Snp genotyping assay

SNPs of TCF7L2 (rs7903146, C/T), CASC8 (rs6983267, G/T), and GREM1 (rs1696981, C/T) were identified using a real-time PCR method based on the ready-made TaqMan^® Genotyping Master Mix (Applied Biosystems, Waltham, MA, USA) and 20× SNP Genotyping Assay (Applied Biosystems) containing target-specific oligonucleotides labeled with a reporter dye at the 5′ end of each probe; VIC dye was linked to the 5′ end of the Allele 1 probe and FAM dye was linked to the 5′ end of the Allele 2 probe (Table 1). The DNA concentration was set between 1 and 10 ng per 10 μL of RT-PCR reaction. Briefly, each 10 μL of RT-PCR reaction consisted of 5 μL TaqMan Genotyping Master Mix (2×), 0.5 μL of TaqMan Genotyping Assay mix (20×), and 4.5 μL of DNA. Samples were incubated in a 7500 Fast Real-Time System (Applied Biosystems) with the following cycle conditions: 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min. The last two steps of denaturing and annealing/extension were repeated 40 times. Allelic discrimination was made with the help of 7500 Fast Real-Time PCR software, version 2.3.

To genotype the TNFA -857C/T SNP, we used the predesigned SNP genotyping assay (part number: C__11918223_10, Foster City, CA, USA) provided by Applied Biosystems. To determine the anticyclic citrullinated peptides antibody (anti-CCP) levels by enzyme-linked immunosorbent assay (ELISA) (DRG, EIA-5653), we followed the methods outlined in Durán-Avelar et al. [16 (link)].

The included individuals were genotyped using Single Nucleotide Polymorphism (SNP) genotyping TaqMan^® assays on a ABI Step-One Plus (Applied Biosystems) real-time PCR instrument using the allelic discrimination method according to manufacturer’s instruction [12 (link)]. All probes were pre-designed SNP Genotyping Assay and purchased from Applied Biosystems. For PiS, the rs1758 (assay ID: C_594695_20) probe was used; for PiZ allele, the rs28929474 (assay ID: C_34508510_10) was used; for the PiM2/M4 allele, the rs709932 (assay ID: C_2895146_20) probe was used and finally for the PiNull allele, rs28929473 (assay ID: C_63321235_20) was used.

Total RNA extraction and SYBR-Green RT–qPCR were performed as previously described in ref. 37 (link). Primers used in this study are reported in Supplementary Table 2. XIST allele-specific expression (Supplementary Fig. 4f) was analysed by Taqman PCR performed with SNP genotyping assay (rs1620574; assay ID C-7626536_10 Applied Biosystems) and Taqman Universal Master Mix No AmpEraseUNG (Applied Biosystems) according to the manufacturer instructions.

Genetic variants selected in the sequencing screening were genotyped in 91 and 131 EA patients from PEARL for discovery and validation phases of the study, respectively.
The selected VIP genetic variants rs12213214, rs140023105, rs35643203, rs3799142, rs7764067, rs3823082, rs12201173, rs71575932, rs74760293, rs149081483 and rs688136 were genotyped using a pre-designed single nucleotide polymorphism (SNP) Genotyping Assays (Part numbers: C__27847302_10, C__27855145_10, C___3250637_10, C__27502877_20, C__29430252_10, C__27491244_10, C__32237894_10, C___3250638_10, C__25962626_10, C_172567893_10, C___3250639_10, respectively), and rs60946248 and rs185451870 were genotyped using a custom SNP Genotyping Assay (Applied Biosystems).
After PCR, the genotype of each sample was determined automatically by measuring allele-specific fluorescence on a CFX Touch Real-Time PCR System using the software CFX 3.1 Manager (BioRad). Duplicate samples and negative controls were included to verify genotyping accuracy.
The genotype of rs12201030, rs7755568 and rs12201140 was obtained by sequencing amplicon 17, as we described above (see amplicon information in Supplementary Table 2). These SNPs are located in a low complexity region with a predominance of A and T bases, making it impossible to obtain specific TaqMan probes.

Genomic DNA was isolated using QIAamp DNA blood midi kit (QIAGEN®, Crawley, UK). Genotyping of the −460 (rs833061) and +405 (rs2010963) VEGF‐A SNPs¹⁵, ¹⁶, ³⁷ was performed using the SNP genotyping assay (Applied Biosystems, Foster City). A detailed description of DNA extraction and genotyping is provided in Supplementary Files S1.