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 FAN1, a 5' flap endonuclease with 5'-3' exonuclease activity, protects against repeat expansion in our mouse model. This is consistent with Genome Wide Association data that identified single-nucleotide polymorphisms in the vicinity of FAN1 as being associated with variability in the age at onset of a variety of diseases thought to share a common mutational mechanism. Our data suggest that variations in FAN1 may be a factor that modifies disease risk in the FXDs. We have also shown that expansions are confined to the early gamete in males, while expansions can occur in the postnatal oocyte as well as in the very early embryo. Since we have previously shown that the repeats are difficult to replicate (Woodford and Usdin, 1995; Yudkin et.al., 2014), it may be that expansions occurring in the non-dividing oocyte are less likely to contract than expansions in sperm. If correct, this could provide an explanation of why large expansions are only maternally transmitted and why males with full mutations in their somatic cells only have premutations in their gametes.
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