Genotyping and quantification of ATP8B4 mRNA expression in PBMCs from patients

Genomic DNA was extracted from peripheral blood samples. Validation genotyping for rs55687265 in ATP8B4 was performed in an independent replication cohort of 415 European American patients with SSc-PAH+ (n = 92) and SSc-PAH− (n = 323) by using TaqMan^® Allelic discrimination Assays on the 7900HT Sequence Detection System (Applied Biosystems). Positive controls representing 3 genotype clusters were run on every plate to ensure accurate clustering and allele calling. We replicated 10% of the samples for quality control. Additional genotyping for controls in the replication cohort was performed as part of the Illumina HumanExome-12v1_A Beadchip array (Supplementary Appendix).

We utilized existing data from high-throughput expression profiling using Illumina Sentrix Human BeadChips (HT12_v3) with mRNA from PBMCs of SSc-PAH− (n = 19) and SSc-PAH+ (n = 42) patients and corresponding controls (n = 41). Control subjects were healthy individuals who had no known cardiovascular, pulmonary and kidney disease. Details on isolation of PBMCs, purification of RNA, the microarray experiment and analysis have been described previously (20). For comparison of gene expression between specified pairs of groups, fold changes, P-values and Benjamini-Hochberg false discovery rates (BH-FDRs) (21) were obtained using custom software written in IDL (Interactive Data Language; Exelis Visual Information Solutions, Boulder, CO) (Supplementary Appendix) (20). For the validation analysis using quantitative RT-PCR (qRT-PCR), we selected 24 samples (12 samples from the 61 cases and 12 from the controls) to represent the entire range of microarray expression values in the full dataset, and the correlation between ATP8B4 mRNA levels as detected by microarray and by RT-PCR was assessed (Supplementary Appendix).