The fragile X locus is prone to spontaneous DNA damage that is preferentially repaired by nonhomologous end-joining to preserve genome integrity.

A long CGG-repeat tract in the FMR1 gene induces the epigenetic silencing that causes fragile X syndrome (FXS). Epigenetic changes include H4K20 trimethylation, a heterochromatic modification frequently implicated in transcriptional silencing. Here, we report that treatment with A-196, an inhibitor of SUV420H1/H2, the enzymes responsible for H4K20 di-/trimethylation, does not affect FMR1 transcription, but does result in increased chromosomal duplications. Increased duplications were also seen in FXS cells treated with SCR7, an inhibitor of Lig4, a ligase essential for NHEJ. Our study suggests that the fragile X (FX) locus is prone to spontaneous DNA damage that is normally repaired by NHEJ. We suggest that heterochromatinization of the FX allele may be triggered, at least in part, in response to this DNA damage.

Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome.

The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.

Genomic and evolutionary classification of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.

Hard wiring of normal tissue-specific chromosome-wide gene expression levels is an additional factor driving cancer type-specific aneuploidies.

BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.

A MutSß-Dependent Contribution of MutSα to Repeat Expansions in Fragile X Premutation Mice?

The fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5' UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutSß, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutSα also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in Msh6-/- mice. This effect is not mediated via an indirect effect of the loss of MSH6 on the level of MSH3. However, since MutSß is required for 98% of germ line expansions and almost all somatic ones, MutSα is apparently not able to efficiently substitute for MutSß in the expansion process. Using purified human proteins we demonstrate that MutSα, like MutSß, binds to substrates with loop-outs of the repeats and increases the thermal stability of the structures that they form. We also show that MutSα facilitates binding of MutSß to these loop-outs. These data suggest possible models for the contribution of MutSα to repeat expansion. In addition, we show that unlike MutSß, MutSα may also act to protect against repeat contractions in the Fmr1 gene.

Heterozygosity for a hypomorphic Polß mutation reduces the expansion frequency in a mouse model of the Fragile X-related disorders.

The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polß, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.

Repeat-mediated genetic and epigenetic changes at the FMR1 locus in the Fragile X-related disorders.

The Fragile X-related disorders are a group of genetic conditions that include the neurodegenerative disorder, Fragile X-associated tremor/ataxia syndrome (FXTAS), the fertility disorder, Fragile X-associated primary ovarian insufficiency (FXPOI) and the intellectual disability, Fragile X syndrome (FXS). The pathology in all these diseases is related to the number of CGG/CCG-repeats in the 5' UTR of the Fragile X mental retardation 1 (FMR1) gene. The repeats are prone to continuous expansion and the increase in repeat number has paradoxical effects on gene expression increasing transcription on mid-sized alleles and decreasing it on longer ones. In some cases the repeats can simultaneously both increase FMR1 mRNA production and decrease the levels of the FMR1 gene product, Fragile X mental retardation 1 protein (FMRP). Since FXTAS and FXPOI result from the deleterious consequences of the expression of elevated levels of FMR1 mRNA and FXS is caused by an FMRP deficiency, the clinical picture is turning out to be more complex than once appreciated. Added complications result from the fact that increasing repeat numbers make the alleles somatically unstable. Thus many individuals have a complex mixture of different sized alleles in different cells. Furthermore, it has become apparent that the eponymous fragile site, once thought to be no more than a useful diagnostic criterion, may have clinical consequences for females who inherit chromosomes that express this site. This review will cover what is currently known about the mechanisms responsible for repeat instability, for the repeat-mediated epigenetic changes that affect expression of the FMR1 gene, and for chromosome fragility. It will also touch on what current and future options are for ameliorating some of these effects.

X inactivation plays a major role in the gender bias in somatic expansion in a mouse model of the fragile X-related disorders: implications for the mechanism of repeat expansion.

