Non-Invasive Assay for Diagnosis and Treatment of Endometriosis

Project Summary AbstractEndometriosis affects up toof women of reproductive age often causing painful symptoms and an increased risk of infertilityA major clinical problem is the lack of a noninvasive and early diagnostic procedureThe diagnostic gold standard is laparoscopic surgery and histology of the recovered lesionsThe goal of this Phase I project is to develop a noninvasive diagnostic test for endometriosis that focuses on early changes in DNA methylation identified from published biomarker discoveryAssays will be based on the MethylMeter platformA fragmented DNA sample will be fractionated into a methylated DNA fraction and an unmethylated DNA fraction with the use of magnetic beads bearing a methyl CpG binding domain proteinThe amount of a targeted biomarker in each fraction will be quantified by Coupled Abscription PCR SignalingCAPSCAPS combines target DNA amplification with signal amplificationAmplicons with linked promoters are created during the PCR stepThe promoters are then subjected to abortive transcription with an RNA polymeraseAbscriptaseto produce a uniform population ofnt long RNAs also known as abscriptsA fluorescent signal is created by each abscript by participating in the opening of a molecular beaconCAPS is more sensitive than qPCR because each amplicon generates multiple signals as opposed to oneAssays will be developed for methylation detection targeted to specific CpG islands and surrounding sequencesAssay development will involve the identification and validation of PCR primers taking into consideration additional sequence for the linked abortive promotersPCR conditions will be optimized to eliminate amplification bias against methylated versions of the targetsConditions for methylated DNA binding will be optimized for a minimum oftargetsPhase II will focus on the clinical validation of the individual target assays and the development of a diagnostic signatureProtocols for sample collection and processing also will be developed Project Narrative Endometriosis affectsof women of reproductive age carrying risks of reduced fertility and painful symptomsCurrently a surgical procedure is the only definitive diagnosticThis projects aims to develop an earlynoninvasive diagnostic based on aberrant DNA methylation in endometriosis