Soltysova Andrea, Dvorska Dana, Ficek Andrej, Pecimonova Martina, Samec Marek, Kasubova Ivana, Horvathova Kajabova Viera, Demkova Lucia, Babal Pavel, Valaskova Jela, Dankova Zuzana, Smolkova Bozena, Furdova Alena
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
Institute for Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia.
Invest Ophthalmol Vis Sci. 2025 Mar 3;66(3):51. doi: 10.1167/iovs.66.3.51.
Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, with prognosis significantly influenced by genetic and epigenetic factors. Reliable and cost-effective methods to detect chromosomal aberrations and DNA methylation changes are essential for improving prognostication and informing treatment strategies in UM. This study evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting UM-specific copy number variations (CNVs) and promoter methylation changes across 25 tumor suppressor genes (TSGs).
DNA from 58 UM tissues was analyzed with the SALSA MLPA Probemix P027 Uveal melanoma kit, and a subset of 18 samples was further assessed using the SALSA MLPA Probemix ME002-C1 Tumour suppressor mix 2 kit to identify key CNVs and methylation alterations linked to poor prognosis. Validation was carried out with a high-resolution comparative genomic hybridization (CGH) array on 10 samples and the Illumina Infinium Methylation EPIC v1.0 BeadChip array on 25 samples.
Our findings indicate that MLPA is a versatile and robust method for detecting CNVs, showing strong correlations with CGH data and highlighting specific CNV patterns linked to clinical outcomes in UM. However, the ME002-C1 kit showed limited utility for comprehensive methylation analysis, as differential methylation was not observed in the studied TSG loci.
Although MLPA effectively identifies CNVs relevant to UM prognosis, integrating additional methylation-specific approaches could broaden the scope of DNA methylation analysis, offering a more comprehensive molecular understanding of UM that may enhance prognostication and personalized treatment.
葡萄膜黑色素瘤（UM）是成人中最常见的原发性眼内恶性肿瘤，其预后受遗传和表观遗传因素的显著影响。可靠且经济高效的检测染色体畸变和DNA甲基化变化的方法对于改善UM的预后评估和指导治疗策略至关重要。本研究评估了多重连接依赖探针扩增（MLPA）在检测25个肿瘤抑制基因（TSG）中的UM特异性拷贝数变异（CNV）和启动子甲基化变化方面的有效性。
使用SALSA MLPA Probemix P027葡萄膜黑色素瘤试剂盒对58个UM组织的DNA进行分析，并使用SALSA MLPA Probemix ME002-C1肿瘤抑制混合试剂盒2对18个样本的子集进行进一步评估，以确定与预后不良相关的关键CNV和甲基化改变。对10个样本使用高分辨率比较基因组杂交（CGH）阵列进行验证，对25个样本使用Illumina Infinium Methylation EPIC v1.0 BeadChip阵列进行验证。
我们的研究结果表明，MLPA是一种检测CNV的通用且强大的方法，与CGH数据显示出强相关性，并突出了与UM临床结果相关的特定CNV模式。然而，ME002-C1试剂盒在全面甲基化分析中的效用有限，因为在所研究的TSG基因座中未观察到差异甲基化。
尽管MLPA有效地识别了与UM预后相关的CNV，但整合额外的甲基化特异性方法可以拓宽DNA甲基化分析的范围，提供对UM更全面的分子理解，这可能会增强预后评估和个性化治疗。
