Fragile X syndrome (FXS) is the most common form of inherited mental retardation after Down syndrome. The expansion of a CGG repeat, located in the 5'-untranslated region (5'-UTR) of the FMR1 (fragile X mental retardation) gene, leads to the hypermethylation of the repeat and the upstream CpG island. Methylation is associated with transcriptional silencing of the FMR1 gene. The lack of FMR1 protein is believed to be responsible for the typical physical and mental characteristics of the syndrome. To analyze the specific phenotype of that syndrome as well as possible associations between the phenotype and the genotype, we examined a group of 49 fragile X boys and a control group of 16 patients with tuberous sclerosis. To determine the cognitive and behavioral phenotype, the Kaufman Assessment Battery for Children (K-ABC), the Child Behavior Checklist (4/18), and a structured psychiatric interview (Kinder DIPS) were used. The genotype was analyzed by the Southern blot method. The phenotype of boys with FXS is characterized by a specific cognitive profile with strengths in acquired knowledge and in simultaneous processing. The psychiatric comorbidity is high and ADHD (attention deficit hyperactivity disorder), oppositional defiant disorder, enuresis, and encopresis predominate. In a group of 24 fragile X boys, no significant correlations between the specific aspects of the phenotype and the genotype were found.

The autonomous expansion of the unstable 5-d(CGG) n -3 repeat in the 5-untranslated region of the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. We have recently described the isolation of a protein p20-CGGBP that binds sequence-specifically to the double-stranded trinucleotide repeat 5-d(CGG)-3 (Deissler, H., Behn-Krappa, A., and Doerfler, W. (1996) J. Biol. Chem. 271, 4327-4334). We demonstrate now that the p20-CGGBP can also bind to an interrupted repeat sequence. Peptide sequence tags of p20-CGGBP obtained by nanoelectrospray mass spectrometry were screened against an expressed sequence tag data base, retrieving a clone that contained the full-length coding sequence for p20-CGGBP. A bacterially expressed fusion protein p20-CGGBP-6xHis exhibits a binding pattern to the double-stranded 5-d(CGG) n -3 repeat similar to that of the authentic p20-CGGBP. This novel protein lacks any overall homology to other known proteins but carries a putative nuclear localization signal. The p20-CG-GBP gene is conserved among mammals but shows no homology to non-vertebrate species. The gene encoding the sequence for the new protein has been mapped to human chromosome 3.DNA sequences containing trinucleotide repeats of the general sequence (CXG) n or (GAA) n appear to be genetically unstable. Amplifications of such repeats were found to be associated with several human diseases, e.g. fragile X (FraX) 1 syndrome, Huntington's disease, myotonic dystrophy, or Friedreich's ataxia (for reviews see Refs. 1 and 2). The number of repeats usually correlates with the severity of the disease.The FraX syndrome is associated with an amplification of the trinucleotide sequence 5Ј-d(CGG) n -3Ј in the 5Ј-UTR of the FMR1 (fragile X mental retardation) gene and extensive 5Ј-d(CG)-3Ј methylation in the repeat as well as in adjacent regions (3-7). As a consequence, the expression of the encoded protein FMRP is reduced or abolished. Fragile sites similar to this locus have been identified on several of the human chromosomes, and all sites characterized so far have shown an expansion of 5Ј-d(CGG) n -3Ј repeats suggesting an important structural function of this DNA sequence. Several studies have demonstrated that single-stranded oligodeoxyribonucleotides of the general sequence 5Ј-d(CXG) n -3Ј with X ϭ A or G can form stable secondary structures in vitro (8 -14). The mechanism of the amplification of unstable DNA sequences is not understood. Expansion might involve DNA slippage or unequal crossing over, but these models do not explain the observed phenomenon sufficiently (for review see Ref. 15). The function of the repeat itself is also unknown. A 5Ј-d(CGG) 4 -3Ј fragment in the rRNA gene promoter (16) and a 5Ј-d(CTG) 25 -3Ј element located in the promoter of the mouse growth inhibitory factor gene (17) are thought to function as regulatory elements.Genetic instability of long 5Ј-d(CXG) n -3Ј tracts with deletion products of triplet repeat sequences has also been observed in Esch...

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Fragile X syndrome (FXS) is the most common form of inherited mental retardation after Down syndrome. The expansion of a CGG repeat, located in the 5'-untranslated region (5'-UTR) of the FMR1 (fragile X mental retardation) gene, leads to the hypermethylation of the repeat and the upstream CpG island. Methylation is associated with transcriptional silencing of the FMR1 gene. The lack of FMR1 protein is believed to be responsible for the typical physical and mental characteristics of the syndrome. To analyze the specific phenotype of that syndrome as well as possible associations between the phenotype and the genotype, we examined a group of 49 fragile X boys and a control group of 16 patients with tuberous sclerosis. To determine the cognitive and behavioral phenotype, the Kaufman Assessment Battery for Children (K-ABC), the Child Behavior Checklist (4/18), and a structured psychiatric interview (Kinder DIPS) were used. The genotype was analyzed by the Southern blot method. The phenotype of boys with FXS is characterized by a specific cognitive profile with strengths in acquired knowledge and in simultaneous processing. The psychiatric comorbidity is high and ADHD (attention deficit hyperactivity disorder), oppositional defiant disorder, enuresis, and encopresis predominate. In a group of 24 fragile X boys, no significant correlations between the specific aspects of the phenotype and the genotype were found.

The isoschizomeric restriction endonucleases Fnu4HI and BsoFI cleave DNA at 5'-GCdecreasesNGC-3' sequences. Fnu4HI has been shown to be inhibited by 5'-CG-3'methylation in the sequences 5'-GmCGGC-3' or 5'-GCGGmCG-3'. We have now investigated the methylation sensitivity of BsoFI by testing its activity on plasmid DNA 5'-CG-3' methylated with the M.SssI DNA methyltransferase or on synthetic (CGG)n repetitive oligodeoxyribonucleotides which have been partly or completely C methylated. The data demonstrate that BsoFI cannot cleave at its recognition sequence when it is completely 5'-CG-3' methylated. These enzymes have proven to be useful in analyses of the methylation status in (CGG)n repeats of the human genome.