block GM-CSF signaling in vivo, reduce macrophage surfactant catabolism, and impair pulmonary surfactant clearance (7, 14–21). Without GM-CSF, alveolar macrophages internalize but do not catabolize surfactant, resulting in the development of large foamy, surfactant-laden macrophages and the accumulation intraalveolar surfactant (22, 23). Patients typically present with progressive dyspnea of insidious onset in the third to fifth decades (15). The current standard therapy is whole-lung lavage, which is used to physically remove the accumulated surfactant (19, 24). Secondary PAP is the next most common clinical form, accounting for approximately 8–9% of cases. It occurs in the context of a very heterogeneous group of underlying diseases that reduce pulmonary surfactant clearance, presumably by reducing either the numbers or intrinsic surfactant clearance capacity of alveolar macrophages (25).
We recently identified PAP in a 6-year-old girl referred to Cincinnati Children's Hospital, Cincinnati, Ohio, for whole-lung lavage therapy (8). She presented with insidious, progressive tachypnea of several years duration and had radiologic and histopathologic manifestations identical to those of autoimmune PAP but had a negative GM-CSF autoantibody test. CSF2RA abnormalities that severely reduced GM-CSF signaling were identified as the cause of PAP. Laboratory evaluation revealed increased GM-CSF levels in lung lavage fluid and serum, consistent with reduced GM-CSF receptor-mediated clearance. Unexpectedly, her asymptomatic 8-year-old sister was found to have the identical CSF2RA mutations, defective GM-CSF signaling, and a milder form of the disease.
Based on these findings, we hypothesized that an increased serum GM-CSF level may identify individuals with PAP caused by GM-CSF receptor dysfunction (26) and then screened sera from our global PAP diagnostic program, established as part of the Rare Lung Diseases Network. We report here on the presentation, clinical features, radiographic appearance, pathogenesis, novel disease biomarkers, and therapy of eight patients with a newly identified hereditary form of PAP caused by CSF2RA mutations.
This study was conducted with the approval of the Cincinnati Children's Hospital Medical Center institutional review board. All participants or their legal guardians gave written informed consent and minors gave assent. The pediatric participants with PAP underwent a complete history and examination, and medical records of clinically performed studies were reviewed, including chest radiographs, high-resolution computed tomography (HRCT) of the chest, and routine blood chemistry and hematologic tests. Some participants underwent pulmonary function testing, bronchoscopy with bronchoalveolar lavage (BAL) with examination of BAL cell cytology as reported (8) or surgical lung biopsy as part of their clinical care. When obtained, lung biopsies were processed and evaluated using standard methods. Disorders of surfactant production involving mutations of SFTPB, SFTPC, or ABCA3 were excluded by mutational analysis (Ambry Genetics or in the DNA Diagnostics Laboratory at the Johns Hopkins University, Baltimore, MD). All symptomatic patients underwent whole-lung lavage therapy (27) as indicated clinically. Case histories of the pediatric patients with PAP and details of the whole-lung lavage procedure are provided in the online supplement or were reported elsewhere (8, 28).
Serum GM-CSF autoantibody levels were measured by ELISA as reported (14, 16, 18). Commercial ELISA kits were used to measure SP-D (BioVendor, Candler, NC), GM-CSF (R&D Systems, Minneapolis, MN), monocyte chemotactic protein-1 (MCP-1; BD Biosciences, San Diego, CA), and macrophage colony–stimulating factor (M-CSF; R&D Systems, Minneapolis, MN) as directed by the manufacturers.
GM-CSF–stimulated levels of CD11b on blood leukocytes were evaluated as reported (14). Briefly, heparinized blood was incubated without or with GM-CSF (Leukine, Genzyme, Cambridge, MA; 10 ng/ml, 30 minutes, 37°C) and then CD11b levels were measured using flow cytometry.
GM-CSF–stimulated phosphorylation of STAT5 in blood leukocytes was evaluated as reported (8). Briefly, heparinized blood was incubated without or with GM-CSF (Leukine; 10 ng/ml, 15 minutes, 37°C). Cell lysates were evaluated by Western blotting using anti-STAT5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti–phospho-STAT5 (Millipore, Billerica, MA), or anti-actin (Santa Cruz Biotechnology) as primary antibodies.
