Single molecule fluorescence detection of fragile X mutations

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DESCRIPTION (provided by applicant): Fragile X Syndrome (FXS), is the most common form of inherited mental retardation, affecting 1 in 4000 males and about 1 in 6000 females of all ethnicities. Additionally, carrier frequencies are quite high. About 1 in 250 females carry Fragile X and about 1 in 800 men carry Fragile X. FXS was one of the first human diseases to be linked to an expansion of triplet nucleotide repeats, consisting of anywhere from 200-2000 CGG units in the 5' untranslated region of the FMR1 gene. Recent research indicates, however, that a constellation of cognitive, motor and reproductive deficits can be attributed to even fewer triplet repeats, in the range of about 60-200. Risk can be stratified by triplet repeat number and the disease manifestations respond well to therapies If identified early. The Fragile X gene is, consequently, a substantial diagnostic and screening opportunity, and testing is recommended for affected individuals, carriers, offspring of carriers and individuals with similar symptoms but no history of the disease. Our belief, which is supported by many in the medical community, is that fragile X screening will soon become more prevalent. But the Fragile X gene poses several problems for current molecular diagnostic procedures. Sizing of genomic DNA using Southern blot and gel electrophoresis is time consuming and low-throughput. Polymerases have trouble reading through the CGG repeat, and so most gene amplification methods (e.g. PCR) have not achieved high fidelity and amplifying FMR1. Additionally, these methods may misdiagnose the heterogeneous genotypes of carriers or mosaics. Our goal is to develop a Fragile X screening method that has adequate fidelity and throughput. This proposal describes an assay that combines our FMR1 PCR procedures, extensively optimized for the triplet repeats, with a novel detection system that can achieve both throughput and resolution of heterogeneous genotypes. Our PCR methods are currently able to reliably amplify over 900 CGG repeats with very high sensitivity and fidelity. Our detection methods are then able to individually survey all PCR products and determine the distribution repeat numbers. This proposal focuses on developing the detection method and integrating it with optimized PCR in order to demonstrate feasibility of high throughput screening for Fragile X gene mutations. PUBLIC HEALTH RELEVANCE: Mutations of the Fragile X gene are a significant cause of mental retardation, memory and motor defects, and reproductive insufficiency. Interventions can be successful but require accurate molecular diagnosis of the disease. Our long-term goal is to develop Fragile X mutation screening assays that will identify individuals at-risk for developing or passing on these genes.