Unique and shared systemic biomarkers for emphysema in Alpha-1 Antitrypsin deficiency and chronic obstructive pulmonary disease

Abstract

Background: Alpha-1 Antitrypsin (AAT) deficiency (AATD), the most common genetic cause of emphysema presents with unexplained phenotypic heterogeneity in affected subjects. Our objectives to identify unique and shared AATD plasma biomarkers with chronic obstructive pulmonary disease (COPD) may explain AATD phenotypic heterogeneity.

Methods: The plasma or serum of 5,924 subjects from four AATD and COPD cohorts were analyzed on SomaScan V4.0 platform. Using multivariable linear regression, inverse variance random-effects meta-analysis, and Least Absolute Shrinkage and Selection Operator (LASSO) regression we tested the association between 4,720 individual proteins or combined in a protein score with emphysema measured by 15th percentile lung density (PD15) or diffusion capacity (DLCO) in distinct AATD genotypes (Pi*ZZ, Pi*SZ, Pi*MZ) and non-AATD, PiMM COPD subjects. AAT SOMAmer accuracy for identifying AATD was tested using receiver operating characteristic curve analysis.

Findings: In PiZZ AATD subjects, 2 unique proteins were associated with PD15 and 98 proteins with DLCO. Of those, 68 were also associated with DLCO in COPD also and enriched for three cellular component pathways: insulin-like growth factor, lipid droplet, and myosin complex. PiMZ AATD subjects shared similar proteins associated with DLCO as COPD subjects. Our emphysema protein score included 262 SOMAmers and predicted emphysema in AATD and COPD subjects. SOMAmer AAT level <7.99 relative fluorescence unit (RFU) had 100% sensitivity and specificity for identifying Pi*ZZ, but it was lower for other AATD genotypes.

Interpretation: Using SomaScan, we identified unique and shared plasma biomarkers between AATD and COPD subjects and generated a protein score that strongly associates with emphysema in COPD and AATD. Furthermore, we discovered unique biomarkers associated with DLCO and emphysema in PiZZ AATD.

Funding: This work was supported by a grant from the Alpha-1 Foundation to RPB. COPDGene was supported by Award U01 HL089897 and U01 HL089856 from the National Heart, Lung, and Blood Institute. Proteomics for COPDGene was supported by NIH 1R01HL137995. GRADS was supported by Award U01HL112707, U01 HL112695 from the National Heart, Lung, and Blood Institute, and UL1TRR002535 to CCTSI; QUANTUM-1 was supported by the National Heart Lung and Blood Institute, the Office of Rare Diseases through the Rare Lung Disease Clinical Research Network (1 U54 RR019498-01, Trapnell PI), and the Alpha-1 Foundation. COPDGene is also supported by the COPD Foundation through contributions made to an Industry Advisory Board that has included AstraZeneca, Bayer Pharmaceuticals, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Novartis, Pfizer, and Sunovion.

Keywords: Alpha-1 antitrypsin deficiency; Emphysema; Plasma biomarker; Protein score; SomaScan.

Figure 1

Study consort. Diagram representation of the individuals recruited in the COPDGene, GRADS, QUANTUM, and Birmingham cohorts but excluded in the current study due to absent SomaScan data and exclusion genotypes. Genotypes not included: COPDGene – Pi*FZ, Pi*SZ, Pi*SS; QUANTUM-1 - Pi*M_heerlenZ and Pi*SZ; GRADS- discordant genotype / phenotype measurements; Birmingham - Pi*M_MaltonNull, Pi*M_procidaZ, Pi*SS, Pi*ZNull, Pi*FZ, Pi*IZ, Pi*M_maltonZ, Pi*SP_Lowell.

Figure 2

SomaScan proteins association with DLCO in PiZZ subjects off augmentation therapy. a. Volcano plot of SomaScan proteins positively and negatively associated with DLCO (N=98) for PiZZ subjects (N=185) off augmentation therapy from the Birmingham, GRADS, QUANTUM cohorts. SomaScan proteins, labeled with their gene abbreviations, are shown in black if significantly (FDR ≤ 0.05, multivariable linear regression) associated with DLCO; non-significant (FDR >0.05, multivariable linear regression) SomaScan proteins are shown in grey. b. Euler plot of SomaScan proteins associated with DLCO that are specific (magenta circle, N=30) and shared (purple circle, N=68) between PiZZ off therapy and PiMM (blue circle, N=1237) subjects. The top five unique proteins keratin 1 (KRT1), myomesin-2 (MYOM2), insulin receptor (INSR), insulin-like growth factor (IGF-1), and hydroxymethylbilane synthase (HMBS) and shared proteins macrophage scavenger receptor types I and II (MSR1), growth/differentiation factor 15 (GDF15), fatty acid-binding protein, adipocyte (FABP4), tissue-type plasminogen activator (PLAT), intelectin-1 (ITLN1) between PiZZ off therapy and PiMM subjects are shown in the insert tables.

