All PBAT analyses were performed with phenotypic information from 378 PI ZZ individuals in 167 families (). Of note, only 22 individuals reported current oral steroid use, so systemic steroid use was not included in any of the models. We investigated genetic association for 75 SNPs in 10 candidate genes selected from the literature demonstrating prior association with COPD and/or asthma intermediate phenotypes (Table 2, and Table E1 in the online supplement). All SNPs were in Hardy-Weinberg equilibrium.
DEMOGRAPHICS FOR 378 PI ZZ INDIVIDUALS
TEN CANDIDATE GENES INCLUDED AS POTENTIAL MODIFIERS OF COPD IN α1-ANTITRYPSIN DEFICIENCY
Nine SNPs in three genes (IL10
, and SERPINE2
) demonstrated significant association with pre- and/or post-bronchodilator FEV1
percent of predicted (P
0.05, ). When an SNP-by-pack-years of smoking interaction term was included, the findings remained robust for SNPs in IL10
0.05, data not shown). In additive models for pre- and post-bronchodilator FEV1
/FVC, 6 SNPs in three genes (IL10
) demonstrated significant association (P
0.05, ). When an SNP-by-smoking interaction term was included, the findings remained significant only for IL10
0.05, data not shown).
PEDIGREE-BASED ASSOCIATION TEST FOR FEV1 (% PREDICTED) AND FEV1/FVC
Approximately 37% of the cohort reported a physician's diagnosis of asthma. These 139 individuals had a mean pre-bronchodilator FEV1
of 54.4 percent predicted (SD ± 28.8). The remaining 239 individuals without a physician's diagnosis of asthma had a mean FEV1
of 72.4 percent predicted (SD ± 34.3); there was no difference in the mean age or pack-years by asthma status. We have previously observed that a physician's diagnosis of asthma is a strong predictor of lower FEV1
in this cohort (35
). Because of the consistency of the PBAT association findings for pre- and post-bronchodilator FEV1
/FVC for multiple SNPs in IL10
, we focused upon IL10
SNPs and repeated the PBAT analysis for the IL10
SNPs after excluding the phenotypic data for the 139 individuals who reported a physician's diagnosis of asthma. SNPs in IL10
remained significantly associated with FEV1
percent of predicted and FEV1
/FVC (Table E2).
The quantitative phenotype analyses performed likely represent association with COPD susceptibility, but they could also be influenced by FEV1
variation within the normal range. Therefore, we subsequently performed a qualitative analysis to investigate whether any of the candidate genes demonstrated associations with severe COPD. For the qualitative COPD phenotype analysis, we excluded the phenotypic data for individuals who were over 60 years of age and who had a history of cigarette smoking (20 individuals excluded from the pre-bronchodilator analysis and 18 individuals excluded from post-bronchodilator analysis), since smokers that have survived to later ages with severe AAT deficiency might actually have protective genes, despite low lung function values at the time of study participation. The mean pre-bronchodilator percent predicted FEV1
for the 138 individuals with moderate-to-severe COPD was 29.4 ± 10.6, versus a mean percent predicted FEV1
of 81.7 ± 26.6 for those without moderate-to-severe COPD. Although there was no difference in the mean age for these two groups (53.3 yr for those with moderate-to-severe COPD versus 51.5 yr for those without, P
= 0.10), subjects affected with moderate-to-severe COPD tended to have more extensive smoking histories (mean pack-year difference 18.8 [± 16.0] among subjects with moderate-to-severe COPD versus 6.9 [± 11.4] in the other subjects, P
< 0.0001) and tended to be male (P
< 0.0001), with men constituting 63% of those individuals affected with moderate-to-severe COPD. Of note, there was no age difference between men and women with moderate-to-severe COPD (52.9 yr for men versus 53.9 yr for women, P
= 0.60.) PBAT analysis of the qualitative COPD phenotype revealed association with SNPs in IL10
= 0.02 and rs1800871 P
0.03, pre- and post-bronchodilator), TNF
= 0.03, pre-bronchodilator), EPHX1
= 0.04, pre-bronchodilator), and NOS1
0.006 and rs816361 P
0.01, pre- and post-bronchodilator) (). Significant associations with any NOS1
SNPs were not observed with FEV1
/FVC as quantitative phenotypes.
