Psq assay design

Genomic DNA was extracted from the liver of the male offspring at 3 months of age using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Primers were designed by PSQ Assay Design (Biotage AB, Uppsala, Sweden). Genomic DNA was modified by sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions [48 (link)]. In hepatocyte nuclear factor 4 A (HNF4A), the CpG sites are located from 159,936,862 to 159,937,373 bp of chromosome 3q42 region (reference sequence was GENBANK No. NC_005102.4, Figure S5). Additionally, these sites include exons of HNF4A isoform 1 (NM_022180.2). The primers used for the analysis via bisulfite amplicon sequencing are represented in Table S1. Amplification was carried out using the following cycling protocol; a cycle of 95 °C for 4 min; 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and finally 72 °C for 7 min. The PCR products were purified using QIAquick PCR columns (Qiagen, Hilden, Germany) and quantified using Picogreen (Invitrogen, Carlsbad, CA, USA).

For SNVs with low allele frequencies or in case primer construction for Sanger sequencing failed, pyrosequencing of both tumor samples and corresponding non-tumor sample was performed. Primer design was performed with PSQ Assay Design (Biotage, Uppsala, Sweden) and assays were established with Pyromark Q24 (Qiagen). PCR reaction was performed with the PyroMark PCR kit (Qiagen) according to manufacturer’s recommendations using 20 pmol primer and 25–50 ng template DNA. The PCRs were performed as described for Sanger sequencing. The resulting PCR products were sequenced with the Pyromark Q24 pyrosequencer using PyroMark Gold Q96 reagents (Qiagen). All assays were run with tumor and non-tumor samples as well as positive (Qiagen human control DNA) and negative control. SNVs with ≥5% difference in mutant AF compared to non-tumor tissue and positive control were assessed as true variants.