Blood was collected by phlebotomy and GM-CSF receptor α and β chain levels on blood leukocytes were evaluated by flow cytometry using GM-CSF receptor α chain (BD Biosciences) or β chain (Santa Cruz Biotechnology) specific antibodies as reported (8).
Receptor-mediated GM-CSF binding was evaluated as reported (8). Briefly, peripheral blood mononuclear cells were isolated and seeded into culture dishes in medium containing recombinant human GM-CSF (Leukine, Berlex) at a concentration of 1 ng/ml. At various times, the GM-CSF concentration was measured in the culture medium by ELISA (R&D Systems) and expressed as a percentage of original GM-CSF concentration.
GM-CSF receptor α in blood leukocytes was evaluated by Western blotting using a GM-CSF receptor α chain–specific antibody (Santa Cruz Biotechnology) as reported (8).
Sequencing was performed in the Genetic Variation and Gene Discovery Core Facility at the Cincinnati Children's Hospital Medical Center. Briefly, genomic DNA and total RNA were obtained from blood leukocytes using commercial kits (QIAGEN, Valencia, CA) and polymerase chain reaction (PCR) was used to prepare DNA spanning each CSF2RA exon (using genomic DNA) or the entire coding sequence (using cDNA reverse transcribed from mRNA). Nucleotide sequences were compared with data reported in GenBank under accession NM_006140.3 (CSF2RA) or NM_000395.2 (CSF2RB).
Numeric data were evaluated for normality and variance using the Shapiro-Wilk and Levene median tests, respectively, and presented as the mean ± SE (parametric data) or median and interquartile range (nonparametric data). Statistical comparisons were made with Student t test, one-way analysis of variance, or Kruskal-Wallis rank-sum test as appropriate and P values less than or equal to 0.05 were considered to indicate statistical significance. Statistical analysis used SigmaPlot software (Systat, San Jose, CA). All experiments were repeated at least twice, with similar results.
Between May 2004 and November 2009, 187 individuals underwent testing for an increased serum GM-CSF autoantibody level to diagnose autoimmune PAP. Of these, 110 had a positive GM-CSF autoantibody test (>3 μg/ml), whereas five with a negative test were children (<18 years old) with PAP and no underlying disease associated with secondary PAP (7). Evaluation of the family members of these five children identified two more children with undiagnosed PAP and 16 healthy family members. One additional recently reported case (28) was included in the tabular analysis. The demographics and results of screening tests of the children with hereditary PAP are shown (Tables 1 and 2, respectively). The serum GM-CSF concentration in these children with GM-CSF autoantibody-negative PAP was higher than that of healthy family members and than that of unrelated healthy controls (Figure 1).
|Origin: country, state||Kuwait||Kuwait||Kuwait||USA, NC||Italy (Macedonia†)||USA, NC||USA, NC||USA, TX|
|Birth history/complications||Term/none||Term/none||Term/none||Term/meconium aspiration||Term/none||Term/none||Term/none||Unknown|
|Medical history/comorbidities||None||Pneumonia and sepsis at 2.5 yr‡||None||None||None||None||None||Turner syndrome, RSV at 1 mo§|
|Age at symptom onset, yr||None||1.5||1.5||8||9||None||4||Unknown|
|Presenting symptoms and signs||None||Progressive tachypnea, cyanosis||Dyspnea, fatigue||Dyspnea||Dyspnea||None||Progressive tachypnea, dyspnea||Respiratory distress, hypoxemia|
|Age at PAP diagnosis, yr||5||2.5||4||10||11||8||6||3|
|Clinical status at presentation|
|Sao2,%, Fio2||100, RA||83, 15 L/min, mask||N.A.||78, RA||90, 100%, mask||97, RA||88, RA||Unknown|
|Respiratory status||Asymptomatic||Continuous O2||Continuous O2||Continuous O2||Continuous O2||Asymptomatic||Symptomatic, no O2||Continuous O2|
|Height, cm (percentile)||121.5 (95th)||97 (67th)||106 (68th)||144 (50th)||140 (29th)||122 (10th)||103.5 (3rd)||Unknown|
