title: Methylation mosaicism of 5'-(CGG)(n)-3' repeats in fragile X, premutation and normal individuals.
abstract: Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.
authors.affiliation: Institute of Genetics, University of Cologne, D-50931 Köln, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Genç
authors.forename: H
authors.initials: H
authors.lastname: Müller-Hartmann
authors.forename: M
authors.initials: M
authors.lastname: Zeschnigk
authors.forename: H
authors.initials: H
authors.lastname: Deissler
authors.forename: B
authors.initials: B
authors.lastname: Schmitz
authors.forename: F
authors.initials: F
authors.lastname: Majewski
authors.forename: A
authors.initials: A
authors.lastname: von Gontard
authors.forename: W
authors.initials: W
authors.lastname: Doerfler
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D004247:DNA / Q000097:blood; D019175:DNA Methylation; D004926:Escherichia coli; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D006580:Genetic Carrier Screening; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009030:Mosaicism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics*; D010375:Pedigree; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012016:Reference Values; D015183:Restriction Mapping; D018911:Trinucleotide Repeats; D014960:X Chromosome
chemical_list: D020121:5' Untranslated Regions; D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; D008156:Luciferases; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497683:FMR1 protein, human