Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements and epigenetic modulation that are not easily captured by short-read sequencing. This study presents a novel approach for simultaneous profiling of long-range genetic and epigenetic changes in matched cancer samples, focusing on clear cell renal cell carcinoma (ccRCC). ccRCC is a common kidney cancer subtype frequently characterized by a 3p deletion and the inactivation of the von Hippel-Lindau (VHL) gene. We performed integrated genetic, cytogenetic, and epigenetic analyses on paired tumor and adjacent nontumorous tissue samples. Optical genome mapping identified genomic aberrations as structural and copy number variations, complementing exome-sequencing findings. Single-molecule methylome and hydroxymethylome mapping revealed a significant global reduction in 5hmC level in both sample pairs, and a correlation between both epigenetic signals and gene expression was observed. The single-molecule epigenetic analysis identified numerous differentially modified regions, some implicated in ccRCC pathogenesis, including the genes VHL, PRCC, and PBRM1. Notably, pathways related to metabolism and cancer development were significantly enriched among these differential regions. This study demonstrates the feasibility of integrating optical genome and epigenome mapping for comprehensive characterization of matched tumor and adjacent tissue, uncovering both established and novel somatic aberrations.

Department of Biomedical Engineering Tel Aviv University 6997801 Tel Aviv Israel

Institute of Experimental Botany of the Czech Academy of Sciences 77900 Olomouc Czech Republic

Institute of Organic Chemistry RWTH Aachen University D 52056 Aachen Germany

Pediatric Nephrology Unit The Edmond and Lily Safra Children's Hospital Sheba Medical Center 52621 Ramat Gan Israel

Pediatric Stem Cell Research Institute Edmond and Lily Safra Children's Hospital Sheba Medical Center 52621 Ramat Gan Israel

School of Chemistry Raymond and Beverly Sackler Faculty of Exact Sciences Tel Aviv University 6997801 Tel Aviv Israel

School of Medicine Faculty of Medical and Health Sciences Tel Aviv University 6997801 Tel Aviv Israel

The Susanne Levy Gertner Oncogenetics Unit The Danek Gertner Institute of Human Genetics Sheba Medical Center 52621 Ramat Gan Israel

Zobrazit více v PubMed

López  JI  Renal tumors with clear cells. A review. Pathology. 2013; 209:137–46.10.1016/j.prp.2013.01.007. PubMed DOI

Bacigalupa  ZA, Rathmell  WK  Beyond glycolysis: hypoxia signaling as a master regulator of alternative metabolic pathways and the implications in clear cell renal cell carcinoma. Cancer Lett. 2020; 489:19–28.10.1016/j.canlet.2020.05.034. PubMed DOI PMC

Gossage  L, Eisen  T, Maher  ER  VHL, the story of a tumour suppressor gene. Nat Rev Cancer. 2015; 15:55–64.10.1038/nrc3844. PubMed DOI

Duns  G, Hofstra  RMW, Sietzema  JG  et al. .  Targeted exome sequencing in clear cell renal cell carcinoma tumors suggests aberrant chromatin regulation as a crucial step in ccRCC development. Hum Mutat. 2012; 33:1059–62.10.1002/humu.22090. PubMed DOI

Le  VH, Hsieh  JJ  Genomics and genetics of clear cell renal cell carcinoma: a mini-review. JTGG. 2018; 2:17.10.20517/jtgg.2018.28. DOI

Moore  LE, Jaeger  E, Nickerson  ML  et al. .  Genomic copy number alterations in clear cell renal carcinoma: associations with case characteristics and mechanisms of VHL gene inactivation. Oncogenesis. 2012; 1:e14.10.1038/oncsis.2012.14. PubMed DOI PMC

Quddus  M, Pratt  N, Nabi  G  Chromosomal aberrations in renal cell carcinoma: an overview with implications for clinical practice. Urol Ann. 2019; 11:6–14. PubMed PMC

Klatte  T, Rao  PN, De  Martino M  et al. .  Cytogenetic profile predicts prognosis of patients with clear cell renal cell carcinoma. JCO. 2009; 27:746–53.10.1200/JCO.2007.15.8345. PubMed DOI

Shenoy  N, Vallumsetla  N, Zou  Y  et al. .  Role of DNA methylation in renal cell carcinoma. J Hematol Oncol. 2015; 8:88.10.1186/s13045-015-0180-y. PubMed DOI PMC

