Evolution of the Friedreich's ataxia trinucleotide repeat expansion: founder effect and premutations

Abstract

Friedreich's ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for approximately 17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through "premutation" intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.

Figure 1

Distribution of the GAA repeat sizes observed in control chromosomes. The GAA repeat is moderately polymorphic among normal alleles. The distribution is bimodal; most (83%) alleles contain around nine repeats (–12), and 17% are LN alleles of 16 repeats or more. Accurate sizing was determined on 26 SN and 65 LN GAA alleles by denaturing polyacrylamide gel migration.

Figure 2

Transition from premutation to pathological expansion documented by BsiHKAI Southern blot analysis. (A and B) Transition of paternal intermediate alleles (not in the disease-causing range) to pathological size expansions in offspring. In both families, haplotype analysis allowed us to exclude false paternity (data not shown). (A) In this family, the father has an allele of ≈60 repeats and his two children are homozygous for expansions in the pathological range. (B) Variable transmission of a premutation within a family. A patient homozygous for GAA expansion inherited an allele from his father carrying a 42 pure GAA repeat allele. A paternal aunt has a 38-GAA allele that increased to 62 GAA upon transmission to her son. Analysis was on a single blot, and the picture was cropped for clarity. (C) Stable transmission of the smallest pathological allele. The half black symbols correspond to the transmission of large expansions and the half shaded symbols to the small expansions, as demonstrated by haplotype segregation (not shown). The small expansion (arrow) was transmitted relatively stably to two siblings (lanes 2 and 3) carrying ≈112 repeats, to the third sister (deduced) and one of her daughters (lane 6) carrying ≈135 repeats. Lanes: 4 and 5, affected children homozygous for larger expansions, in the same family; 1, unrelated individual heterozygous for the GAA expansion; and 7, unrelated control. Samples were analyzed on two separate blots, and unrelated lines were cut out.

Figure 3

Localization of the markers used for haplotype analysis. Transcription map of the frataxin gene (6) and ZO2 gene (16) is represented. The GAA repeat in intron 1 is indicated (▿). The localization of the five polymorphic markers used for linkage disequilibrium analysis is indicated (arrows). FAD1 is located in a 5′ exon of the ZO2 gene (15). The exons and splicing pattern of the frataxin (X25) gene are represented.