Retrospective fragile X study by capillary and agarose gel electrophoresis

Fragile X Syndrome (FxS) is the most common cause of inherited mental disability and autism spectrum disorder. Estimated incidence in males is 1 in 4000 and 1 in 7000 for females. The gene is on the distal end of the long arm of the X chromosome and contains a trinucleotide cytosine, guanine, guanine (CGG) microsatellite repeat in the 5' untranslated region (5'UTR) of the gene. When the repeat region is fully expanded the CpG island near the gene promoter site becomes methylated, and the result is silencing of the gene and loss of protein product. Health issues that correspond to a partially expanded trinucleotide repeat are Fragile X Tremor and Ataxia Syndrome (FXTAS) and Primary Ovarian Insufficiency (POI). Current molecular detection involves Polymerase Chain Reaction (PCR) amplification of the CGG region, followed by capillary electrophoresis (CE) separation and fluorescent detection of the PCR amplicon with very high sizing resolution for normal and lower range premutation repeats. For larger pre-mutation repeats and full mutations, detection is done with digestion of genomic DNA with methylation sensitive enzymes, followed by Southern blotting. The Southern blots have some significant disadvantages; they are labor intensive, require large amounts of genomic DNA, blot sizing of the CCG repeat region has very imprecise resolution, reagents are expensive and they have a long processing time. Fluorescent detection of PCR product by CE is faster, less expensive, and has iv much higher resolution, but it has not been useful for the detection of full mutations due to PCR inefficiency for large CGG repeats and difficulty of detecting a very large CG rich trinucleotide repeat PCR product. The purpose of this thesis is an overview of the current molecular paradigms of FxS, FXTAS and POI, molecular testing and a comprehensive evaluation of the Celera Fragile X assay and the possibility that use of their 1.5% agarose recipe, gel electrophoresis (GE) and a long injection capillary electrophoresis protocol can detect all premutation and full mutation samples and reduce the need for Southern blotting for molecular detection of full mutation patients.