Somatic Sequence Variation at the Friedreich Ataxia Locus Includes Complete Contraction of the Expanded GAA Triplet Repeat, Significant Length Variation in Serially Passaged Lymphoblasts and Enhanced Mutagenesis in the Flanking Sequence

Abstract

The vast majority of Friedreich ataxia patients are homozygous for large GAA triplet repeat expansions in intron 1 of the X25 gene. Instability of the expanded GAA repeat was examined in 23 chromosomes bearing 97-1250 triplets in lymphoblastoid cell lines passaged 20-39 times. Southern analyses revealed 18 events of significant changes in length ranging from 69 to 633 triplets, wherein the de novo allele gradually replaced the original over 1-6 passages. Contractions and expansions occurred with equal frequency and magnitude. This behavior is unique in comparison with other large, non-coding triplet repeat expansions [(CGG)(n)and (CTG)(n)] which remain relatively stable under similar conditions. We also report a rare patient who, having inherited two expanded alleles, showed evidence of contracted GAA repeats ranging from nine to 29 triplets in DNA from two independent peripheral blood samples. The GAA triplet repeat is known to adopt a triplex structure, and triplexes in transcribed templates cause enhanced mutagenesis. The poly(A) tract and a 135 bp sequence, both situated immediately upstream of the GAA triplet repeat, were therefore examined for somatic mutations. The poly(A) tract showed enhanced instability when in cis with the GAA expansion. The 135 bp upstream sequence was found to harbor a 3-fold excess of point mutations in DNA derived from individuals homozygous for the GAA triplet repeat expansion compared with normal controls. These data are likely to have important mechanistic and clinical implications.

Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length

“…Sizes of expanded GAA-TR alleles seen in early passages of lymphoblastoid cell lines when compared with the blood samples they were derived from are either not different in length (40%) or are shorter by ~100 triplets (60%). 20 This suggests that our estimate of a threshold length of ~400 triplets in the shorter of the two expanded GAA-TR alleles will likely be comparable to that seen in peripheral blood leucocytes. It also remains to be seen how well the expanded GAA-TR allele lengths as measured in lymphoblastoid cell lines or peripheral blood leucocytes correlate with those seen in disease-relevant tissues such as dorsal root ganglia (DRG).…”

The contribution of <i>cis</i>-elements to disease-associated repeat instability: clinical and experimental evidence

“…Instability in the EBV-transformed DM1 lymphoblastoid cells, which was expansion biased, could only be detected by the sensitive small-pool PCR method indicating that length changes were rare (Ashizawa et al, 1996). In contrast, EBV-transformed FRDA lymphoblastoid cells displayed frequent contractions and expansions of the GAA tract (Bidichandani et al, 1999). It is noteworthy that virally transformed cells and spontaneously transformed murine cells can display different mechanisms of replication, repair and epigenetic regulation compared to their progenitor cells (Meek et al, 1977;Oppenheim et al, 1981;Aiyar et al, 1998;Habib et al, 1999).…”

SCA10 and ATTCT repeat expansion: clinical features and molecular aspects

“…The large pentanucleotide repeat expansion could form a weak triplex, which might impose a structural constraint and suppress SCA10 transcription. This mechanism has been proposed for Friedreich's ataxia, where an intron GAA trinucleotide repeat interferes with transcription of the FRDA gene by DNA triplex formation (Ohshima et al, 1998;Bidichandani et al, 1999;Sakamoto et al, 1999). Alternatively, intron ATTCT expansion might compromise the processing efficiency of the SCA10 transcript.…”