SBIR-STTR Award

Fragile X Syndrome (FRAX), is the most common cause of mental retardation in males. The incidence of FRAX is 1 per 4000 in males and 1 per 8000 in females. FRAX is caused by the expansion of a CGG trinucleotide repeat of the 5' untranslated region (UTR) of FMR1 gene. This gene is located at chromosome Xq27.3. In normal individuals, the 5' UTR of the FMR1 gene contains 5 to 45 CGG repeats. However, over 200 repeats are found in individuals with FRAX. Presently, Southern Blot analysis is used to examine the size of the repeat segment and methylation status of the FRAX gene. Yet, this test only detects the gross size of CGG repeats and is highly labor intensive and expensive. PCR analysis can be used to examine the size of CGG repeats. This approach is limited, as the PCR reaction fails to amplify long stretches of CGG expansions. Recently, we developed a novel, highly efficient, accurate, test for diagnosing FRAX. This test relies on amplifying CGG repeat expansions by Site Specific Multiple Displacement Amplification (SSMDA), which capably amplifies very long stretches of GC-rich regions in the genome. SSMDA is followed by quantitative assessment of the numbers of CGG triplet repeats using real-time Polymerase Chain Reaction. We hypothesize that using this new molecular-based screening method, we can develop an effective, rapid and low-cost screening test for FRAX with broad commercial application. We anticipate that this Phase 1 application will lead to the development of a new screening procedure for FRAX that is sensitive, accurate, inexpensive, and adaptable for high-throughput use. If successful, we anticipate these Phase 1 studies will lead to Phase 2 studies. If broadly applied, this strategy will be applicable for diagnosing carriers of FRAX and individuals with FRAX.

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Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. FRAX is caused by the expansion of a CGG trinucleotide repeat of the 5' untranslated region (UTR) of the FMR1 gene. In normal individuals, the 5' UTR of the FMR1 gene contains 5 to 45 CGG repeats; however, individuals with FRAX have over 200 repeats. Presently, Southern Blot analysis is used to determine the size of the repeat segment and methylation status of the FRAX gene. This test only detects the gross size of CGG repeats and is labor intensive and expensive. PCR and gel electrophoresis is typically used to determine the size of the CGG expansion. This approach is limited, as PCR reactions typically fail to amplify long stretches of CGG expansions (>25 repeats) and molecular weight determination by electrophoresis via capillary or slab gels is labor intensive. In our Phase I SBIR award, we developed a novel, highly efficient and accurate screening test for diagnosing FRAX. In Step 1, Whole Genome Multiple Displacement Amplification using 7-deaza-2-Guanosine (7-deaza GTP) nucleotide analog is incorporated into multiple copies of the CGG FMR1 expansion. In Step 2, Site Specific Multiple Displacement Amplification (SSMDA) using 7-deaza GTP is performed to weaken the GC base pairings, making the GCC expansion more accessible to Taq DNA Polymerase in real-time PCR. In Step 3, SSMDA is followed by quantitative assessment of the numbers of CGG triplet repeats using TaqMan real-time PCR without the need for sizing by gel electrophoresis or Southern blotting. We hypothesize that using this new molecular-based method, we can develop an effective, rapid and low-cost screening test for FRAX with broad commercial application. In this Phase II application we propose to: SA1: Optimize our assay for CGG repeat copy number determination using a large number of DNA samples with varying degrees of CGG repeats. SA2: Test our assay in a clinical FRAX diagnostic laboratory. SA3: Test our assay in blood spots for potential newborn screening. We anticipate that this Phase II application will lead to the optimization and implementation of a test that is suitable for low cost high-throughput screening for FRAX. As such, this test has the potential to markedly change how we currently screen for FRAX. These studies will involve the combined expertise of JS Genetics, which has been developing novel DNA-based diagnostic tests, and Dr. Bai-Lin Wu of Boston Children's Hospital and Harvard Medical School, who an extensive track record of developing clinical DNA diagnostic studies and in clinical laboratory testing of FRAX.

Public Health Relevance:
Fragile X Syndrome (FRAX) is the most common genetic cause of mental retardation in males. We propose the development of a new molecular-based method, for rapid and low-cost screening of FRAX with broad commercial application.