|Weight, kg (percentile)||23.1 (83rd)||15.6 (78th)||17.2 (59th)||54 (96th)||30 (11th)||22.7 (10th)||13.6 (3rd)||Unknown|
|Diagnostic studies performed|
|Chest CT scan||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes|
|Pulmonary function tests||No||No||No||Yes, spirometry only||Yes, after WLL||Yes||Yes||No|
|FEV1, % predicted||N.D.||N.D.||N.D.||36||41||90||44||N.D.|
|FVC, % predicted||N.D.||N.D.||N.D.||39||46||88||44||N.D.|
|TLC, % predicted||N.D.||N.D.||N.D.||N.D.||62||N.D.||N.D.||N.D.|
|DlCO, % predicted||N.D.||N.D.||N.D.||N.D.||56||57||N.D.||N.D.|
|Bronchoscopy and BAL||Yes||Yes||Yes||Yes||Yes||No||Yes||Yes|
|Genetic testing for DSP¶, result||No||Yes, negative||Unknown||No||No||No||Yes, negative||Yes, negative|
|GM-CSF autoantibody test**, result||Yes, negative||Yes, negative||Yes, negative||Yes, negative||Yes, negative||Yes, negative||Yes, negative||Yes, negative|
|Total number/time period, yr††||N.A.||17/1.2||11/1||11/2.5||4/0.25||N.A.||2, at diagnosis||Unknown|
|Maximum volume, L‡‡||N.A.||7.2||8.4||12.5||6.5||N.A.||?||Unknown|
|Worst respiratory status§§||Asymptomatic||Respiratory failure||Continuous O2||Respiratory failure||Respiratory failure||Asymptomatic||Continuous O2||Death|
|Best respiratory status‖‖||Asymptomatic||Symptomatic, O2 with activity and with sleep||Symptomatic, O2 with activity and with sleep||Symptomatic, O2 with activity||Asymptomatic, no O2||Asymptomatic||Symptomatic, no O2||Unknown|
|Current age, yr||5.9||4.2||5.3||13||12||11||9||N.A.|
|Current respiratory status||Asymptomatic||Symptomatic, continuous O2||Symptomatic, continuous O2||Continuous O2||Asymptomatic, no O2||Asymptomatic||Symptomatic, no O2||N.A.|
| Future WLL anticipated||Unknown||Yes||Yes||Yes||Yes||Unknown||Yes||N.A.|
|Serum GM-CSF, pg/ml||16.1||63||226||59||363||19.8||54.7||1,573*|
|Serum SP-D, ng/ml||72||200||750||215||904||180||240||N.D.|
|Lung GM-CSF, pg/ml BAL†||828||838||1,062||1,749||6,437||573||N.D.||N.D.|
|Lung M-CSF, pg/ml BAL‡||316||1,345||5,037||6,485||16,757||N.D.||8,313||N.D.|
|Lung MCP-1, pg/ml BAL§||13,921||21,776||25,747||38,175||23,992||N.D.||25,085||N.D.|
|CD11b SI (% control)‖||1.8 ± 4.8||2.8 ± 1.7||0.2 ± 1.3||2 ± 2||0.5 ± 1.1||N.D.||5.3 ± 1.4||Negative|
|GM-CSF receptor α||Absent||Absent||Absent||Absent||Absent||Present||Present||Absent|
|GM-CSF receptor β||Present||Present||Present||Present||Present||Present||Present||Present|
|Defect reproduced by cloning¶||N.D.||N.D.||N.D.||N.D.||N.D.||Yes||Yes||N.D.|
Eight children with PAP associated with recessive CSF2RA abnormalities (hereafter, patients) contributed to the clinical phenotype reported here (Table 1). Sixteen healthy family members of these patients were also evaluated, including individuals heterozygous for the molecular defects identified (Figure 2). All patients were full-term infants without any need for respiratory support at birth. Six (patients B–E, G, and H) developed progressive dyspnea, exercise intolerance, or tachypnea of insidious onset at various ages ranging from 1.5 to 9 years and two remained asymptomatic at age 5 (patient A) or 8 (patient F) years (Table 1). The mean ages at symptom onset, diagnosis of PAP, and report submission were 4.8 ± 1.6, 6.4 ± 1.2, and 8.6 ± 1.3, respectively. Disease severity at the time of diagnosis varied markedly, ranging from hypoxemic respiratory failure to asymptomatic. Poor weight gain and linear growth occurred in three (patients E–G) (Table 1). All patients had normal routine blood chemistry and hematologic indices. All symptomatic and one of two asymptomatic patients (patient F) had increased serum SP-D (Table 2). In addition to GM-CSF, the concentration of M-CSF and MCP-1 was increased in lung BAL fluid in all patients tested (Table 2). All symptomatic patients received whole-lung lavage therapy and experienced significant, although incomplete, clinical improvement (Table 1). Wide intersubject variability occurred in the number and frequency of whole-lung lavage therapy procedures needed.
