Molecular analysis of mutations in the gene FMR-1 segregating in fragile X families

Abstract

Molecular genetic analysis of the transmission of mutations in 73 families with fragile X (one of the largest samples evaluated so far) has confirmed previous hypotheses that the fragile X syndrome results from two consecutive mutational steps, designated "premutation" and "full fragile X mutation". These mutations give rise to expansions of restriction fragments, most probably by amplification of the FMR-1 CGG repeat. Premutations are identified by small expansions that apparently have no effect on either the clinical or the cellular phenotype. Full mutations are reflected by large expansions and hypermethylation of the expanded gene region. All males showing large expansions were affected. Individuals with full mutations also expressed the fragile X, with only one exception. An affected "mosaic" male, showing a predominance of premutated fragments in his leukocytes, was shown to be fragile-X-negative on different occasions. About 50% of heterozygotes with full mutations were reported by clinicians to be mentally retarded. Conversion of the premutation to the full mutation may occur at oogenesis, as previously suggested, or after formation of a zygote at an early transitional stage in development when the CGG repeat behaves as a mitotically unstable element on maternally derived/imprinted X chromosomes carrying a premutation of sufficient repeat length.

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

“…Aliquots (20 μg) were cleaved with restriction endonuclease Eco RI plus Eag I or with Pst I, size separated by electrophoresis through 0.8% agarose gels, blotted onto a positively charged nylon membrane, and hybridised to [α- 32 P]dCTP oligolabelled probes Ox0.55 or Ox1.9, respectively, as described previously 6. Expansion size was measured as CGG repeat index50 given by the difference in size (base pairs) of normal and mutant bands, dividing by 3, and adding 30, the most common CGG repeat number of normal alleles in the German population.…”

Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats.

“…This results in downregulation of transcription8 and absence of FMR1 protein (FMRP) in cells normally expressing the FMR1 gene. "-1' Most patients with full mutations have somatic mosaicism of the repeat expansion,12 13 and some show mosaicism of methylation in that a proportion of cells have an unmethylated promoter allowing for apparently normal transcription of the FMR1 gene despite the repeat expansion.' 4 15 In the latter cases, however, expression of FMRP is markedly reduced in the presence of more than 200 CGGs, probably because of an impediment of the linear migration of the 40S ribosomal subunit along the 5' untranslated mRNA sequence by trinucleotide expansion.…”

“…DNA was digested with HindIII (1-4,[10][11]14), EcoRI + EagI (5-9), or PstI(12,13,15) and hybridised to probe OxO.55. The lanes are as follows: 1, 2, 5, 6: GZ (family A, II.3); 3, 7:fragile X male with methylation mosaicism offull mutations; 4, 8: controlfemale; 9, 11, 12: KK (family B, II.…”

Clinical utility gene card for: fragile X mental retardation syndrome, fragile X-associated tremor/ataxia syndrome and fragile X-associated primary ovarian insufficiency

“…The mitotic instability of the repeat of the full mutation in somatic cells in early embryogenesis before the methylation of expanded CGG repeat leading to their stabilization [80][81][82] causes somatics mosaicism in most individuals. 83 As the expansion size of a methylated full mutation does not have an influence on the severity of the clinical phenotype, 36,84 somatic mosaicism is of no consequence for the clinical expression when the expansions are in the full mutation range. There are, however, two special subclasses of mosaicism based on size and methylation status.…”