Development of Novel Diagnostics for Fragile X Syndrome

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DESCRIPTION (provided by applicant): Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. FRAX is caused by the expansion of a CGG trinucleotide repeat of the 5' untranslated region (UTR) of the FMR1 gene. In normal individuals, the 5' UTR of the FMR1 gene contains 5 to 45 CGG repeats; however, individuals with FRAX have over 200 repeats. Presently, Southern Blot analysis is used to determine the size of the repeat segment and methylation status of the FRAX gene. This test only detects the gross size of CGG repeats and is labor intensive and expensive. PCR and gel electrophoresis is typically used to determine the size of the CGG expansion. This approach is limited, as PCR reactions typically fail to amplify long stretches of CGG expansions (>25 repeats) and molecular weight determination by electrophoresis via capillary or slab gels is labor intensive. In our Phase I SBIR award, we developed a novel, highly efficient and accurate screening test for diagnosing FRAX. In Step 1, Whole Genome Multiple Displacement Amplification using 7-deaza-2-Guanosine (7-deaza GTP) nucleotide analog is incorporated into multiple copies of the CGG FMR1 expansion. In Step 2, Site Specific Multiple Displacement Amplification (SSMDA) using 7-deaza GTP is performed to weaken the GC base pairings, making the GCC expansion more accessible to Taq DNA Polymerase in real-time PCR. In Step 3, SSMDA is followed by quantitative assessment of the numbers of CGG triplet repeats using TaqMan real-time PCR without the need for sizing by gel electrophoresis or Southern blotting. We hypothesize that using this new molecular-based method, we can develop an effective, rapid and low-cost screening test for FRAX with broad commercial application. In this Phase II application we propose to: SA1: Optimize our assay for CGG repeat copy number determination using a large number of DNA samples with varying degrees of CGG repeats. SA2: Test our assay in a clinical FRAX diagnostic laboratory. SA3: Test our assay in blood spots for potential newborn screening. We anticipate that this Phase II application will lead to the optimization and implementation of a test that is suitable for low cost high-throughput screening for FRAX. As such, this test has the potential to markedly change how we currently screen for FRAX. These studies will involve the combined expertise of JS Genetics, which has been developing novel DNA-based diagnostic tests, and Dr. Bai-Lin Wu of Boston Children's Hospital and Harvard Medical School, who an extensive track record of developing clinical DNA diagnostic studies and in clinical laboratory testing of FRAX. PUBLIC HEALTH RELEVANCE: Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. We propose the development of a new molecular-based method, for rapid and low-cost screening of FRAX with broad commercial application.