Expansion andMethylation Status atFRAXECanBeDetected on EcoRlBlots UsedforFRAXADiagnosis: Analysis ofFourFRAXE Families withMildMentalRetardation inMales

Summary Theoriginal test fortheanalysis oftheCCGexpansion attheFRAXElocus involves Southern blot analysis of HindIl digests. We showthat, byusing adifferent probe, theFRAXEmutation canbedetected easily on thesameEcoRIorEagI+EcoRP blots asareusedfor detection ofFRAXA.Unexpectedly, wefound that both theexpansion andmethylation status canbedetermined onasingle EcoRIdigest, because ofthepresence ofa methylation-sensitive EcoRIsite veryclose totheCCG repeat. Wethus detected inaseries ofmentally retarded individuals previously tested forFRAXAexpansion a FRAXEproband wholedtotheidentification ofalarge sibship (7of10children carrying amutation). Wealso showthat twofragile Xfamilies without FRAXAmutation that previously havebeendescribed byOberle et al. havetheFRAXEexpansion. Inanother family also ascertained initially bycytogenetic finding ofafragile X site, weperformed thecombined cytogenetic andmolecular prenatal diagnosis ofamutated malefetus. All ninemales (>3years old) inwhomwefound amethylated mutation hadmild mental retardation. Ourresults suggest that thethreshold ofrepeat length forabnormal methylation andfragile-site expression maybesmaller atFRAXEthan atFRAXA.