HRCT of the chest was performed in all patients. Symptomatic individuals had characteristic diffuse bilateral ground-glass opacifications with sharply delimited margins bordered by fine lines forming polygonal shapes 3–10 mm in diameter (Figure 3). This “geographic” pattern reflected lobular and lobar boundaries and varied in intensity with disease severity. Patient A (asymptomatic at age 5) had limited abnormalities (Figure 3A) and patient F (asymptomatic at age 8) had scattered involvement of secondary lobules in each lobe (8). The chest radiographs of symptomatic patients showed bilateral, diffuse, patchy airspace disease (Figures 3B–E) similar to that reported for patient G (8).
Pulmonary function testing was performed in patients D, F, and G at presentation and in patient E after an initial “rescue” whole-lung lavage treatment. All symptomatic patients evaluated had a restrictive ventilatory defect with proportionate impairment of the FVC, FEV1, and TLC, and a disproportionately more severe reduction of the diffusing capacity for carbon monoxide (DlCO) (Table 1). The DlCO could not be performed in patient G, who had refused any breathholding maneuvers for several years before diagnosis. Patient F was asymptomatic and had normal spirometry but moderately reduced DlCO (Table 1).
All except patient F underwent bronchoscopy with BAL or whole-lung lavage with evaluation of BAL cytology (Table 1). Patients D, G, and H also underwent surgical lung biopsy. In all patients evaluated, the BAL fluid had a milky appearance with a substantial amount of sediment. In most, a dense accumulation of surfactant and cellular debris precluded adequate differential cytometry. In the asymptomatic patient who had bronchoscopy with BAL for diagnosis (patient A), the BAL cell differential included 79.6% macrophages, 4.2% neutrophils, 16% lymphocytes, and 0.2% eosinophils (n = 500 cells). Cytology also showed increased debris and numerous, enlarged foamy alveolar macrophages (Figure 4A) that stained positively with periodic acid–Schiff (PAS) reagent (Figure 4B), and oil red O (Figure 4C). Ultrastructural evaluation revealed that the foamy appearance of the alveolar macrophages was caused by the accumulation of numerous intracellular lamellar and lipid droplet inclusions (Figure 4D). Copious extracellular surfactant was also present (Figure 4D).
Microscopic examination of surgical lung biopsies showed that alveoli were filled with a fine granular, eosinophilic material staining strongly positive with PAS reagent (Figure 4E) and for SP-A, SP-B, and SP-D (8). The abnormal alveolar filling was patchy with completely filled alveoli adjacent to completely empty alveoli (Figure 4F), paralleling the geographic pattern of involvement visualized radiographically (Figure 3). Alveolar walls were well preserved and without abnormality (Figure 4G), similar to results for patients with autoimmune PAP (7), primates with PAP caused by injection of human PAP patient-derived GM-CSF autoantibodies (17), and mice deficient in GM-CSF (22) or its receptor (29). Less severely involved regions also showed intact, enlarged foamy alveolar macrophages in alveoli with no or only minor amounts of alveolar filling with the granular material (Figure 4H). Scattered regions of increased lymphocytes were occasionally seen (8). These results contrast sharply with lung biopsies of patients with surfactant production disorders caused by mutations in SFTPB, SFTPC, or ABCA3, which routinely show gross parenchymal distortion, epithelial cell hyperplasia, and variable accumulation of dysfunctional surfactant (30).
All symptomatic patients underwent whole-lung lavage therapy as described in the online supplement, which resulted in clinical and radiographic improvement. Radiographic clearing was evident by comparison of routine chest radiographs before (Figure 5A) and after (Figure 5B) whole-lung lavage therapy. The degree of radiographic improvement was more clearly defined on HRCT scans of the chest before (Figure 5C) and after (Figure 5D) whole-lung lavage therapy. Although all patients improved with this therapy, the degree of overall improvement varied between washes, among patients, and between lavage of the right and left lungs (the right was generally more difficult). Patients received a mean (± SEM) number of whole single-lung lavages of 8 ± 2.3 with 10.5 ± 1.4 L per lung each over a period of 1.8 ± 0.4 years. Importantly, the number of lavages required did not correlate with the age at diagnosis or duration of disease (R2 = −0.303, −0.273, respectively; patients A–G; P = 0.491; Spearman rank order correlation).
