Solid-state nanopore analysis of human genomic DNA shows unaltered global 5-hydroxymethylcytosine content associated with early-stage breast cancer

• Published in: Nanomedicine Vol. 35; p. 102407
• Main Authors: Zahid, Osama K., Rivas, Felipe, Wang, Fanny, Sethi, Komal, Reiss, Katherine, Bearden, Samuel, Hall, Adam R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2021
• Subjects: 5-Methylcytosine - analogs & derivatives; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Cancer; DNA, Neoplasm - genetics; DNA, Neoplasm - metabolism; Epigenetics; Female; Genome, Human; Humans; Hydroxymethylcytosine; MCF-7 Cells; Nanopore Sequencing; Neoplasm Staging; Solid-state nanopore; Epigenetics; Hydroxymethylcytosine; Solid-state nanopore; Cancer
• DOI: 10.1016/j.nano.2021.102407
• ISSN: 1549-9634

5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease. A selective solid-state nanopore assay is used to examine global 5-hydroxymethylcytosine (5hmC) content in human genomic DNA. 5hmC has been shown to be strongly downregulated in diverse cancers, but it is less clear whether the decrease is a cause or an effect of disease initiation. To investigate this, we compare DNA from healthy breast tissue with that derived from stage 1 breast tumor tissue. We show that no statistical difference is observed between the two even after implementing technical corrections to maximize quantitative accuracy. Our results suggest that 5hmC downregulation is not a driver of tumorigenesis but a result, indicating that the potential for the modification as a cancer biomarker may be diminished in early stage disease. [Display omitted]