Cancer Genetics and Epigenetics 2024, Vol.12, No.4, 223-233 http://medscipublisher.com/index.php/cge 226 epigenetic changes in the VHL gene, which are crucial for understanding the molecular pathogenesis of ccRCC (Banks et al., 2006; Sato et al., 2013). 3.2 Advances in high-throughput screening High-throughput screening technologies have significantly advanced the detection of VHL mutations. Next-generation sequencing (NGS) has emerged as a powerful tool, allowing for comprehensive analysis of the VHL gene across numerous samples simultaneously. NGS can identify a wide range of mutations, including those present at low frequencies, which are often missed by conventional methods (Coppin et al., 2019). This technology has been optimized to detect mosaic mutations in the VHL gene, which are particularly challenging to identify (Coppin et al., 2019). The integration of NGS with other techniques, such as SNaPshot and droplet digital PCR, enhances the sensitivity and accuracy of mutation detection, making it a valuable approach in clinical diagnostics (Coppin et al., 2019) (Table 1). Table 1 Proportion of VHL Mutated Event Detected by SNaPshot and ddPCR for Each Individual Plasmid Mutant (Adopted from Coppin et al., 2019) Event SNaPshot ddPCR Theoretical proportion of mutated event,% 10 5 1 10 5 1 0bserved proportion of mutated event (forward +reverse),% Threshold Threshold c.194C>G p. (Ser65Trp) 17.7 8.43 ND 5 ND ND ND ND c.226_228del p. (Phe76del) 7.8 3.37 ND 5 6.38 2.95 0.69 1 c.232A>C p. (Asn78His) 20.0 12.9 5.5 <5 4.71 2.04 0.053 1 c.341-1G>C 25.8 18.21 ND 5 13.61 6.8 1.01 1 c.467A>G p. (Tyr156Cys) 12.1 7.02 ND 5 21.29 7.99 1.19 1 c.481C>T p. (Arg161*) 13.2 ND ND >5 24.65 9.98 2.89 1 c.482G>A p. (Arg161Gln) 13.3 5.1 ND 5 15.19 8.73 2.32 1 c.500G>A p. (Arg167Gln) 7.9 ND ND >5 21.03 7.48 4.36 1 Table caption: ddPCR: Droplet digital PCR; ND: Not detected (Adopted from Coppin et al., 2019) Next-generation sequencing (NGS) plays a pivotal role in improving the diagnostic accuracy of VHL mutations. By providing high-resolution data, NGS allows for the detection of both common and rare mutations, as well as complex genetic alterations such as copy number variations and intragenic deletions (Nickerson et al., 2008; Coppin et al., 2019). The ability to sequence entire exomes or genomes enables a comprehensive analysis of the VHL gene and its regulatory regions, uncovering mutations that may contribute to tumorigenesis (Sato et al., 2013). Furthermore, NGS can be used to analyze circulating tumor DNA (ctDNA) in blood samples, offering a non-invasive method to monitor VHL mutations and assess tumor dynamics in real-time (Sumiyoshi et al., 2021). This approach not only enhances diagnostic precision but also facilitates personalized treatment strategies for patients with ccRCC (Dizman et al., 2020). 3.3 Epigenetic analysis andVHLgene silencing Epigenetic modifications, particularly promoter hypermethylation, play a crucial role in the silencing of the VHL gene in renal cell carcinoma. Hypermethylation of CpG islands in the VHL promoter region leads to the suppression of gene expression, contributing to the inactivation of this tumor suppressor gene (Herman et al., 1994). This epigenetic alteration is a significant mechanism by which the VHL gene is silenced in ccRCC, alongside genetic mutations (Patard et al., 2009). Studies have shown that hypermethylation of the VHL promoter is present in a substantial proportion of ccRCC cases, indicating its importance in the pathogenesis of this cancer (Herman et al., 1994; Banks et al., 2006). Several techniques are employed to detect epigenetic changes such as promoter hypermethylation in the VHLgene. Methylation-specific PCR (MSP) is a widely used method that differentiates between methylated and unmethylated DNA sequences, allowing for the detection of hypermethylation in the VHL promoter (Herman et
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