Pulmonary alveolar proteinosis: An autoimmune disease lacking an HLA association
Pulmonary alveolar proteinosis: An autoimmune disease lacking an HLA association
Kirsten AndersonID 0 1
Brenna Carey 1
Allison Martin 0 1
Christina Roark 1
Claudia Chalk 1
Marchele Nowell-Bostic 1
Brian Freed 0 1
Michael Aubrey 0 1
Bruce Trapnell 1
Andrew Fontenot 0 1
0 University of Colorado Anschutz Medical Campus, Department of Medicine, Aurora, CO, United States of America, 2 ClinImmune Labs Aurora, CO, United States of America, 3 Cincinatti Children's Hospital Medical Center (CCHMC) Cincinnati , OH , United States of America
1 Editor: Gernot Zissel , Universitatsklinikum Freiburg , GERMANY
Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of pulmonary surfactant in alveolar macrophages and alveoli, resulting in respiratory impairment and an increased risk of opportunistic infections. Autoimmune PAP is an autoimmune lung disease that is caused by autoantibodies directed against granulocyte-macrophage colony-stimulating factor (GM-CSF). A shared feature among many autoimmune diseases is a distinct genetic association to HLA alleles. In the present study, we HLA-typed patients with autoimmune PAP to determine if this disease had any HLA association. We analyzed amino acid and allele associations for HLA-A, B, C, DRB1, DQB1, DPB1, DRB3, DRB4 and DRB5 in 41 autoimmune PAP patients compared to 1000 ethnic-matched controls and did not find any HLA association with autoimmune PAP. Collectively, these data may suggest the absence of a genetic association to the HLA in the development of autoimmune PAP.
Data Availability Statement: The data underlying
the results presented in the study are available in
the Supplemental Materials along with the data
used to generate the results.
Funding: BT received funding from the National
Institute of Health for this research under grant
numbers R01 HL085453 and U54HL127672.
https://www.nih.gov. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Pulmonary alveolar proteinosis (PAP) is a rare syndrome comprising a heterogeneous group
of diseases characterized by the accumulation of pulmonary surfactant in alveolar
macrophages and the alveolar space [
]. Eventually, surfactant accumulation results in respiratory
impairment and/or failure as well as an increased risk of opportunistic infections . This
syndrome occurs in individuals from ages 8 to 90 years, but it is most common in male smokers
in the third to fourth decade [
]. The catabolism of pulmonary surfactant in alveolar
macrophages is controlled by granulocyte-macrophage-colony stimulating factor (GM-CSF) .
GM-CSF is a cytokine that modulates the survival, differentiation, proliferation, and priming
of myeloid cells [
]. GM-CSF signaling can be disrupted by mutations in the GM-CSF gene [
] or its receptors [
], as well as by neutralizing autoantibodies [
2, 14, 15
]. In this regard,
Competing interests: The authors have declared
that no competing interests exist.
autoimmune PAP is the disease that results from autoantibodies directed against GM-CSF,
and the identification of neutralizing, polyclonal anti-GM-CSF autoantibodies in autoimmune
PAP is an essential component of the disease diagnosis.
In most autoimmune diseases, CD4+ T cells are required to assist B cells in isotype
switching, which is necessary for the generation of autoantibodies such as those found in
autoimmune PAP patients. Few studies have investigated the role of the adaptive immune response,
and no studies to date have investigated the association between the genetic susceptibility to
autoimmune PAP and HLA. In the vast majority of immune-mediated diseases, genetic
susceptibility has been most strongly associated with the HLA region on the short arm of
chromosome 6 [
]. For example, the major genetic contribution to rheumatoid arthritis (RA)
involves DRB1 alleles such as DRB1 04:01 and 04:04 [
], whereas HLA-DQ alleles, especially
DQB1:02:01 and DQB1:03:02, provide the major genetic contribution to type 1 diabetes (T1D)
]. In the case of chronic beryllium disease, genetic susceptibility is strongly linked to
HLA-DPB1 alleles possessing a glutamic acid at position 69 of the ?-chain [
]. Thus, the
impetus for this study was to identify to HLA alleles that confer susceptibility to the generation
of autoimmune PAP.
