Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure

The normal human FMR1 gene contains a genetically stable (CGG)(n) trinucleotide repeat which usually carries interspersed AGG triplets. An increase in repeat number and the loss of interspersions results in array instability, predominantly expansion, leading to FMR1 gene silencing. Instability is directly related to the length of the uninterrupted (CGG)(n) repeat and is widely assumed to be related to an increased propensity to form G-rich secondary structures which lead to expansion through replication slippage. In order to investigate this we have cloned human FMR1 arrays with internal structures representing the normal, intermediate and unstable states. In one replicative orientation, arrays show a length-dependent instability, deletions occurring in a polar manner. With longer arrays these extend into the FMR1 5'-flanking DNA, terminating at either of two short CGG triplet arrays. The orientation-dependent instability suggests that secondary structure forms in the G-rich lagging strand template, resolution of which results in intra-array deletion, These data provide direct in vivo evidence for a G-rich lagging strand secondary structure which is believed to be involved in the process of triplet expansion in humans.