TY - JOUR
T1 - Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia
AU - Pollard, Laura M.
AU - Sharma, Rajesh
AU - Gómez, Mariluz
AU - Shah, Sonali
AU - Delatycki, Martin B.
AU - Pianese, Luigi
AU - Monticelli, Antonella
AU - Keats, Bronya J.B.
AU - Bidichandani, Sanjay I.
N1 - Funding Information:
We are grateful to the patients and their families for participating in this study. We would like to thank Dr Gillian Dalgliesh for critically reviewing this manuscript. This research was supported in part by grants from the NIH/NINDS (NS047596), American Diabetes Association and OCAST to S.I.B. S.S. was supported by a fellowship from the Presbyterian Health Foundation.
PY - 2004
Y1 - 2004
N2 - Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.
AB - Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)n sequences (9-105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8-107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.
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U2 - 10.1093/nar/gkh933
DO - 10.1093/nar/gkh933
M3 - Article
C2 - 15534367
AN - SCOPUS:10244264815
SN - 0305-1048
VL - 32
SP - 5962
EP - 5971
JO - Nucleic acids research
JF - Nucleic acids research
IS - 19
ER -