Having established that GM-CSF signaling dysfunction was not caused by neutralizing GM-CSF autoantibodies, we evaluated the patients for GM-CSF receptor abnormalities based on the hypothesis that this might cause GM-CSF accumulation as it does in mice with GM-CSF receptor defects (8). GM-CSF receptor function was evaluated by measuring leukocyte levels of either phosphorylated STAT5 (8) or cell-surface CD11b (14) in whole blood incubated without and with GM-CSF. Patients A–G had no GM-CSF–stimulated increase in phosphorylated STAT5 (Figure 6A; see Figure E1 in the online supplement) (8) and none of the patients had an increase in GM-CSF–stimulated CD11b levels (Table 2). In contrast, controls and healthy family members all had readily detectable GM-CSF–stimulated increases in phosphorylated STAT5 (Figure 6A; see Figure E1) or CD11b (not shown) (8, 28) in blood leukocytes.
To determine if the absence of either GM-CSF receptor α or β chains was the basis for the absence of GM-CSF signaling, blood leukocytes were evaluated by flow cytometry. GM-CSF receptor β was present on blood leukocytes from all patients and the mean fluorescence intensity was similar to that of healthy family members and healthy controls (Table 2). We confirmed the function of the β chain in patients by stimulation of blood leukocytes with IL-3, which resulted in STAT5 phosphorylation as expected (31).
In contrast, GM-CSF receptor α was not detectable in patients A–E or patient H, but was present on leukocytes from all healthy family members and controls evaluated (Figure 6B; see Figure E2). GM-CSF receptor α was also evaluated by Western blotting of leukocyte lysates, which confirmed its absence in patients B–E and presence in family members and healthy controls (Figure 6C; see Figure E3). The GM-CSF receptor α was detectable on leukocytes from patients F and G but had a reduced molecular mass on polyacrylamide gels because of the consequences of disruption of a single glycosylation site (8). Clearance of GM-CSF by blood leukocytes was absent in patients B and C and markedly reduced in patients F and G (Table 2), consistent with a defect in GM-CSF binding.
Thus, the absence (patients A–E and H) or structural abnormality (patients F and G) of the GM-CSF receptor α protein provided an explanation for the impaired GM-CSF signaling in these patients: both a severe reduction and the complete absence of GM-CSF signaling are associated with the development of PAP.
Nucleotide sequence analysis of genomic DNA and mRNA from blood leukocytes of each patient, their family members, and healthy controls was done to determine the molecular nature of abnormal CSF2RA gene expression. The sequencing strategy and primers used are shown (Figure 7A and Table E1, respectively). For clarity, results from the propositus in each family are described together with their respective family members. Patient B was homozygous for a single nucleotide substitution (C to T) at position 649 (i.e., c.649C > T) in CSF2RA exon 8 (Figure 7B). This point mutation introduced a premature stop codon at position 217 (i.e., Arg217X). Analysis of mRNA confirmed the genomic DNA abnormality in patient B (not shown). Both healthy parents and two healthy siblings (AIII1, AIII2, AIV1, and AIV2, respectively; Figure 2) were heterozygous for this point mutation and the other healthy sibling (AIV5) had only a normal sequence. Patient A, the brother of patient B (Figure 2), was homozygous for this mutation in genomic DNA and mRNA (not shown, but identical to Figure 7B). Patient C, a first cousin of patients A and B, was homozygous for this mutation (not shown, but similar to Figure 7B), whereas of both her healthy parents and two healthy siblings (AIII3, AIII4, AIV7, and AIV8, respectively; Figure 2) were heterozygous for it and a normal CSF2RA sequence (not shown). The remainder of the CSF2RA coding sequence of mRNA from patients A–C was normal (not shown).
Analysis of leukocyte mRNA from patient D revealed a single nucleotide sequence containing a two-nucleotide (GC) duplication immediately after nucleotide position 921 (i.e., c.920_921dupGC) in exon 10, resulting in a reading frame shift at position 308 and introduction of a premature stop codon at position 332 in exon 11 (Figure 7C). Analysis of genomic DNA confirmed the mRNA abnormality in patient D (not shown). The remainder of the CSF2RA coding sequence of mRNA from patient D was normal (not shown). Analysis of genomic DNA and mRNA sequences showed that her mother (BI2; Figure 2) and grandmother (not shown) did not harbor this mutation. The father was unavailable for evaluation, and CSF2RA allelic heterozygosity analysis was not performed.