Hu  CY, Mohtat  D, Yu  Y  et al. .  Kidney cancer is characterized by aberrant methylation of tissue-specific enhancers that are prognostic for overall survival. Clin Cancer Res. 2014; 20:4349–60.10.1158/1078-0432.CCR-14-0494. PubMed DOI PMC

Arai  E, Chiku  S, Mori  T  et al. .  Single-CpG-resolution methylome analysis identifies clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas. Carcinogenesis. 2012; 33:1487–93.10.1093/carcin/bgs177. PubMed DOI PMC

Lasseigne  BN, Brooks  JD  The role of DNA methylation in renal cell carcinoma. Mol Diagn Ther. 2018; 22:431–42.10.1007/s40291-018-0337-9. PubMed DOI PMC

Joosten  SC, Odeh  SNO, Koch  A  et al. .  Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers. Clin Epigenet. 2021; 13:103.10.1186/s13148-021-01084-8. PubMed DOI PMC

Wang  J, Zhang  Q, Zhu  Q  et al. .  Identification of methylation-driven genes related to prognosis in clear-cell renal cell carcinoma. J Cell Physiol. 2020; 235:1296–308.10.1002/jcp.29046. PubMed DOI PMC

Shi  DQ, Ali  I, Tang  J  et al. .  New insights into 5hmC DNA modification: generation, distribution and function. Front Genet. 2017; 8:100.10.3389/fgene.2017.00100. PubMed DOI PMC

Jin  SG, Jiang  Y, Qiu  R  et al. .  5-hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations. Cancer Res. 2011; 71:7360–5.10.1158/0008-5472.CAN-11-2023. PubMed DOI PMC

Margalit  S, Avraham  S, Shahal  T  et al. .  5-Hydroxymethylcytosine as a clinical biomarker: fluorescence-based assay for high-throughput epigenetic quantification in human tissues. Intl J Cancer. 2020; 146:115–22.10.1002/ijc.32519. PubMed DOI

Chen  K, Zhang  J, Guo  Z  et al. .  Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer. Cell Res. 2016; 26:103–18.10.1038/cr.2015.150. PubMed DOI PMC

Chen  S, Zhou  Q, Liu  T  et al. .  Prognostic value of downregulated 5-hydroxymethyl-cytosine expression in renal cell carcinoma: a 10 year follow-up retrospective study. J Cancer. 2020; 11:1212–22.10.7150/jca.38283. PubMed DOI PMC

Yu  M, Han  D, Hon  GC  et al. .  Tet-assisted bisulfite sequencing (TAB-seq). Methods Mol Biol. 2018; 1708:645–63.10.1007/978-1-4939-7481-8_33. PubMed DOI PMC

Booth  MJ, Ost  TWB, Beraldi  D  et al. .  Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc. 2013; 8:1841–51.10.1038/nprot.2013.115. PubMed DOI PMC

Gabrieli  T, Sharim  H, Nifker  G  et al. .  Epigenetic optical mapping of 5-hydroxymethylcytosine in nanochannel arrays. ACS Nano. 2018; 12:7148–58.10.1021/acsnano.8b03023. PubMed DOI PMC

Michaeli  Y, Shahal  T, Torchinsky  D  et al. .  Optical detection of epigenetic marks: sensitive quantification and direct imaging of individual hydroxymethylcytosine bases. Chem Commun. 2013; 49:8599–601.10.1039/c3cc42543f. PubMed DOI

Nifker  G, Levy-Sakin  M, Berkov-Zrihen  Y  et al. .  One-pot chemoenzymatic cascade for labeling of the epigenetic marker 5-hydroxymethylcytosine. ChemBioChem. 2015; 16:1857–60.10.1002/cbic.201500329. PubMed DOI

Shahal  T, Gilat  N, Michaeli  Y  et al. .  Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA. Anal Chem. 2014; 86:8231–7.10.1021/ac501609d. PubMed DOI

Jeffet  J, Margalit  S, Michaeli  Y  et al. .  Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale Jonathan. Essays Biochem. 2021; 65:51–66.10.1042/EBC20200021. PubMed DOI PMC

Sharim  H, Grunwald  A, Gabrieli  T  et al. .  Long-read single-molecule maps of the functional methylome. Genome Res. 2019; 29:646–56.10.1101/gr.240739.118. PubMed DOI PMC

Mak  ACY, Lai  YYY, Lam  ET  et al. .  Genome-wide structural variation detection by genome mapping on nanochannel arrays. Genetics. 2016; 202:351–62.10.1534/genetics.115.183483. PubMed DOI PMC

