Study subjects

The 885 healthy subjects in the GWAS and the 1100 healthy subjects in the replication study were randomly selected from the Taiwan Han Chinese Cell and Genome Bank in Taiwan. Individuals range in age from 21 to 93 years (with a mean of 51 years) and they consist of 997 women and 988 men. The studies were approved by the Institutional Review Boards and the Ethics Committees of Academia Sinica in Taiwan. Written informed consent was obtained from the subjects in accordance with institutional requirements and Declaration of Helsinki principles.

Plasma sample collection and human anti-PEG assay

Plasma samples of healthy subjects were enrolled from a prior project that had been collected, centrifuged, and stored at the National Center for Genome Medicine, Academia Sinica25. All subjects of this study agreed to offer the remaining centrifugal plasma for other research in a de-linked fashion. Frozen plasma aliquots were stored at -80°C until anti-PEG antibody determinations. The human anti-PEG assay was used to determine the plasma levels of anti-PEG IgG and IgM according to our previous study14. Maxisorp 96-well microplates (Nalge-Nunc International, Rochester, NY) were coated with NH 2 -PEG 10000 -NH 2 overnight at 4 °C. Human plasma samples at dilutions of 25, 50, and 100-fold and serially diluted c3.3-IgG or cAGP4-IgM antibody standards (in duplicate) were added to separate plates at room temperature for 1 h. The horseradish peroxidase (HRP)-conjugated goat F(ab′) 2 antihuman IgG Fc or HRP-conjugated goat F(ab′) 2 antihuman IgM Fc 5μ were added to the IgG or IgM detection plates, respectively, for 1 h at room temperature. The plates were washed before adding ABTS substrate for 30 min at room temperature. The absorbance (405 nm) of wells was measured in a microplate reader (Molecular Devices). Positive responses were defined as samples with absorbance values at least 3 times greater than the mean background absorbance and more than 35% reduction of absorbance reading by PEG-liposomes competition assay. The relative concentrations of anti-PEG IgG or IgM in positive samples was calculated by comparison with c3.3-IgG or cAGP4-IgM standard curves, respectively.

Phenotype definition of healthy subjects

Antibodies against polyethylene glycol (PEG) were quantified in plasma samples from random selected Han Chinese residing in Taiwan by direct ELISA with confirmation by a competition assay14. A summary of the age and sex distribution in the GWAS and replication cohort is presented in Supplementary Table 6. The total number of anti-PEG positive and negative in the GWAS and replication cohort is presented in Supplementary Table 5. In order to get clear association signals with anti-PEG IgM only and IgG only, we excluded subjects double positive for anti-PEG IgM and IgG from anti-PEG IgM and IgG populations, and used subjects without either anti-PEG IgM or IgG as controls. In the GWAS cohort, 177 and 140 individuals out of 885 subjects are defined as positive for anti-PEG IgM only and IgG only, respectively, and 492 individuals without either anti-PEG IgM or IgG are defined as controls. In the replication cohort with a total of 1100 subjects, there were 211 subjects positive for anti-PEG IgM only, 192 subjects for anti-PEG IgG only, and 596 subjects as controls.

Genotyping and quality control

Genomic DNA was extracted from blood using the Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, MN). Each individual was genotyped using the Affymetrix Genome-Wide Human SNP Axiom CHB1 array (with a total of 628,132 SNPs) according to the manufacturer’s protocols by the National Center for Genome Medicine (NCGM) at Academia Sinica. The call rates of all samples were greater than 95%. First-degree relatives (parent-offspring and full sibling pairs) in healthy subjects classified in case and control groups by the human anti-PEG assay were identified by kinship analysis and were excluded from further analysis. Genotyping quality control for each SNP was further evaluated by determining the total call rate (successful call rate) and minor allele frequency (MAF) in cases and controls. SNPs were excluded from further analysis if only one allele appeared in cases and controls, the total call rate was less than 0.95 or the total MAF was less than 0.05. SNPs departing significantly from Hardy-Weinberg equilibrium (P < 1 × 10−4) were also excluded from further analysis.

Statistical analysis

To access population stratification that could influence association tests, principle component analysis was carried out using EIGENSTRAT 2.026. We also estimated the variance inflation factor for genomic control. The Cochran-Armitage trend test for genome-wide association analysis was used to compare allele and genotype frequencies between cases and controls. A quantile–quantile plot was used to examine the P-value distribution. SNPs with P-values <8.97 × 10−8 were considered to be significantly associated with the traits. Logistic regression model of two-point analyses were used to estimate the affected status of two SNPs and their interaction. SNPs were coded as 0, 1 and 2 for the number of minor alleles and were treated as continuous variables. Heterogeneity tests (I 2 and P-values of the Q statistics) were performed for each SNP before combination of GWAS and replication analysis. The association tests and heterogeneity tests were performed using PLINK 1.9 (http://pngu.mgh.harvard.edu/~purcell/plink). Logistic regression was carried out using SAS software (SAS Institute, Inc., Cary, NC, USA.). The power calculation of effect size was performed using CaTS with parameters estimated from the current sample27: prevalence of the trait = 20%, risk allele frequency in case = 60%, and relative risk = 2.3. The sample sizes for the two-stage GWAS reached a statistical power of 99% in the joint analysis.

Validation and replication

The top SNPs (P < 1 × 10−5) from the genome-wide association analysis of anti-PEG IgM and IgG were further validated in 156 subjects using MALDI-TOF mass spectrometry (MassARRAY; Sequenom, San Diego, CA, USA).The SNP genotypes with over 96% successful rate and over 99% concordance rate between the two platforms were then genotyped in an additional independent Han Chinese replication cohort.

Correlation analysis of genotypes and anti-PEG IgM response

The association between genotypes of the most significant SNP (rs12590237) and anti-PEG IgM was conducted with subjects in the GWAS and replication stage. Significance of differences in positive IgM frequencies among different genotypes was calculated using the z-score calculator for two population proportions. Overall comparisons between the genotype classes and concentrations of anti-PEG IgM were performed by using the Kruskal–Wallis test. Statistical analyses were performed by using Prism 4 software (Graphpad Software, Inc.). The statistical significance was set at P < 0.05.

Data availability

The data that support the findings of this study are available from the corresponding author on request.