Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: Implications for molecular diagnosis of the Fragile X syndrome

The Fragile X syndrome is a common form of X-linked mental retardation, aff ecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosi s of Fragile X syndrome has become the gold standard. Numerous molecular di agnostic centers worldwide use PCR and Southern blotting to characterize th e size of the CGG repeats within the gene, expansion of which has been show n to be associated with the vast majority of cases of Fragile X syndrome. I nstability of this repeat through successive generations has been demonstra ted in many patients and has been associated with numerous factors, includi ng repeat length and molecular structure of the repeat. Nine males with nor mal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripher al lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecu lar causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exh ibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an int eresting molecular phenomenon and raise many questions relating to the fact ors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.