Enabling use of blood spot cards for accurate high-throughput Fragile X screening

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop accurate, rapid, high-throughput and cost-effective diagnostic tests for Fragile X syndrome (FXS) and related disorders in anticipation of population screening needs. Newborn screening (NBS) for Fragile X is timely due to the disease prevalence, lack of a clear phenotype at birth, current and emerging options for early interventions, and the impact of associated syndromes. Access to an inexpensive and effective high-throughput screening technology is one of the most significant hurdles to NBS implementation. To address this technology gap, Asuragen has formulated a PCR reagent set that can reproducibly detect the full spectrum of Fragile X triplet repeat expansions, including those with gt1000 CGG repeats, in purified DNA from clinical samples. In this proposal, investigations are described that will leverage these PCR innovations to enable direct amplification from blood spot cards and support the accurate detection of clinically relevant Fragile X samples. Fragile X is the most common form of inherited mental retardation, affecting roughly 1 in 4,000 males and 1 in 6,000 females, with carrier rates as high at 1 in 130 in females and 1 in 250 in males. The disease arises from expansion of a CGG trinucleotide sequence in the 5'-untranslated region of the FMR1 gene. Disease severity is strongly linked to the number of CGG repeats in this region of the gene. Patients with gt200 CGG often manifest FXS. More modest expansions areassociated with reproductive, and cognitive/motor disorders. Improved diagnostic screening is particularly relevant in the face of promising pharmaceutical and behavioral therapeutics for FXS. A commercialized, and published the validation of FMR1 PCR technologies have been successfully developed that can detect all categories of Fragile X alleles, including very large full mutation expansions previously detectable only by Southern blot analysis, and resolve other longstanding technical issues (such as female zygosity) that confound existing PCR-based diagnostic methods. For this project, these PCR advances will be extended to develop a prototype high-throughput NBS assay that can accurately identify newborns at risk for fragile X disorders. The specific aims of the proposal are to: 1) Optimize procedures for blood spot processing with Whatman #903 paper; 2) Optimize FMR1 CGG RP PCR reagents and CE detection for use with processed blood spot card samples; and 3) Automate optimized blood spot processing and FMR1 PCR. The outcome of this research will be demonstrated feasibility for an accurate, automated, and cost- effective platform for fragile X detection in newborns, and address a longstanding unmet need for scalable technologies that can identify fragile Xdisorders in both males and females in the broader population. PUBLIC HEALTH RELEVANCE: We are developing an automated, rapid, and cost-effective molecular test for screening newborns to detect the characteristic genetic mutation associated with Fragile X Syndrome. Fragile X is one of the most commonly inherited forms of mental retardation and can also cause conditions such as ADHD and autism. The diagnostic tests detects DNA from a small drop of blood spotted on paper and will support early diagnosis which may be beneficial because of current and emerging options for early interventions.