Detinis  ZurT, Margalit  S, Jeffet  J  et al. .  Single-molecule toxicogenomics: optical genome mapping of DNA-damage in nanochannel arrays. DNA Repair. 2025; 146:103808.10.1016/j.dnarep.2025.103808. PubMed DOI

Margalit  S, Abramson  Y, Sharim  H  et al. .  Long reads capture simultaneous enhancer–promoter methylation status for cell-type deconvolution. Bioinformatics. 2021; 37:i327–33.10.1093/bioinformatics/btab306. PubMed DOI PMC

Gabrieli  T, Michaeli  Y, Avraham  S  et al. .  Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping. Nucleic Acids Res. 2022; 50:e92.10.1093/nar/gkac460. PubMed DOI PMC

Uppuluri  L, Jadhav  T, Wang  Y  et al. .  Multicolor whole-genome mapping in nanochannels for genetic analysis. Anal Chem. 2021; 93:9808–16.10.1021/acs.analchem.1c01373. PubMed DOI PMC

Langmead  B, Salzberg  SL  Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9:357–9.10.1038/nmeth.1923. PubMed DOI PMC

Van  der Auwera GA, O'connor  BD  Genomics in the Cloud: Using Docker, GATK, and WDL in Terra. 2020; 1st EditionO’Reilly Media.

Danecek  P, Bonfield  JK, Liddle  J  et al..  Twelve years of SAMtools and BCFtools. Gigascience. 2021; 10:giab008.10.1093/gigascience/giab008. PubMed DOI PMC

Cingolani  P, Platts  A, Wang  LL  et al. .  A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly. 2012; 6:80–92.10.4161/fly.19695. PubMed DOI PMC

Sherry  ST, Ward  MH, Kholodov  M  et al. .  DbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 2001; 29:308–11.10.1093/nar/29.1.308. PubMed DOI PMC

Grunwald  A, Dahan  M, Giesbertz  A  et al. .  Bacteriophage strain typing by rapid single molecule analysis. Nucleic Acids Res. 2015; 43:e117.10.1093/nar/gkv563. PubMed DOI PMC

Frankish  A, Diekhans  M, Ferreira  AM  et al. .  GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 2019; 47:D766–73.10.1093/nar/gky955. PubMed DOI PMC

Cao  Q, Anyansi  C, Hu  X  et al. .  Reconstruction of enhancer-target networks in 935 samples of human primary cells, tissues and cell lines. Nat Genet. 2017; 49:1428–36.10.1038/ng.3950. PubMed DOI

Haeussler  M, Zweig  AS, Tyner  C  et al. .  The UCSC Genome Browser database: 2019 update. Nucleic Acids Res. 2019; 47:D853–8.10.1093/nar/gky1095. PubMed DOI PMC

Amemiya  HM, Kundaje  A, Boyle  AP  The ENCODE Blacklist: identification of problematic regions of the genome. Sci Rep. 2019; 9:9354.10.1038/s41598-019-45839-z. PubMed DOI PMC

Yao  X, Tan  J, Lim  KJ  et al. .  VHL deficiency drives enhancer activation of oncogenes in clear cell renal cell carcinoma. Cancer Discov. 2017; 7:1284–305.10.1158/2159-8290.CD-17-0375. PubMed DOI

Quinlan  AR, Hall  IM  BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26:841–2.10.1093/bioinformatics/btq033. PubMed DOI PMC

Neph  S, Kuehn  MS, Reynolds  AP  et al. .  BEDOPS: high-performance genomic feature operations. Bioinformatics. 2012; 28:1919–20.10.1093/bioinformatics/bts277. PubMed DOI PMC

Hao  Z, Lv  D, Ge  Y  et al. .  RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput Sci. 2020; 6:e251.10.7717/peerj-cs.251. PubMed DOI PMC

Trapnell  C, Pachter  L, Salzberg  SL  TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25:1105–11.10.1093/bioinformatics/btp120. PubMed DOI PMC

Leinonen  R, Sugawara  H, Shumway  M  The sequence read archive. Nucleic Acids Res. 2011; 39:D19–21.10.1093/nar/gkq1019. PubMed DOI PMC

Anders  S, Pyl  PT, Huber  W  HTSeq-A Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31:166–9.10.1093/bioinformatics/btu638. PubMed DOI PMC

