Massarray analyzer 4 system

All samples were genotyped for 67 positions (Table 1) using iPLEX Pro chemistry on the MassARRAY® Analyzer 4 System (Agena Biosciences, San Diego, CA, USA). Twenty-nine SNP and indel variants as well as copy number status of the CYP2D6 gene were analyzed using the Agena iPLEX® ADME CYP2D6 Panel v1.0, which is a 3-well assay combining genotyping for SNP and indel variants as well as five assays to determine genomic copy number. This genotyping platform and assay have been previously shown to accurately genotype pharmacogenetic loci in Coriell reference samples.^{32 (link)}CYP2D6 diplotypes and CNV calling for MassArray data were determined using the Agena PGx Report 2.0 Reporter plugin for the Typer Analyzer software (Agena Bioscience). CYP2D6 copy number was estimated from the five copy number assays that are integrated in the CYP2D6 genotyping panel, which is calculated from informative polymorphisms between CYP2D6 and CYP2D7. Variants in the remaining 18 genes in this study were also typed on the MassARRAY® Analyzer 4 System with custom-designed primers using a combination of Agena’s Assay Design Suite (ADS) and Primer3 (ref 33 (link)).

Measurements of all patient data on PRDM8 and its two CpG sites cg19409579 and cg27242132 have been performed with MassARRAY analysis instead of pyrosequencing. This is due to the fact that the PRDM8 region has many CpG sites, and pyrosequencing primers overlapping with CpGs were temperature sensitive and did not provide reliable measurements. With MassARRAY, longer amplicons can be measured thus making primer design outside of CpG-rich regions and analysis of these PRDM8 regions possible. MassARRAY analyses were performed with a MassARRAY Analyzer 4 System (Agena Bioscience, Hamburg, Germany) as previously described [18 (link)] at Varionostic GmbH (Ulm, Germany).

The single-nucleotide polymorphism (SNP) rs352140 in the TLR9 gene was screened for the association analysis based on the following principles: (1) minor allele frequencies in the Asian population >0.05; (2) p value of Hardy–Weinberg equilibrium >0.05; and (3) reported to be highly associated with the susceptibility to other autoimmune diseases, including SLE and GD, in the Chinese population. The SNP genotypes of individuals were identified by BGI (Beijing Genomics Institute, Shenzhen, China) through the MassARRAY^® Analyzer 4 System (Agena Bioscience, San Diego, CA, USA). The following primers were used for amplification in the study: forward, 5’-ACGTTGGATGTGGCTGTTGTAGCTGAGGTC-3’; reverse, 5’-ACGTTGGATGATAAGCTGGACCTCTACCAC-3’. The single base extension primer was 5’-GTCCAGGGCCTCCAGTCG-3’.