Sequence analysis of the fragile X trinucleotide repeat: implications for the origin of the fragile X mutation

Abstract

This study addresses mechanism of instability of the FMR-1 (CGG)n-repeat, and investigates features which may distinguish between normal stable and fragile X unstable repeats. To achieve this, we have sequenced 178 alleles to analyze patterns of AGG interruptions within the CGG repeat, and have typed the (CA)n-repeat at DXS548 for 204 chromosomes. Overall, our data is consistent with the idea that the length of uninterrupted CGG repeats determines instability. We predict that certain sequence configurations [no AGG, and (CGG)9-11AGG(CGG) > or = 20] present in the general population, are predisposed towards replication slippage. Association between these proposed predisposing repeats and DXS548 alleles may explain the previously reported frequencies of fragile X mutations and large-size normal repeats on specific haplotype backgrounds. We propose that predisposing alleles arise in the general population by as yet undefined mechanism(s) which introduce a relatively long stretch of pure CGG repeat at the 3'-end (relative to the direction of transcription) of the FMR-1 repeat region. The 3' pure repeat may then be susceptible to further expansion by replication slippage. Slippage on these predisposing chromosomes could accumulate over many generations until a threshold size is reached, at which point the repeat is susceptible to greater instability (i.e. premutation stage). Thus, results suggest that evolution of fragile X full mutations could involve 4 definable stages: 1) ancestral events leading to the formation of predisposing alleles which have large total repeat length (e.g. between 35 to 50) but no AGG or 1 AGG; 2) gradual slippage of these predisposing alleles to small premutations (S alleles); 3) conversion from S alleles to larger premutations (Z); 4) massive expansion from a Z allele to a full mutation (L).

Distribution of AGG interruption patterns within nine world populations

“…In intermediate and premutation alleles the AGG interruptions tend to occur at the 5' end of the locus and the pure CGG stretch, defined as the longest stretch of uninterrupted CGG repeats, is located at the 3' end (8,9). The loss of AGG interruptions appear to have occurred multiple times during human evolution (10) but can be a late event in the mutation pathway that leads to expansion (11).…”

Analysis of FMR1 and flanking microsatellite markers in normal and fragile X chromosomes in Portugal: evidence for a “protector” haplotype

“…[3][4][5] The number of repeats is polymorphic in normal individuals, usually in the range of 6-60 triplets (with a mode of 30), frequently interrupted by 1-3 AGG triplets. [6][7][8] Premutation alleles of nonpenetrant carriers of the fragile X syndrome have between 60 and 200 repeats, which tend to increase in size when maternally transmitted to the offspring. The expansion of fragile X premutation alleles to a full mutation (CGG > 200) occurs exclusively during female transmission.…”

DNA Secondary Structures and the Evolution of Hypervariable Tandem Arrays

“…These changes tend to occur at one end of the array, and for this reason are said to show polarity. Tandem arrays are also prone to the acquisition of point mutations, and the distribution of these mutations shows a similar polarity (9,12,16,17). This has led to the suggestion that either flanking sequences are important in imparting polarity to an otherwise non-polar process (12) or a mechanism that has an inherent polarity such as replication slippage (16) is involved.…”