title: Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape.
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 (R(2) = 0.985), MAL (R(2) = 0.986) and hsa-miR-124-2 (R(2) = 0.944).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. r.steenbergen@vumc.nl
authors.forename: Suzanne
authors.initials: S
authors.lastname: Snellenberg
authors.forename: Lise M A
authors.initials: LM
authors.lastname: De Strooper
authors.forename: Albertus T
authors.initials: AT
authors.lastname: Hesselink
authors.forename: Chris J L M
authors.initials: CJ
authors.lastname: Meijer
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Snijders
authors.forename: Daniëlle A M
authors.initials: DA
authors.lastname: Heideman
authors.forename: Renske D M
authors.initials: RD
authors.lastname: Steenbergen
mesh_terms: D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002584:Cervix Uteri / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D019175:DNA Methylation; D005260:Female; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics; D008297:Male; D008403:Mass Screening / Q000379:methods; D035683:MicroRNAs / Q000235:genetics; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins / Q000235:genetics; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000235:genetics* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis / Q000235:genetics / Q000821:virology
chemical_list: C403514:CADM1 protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D007136:Immunoglobulins; C569026:MAL protein, human; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins