BMC Cancer (Nov 2012)

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

  • Snellenberg Suzanne,
  • Strooper Lise MA,
  • Hesselink Albertus T,
  • Meijer Chris JLM,
  • Snijders Peter JF,
  • Heideman Daniëlle AM,
  • Steenbergen Renske DM

DOI
https://doi.org/10.1186/1471-2407-12-551
Journal volume & issue
Vol. 12, no. 1
p. 551

Abstract

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Abstract Background Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. Methods Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. Results Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R2=0.985), MAL (R2=0.986) and hsa-miR-124-2 (R2=0.944). Conclusion Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions.

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