Apparently Unstable Normal FMR1 Alleles in Nine Developmentally Delayed Patients: Implications for Molecular Diagnosis of the Fragile X Syndrome

Abstract

The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.

Extra Alleles in FMR1 Triple-Primed PCR

“…In 2000, nine males with developmental delay were reported to have two normal alleles by FXS PCR. 41 These boys had normal karyotypes, and typing of FMR1 flanking microsatellites revealed a single FMR1 locus in their genomes.…”

CpG Methylation Modifies the Genetic Stability of Cloned Repeat Sequences

“…Tracts >33 pure repeats are unstable. Length heterogeneity is routinely observed in the expanded (n = 200-1000) repeats of FRAXA patients (Wohrle et al 1998), as well as in premutation individuals (Nolin et al 1999) and rare cases of length mosaicism within normal range (n = 29-51) that have been reported as having apparent somatic deletions (n = 11-34) (Brown and Nolin 2000;Tzountzouris et al 2000). "Highfunctioning" FRAXA patients display high levels of CGG length heterogeneity, with alleles lacking aberrant CpG methylation frequently containing 130 to 300 repeats, whereas longer tracts within the same tissue were methylated (Wohrle et al 1998).…”