Mutation spectra in fragile X syndrome induced by deletions of CGG*CCG repeats

Abstract

The fragile X syndrome results from expansions as well as deletions of the repeating CGG.CCG DNA sequence in the 5'-untranslated region of the FMR1 gene on the X chromosome. The relative frequency of disease cases promoted by these two types of mutations cannot be ascertained at present because the routine clinical assay monitors only expansions. At least 30 articles have been reviewed that document the involvement of deletions of part or all of the CGG.CCG repeats along with varying extents of DNA flanking regions as well as very small mutations including single base pair changes. Studies of deletion mutants of CGG.CCG tracts in Escherichia coli plasmids revealed a similar spectrum of mutagenic products. The triplet repeat tract in a non-B conformation is the mutagen, not the sequence per se in the right-handed B helix. Hence, molecular investigations in a simple model organism may generate useful initial information toward therapeutic strategies for this disease.

FIGURE 1.

Mutagenic spectra found in fragile X clinical phenotypes. This schematic diagram is not drawn to scale.

FIGURE 2.

Upper panel, restriction maps of the mutant clones induced by long tracts of CGG·CCG repeats in plasmids in E. coli. Amp^R, ampicillin resistance gene; Ori, pUC19 origin of replication; Ter, transcription terminator cassette; GFP, LacZ-green fluorescent protein fusion gene; CGG·CCG, (CGG·CCG)_n tract where n = 0 (control), 24, 44, and 73 repeats. The CGG·CCG tracts were cloned into the pGFPT vector. Microinsertions are designated with asterisks. For further details, see Fig. 2 in Ref. . kbp, kilobase pairs. Lower panel, non-B DNA structures predicted from the sequences flanking the healed junctions of clones with single (clone 54) or double (clone 18) deletions. The sequences read from the 5′- to the 3′-ends of the top strands. The arrows show the directions of the sequences that were deleted, and the numbers at these arrows designate their healed junction positions. Nucleotides in shaded boxes indicate homology at breaks identified by the DNA sequencing data. The dashed lines between the nucleotides present the continuous intervening sequences. However, the slipped structures and the deletions may be anywhere inside the CGG·CCG repeat tracts. The numbers beside the lines indicate the base pairs with direct repeat homology shown for both DNA strands, and the numbers above the boxes present base pairs with inverted repeat homology. Clone 54 shows a cruciform and a slipped strand structure, whereas clone 18 derived from pRW5501 presents a cruciform and two slipped structures. These conformations are representative of the types of non-B structures found. Other types of non-B structures are found at other breakpoint junctions (46, 47, 50). For further details, see supplemental Fig. 1 in Ref. .