In patient E, reverse transcription and PCR amplification of mRNA showed that the CSF2RA coding sequence was approximately 200 nucleotides shorter than that of her brother (or a healthy control), and that both parents (CII1, CI1, and CI2, respectively; Figure 2) had PCR products similar in size to the patient and to control (Figure 7D). This suggested a CSF2RA deletion mutation may be segregating in this family. All CSF2RA exons were detected in genomic DNA from patient E and her family members (and a healthy control) except for exon 7, which was detected in all except the patient (Figure 7E). Targeted PCR amplification showed patient E was homozygous for a 1.7-kb deletion encompassing exon 7 and that both parents were heterozygous carriers of this mutation, whereas her brother was homozygous normal (Figure 7F). Analysis of genomic DNA sequence showed the deletion included 1,794 nucleotides (c.474–1007_646+614del1,794) encompassing exon 7 (Figure 7G). Sequence analysis revealed two tandem Alu family repetitive DNA sequences flanking exon 7 in the parents and brother of patient E and a healthy control (Figure 7A). These Alu repeats were 90% homologous with each other and contained a central 10-nucleotide region of 100% homology (Figure 7G, overlapping region of red and black arrows) among the family members and control. Patient E had a deletion extending from within the central homologous region in the upstream 5′ flanking Alu repeat to within this same central sequence in the downstream 3′ flanking Alu repeat. Analysis of mRNA sequence revealed that leukocytes from patient E expressed two CSF2RA mRNA transcripts encoding distinct protein abnormalities (Figure 7H). Transcript 1 comprised a precise deletion of exon 7, substitution of threonine for arginine at codon 159, followed by a frame shift introducing a premature stop codon 56 codons downstream within exon 10 (i.e., p.Arg159ThrfsX56). Transcript 2 comprised the deletion of exon 7 and the first 19 nucleotides of exon 8, resulting in substitution of asparagine for lysine at position 158 followed by an in-frame deletion of the next 65 codons (i.e., p.Lys158_Ser222delinsAsn). Comparison of genomic DNA and mRNA sequence revealed these were alternatively spliced transcripts (Figure 7I): transcript 1 used the natural donor site of exon 6 and the natural splice acceptor site for exon 8 and transcript 2 used this same donor and a cryptic splice acceptor site within exon 8, 19 nucleotides downstream from the 5′ end of exon 8. Together, these findings strongly suggest that homologous recombination between these two Alu repeats in an ancestral CSF2RA gene (as observed in γo-βo-thalassemia [32, 33]) is the mechanism underlying the CSF2RA exon 7 deletion mutation in this family (Figure 7I).
These results identify that a wide range of genetic CSF2RA abnormalities and their corresponding protein abnormalities (Figure 8) are associated with development of hereditary PAP. These included CSF2RA missense, nonsense, duplication, deletion, and frameshift mutations; aberrant alternative splicing; gene deletions; and chromosomal rearrangements with partial gene deletion (Table 3).
