![]() ![]() In contrast, for recessive diseases, only couples in which both parents are carriers are at risk. 9, 10 Because FXS is an X-linked disorder, every woman found to be a carrier is at risk for having affected offspring regardless of the genotype of her husband. Observed carrier frequencies for FXS vary by population with 1:113 in Israel, 7 1:259 in Quebec, Canada, 8 and 1:317–1:382 in the United States. Several studies have examined the carrier frequency of expanded alleles (≥55 repeats) in various populations. Thirty to 40% of male carriers of a premutation allele will suffer from fragile X-associated tremor and ataxia (FXTAS) by age 50 years, 4, 5 whereas 20% percent of female premutation carriers are affected with premature ovarian insufficiency (POI). FXS is diagnosed more frequently in males than females. ![]() Men with FXS have profoundly delayed speech, mild to moderate mental retardation, and distinctive physical and behavioral traits. Symptoms are usually milder in affected females and may consist only of attention deficit disorder or personality disorder. ![]() ![]() Premutation alleles are defined as 55-200 CGG. Intermediate alleles of between 45 and 54 repeats almost never expand to full mutations in a single meiosis. The American College of Medical Genetics 2, 3 defines a normal repeat length as between 5 and 44. Methylation of the expanded CGG tract leads to silencing of expression of the FMR1 gene. 1 The disease is caused by the expansion of a trinucleotide CGG repeat in the 5′-untranslated region of the FMR1 gene. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals.įragile X syndrome (FXS, OMIM 300624) is the most common inherited mental retardation syndrome, affecting ∼1:4000 men and ∼1:8000 women. The method distinguishes normal homozygous females from full mutation carrying females. This method also detected the full mutation alleles in DNA isolated from blood spots.Ĭonclusion: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths ≥55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Results: A distinctive pattern of tapering or “stutter” polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Amplicons are resolved by capillary electrophoresis. Methods: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths >200. Purpose: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5′-untranslated region. ![]()
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