Patients and controls
We collected DNA from 41 Caucasian subjects with autoimmune PAP defined by a serum
level of GM-CSF autoantibody greater than 5.6 ?g/mL. These patients were recruited from the
Translational Pulmonary Science Center (TPSC) at Cincinnati Children?s Hospital Medical
Center (CCHMC), the University of Cincinnati Medical Center, and other Rare Lung Disease
Network (RLDN) centers/affiliates. The Cincinnati Children?s Hospital Medical Center Office
of Research Compliance and Regulatory Affairs issued for this study IRB # 2011?0147. Written
consent was obtained from all patients enrolled in the study. The patients were all Caucasians
with a positive GM-CSF autoantibody test [
], with a mean age of 44.3+/-6.7 years (+/-SEM),
and comprised of 40.9% female and 59.1% male, from various regions in the United States
(84.1%), Canada (2.3%), Turkey (13.6%) Subjects and demographic information are available
in S1 Table.
High resolution molecular typing for HLA-A, B, C, DRB1, DPB1, DQB1, DRB3, DRB4 and
DRB5 was performed at ClinImmune Laboratories at the University of Colorado Anschutz
Medical Campus. 1,000 control subjects for HLA analysis were acquired from the National
Marrow Donor Program (NMDP) Full HLA types for patients and controls are published in
S2 Table. For validation of small sample sizes in HLA Epitope Analysis Program (HEAP), we
used data from patients with RA (patient data from IHWG) and T1D (data from the T1D
HLA analysis and statistics
The number of patients carrying at least one copy of each allele was counted and compared
with the number of controls carrying at least one copy of the same allele. The significance of
each was calculated using a Chi-square test or Fisher?s exact test as appropriate depending on
the number of subjects in each contingency table. The resulting p-values were corrected for
multiple comparisons using the false discovery rate method [
]. We additionally performed
an equivalence test for all alleles using R software version 3.4.4 and the package TOSTER
HLA amino acid association analysis was performed using the R software package version
2.6.1. Combinations of 1?4 polymorphic amino acids at positions 8?93 of HLA-DRB1, DPB1
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and DQB1, as well as combinations of up to 4 amino acids at positions 2?192 of HLA-A, B and
C were analyzed for association with PAP. Chi-square or Fisher?s exact tests were calculated as
appropriate, and the P-values were adjusted for multiple comparisons.
HEAP validation for small sample sizes
In small sample sizes, allele association studies are likely to miss true positive associations due
to polymorphisms in the HLA region. Rare alleles that may be truly disease susceptible alleles
cannot reach statistical significance due to their low frequency in both patient and control
groups. Amino acid association tests, on the other hand, benefit small studies by identifying
shared features between disparate alleles, including rare alleles.
To test whether we could detect a true positive HLA association with small patient
numbers, we analyzed known HLA amino acid associations from RA and T1D. RA is strongly
associated with alleles of DRB1 04, which in single amino acid analysis can be identified by the
presence of histidine at position 13 [
]. T1D has strong single amino acid associations in both
HLA-DRB1 (histidine at position 13) and HLA-DQB1 (alanine at position 57) [
]. In T1D,
the DQB1 association is stronger than the association in DRB1 [
]. We determined whether
we could correctly identify the known HLA amino acid associations in RA and T1D through
random sampling of 50, 40, 30 and 20 patients. We performed each random sampling ten
times for each level of patient numbers and with 1,000 NMDP controls each. With these data,
we determined how frequently HEAP could identify the known amino acid association for RA
and T1D. Statistics were performed as described above for PAP association.