DNA Sequence Change*
Location of Mutation (exon)
Type of Mutation
Amino Acid Change* (three letter code)
Granulocyte-Macrophage Colony–Stimulating Factor Signaling
Found in Patients
No. of Healthy Carriers
|c.649C > T||8||Nonsense||p.Arg217X||R217X||Absent||Absent||A–C||8|
|c.920_921dupGC||10||Duplication and frameshift||p.Ser308AlafsX25||920dupGC||Absent||Absent||D||0|
|c.474-1007_646+614del1,794||7||Deletion and frameshift||p.Arg159ThrfsX56||ΔEx7||Absent||Absent||E||2|
|c.474-1007_646+614del1,794||7 – 8 (partial)||Deletion in frame||p.Lys158_Ser222delinsAsn||ΔEx7-8||Absent||Absent||E||2|
|c.586G > A||7||Missense||p.Gly196Arg||G196R‡||Present||Reduced||F and G||1|
|Xp22.33del1.6mb||1 – 13||Deletion||No protein||XpΔ1.6||Absent||Absent||F and G||1|
|Xp22.33del0.41mb§||6 – 13||Deletion||No protein||XpΔ0.41||Absent||Absent||H||2|
Here, we report eight individuals with a newly identified genetic lung disease causing PAP in children, including their clinical presentation, laboratory findings, histopathology, pathogenesis, diagnosis, and response to therapy. Six patients presented between the ages of 1.5 and 9 years with progressive dyspnea of insidious onset manifesting as tachypnea or exercise intolerance, whereas two were asymptomatic at ages 5 and 8 years and diagnosed only after the identification of affected siblings. All were negative for GM-CSF autoantibodies, had increased serum levels of GM-CSF and SP-D, and GM-CSF signaling dysfunction caused by various recessive homozygous or compound heterozygous CSF2RA abnormalities. GM-CSF, M-CSF, and MCP-1 were markedly increased in BAL fluid. Symptomatic patients were treated with whole-lung lavage therapy and responded well with clinical and radiographic improvement. Notwithstanding, repeated treatments were necessary in some to keep up with ongoing surfactant accumulation.
Our results support the conclusions that GM-CSF signaling is critical for surfactant homeostasis in humans and that CSF2RA mutations cause a genetic form of PAP. The disruption of GM-CSF signaling was supported by multiple lines of evidence including (1) inability to detect GM-CSF receptor α protein, (2) absence of ligand binding, (3) absence of downstream signaling events, (4) identification of function-altering CSF2RA mutations for which all patients were either homozygous or compound heterozygous and for which all healthy family members were heterozygous or not carriers, and (5) use of gene cloning to reproduce the CSF2RA mutation and GM-CSF signaling dysfunction observed in patients F and G (8). These conclusions are supported by similarities with other forms of PAP that involve disruption of GM-CSF signaling, including mice deficient in expression of GM-CSF (22, 23) or its receptor (29, 34, 35), humans with autoimmune PAP caused by neutralizing GM-CSF autoantibodies (7, 14–16, 18), and the development of PAP in healthy primates after injection with human PAP patient-derived GM-CSF autoantibodies (17). Several biomarkers of PAP were similarly increased in our patients, autoimmune PAP patients, and GM-CSF–deficient mice including increased MCP-1 and M-CSF in the lungs and increased SP-D in the serum (4, 7, 8, 36–39). The histopathologic features of PAP in these children (structurally intact alveoli filled with foamy, lipid- or surfactant-laden macrophages and secreted functional surfactant) were similar to those of patients with autoimmune PAP (7, 8, 19) and mice deficient in GM-CSF or its receptor (22, 23, 29). However, these histopathologic features contrast with those seen in disorders of surfactant production caused by mutations in SFTPC, or ABCA3, which include marked distortion of alveolar wall architecture by cellular infiltration, epithelial cell hyperplasia and fibrosis, and variable accumulation of dysfunctional surfactant (30). Although the histopathology has some overlap with that reported in SFTPB mutation cases, the clinical presentation is quite distinct, because most SFTPB mutation cases present with neonatal respiratory failure and our patients presented with insidious symptoms well beyond the neonatal period. Finally, whole-lung lavage was successful in all of the symptomatic patients, similar to results in autoimmune PAP patients (7, 19, 24). Together, these results and published reports highlight the pathogenic similarities of PAP caused by disruption of GM-CSF signaling at different points (ligand production, intercellular signaling, and receptor activation) and in different species (humans, monkeys, and mice), suggesting the pathogenesis may be similar in each.
