The overall goal of this project is to develop a cost-effective multiplexed diagnostic test for spinal muscular atrophy (SMA) to rapidly and accurately analyze multiple classes of disease-causing, disease-modifying DNA variants, and carrier status markers. Ultimately, the proposed test will have a far-reaching impact by expanding the identification and stratification of patients who may benefit from new treatments, and by improving the detection of SMA carriers. The proposed test will detect an expanded panel of variants that are not assessed by conventional testing. The broader content is important because age of onset, disease severity and progression correlations with the nature of the SMN1 mutations, and the compensatory and modifying effects of SMN2 and other genes. The expanded SMA test will not only extend the molecular testing to benefit more prospective parents and patients with atypical symptoms including adult onset cases, but will also address SMA testing needs of underserved populations. The expanded SMA test panel will build upon Asuragenâs AmplideX® PCR/CE SMN1/2 Kit (RUO and CE- IVD) and take advantage of a novel strategy Multiplexed & Efficient Resolution of Genetic Elements (MERGE)- PCR to assess: 1) copy number variants (CNVs) of SMN1 and SMN2, 2) pathogenic mutations in SMN1, 3) disease-modifying mutations, and 4) silent carrier markers. We will also incorporate a novel gene-tagged genotyping strategy to determine whether a mutation is in the SMN1 or SMN2 gene. The specific aims are: Aim 1. Design and optimize individual assays for a unified multiplex MERGE-PCR/CE test to detect multiple classes of disease-causing, disease-modifying DNA variants and carrier markers. Aim 2. Develop algorithms to support assay signal deconvolution and enable accurate genotype classification of CNV, SNV, and INDEL signals from multiple fluorophore channels. Aim 3. Develop and verify a single-tube multiplex PCR-based test. In Phase II, we will advance the technology into a cGMP kit development process, enable market-ready software for automatic variant calling, and develop controls and standards for an in vitro diagnostic product. The project will benefit from Asuragenâs years of experience optimizing multiplexing PCR chemistries to develop and commercialize high-performance diagnostic PCR/CE assays.
Public Health Relevance Statement: Project Narrative We propose to develop a novel diagnostic test kit for spinal muscular atrophy that will support informed treatment decisions for more patients. The assay has a novel design approach that will enable the detection of a broad panel of genetic variants and the format will make broad, reliable, and cost effective testing readily accessible through most clinical laboratories.
NIH Spending Category: Bioengineering; Biotechnology; Genetic Testing; Genetics; Neurodegenerative; Neurosciences; Pediatric; Rare Diseases; Spinal Muscular Atrophy
Project Terms: Address; Adult; African American; Age of Onset; Algorithms; Alleles; Anterior; Asians; base; Biological Assay; Capillary Electrophoresis; carrier status; Cell Line; Cells; Cessation of life; Chemistry; Classification; Clinical; Computer software; Copy Number Polymorphism; cost effective; cost effectiveness; Counseling; Cyclic GMP; design; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; digital; Disease; Disease Progression; DNA; Early Diagnosis; experience; Family history of; fluorophore; follower of religion Jewish; Genes; Genetic; genetic element; genetic variant; Genotype; Goals; Horns; Hour; improved; In Vitro; Individual; infant death; instrument; Laboratories; loss of function mutation; Methods; Modeling; Molecular; molecular diagnostics; motor function improvement; Motor Neurons; Mutation; Nature; next generation sequencing; novel; novel diagnostics; novel strategies; outcome forecast; Parents; Pathogenicity; patient stratification; Patients; Performance; Phase; Population; Prenatal care; Process; prospective; prospective test; Proteins; Reporting; Resolution; Respiratory Failure; Running; Sampling; screening; Severities; Severity of illness; signal processing; Signal Transduction; skeletal muscle wasting; SMN2 gene; Spinal cord grey matter structure; Spinal Muscular Atrophy; survival motor neuron gene; Symptoms; synthetic construct; Technology; Testing; Tube; Underserved Population; Variant; Whole B