HLA Allele frequency and amino acid polymorphism in autoimmune PAP
We collected DNA from 47 Caucasian patients with autoimmune PAP from Cincinnati
Children?s Medical Center as part of the RLDN. We performed high resolution typing of all
classical loci of HLA class I and class II. We analyzed both allele frequencies and amino acid
frequencies between patients and 1000 ethnic-matched controls provided by the NMDP. To
identify potential shared amino acid sequences, we used HEAP, which compares all
polymorphic amino acids in a locus [
]. We examined potential HLA shared amino acids from 1
to 4 non-contiguous amino acids and analyzed a total of 5,699,818 possible amino acid
combinations. However, we did not find any significant associations at the amino acid level (S9?S32
Tables). We also calculated allele frequencies for all loci and performed a chi-square analysis
between patients and controls. In our control and patient populations, we identified 203
different alleles in HLA-A, B, C, DRB1, DPB1 and DQB1 (Tables 1?6), and none were significantly
associated with autoimmune PAP. Many of the alleles tested were rare (<1% in both patient
and control groups) and were not found to be associated with PAP. For full allele results, see
DRB3, DRB4 and DRB5 are three protein-coding loci in tight linkage disequilibrium with
DRB1. These alleles pair with DR?, are expressed on the cell surface, and present peptides to T
cells as any other class II allele. We imputed these alleles based on linkage analysis and
examined the frequencies of these loci for potential association with PAP; however, no associations
were identified (Table 7). Collectively, with our cohort, our data show that autoimmune PAP
is unlikely to have an HLA association that can explain disease development.
Given the small sample size, we performed a test of equivalence. In traditional t-tests, the
null hypothesis is that the means in two groups are not different. Our initial analysis suggested
that we cannot reject this null hypothesis. However, this does not demonstrate unequivocally
that the groups are the same. In an equivalence test, the null hypothesis is that the two groups
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are different. A significant p-value in an equivalence test would allow us to reject this null
hypothesis and indicates that the means between our two groups are equivalent. An
equivalence test was performed for all alleles, and for each one we obtain a highly significant p-value,
suggesting that there is no difference between the frequencies of HLA alleles between patients
and controls. The maximum p-value we observed over all alleles tested was 4.23 x 10?12.
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Adequacy of sample size
Due to the small number of autoimmune PAP subjects enrolled in the present study, we
queried whether an association between HLA and similar numbers of subjects with diseases of
known HLA associations (e.g., T1D and RA) could be detected. As shown in Fig 1, we found
that HEAP could consistently identify a strong HLA association with as few as 20 patients in
both T1D and RA. Thus, our findings suggest that our sample size of 47 should be adequate to
detect an HLA association in autoimmune PAP, if a strong association exists.
Based on the autoimmune nature of PAP, we hypothesized that a link existed between this
disease and HLA. However, our study suggests that no such link exists. It remains possible that
the lack of an association was due to the small sample size (47 patients). However, we did not
note any trends towards association. As discussed above, our sample size should be sufficient
to identify a strong positive association. However, it may not be large enough to definitively
conclude the absence of an association. Previous studies in our laboratory have conclusively
demonstrated the lack of an HLA association with as few as 150 patients [
]. However, it
should be noted that our methodology can identify a strong positive HLA association with
only 50 patients [
]. Our HEAP analysis consistently identified a strong, single amino acid
HLA association in T1D and RA with as few as 20 subjects. Collectively, these data suggest that
our sample size should be capable of detecting an association if one were present. However,
due to sample size considerations, we were forced to limit our study to Caucasian patients. A
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Fig 1. HEAP limits of detection by patient number. Identification of significant p-values with known disease
associated epitopes by number of patients in the sample. Y-axis shows?Log(p-value) from the epitope analysis, X-axis
shows the number of patient samples included in each analysis. For highly significant HLA associations, HEAP can
consistently identify association with as few as 20 patients.
larger study with a more diverse population would allow a more conclusive assessment and to
include other ethnic minorities who represent more diverse HLA alleles.
HLA-linked autoimmune diseases generally have a T cell-mediated component. Our initial
hypothesis was based on the finding of an increased number of CD4+ and CD8+ T cells in the
bronchoalveolar lavage of patients with PAP [
]. This suggests HLA involvement in
presenting self-antigen to T cells which may then lead to CD4+ T cells helping B cells to class-switch.
This would be consistent with our observations that antibodies against GM-CSF in
autoimmune PAP patients are IgG [
]. However, it is possible that the increased number of T cells
observed in the lungs of PAP patients was secondary to infection and can still account for the
class-switched antibodies against GM-CSF. The absence of an HLA association in this cohort
of autoimmune PAP subjects raises the possibility that the T cell alveolitis may be a direct
consequence of pulmonary infection.
