Chromas version 2

Bisulfite sequencing PCR (BSP) was used as a quantitatively analyzed method for detecting the methylation rate of the RNF180 promoter area. The gene sequence for this study was obtained from Genebank. The MethylKIT package (http://www.bioconductor.org/packages/release/bioc/html/methylKit.html) of R software was used to analyze methylation data, all CpG sites and transcription initiation site (TSS). Genomic DNA sodium bisulfite modification was performed by Zymo DNA Methylation-Gold kit (Cat. Nos. D5005 or D5006, Zymo Research, US) according to the manufacturers’ instructions. Polymerase chain reaction (PCR) was performed by the enzyme TaKaRa Taq™ Hot Start Version (Code No. R007A Takara, Japan). The primers were as follows: F:5′-GTGGTTTTGGTAAGGGGATGAT-3′; R: 5′-AACAACCAAACTCTAAAAACTC-3′.11 (link) The condition was as follows: 94°C for 3 mins for initial denaturation, run 45 cycles (94°C 20 s, 58.5°C 30 s, 72°C 45 s); after the end of the cycles followed by an extension 10 mins at 72°C, a final termination at 4°C. PCR purification products were used for forward sequence analysis. Chromas version 2.6.6 (Technelysium Pty Ltd., Australia) was used to analyze sequencing results. According to the theory of bisulfite modification, methylation rate calculation formula was as follows: Meth%=C/(C+T)*100%.12 (link)

All substitutions or indels identified through WES were confirmed by Sanger sequencing, and available family members were sequenced for segregation analysis (Supplementary Figs. S1–S7). Potential de novo variants were confirmed by sequencing of parental DNA and excluding nonpaternity. Polymerase chain reaction (PCR) of CYP1B1 and FOXC1 was performed on Veriti 96 Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA). The protocol used for CYP1B1 amplification has previously been published.¹⁵ (link) PCR for FOXC1 was performed according to PCR Phusion High-Fidelity DNA Polymerase Protocol (Thermofisher Scientific, Waltham, MA, USA) using the GC-buffer and 2x S-Solution (Solis BioDyne, Tartu, Estonia). Big Dye Terminator Cycle Sequencing Kit version 1.1/3.1 (Thermofisher Scientific) was used for the Sanger reactions and a 3130xl Genetic Analyzer (Applied Biosystems) performed capillary sequencing. Sequences were visualized by Chromas version 2.6.6 (Technelysium, Brisbane, Australia). Primers are available on request. Multiplex ligation-dependent probe amplification (MLPA) was used to confirm FOXC1 CNVs using the SALSA MLPA P054-B2 FOXL2-TWIST1 probemix (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions. MLPA amplicons were analyzed using a 3130xl Genetic Analyzer (Applied Biosystems) and Sequence Pilot version 5.0 (JSI medical systems).

Direct sequencing was performed using the Sanger method to detect hotspot mutations on exons 9, 13, and 14 of the POLE gene. Genomic DNA was extracted from tumor specimens of 10‐μm FFPE slices resected during radical surgery using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's protocol. The samples were enriched using the GoTaq Colorless Master Mix (Promega). The primers used for PCR amplification and the PCR conditions are listed in Tables S1 and S2. An Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific) was used to analyze the samples. Two researchers (H.Y and Y.H.) evaluated the results using Chromas Version 2.6.6 (Technelysium Pty Ltd) and confirmed suspected mutations using reverse primers.