Background: The Repeat Expansion Diseases are caused by increases in the number of repeat units present in a specific tandem repeat. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor-ataxia syndrome (FXTAS), and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females, the PM allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation (FM) and, with very few exceptions, all individuals who inherit such alleles have Fragile X syndrome (FXS), the leading heritable cause of intellectual disability and autism. FXS symptoms arise because repeat expansion leads to gene silencing and the subsequent absence of FMRP, the FMR1 gene product, a protein important in many pathways including insulin signaling. The mechanism by which is expansion occurs is thought to differ from the generalized microsatellite instability (MSI) seen in many different cancers in that the instability is confined to a single genetic locus, it shows a strong expansion bias and our work has now shown that genes that normally protect against MSI are actually required to generate the FX mutation. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. There is reason to think that this fragile site is responsible for the high frequency loss of the affected chromosome resulting in Turner syndrome (45, X0) in female carriers of a FM allele. Progress report: Our work over the past few years has shown that components of 3 different DNA repair pathways that normally act to prevent mutations, are actually responsible for the mutation that results in the FXDs (Lokanga et. al., 2012; 2015; Zhao et. al., 2014; 2015; and Zhao and Usdin, 2015). In this reporting period, we have extended our studies to examine other proteins in these pathways in an attempt to better understand how normal DNA repair processes become subverted to produce these mutations. Amongst our findings in this reporting period is the discovery that MutSalpha, a protein involved in mismatch repair, plays a previously unsuspected role in the expansion process. It is directly responsible for 2% of expansions, but is also able to act in conjunction with a second mismatch repair protein, MutSbeta, to facilitate a much larger number of expansions. Some of this work was complicated by the fact that large FMR1 alleles are very difficult to analyze by PCR. The difficulty with PCR likely arises because, as we demonstrated many years ago, the repeats form very stable secondary structures that block DNA polymerases (Usdin and Woodford, 1995). We have now developed a robust and inexpensive PCR assay that is able to reliably analyze very large FMR1 alleles (Hayward et. al., 2016). In addition, AGG interruptions to the CGG repeat tract are commonly seen in humans, where they have been shown to be critical modulators of expansion risk. However, using published assays, the number of AGG interruptions can be difficult to determine unambiguously. We have now developed a simple assay to easily and reliably determine the number of AGG interruptions to the CGG-repeat tract (Hayward and Usdin, 2017). The assays we have developed may be useful both in the lab for research purposes and in clinic for risk assessment and new born screening programs.
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