Despite being found predominantly in patients with autoimmune PAP, anti-GM-CSF
antibodies are also found in the serum of healthy subjects. It is not entirely unusual to observe
autoantibodies in healthy individuals and suggests that the progression to disease state involves
interactions between specific genes and the environment. In this case, it is possible that
autoantibodies directed against GM-CSF function in normal, healthy individuals to prevent
excessive inflammatory responses; thus, protecting the lung from damage. Genetically susceptible
individuals would have difficulty controlling the amount of anti-GM-CSF antibodies. In this
regard, it is possible that autoimmune PAP represents an improper control of anti-GM-CSF
antibodies. autoimmune PAP patients may have a genetic link that makes them more
susceptible to lung infection but unfortunately, in this study, no link between HLA and autoimmune
PAP was identified.
S1 Table. PAP patients included in study. Supplemental information for PAP patients
including: age, gender, race, ethnicity and GMab levels.
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S2 Table. HLA types of PAP patients and 1000 NMDP controls. HLA types of all loci A, B,
C, DRB1, DPB1 and DQB1 for patients and controls.
S3 Table. HLA-A allele test full results.
S4 Table. HLA-B allele test full results.
S5 Table. HLA-C allele test full results.
S6 Table. HLA-DRB1 allele test full results.
S7 Table. HLA-DQB1 allele test full results.
S8 Table. HLA-DPB1 allele test full results.
S9 Table. HLA-A 1 amino acid association test.
S10 Table. HLA-A 2 amino acid association test.
S11 Table. HLA-A 3 amino acid association test.
S12 Table. HLA-A 4 amino acid association test.
S13 Table. HLA-B 1 amino acid association test.
S14 Table. HLA-B 2 amino acid association test.
S15 Table. HLA-B 3 amino acid association test.
S16 Table. HLA-B 4 amino acid association test.
S17 Table. HLA-C 1 amino acid association test.
S18 Table. HLA-C 2 amino acid association test.
S19 Table. HLA-C 3 amino acid association test.
S20 Table. HLA-C 4 amino acid association test.
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S21 Table. HLA-DRB1 1 amino acid association test.
S22 Table. HLA-DRB1 2 amino acid association test.
S23 Table. HLA-DRB1 3 amino acid association test.
S24 Table. HLA-DRB1 4 amino acid association test.
S25 Table. HLA-DQB1 1 amino acid association test.
S26 Table. HLA-DQB1 2 amino acid association test.
S27 Table. HLA-DQB1 3 amino acid association test.
S28 Table. HLA-DQB1 4 amino acid association test.
S29 Table. HLA-DPB1 1 amino acid association test.
S30 Table. HLA-DPB1 2 amino acid association test.
S31 Table. HLA-DPB1 3 amino acid association test.
S32 Table. HLA-DPB1 4 amino acid association test.
Conceptualization: Kirsten Anderson, Brenna Carey, Claudia Chalk, Marchele Nowell-Bostic,
Brian Freed, Michael Aubrey, Bruce Trapnell, Andrew Fontenot.
Data curation: Brenna Carey, Allison Martin, Claudia Chalk, Marchele Nowell-Bostic, Brian
Freed, Bruce Trapnell, Andrew Fontenot.
Formal analysis: Kirsten Anderson, Christina Roark, Brian Freed, Michael Aubrey.
Investigation: Kirsten Anderson.
Methodology: Kirsten Anderson, Michael Aubrey.
Project administration: Allison Martin, Claudia Chalk, Marchele Nowell-Bostic, Bruce
Trapnell, Andrew Fontenot.
Resources: Brian Freed, Andrew Fontenot.
Supervision: Brian Freed, Bruce Trapnell, Andrew Fontenot.
Validation: Kirsten Anderson, Michael Aubrey.
Visualization: Kirsten Anderson.
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Writing ? original draft: Kirsten Anderson.
Writing ? review & editing: Kirsten Anderson, Brenna Carey, Allison Martin, Brian Freed,
Michael Aubrey, Bruce Trapnell, Andrew Fontenot.
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