Development of a reliable and standardized molecular assay for fragile x protein

DESCRIPTION provided by applicant The goal of this project is to develop a validated quantitative assay for the fragile X mental retardation protein FMRP to aid in the clinical management of those with fragile X syndrome FXS autism and other disorders and to advance FXS research FXS is the leading form of inheritable intellectual disability ranging from mild to severe and is the most common known mutation in autism It is caused by CGG repeat expansion in the X linked FMR gene In non affected individuals to repeats occur in the FMR andapos UTR whereas in those with FXS andgt repeats are observed causing substantially reduced levels of FMRP and intellectual disability Expansion to repeats results in normal or much smaller reductions in FMRP These individuals are unaffected or present a varying less serious degree of cognitive behavioral and emotional dysfunctions Individuals with fewer than repeats almost never manifest FMR related clinical features FMRP diagnostic testing is already recognized in the ACMG fragile X guidelines However reliable correlation between FMRP levels and patient diagnosis and prognosis has been undermined by both biological and technical issues There is a critical need for a well validated and standardized quantitative FMRP assay to support researchers and clinicians in their efforts to understand FXS biology and to aid in patient management Biological challenges to the reliable interpretation of FMR molecular tests include somatic mosaicism or the presence of varying genotypes and or methylation states in different tissues Importantly case studies have revealed tissue specific differences for fragile X mutations wherein buccal cells that share an ectodermal developmental lineage with brain tissue may be more clinically informative than blood We will test the feasibility of using BCs to assay FMRP because they may better represent fragile X biology in the brain Technical challenges include pre analytical limitations substandard antibodies and the lack of quantitative FMRP standards Recently an FMRP assay was published that addresses these issues including compatibility with dried blood spots DBS and a workflow on the Luminex platform that is amendable to routine testing Our overall objective is to make improvements to this assay by reducing sources of variation validating the test with annotated clinical samples and then launching the assay for the analysis of blood and buccal cells stabilized on andquot andquot paper This technology will enable the accurate quantification of FMRP and FMR genotyping from a single sample thereby simplifying the collection of a comprehensive FXS dataset enabling diagnostic and prognostic applications and improving our overall understanding of FXS biology Specific Aim Complete a multi site validation of the published assay using common reagents and samples Specific Aim Develop the protocols and reagents to normalize for the FMRP producing blood cells in a DBS sample Specific Aim Demonstrate the feasibility of using buccal cells stored on papers for assaying FMRP and perform a pilot clinical study PUBLIC HEALTH RELEVANCE We are developing a test to improve the diagnosis and screening for fragile X syndrome and other related disorders Fragile X is one of the most commonly inherited forms of mental retardation and can also cause conditions such as ADHD and autism Alterations in the fragile X gene are associated with changes in the fragile x protein The test detects this protein from a small drop of blood spotted on paper to support diagnosis and to help guide decisions for current and emerging treatments