The Fragile X-related disorders are X-linked disorders resulting from the inheritance of FMR1 alleles with >54 CGG/CCG repeats in their 5' UTR. The repeats expand both somatically and on intergenerational transmission and increased repeat numbers are associated with increased risk of disease and increased risk of further expansion. The mechanism responsible for expansion is unknown. Here, we show in a knockin mouse model of these disorders that somatic expansion is much less common in females than in males. We show that this is due in large part to the fact that expansions occur only when the repeat is on the active X chromosome. However, even when this is taken into account, expansions in females are still less common than expected. This additional gender effect is not due to a protective effect of estrogen, a deleterious effect of testosterone or to differences in the expression of the Fmr1 gene or a variety of X-linked and autosomal DNA repair genes. However, our data do suggest that a higher level of expression of genes that protect against oxidative damage in females may contribute to their lower levels of expansion. Whatever the basis, our data suggest that the risk for somatic expansion may be lower in women than it is in men. This could help explain the reduced penetrance of some aspects of disease pathology in women. The fact that expansion only occurs when the Fmr1 allele is on the active X chromosome has important implications for the mechanism of repeat expansion.

The mismatch repair protein MSH2 is rate limiting for repeat expansion in a fragile X premutation mouse model.

Fragile X-associated tremor and ataxia syndrome, Fragile X-associated primary ovarian insufficiency, and Fragile X syndrome are Repeat Expansion Diseases caused by expansion of a CGG•CCG-repeat microsatellite in the 5 UTR of the FMR1 gene. To help understand the expansion mechanism responsible for these disorders, we have crossed mice containing∼147 CGG•CCG repeats in the endogenous murine Fmr1 gene with mice containing a null mutation in the gene encoding the mismatch repair protein MSH2. MSH2 mutations are associated with elevated levels of generalized microsatellite instability. However, we show here for the first time that in the FX mouse model, all maternally and paternally transmitted expansions require Msh2. Even the loss of one Msh2 allele reduced the intergenerational expansion frequency significantly. Msh2 is also required for all somatic expansions and loss of even one functional Msh2 allele reduced the extent of somatic expansion in some organs. Tissues with lower levels of MSH2 were more sensitive to the loss of a single Msh2 allele. This suggests that MSH2 is rate limiting for expansion in this mouse model and that MSH2 levels may be a key factor that accounts for tissue-specific differences in expansion risk.

Somatic expansion in mouse and human carriers of fragile X premutation alleles.

Repeat expansion diseases result from expansion of a specific tandem repeat. The three fragile X-related disorders (FXDs) arise from germline expansions of a CGG•CCG repeat tract in the 5' UTR (untranslated region) of the fragile X mental retardation 1 (FMR1) gene. We show here that in addition to germline expansion, expansion also occurs in the somatic cells of both mice and humans carriers of premutation alleles. Expansion in mice primarily affects brain, testis, and liver with very little expansion in heart or blood. Our data would be consistent with a simple two-factor model for the organ specificity. Somatic expansion in humans may contribute to the mosaicism often seen in individuals with one of the FXDs. Because expansion risk and disease severity are related to repeat number, somatic expansion may exacerbate disease severity and contribute to the age-related increased risk of expansion seen on paternal transmission in humans. As little somatic expansion occurs in murine lymphocytes, our data also raise the possibility that there may be discordance in humans between repeat numbers measured in blood and that present in brain. This could explain, at least in part, the variable penetrance seen in some of these disorders.

Chromatin changes in the development and pathology of the Fragile X-associated disorders and Friedreich ataxia.

The Fragile X-associated disorders (FXDs) and Friedreich ataxia (FRDA) are genetic conditions resulting from expansion of a trinucleotide repeat in a region of the affected gene that is transcribed but not translated. In the case of the FXDs, pathology results from expansion of CGG•CCG-repeat tract in the 5' UTR of the FMR1 gene, while pathology in FRDA results from expansion of a GAA•TTC-repeat in intron 1 of the FXN gene. Expansion occurs during gametogenesis or early embryogenesis by a mechanism that is not well understood. Associated Expansion then produces disease pathology in various ways that are not completely understood either. In the case of the FXDs, alleles with 55-200 repeats express higher than normal levels of a transcript that is thought to be toxic, while alleles with >200 repeats are silenced. In addition, alleles with >200 repeats are associated with a cytogenetic abnormality known as a fragile site, which is apparent as a constriction or gap in the chromatin that is seen when cells are grown in presence of inhibitors of thymidylate synthase. FRDA alleles show a deficit of the FXN transcript. This review will address the role of repeat-mediated chromatin changes in these aspects of FXD and FRDA disease pathology. This article is part of a Special Issue entitled: Chromatin in time and space.