An important finding of our study was the marked variable penetrance, which ranged from asymptomatic with mild cytologic abnormalities to severe dyspnea with dense bilateral consolidative opacification throughout both lungs. These differences could not be attributed to specific CSF2RA mutations because the greatest variability occurred among patients A and B, who were siblings of a consanguineous marriage and homozygous for the same CSF2RA mutation. Further, patients E and F had markedly differing disease severity and were siblings of a nonconsanguineous marriage with identical compound heterozygous CSF2RA mutations. Nor could the variable penetrance be attributed to differences in the level of impairment of GM-CSF signaling, because patient A, who had no detectable GM-CSF signaling, had a nearly normal HRCT, whereas patient F, who had a minimal but nonzero residual GM-CSF signaling, had dense bilateral diffuse consolidative opacification. Finally, these differences could not be attributed to differences in duration of disease (i.e., age), because patient A had a nearly normal HRCT at age 5 years (and patient F had only mild HRCT changes at age 8 years), whereas patients B and C had among the most severe HRCT abnormalities at 3 to 4 years of age. Among symptomatic patients, the long delay from birth to the onset of symptoms (4.8 ± 1.6 yr) suggests that net surfactant accumulation within alveoli occurs very slowly. Longitudinal assessment of the two asymptomatic patients should be useful in assessing this hypothesis. Based on these findings, we conclude that whereas loss of GM-CSF signaling was necessary for development of PAP, other factors are important in determining disease severity. These may include differences in surfactant production or uptake and recycling or catabolism of surfactant in type II alveolar epithelial cells, all of which are poorly understood. Future studies are needed to evaluate the potential role of these and other factors as determinants of disease severity in hereditary PAP.
Patients with hereditary PAP presented with clinical manifestations because of the effects of surfactant accumulation on gas exchange analogous to the presentation of autoimmune PAP patients (7) except at a younger age (51 [n = 223] vs. 6.4 ± 1.2 [n = 6] years, respectively). Although not designed to study prevalence, we identified 110 cases of autoimmune PAP and seven cases of hereditary PAP among 187 sera screened for autoimmune PAP suggesting that hereditary PAP represents a relatively small proportion of total cases of PAP caused by loss of GM-CSF signaling (∼6% in our series). The disease occurred in both sexes, white and black individuals, and on three different continents. The radiologic appearance was similar among symptomatic patients and identical to that of autoimmune PAP patients (7, 19). Of diagnostic importance was the observation that the radiograph in hereditary PAP appeared worse than expected based on the clinical findings. Our data suggest that radiographic changes in hereditary PAP progress slowly, likely preceding the development of symptoms by months or years. Although pulmonary function tests (especially DlCO) can be useful in assessing disease severity, they are difficult to perform in children less than 6 years old. Routine laboratory tests were of little help. However, several novel biomarkers were diagnostically useful. Serum GM-CSF autoantibody levels, which are increased in all patients with autoimmune PAP (7, 15), were low in all patients with hereditary PAP. In contrast, GM-CSF levels were elevated in all patients with hereditary PAP and low in patients with autoimmune PAP (16). A substantial delay in diagnosis (∼2 yr) and misdiagnosis as asthma (before radiography) or pneumonia (after radiography) underscores the importance of a high degree of clinical suspicion in making a timely and accurate diagnosis of hereditary PAP. Our results support the value of performing HRCT of the chest, serum GM-CSF autoantibody, GM-CSF, and SP-D testing in pediatric patients with PAP at the time of diagnostic assessment. If bronchoscopy with BAL is performed, elevated levels of GM-CSF, MCP-1, or M-CSF may be useful. Evaluation of GM-CSF–stimulated increases in leukocyte CD11b levels or STAT5 phosphorylation by flow cytometry are convenient screening tests to evaluate for GM-CSF signaling dysfunction. If this strategy is used, patients with hereditary PAP can be effectively identified without the need for surgical lung biopsy. Lastly, HRCT may be a useful clinical outcome measure in the evaluation of novel therapies for PAP because it directly measures the accumulation of the pathogenic material (surfactant) in the lungs. This conclusion is supported by a recent report using quantitative CT densitometry to measure the response to GM-CSF therapy in autoimmune PAP (40).
Patients with hereditary PAP responded well to whole-lung lavage, similar to results for patients with autoimmune PAP (7, 19, 24) and in contrast to patients with disorders of surfactant production who do not respond well. Of technical importance, the narrower airway in children precludes the use of a standard Carlen double-lumen tube and special materials and procedures were needed (see the online supplement). No formal criteria were developed to measure the therapy responses in our study and these should be developed and evaluated in future studies. Based on our prior use of quantitative CT densitometry to monitor outcomes (40), we believe a limited scan chest HRCT protocol may be useful. Serial serum SP-D measurement may also be of use. Bone marrow transplantation was attempted in one patient who died shortly after the procedure of uncontrolled respiratory infection (28). This approach has been considered for some of our current patients; however, suitable donors have been difficult to find. We successfully demonstrated the feasibility of gene therapy for GM-CSF receptor defects in mice (41) and in preliminary in vitro human studies (8). Further studies are required to evaluate these potential therapies.
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