Ramírez  F, Dündar  F, Diehl  S  et al. .  DeepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 2014; 42:W187–91.10.1093/nar/gku365. PubMed DOI PMC

Benjamini  Y, Hochberg  Y  Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995; 57:289–300.10.1111/j.2517-6161.1995.tb02031.x. DOI

Yu  G, Wang  LG, Han  Y  et al. .  ClusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16:284–7.10.1089/omi.2011.0118. PubMed DOI PMC

Jonasch  E, Walker  CL, Rathmell  WK  Clear cell renal cell carcinoma ontogeny and mechanisms of lethality. Nat Rev Nephrol. 2021; 17:245–61.10.1038/s41581-020-00359-2. PubMed DOI PMC

Gandawijaya  J, Bamford  RA, Burbach  JPH  et al. .  Cell adhesion molecules involved in neurodevelopmental pathways implicated in 3p-deletion syndrome and autism spectrum disorder. Front Cell Neurosci. 2021; 14:611379.10.3389/fncel.2020.611379. PubMed DOI PMC

Di  Nunno V, Mollica  V, Brunelli  M  et al. .  A meta-analysis evaluating clinical outcomes of patients with renal cell carcinoma harboring chromosome 9P loss. Mol Diagn Ther. 2019; 23:569–77.10.1007/s40291-019-00414-0. PubMed DOI

Monzon  FA, Alvarez  K, Peterson  L  et al. .  Chromosome 14q loss defines a molecular subtype of clear-cell renal cell carcinoma associated with poor prognosis. Mod Pathol. 2011; 24:1470–9.10.1038/modpathol.2011.107. PubMed DOI PMC

Ueno  K, Hirata  H, Shahryari  V  et al. .  Tumour suppressor microRNA-584 directly targets oncogene Rock-1 and decreases invasion ability in human clear cell renal cell carcinoma. Br J Cancer. 2011; 104:308–15.10.1038/sj.bjc.6606028. PubMed DOI PMC

Benayoun  BA, Caburet  S, Veitia  RA  Forkhead transcription factors: key players in health and disease. Trends Genet. 2011; 27:224–32.10.1016/j.tig.2011.03.003. PubMed DOI

Song  C-X, Szulwach  KE, Fu  Y  et al. .  Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol. 2011; 29:68–72.10.1038/nbt.1732. PubMed DOI PMC

Avraham  S, Schütz  L, Käver  L  et al. .  Chemo-enzymatic fluorescence labeling of genomic DNA for simultaneous detection of global 5-methylcytosine and 5-hydroxymethylcytosine. ChemBioChem. 2023; 24:e202300400.10.1002/cbic.202300400. PubMed DOI

Uroshlev  LA, Abdullaev  ET, Umarova  IR  et al. .  A method for identification of the methylation level of CpG islands from NGS data. Sci Rep. 2020; 10:8635.10.1038/s41598-020-65406-1. PubMed DOI PMC

Margalit  S, Tulpová  Z, Zur  TD  et al. .  Long-read structural and epigenetic profiling of a kidney tumor-matched sample with nanopore sequencing and optical genome mapping. NAR Genomics Bioinform. 2025; 7:lqae190.10.1093/nargab/lqae190. PubMed DOI PMC

Balog  J, Miller  D, Sanchez-Curtailles  E  et al. .  Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47. Eur J Hum Genet. 2012; 20:185–91.10.1038/ejhg.2011.150. PubMed DOI PMC

Pook  MA  DNA methylation and trinucleotide repeat expansion diseases. DNA Methylation - From Genomics to Technology. 2012; InTech; 193–208.

Hansen  KD, Timp  W, Bravo  HC  et al. .  Increased methylation variation in epigenetic domains across cancer types. Nat Genet. 2011; 43:768–75.10.1038/ng.865. PubMed DOI PMC

Stults  DM, Killen  MW, Pierce  HH  et al. .  Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Res. 2008; 18:13–8.10.1101/gr.6858507. PubMed DOI PMC

Hakimi  AA, Reznik  E, Lee  C-H  et al. .  An integrated metabolic atlas of clear cell renal cell carcinoma. Cancer Cell. 2016; 29:104–16.10.1016/j.ccell.2015.12.004. PubMed DOI PMC

Monjaras-Avila  CU, Lorenzo-Leal  AC, Luque-Badillo  AC  et al. .  The tumor immune microenvironment in clear cell renal cell carcinoma. Int J Mol Sci. 2023; 24:7946.10.3390/ijms24097946. PubMed DOI PMC

Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping