Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases have been identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. The FXDs are a group of 3 different disorders: 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 and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency. Furthermore, in females, the premutation 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 and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is the leading heritable cause of intellectual disability. The mechanism by which is expansion occurs is unknown. It is thought to differ from the generalized microsatellite instability seen in many different cancers in that the instability is confined to a single genetic locus and shows a strong expansion bias. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. This site, which gives the disorder its name, is one of many fragile sites present on the human genome. These sites are prone to breakage and in some cases are associated with deleterious chromosome deletions and translocations. In the case of the FX fragile site, there is reason to believe that it 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: We have shown previously that the mismatch repair protein MSH2 is absolutely required for all intergenerational and somatic expansions in a knockin mouse model for these disorders (Lokanga, Zhao and Usdin, 2014). We also showed that expansion occurs independently of chromosomal duplication in a process that is dependent on transcription or the presence of the predisposed allele in a transcriptionally competent region of the genome (Lokanga, Zhao, Entezam and Usdin, 2014). However, this requirement for transcriptional competence does not reflect a role for Transcription Coupled Repair (TCR), a form of nucleotide excision repair that is confined to actively transcribed genes (Zhao and Usdin, 2014). In fact we have shown that although CSB, a protein that is essential for TCR, plays an auxiliary role in promoting expansions (Zhao and Usdin, 2014) it also protects the genome against expansions, presumably via its participation in another DNA repair pathway (Zhao and Usdin, 2015). In this reporting period we also showed that a hypomorphic mutation in Pol β, a key polymerase involved in Base Excision Repair (BER), reduces the expansion frequency. This implicates central events in BER in the expansion process. Since BER is the major pathway by which oxidative damage to DNA is repaired in mammalian cells, this observation is consistent with our earlier observation that oxidative stress increases expansion risk (Entezam et al., 2010). In the last reporting period we provided evidence that Fragile X chromosome fragility arises from a problem with replication through the repeat region (Yudkin et al., 2014). Since many of the repeats responsible for other Repeat Expansion Diseases, like the FX repeat, also impair replication in bacteria or yeast, it has long been debated whether these other repeats also result in chromosome fragility in human cells. The Frataxin (FXN) locus contains an expansion-prone GAA/TTC-repeat that causes the Repeat Expansion Disease, Friedreich ataxia (FRDA). We examined the FXN gene in normal and FRDA cells using fluorescent in situ hybridization in the presence and absence of an ATM inhibitor, since we had previously shown that the FX repeat forms fragile sites in the presence of this compound (Kumari et. al., 2009). Unexpectedly, we found a high frequency of chromosomal rearrangements even in normal cells without inhibitor (Kumari et. al., 2015). Bioinformatic analysis suggests that this region does not have some of the characteristics thought to confer fragility to other sites such as a high twist-flex ratio or a paucity of sites of origins of replication. Rather our data suggested that the proximity of this region to one of the human genomes largest stretch of pericentric heterochromatin may be responsible. While both normal and patient cells show a high frequency of past rearrangement events, only patient cells showed an increase in the presence of the inhibitor (Kumari et. al., 2015). This would be consistent with the expanded FXN locus being prone to breakage and rearrangements, one of the hallmarks of fragile sites. However, no gaps or constrictions of the chromosome that are diagnostic of fragile sites were observed in metaphase spreads. However, most fragile sites initiate replication late in the cell cycle, while replication of the FXN locus begins very early. Thus problems encountered during replication may be resolved by the time the cells reach metaphase, with the net result being that chromosome rearrangements rather than unrepaired chromosomes are apparent. Our data thus raise the possibility that other repeats responsible for some of the other Repeat Expansion Diseases may also confer fragility on the regions in which they are located.

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Zhao, Xiao-Nan; Lokanga, Rachel; Allette, Kimaada et al. (2016) A MutS?-Dependent Contribution of MutS? to Repeat Expansions in Fragile X Premutation Mice? PLoS Genet 12:e1006190
Hayward, Bruce E; Zhou, Yifan; Kumari, Daman et al. (2016) A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X-Related Disorders. J Mol Diagn 18:762-774
Zhao, Xiao-Nan; Usdin, Karen (2016) Ups and Downs: Mechanisms of Repeat Instability in the Fragile X-Related Disorders. Genes (Basel) 7:
Kumari, Daman; Hayward, Bruce; Nakamura, Asako J et al. (2015) Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia. Mutat Res 781:14-21
Zhao, Xiao-Nan; Usdin, Karen (2015) The Repeat Expansion Diseases: The dark side of DNA repair. DNA Repair (Amst) 32:96-105
Zhao, Xiao-Nan; Usdin, Karen (2015) The transcription-coupled repair protein ERCC6/CSB also protects against repeat expansion in a mouse model of the fragile X premutation. Hum Mutat 36:482-7
Usdin, Karen; House, Nealia C M; Freudenreich, Catherine H (2015) Repeat instability during DNA repair: Insights from model systems. Crit Rev Biochem Mol Biol 50:142-67
Lokanga, Rachel Adihe; Senejani, Alireza Ghodsi; Sweasy, Joann Balazs et al. (2015) Heterozygosity for a hypomorphic Pol? mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders. PLoS Genet 11:e1005181
Zhao, Xiao-Nan; Kumari, Daman; Gupta, Shikha et al. (2015) Muts? generates both expansions and contractions in a mouse model of the Fragile X-associated disorders. Hum Mol Genet 24:7087-96
Yudkin, Dmitry; Hayward, Bruce E; Aladjem, Mirit I et al. (2014) Chromosome fragility and the abnormal replication of the FMR1 locus in fragile X syndrome. Hum Mol Genet 23:2940-52

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