Additional file 1 of Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing
Creators: name: Demidova, Elena V.
name: Serebriiskii, Ilya G.
name: Vlasenkova, Ramilia
name: Kelow, Simon
name: Andrake, Mark D.
name: Hartman, Tiffiney R.
name: Kent, Tatiana
name: Virtucio, James
name: Rosen, Gail L.
name: Pomerantz, Richard T.
name: Dunbrack, Roland L.
name: Golemis, Erica A.
name: Hall, Michael J.
name: Chen, David Y. T.
name: Daly, Mary B.
name: Arora, Sanjeevani
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Peripheral blood lymphocyte DNA analysis: whole exome sequencing, gene variants annotation and prioritization. Supplementary Figure 1. Pathway enrichment for the genes with identified DDR germline variants according to over-representation analysis (ORA). FDR is provided inside the boxes of the heat map. Supplementary Figure 2. A. siRNA depletion of POLD1, POLE, POLH, POLK, RRM2B and ATM genes in Caki RCC cell line. A. Cells were transfected with the designated siRNAs (two per gene), or GL2 control or WRN positive control. Cells were fixed, permeabilized, blocked and stained for γH2AX antibody. Cells were scored for γH2AX foci and the data are plotted as relative induction of γH2AX to GL2 control from 2 independent experiments. *** for p&lt;0.001, ** for p&lt;0.01, * for p&lt;0.05 and NS for p&gt;0.05, Wilcoxon signed rank test. Supplementary Figure 3. Representative cell cycle data from immortalized B-cell lines from Pt #1 (POLD1, POLH), Pt#2 (POLE), Pt#16 (POLK) PBMCs and corresponding immortalized B-cell lines from matched control. A. Percent positive gated cells is presented for each cell cycle phase for each cell line (controls on the left in blue, cases on the right in red). Data for 3 independent repeats are presented. *** for p&lt;0.001, ** for p&lt;0.01, * for p&lt;0.05, NS for p&gt;0.05, Wilcoxon signed rank test. Supplementary Figure 4. Difference in DNA replication fork elongation/restoration in EBV-transformed cell line with POLK (Pt #16) variant, and representative pictures of DNA fibers for the main figures 2E and 2F. For all graphs: *** for p&lt;0.001, ** for p&lt;0.01, * for p&lt;0.05 and NS for p&gt;0.05, unpaired, non-parametric t-test, Mann-Whitney criteria. Means with SD are plotted. Data for 3 independent repeats are presented as IdU tract length or CldU/IdU tract length ratio. A. Difference in DNA replication fork elongation/restoration in EBV-transformed cell line with POLK E29K variant at the baseline and replication stress was assessed using DNA fiber assay. At baseline the EBV-transformed cells were labeled with IdU for 20 min, for fork restoration cells then were treated with 100 μM aphidicolin and then labeled with CldU for 40 min. For all conditions, after labeling, cells were lysed, and DNA fibers stretched onto glass-slides, fixed, denatured, blocked, and stained with corresponding antibodies. Fiber images were captured using the Nikon TS2R Inverted Microscope and analyzed in ImageJ software. B-D. Additional representative pictures for each DNA fiber experiment: B for IdU 20 min labeling (main figures 2E, 2F, current figure A); C for short aphidicolin treatment (main figures 2E, 2F, current figure A); D for treatment with MNNG (main figure 2E). Scale bar = 5 μm. Supplementary Figure 5. Pol δ complex primer extension competition assay with quantification. Representative gel image showing reactions performed with 20 nM Cy-3 labeled DNA-duplex template (SA#1), 20 nM of indicated proteins (wild type complex, V759I complex and both wild type + V759 variant complexes in ratio 1:1) and 500 uM dNTPs. Under presence of the both wild type and PolD1 V759I Pol δ complexes, DNA-template was extended less efficiently compared to wild type and more efficiently compared to V759I complexes alone. Data for 3 independent repeats are presented. *** for p&lt;0.001, ** for p&lt;0.01, * for p&lt;0.05 and NS for p&gt;0.05, unpaired, non-parametric t-test, Mann-Whitney criteria. Supplementary Figure 6. Raw uncropped gel images, corresponding to sub-figures in Figure 2B: A, B and C for PolD1, Pol η and loading control exposures used to generate the main figure, D for Pol ε, E for Pol κ. Supplementary Figure 7. Raw uncropped gel images, corresponding to sub-figures in Figure 3 and Supplementary Figure 5. A for Figure 3A for Pol δ complex purification, B for Figure 3B Pol η purification, C for Figure 3A on right- Pol δ complex primer extension assay, D for 3B on right- Pol η primer extension assay, E for 3C - Pol η lesion bypass assay, F for Supplementary Figure 5. Supplementary Figure 8. Mutational signature analysis in tumors from Pts 1 and 2. A. SBS signatures are reported in tumors from Pts 1 and 2. B. DBS signatures are reported in tumors from Pts 1 and 2. Supplementary table 1. List of candidate genes for WES analysis (Excel file). Supplementary table 2. Annotation of candidate variants identified in the 22 eoRCC patients (Excel file). Supplementary table 3. Complete summary of results for the DNA polymerase variants identified in the eoRCC patients. Supplementary table 4. Protein stability or ddG values for the PolD1 V759I and Pol η G209V variant proteins that were assessed biochemically in the study. Supplementary table 5. POLE and POLD1 variants in hypermutated ccRCC in TCGA. The table shows the variants from TCGA with allele counts typically observed in the GnomAD database, TMBs observed in association with these variants in other studies [60], and finally protein stability or ddG values for the variant proteins. Supplementary table 6. DNA substrates used in biochemical assays. The following are the references for the DNA substrates [47, 49, 50]. Supplementary table 7. ddG application file. Supplementary Table 8. Results from analysis of CNVs, SNVs and Indels using tumor sequencing data from Pt #1 (POLD1, POLH), and Pt #2 (POLE) (Excel file).