PEDIGREE-BASED ASSOCIATION TEST P VALUES FOR THOSE INDIVIDUALS AFFECTED WITH MODERATE-TO-SEVERE COPD
Given the consistent findings for IL10 SNPs for quantitative and qualitative airflow obstruction phenotypes, we performed a sliding window haplotype analysis using the 11 SNPs genotyped in IL10, using 8, 4, 3, and 2 adjacent SNP sliding windows (Figure E1). This analysis revealed the most robust association with the IL10 promoter SNP rs1800871 and the rs1518110 intronic SNP, which are in tight but not complete LD. A two-SNP haplotype analysis was also performed for rs1800896 (nucleotide −1082) and rs1800871 (nucleotide −819), the two nonadjacent IL10 promoter SNPs that demonstrated significant association. The most highly associated haplotype was A at rs1800896 and T at rs1800871 with a haplotype frequency of 0.20; this haplotype was associated with the quantitative phenotypes FEV1 percent predicted and FEV1/FVC (AT haplotype individual P value < 0.001). Twenty-six individuals were homozygous at both the rs1800896 (AA, homozygous wild-type allele) and rs1800871 (TT, homozygous minor allele) loci (therefore AT haplotypes); the mean pre-bronchodilator FEV1 of these individuals was 57.1 percent predicted versus 65.0 percent predicted for all other individuals, consistent with the direction of the effect of each individual allele (decrease in lung function for the rs1800896 wild-type allele and for the rs1800871 minor allele, ) in the haplotype. demonstrates the high but not complete LD between all SNPs included in the haplotype analyses.
Linkage disequilibrium (LD) between the 11 SNPs analyzed for IL10. LD values are presented as r2. r2 between the rs1800871 promoter SNP and the rs1518110 intronic SNP was 0.82, suggesting tight but not complete LD.
In our cohort the direction of effect of the −1082 A/G promoter polymorphism was for lower lung function with the wild-type allele and higher lung function with the mutant G (minor) allele (). The −1082 rs1800896 SNP has been associated with a functional effect on IL10 protein levels, with the A wild-type allele being associated with lower IL10 levels, and the G mutant allele being associated with higher IL10 levels. We measured serum IL10 levels in this cohort. In serum samples from 266 PI ZZ subjects, the overall mean CV of all of our samples was 3.8% (range of 0–9.6) after exclusion of 41 samples with CVs greater than 10% and 5 samples with values greater than our highest standard. The CV values for individuals on and not on augmentation therapy were not statistically different, at 4.1% (0–9.6%) and 3.5% (0–9.3%), respectively. After data quality assurance, we had measured IL10 levels in 266 individuals, 257 of whom had genotypes at the −1082 locus. The mean IL10 concentration in the overall cohort of 257 individuals was 2.40 (± 0.26) pg/ml (range 0–39.31 pg/ml). When parsed by use of AAT augmentation status, individuals on augmentation therapy had a mean IL10 concentration of 3.03 (± 0.50) pg/ml versus 1.80 (± 0.22) pg/ml for those not on augmentation therapy (P = 0.02). Individuals with the AA −1082 genotype (wild type) had low IL10 levels regardless of augmentation therapy use. However, individuals with one or two copies of the minor G allele had higher IL10 levels when compared with individuals not on augmentation therapy (, Table E3).
Figure 2. Serum IL10 levels in 257 individuals who are homozygous for the Z deficiency allele parsed by the IL10 -1082 promoter SNP genotypes. These data demonstrated a trend for higher IL10 levels in individuals with one or two copies of the minor G allele, especially (more ...)