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0-11 Years Old; 21+ Years Old; 5' Untranslated Regions; 5'utr; Adult; Assay; Autism; Autism, Early Infantile; Autism, Infantile; Autistic Disorder; Award; Base Pairing; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Blood; Blood Capillaries; Blotting, Southern; Boston; Cgg Repeat; Cgg Repeat Expansion; Cannot Achieve A Pregnancy; Capillaries; Capillary; Capillary, Unspecified; Causality; Child; Child Youth; Children (0-21); Chromosomal Bands; Chromosome Band; City Of Boston; Clinical; Counselor; Dna; Dna Alteration; Dna Blotting; Dna Mutation; Deoxyribonucleic Acid; Development; Diagnosis; Diagnostic; Diagnostic Tests; Difficulty Conceiving; Disease; Disorder; Electrophoresis; Epidemiology, Family Medical History; Escalante Syndrome; Etiology; Fmr-1 Protein; Fmr1; Fmr1 Gene; Fmr1 Protein; Fmr1 Protein, Fragile X; Fmrp Protein; Fxtas; Family Medical History; Family History Of; Female; Fetus; Fmr1 Gene,; Fmr1,; Fractionation, Electrophoretic; Fragile X; Fragile X Gene; Fragile X Mental Retardation 1 Gene; Fragile X Mental Retardation Protein; Fragile X Syndrome; Fragile X Premutation-Associated Tremor Ataxia Syndrome; Future Generations; Gtp; Gel; Gene Alteration; Gene Mutation; Genes; Genetic; Genetic Mutation; Genetic Screening Method; Genome; Guanosine; Guanosine 5'-(Tetrahydrogen Triphosphate); Guanosine Triphosphate; High Throughput Assay; Human, Adult; Human, Child; Incidence; Individual; Infertility; Kanner's Syndrome; Laboratories; Language; Lead; Learning Disabilities; Learning Disability; Martin-Bell Syndrome; Martin-Bell-Renpenning Syndrome; Mental Retardation; Methods; Methylation; Molecular; Molecular Interaction; Molecular Weight; Motor; Neonatal Screening; Nerve Cells; Nerve Unit; Neural Cell; Neural Development; Neurocyte; Neurons; Newborn Infant Screening; Prov; Parkinsonian; Parkinsonian Condition; Parkinsonian Diseases; Parkinsonian Disorders; Parkinsonian Syndrome; Parkinsonism; Pb Element; Pediatric Hospitals; Pervasive Development Disorder; Pervasive Developmental Disorder; Phase; Primary Care; Primary Health Care; Primary Healthcare; Professional Counselor; Programs (Pt); Programs [publication Type]; Promoter Regions; Promoter Regions (Genetics); Promotor Regions; Promotor Regions (Genetics); Protein Methylation; Proteins; Provider; Reaction; Renpenning Syndrome 2; Reticuloendothelial System, Blood; Risk; Sbir; Sbirs (R43/44); Sampling; Screening Procedure; Sequence Alteration; Site; Small Business Innovation Research; Small Business Innovation Research Grant; Southern Blotting; Speech; Spottings; Stretching; Syndrome; Taq Dna Polymerase; Taq Polymerase; Taq1 Polymerase; Testing; Thermus Aquaticus Polymerase; Time; Translating; Translatings; Translations; Tremor; Trinucleotide Repeats; Triplet Repeats; Utrs; Untranslated Regions; Woman; X Chromosome; X-Linked Mental Deficiency-Megalotestes Syndrome; X-Linked Mental Retardation With Fragile X Syndrome; X-Linked Mental Retardation-Fragile Site 1 Syndrome; Adult Human (21+); Autism-Fragile X (Afrax) Syndrome; Autism-Fragile X Syndrome; Base; Capillary; Children; Codon Reiteration; Commercial Application; Cost; Design; Designing; Disease Causation; Disease Etiology; Disease/Disorder; Disease/Disorder Etiology; Disorder Etiology; Equilibration Disorder; 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; Gel Electrophoresis; Gene Product; Genetic Promoter Element; Genetic Testing; Heavy Metal Pb; Heavy Metal Lead; High Throughput Screening; Infertile; Language Translation; Mrna Leader Sequences; Macro-Orchidism-Marker X (Momx) Syndrome; Macro-Orchidism-Marker X Syndrome; Male; Mar(X) Syndrome; Marker X Syndrome; Medical Schools; Meetings; Mental Retardation-Macroorchidism Syndrome; Neurodevelopment; Neuronal; New Diagnostics; Newborn Screening; Next Generation Diagnostics; Novel; Novel Diagnostics; Nucleotide Analog; Pregnant; Prenatal Testing; Programs; Public Health Relevance; Rapid Method; Rapid Technique; Screening; Screenings; Tool; Unable To Bear Children; Youngster