The overall objective for this project is to develop and implement a comprehensive set of technologies to improve screening and diagnosis of conditions associated with Fragile X Syndrome (FXS). FXS is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 5'-untranslated region of the FMR1 gene. FXS affects 1/4000 men and 1/6000 women. Full expansion to greater than 200 repeats is associated with hypermethylation of the FMR1 gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats is associated with Fragile X-associated Tremor/Ataxia Syndrome (FX-TAS) in older men and primary ovarian insufficiency (FX-POI) in women. Recently published guidelines suggest follow-up chromosome and Fragile X testing for a diagnosis of autism spectrum disorder (ASD), which impacts 1/150 individuals-roughly 33 times the population incidence of Fragile X. Furthermore, promising drugs are currently in clinical trials and will require accurate and early identification of Fragile X patients. Improvements in testing for Fragile X will have important implications for a broad range of individuals of all ages across multiple mental and health conditions associated with this disorder. The current diagnostic approach for Fragile X testing is to perform PCR, followed by Southern blot as necessary, to determine the CGG repeat number and FMR1 methylation state. However, a major limitation of PCR is the inability to amplify full mutations and even many pre-mutation alleles. All current PCR methods lack the ability to resolve homozygosity. As a result, nearly 50% of clinical laboratories currently reflex all samples to the more expensive, laborious, and low-throughput Southern blot assay. Therefore, despite strong arguments for population-based screening for FXS (either newborn and/or carrier screening), the inability to reliably amplify full mutation repeat expansions makes wide-spread screening too expensive and inaccurate for implementation. Asuragen has undertaken an effort to improve the amplification of long GC-rich repeats in order to better serve the important clinical goals of robust diagnosis and screening for Fragile X mutations. In our preliminary data, we now demonstrate PCR amplification of up to at least ~1000 CGG repeats, the longest repeat length that is commercially available or published. This capability is the foundation for a PCR-based assay that can report both premutation and full mutation alleles and dramatically reduce or eliminate the number of cases reflexed to Southern blots. In this phase I SBIR project, we will establish efficient, automatable testing, develop a PCR_based test to resolve zygosity in female samples, and develop a PCR-based method for determining methylation status of the FMR1 gene. This will result in a diagnostic workflow that can support routine screening, and thus enable earlier interventions and improved treatment options for patients.
Public Health Relevance: The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.
Public Health Relevance Statement: Project Narrative The long-term goal for this project is to improve screening and diagnosis of Fragile X Syndrome and related conditions. In this project, we will leverage our breakthrough in PCR of the FMR1 gene to develop a set of robust and accurate tests that are cost-effective and efficient. This will enable widespread screening to identify carriers and permit earlier diagnosis and intervention for Fragile X Syndrome patients.
Project Terms: 0-6 weeks old; 2(1H)-Pyrimidinone, 4-amino-; 2-Amino-6-Hydroxypurine; 5' Untranslated Regions; 5'UTR; 6H-Purin-6-one, 2-amino-1,7-dihydro-; Active Follow-up; Address; Affect; Age; Alleles; Allelomorphs; Assay; Bioassay; Biologic Assays; Biological Assay; Blinded; Blood; Blotting, Southern; Chromosomes; Clinical; Clinical Trials; Clinical Trials, Unspecified; Cytosine; DNA; DNA blotting; Data; Deoxyribonucleic Acid; Detection; Development; Diagnosis; Diagnostic; Disease; Disorder; Drugs; Early Diagnosis; Early identification; Early treatment; Environment; Escalante syndrome; FMR-1 Protein; FMR1; FMR1 Gene; FMR1 Protein; FMR1 protein, fragile X; FMRP protein; FXTAS; Female; Fmr1 gene,; Fmr1,; Foundations; Fragile X; Fragile X Mental Retardation 1 Gene; Fragile X Mental Retardation Protein; Fragile X Syndrome; Fragile X premutation-associated tremor ataxia syndrome; Genetic Alteration; Genetic Change; Genetic defect; Goals; Guanine; Guidelines; Hypermethylation; Incidence; Individual; Infant, Newborn; Intervention; Intervention Strategies; Laboratories; Length; Martin-Bell Syndrome; Martin-Bell-Renpenning syndrome; Medication; Mental Health; Mental Hygiene; Mental Retardation; Method LOINC Axis 6; Methodology; Methods; Methylation; Monosomy X; Morgagni-Turner syndrome; Morgagni-Turner-Albright syndrome; Mutation; Newborn Infant; Newborns; Patients; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Phenotype; Population; Production; Protein Methylation; Psychological Health; Publishing; Reagent; Reflex; Reflex action; Renpenning syndrome 2; Reporting; Research Specimen; Resolution; Reticuloendothelial System, Blood; SBIR; SBIRS (R43/44); Sampling; Screening procedure; Shereshevskii-Turner syndrome; Small Business Innovation Research; Small Business Innovation Research Grant; Southern Blotting; Specimen; Spottings; Technology; Testing; Time; Translations; Tremor/Ataxia Syndrome; Trinucleotide Repeats; Triplet Repeats; Turner syndrome (TS); Turner's Syndrome; Turner-Albright syndrome; Ullrich-Turner syndrome; Ullrich-Turner syndrome (UTS); Woman; X-linked mental deficiency-megalotestes syndrome; X-linked mental retardation with fragile X syndrome; X-linked mental retardation-fragile site 1 syndrome; XO syndrome; autism spectrum disorder; autism-fragile X (AFRAX) syndrome; autism-fragile X syndrome; base; chromosome XO syndrome; clinical investigation; codon reiteration; cost; disease/disorder; drug/agent; early detection; follow-up; fra(X) syndrome; fra(X)(28) syndrome; fra(X)(q27) syndrome; fra(X)(q27-28) syndrome; fragile X associated tremor ataxia syndrome; fragile X mental retardation 1; fragile X mental retardation-1 protein; fragile X-associated tremor/ataxia syndrome; fragile X-mental retardation syndrome; fragile Xq syndrome; fragile site mental retardation 1; fragile x [{C0016667}]; fragile x syndromes; genital dwarfism; genome mutation; improved; interventional strategy; mRNA Leader Sequences; macro-orchidism-marker X (MOMX) syndrome; macro-orchidism-marker X syndrome; mar(X) syndrome; marker X syndrome; men; men's; mental retardation-macroorchidism syndrome; monosomy X syndrome; newborn human (0-6 weeks); older men; ovarian dwarfism; ovarian short stature syndrome; population based; primary ovarian insufficiency; pseudonuchal infantilism; public health relevance; screening; screenings