Figure 3

Cellular component pathways enriched in the SomaScan proteins associated with DLCO in PiZZ off augmentation therapy and PiM subjects. a. Cellular component pathways enriched within the 671 nominally-significant SomaScan proteins associated with DLCO in PiZZ subjects off therapy. b. Cellular component pathways enriched within the 1305 nominally-significant SomaScan proteins associated with DLCO in PiMM subjects. Individual cellular component pathways are color-coded based on the significance of enrichment p-values depicted at the bottom, light yellow p<0.00005, light orange p<0.000005, dark orange p<0.00000005, red p<0.000000005 (Pathway enrichment analysis, GOrilla). The graphical representation of Directed acyclic graph (DAG) was created using GOrilla.

Figure 4

Somascan proteins association with DLCO and spirometry in PiMZ subjects. a. Volcano plot of SomaScan proteins positively and negatively associated with DLCO (N=10) in PiMZ subjects (N=215) from two cohorts, COPDGene and GRADS studies. SomaScan proteins, labeled with their gene abbreviations, are shown in black if significantly (FDR ≤ 0.05, multivariable linear regression) associated with DLCO; non-significant (FDR > 0.05, multivariable linear regression) SomaScan proteins are shown in grey. b. Euler plot of SomaScan proteins associated with DLCO that are shared (green circle, N=10) between PiMZ and PiMM (blue circle, N=1295) subjects. The top five shared proteins between PiMZ and PiMM subjects are Retinoic acid receptor responder protein 2 (RARRES2), Chordin-like protein 1 (CHRDL1), R-spondin-1 (RSPO1), Fibroblast growth factor-binding protein 1 (FGFP1), and pleiotrophin (PTN). c. Forest plot of natural log CRP (RFU, natural log) negative association with FEV₁ in PiMZ subjects (N=215) from two cohorts, COPDGene and GRADS studies.

Figure 5

Somascan individual proteins and emphysema protein score association with adjusted lung density. a-b. Forest plots of cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) and betacellulin (RFU, natural log) positive association with adjusted lung density (PD15, g/L) in PiZZ individuals (N=63) off augmentation therapy from two, GRADS and QUANTUM study cohorts. c-f. Scatter plots showing the association between measured adjusted lung density (PD15, y axis) vs. PD15 predicted by the emphysema protein score (x axis) in c) COPDGene PiMM (R²=0.44, rho=0.66, LASSO); d) COPDGene PiMZ (R²=0.49, rho=0.70, LASSO); e) GRADS PiZZ off therapy (R²=0.37, rho=0.61, LASSO); and f) PiZZ on therapy individuals (R²=0.23, rho=0.48, LASSO). Emphysema protein score (N=262 proteins) was developed using LASSO in the COPDGene PiMM subjects and the other genotypes were used for validation.

Figure 6

SomaScan Alpha-1 Antitrypsin relative levels in various Alpha-1 genotypes. a. Beeswarm plot of AAT relative fluorescence units (RFU, natural log, y axis) by genotype (x axis). AAT relative levels in pooled PiMM (10.63 ±0.23, N=5101), PiMS (10.37 ± 0.21, N=347), PiMZ (9.98 ± 0.25, N=215), PiSZ (9.10 ± 0.16, N=24), PiZZ off therapy (6.32 ± 0.36, N=185), and PiZZ on AAT augmentation therapy (10.69 ± 0.54, N=52) subjects from all 4 study cohorts. b. Histogram showing the percent distribution of PiZZ (N = 185), PiSZ (N=24), PiMZ (N=215), PiMS (N=347), and PiMM (N=5101) subjects from all 4 study cohorts. c. Receiving operator curves (ROC) of AAT (RFU, natural log) for the following genotype comparisons are shown: PiSZ vs. PiMZ, PiMZ vs. PiMS, PiMZ vs. PiMM, and PiMS vs. PiMM. The ROCs characteristics for the genotype comparisons not graphically depicted are shown in the table insert. *If AUC is equal to 1.0 it is a midpoint of the range of complete separation. If AUC less than 1.0 the value is the cut-point determined by the Youden Index.