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Microsatellite instability is responsible for the human repeat expansion diseases (REDs). The mutagenic process differs from classical cancer-associated microsatellite instability (MSI) in that it requires the mismatch repair proteins that normally protect against MSI. LIG4, an enzyme essential for non-homologous end-joining (NHEJ), the major pathway for double-strand break repair (DSBR) in mammalian cells, protects against expansion in mouse models. Thus, NHEJ may compete with the expansion pathway for access to a common intermediate. This raises the possibility that expansion involves an NHEJ-independent form of DSBR. Pol θ, a polymerase involved in the theta-mediated end joining (TMEJ) DSBR pathway, has been proposed to play a role in repeat expansion. Here we examine the effect of the loss of Pol θ on expansion in FXD mouse embryonic stem cells (mESCs), along with the effects of mutations in Rad52 , Rad54l and Rad54b , genes important for multiple DSBR pathways. None of these mutations significantly affected repeat expansion. These observations put major constraints on what pathways are likely to drive expansion. Together with our previous demonstration of the protective effect of nucleases like EXO1 and FAN1, and the importance of Pol β, they suggest a plausible model for late steps in the expansion process.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive neurological disorder, the clinical and immunological features of which parallel those of congenital viral infection. Here we define the composition of the human ribonuclease H2 enzyme complex and show that AGS can result from mutations in the genes encoding any one of its three subunits. Our findings demonstrate a role for ribonuclease H in human neurological disease and suggest an unanticipated relationship between ribonuclease H2 and the antiviral immune response that warrants further investigation.

Aicardi-Goutières syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection 1 , 2 . Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype 3 . Here, we show that TREX1 , encoding the major mammalian 3′ → 5′ DNA exonuclease 4 , is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1 −/− mouse leads to an inflammatory phenotype 5 . Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.

Carriers of the FMR1 premutation (PM) allele are at risk of one or more clinical conditions referred to as FX premutation-associated conditions (FXPAC). Since the FMR1 gene is on the X chromosome, the activation ratio (AR) may impact the risk, age of onset, progression, and severity of these conditions. The aim of this study was to evaluate the reliability of AR measured using different approaches and to investigate potential correlations with clinical outcomes. Molecular and clinical assessments were obtained for 30 PM female participants, and AR was assessed using both Southern blot analysis (AR-Sb) and methylation PCR (AR-mPCR). Higher ARs were associated with lower FMR1 transcript levels for any given repeat length. The higher AR-Sb was significantly associated with performance, verbal, and full-scale IQ scores, confirming previous reports. However, the AR-mPCR was not significantly associated (p > 0.05) with these measures. Similarly, the odds of depression and the number of medical conditions were correlated with higher AR-Sb but not correlated with a higher AR-mPCR. This study suggests that AR-Sb may be a more reliable measure of the AR in female carriers of PM alleles. However, further studies are warranted in a larger sample size to fully evaluate the methylation status in these participants and how it may affect the clinical phenotype.

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1 – 4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers. In cells with microsatellite instability, expanded TA-dinucleotide repeats form cruciform structures that stall replication forks and cause chromosome shattering in the absence of the WRN helicase.

Imprinted genes are expressed differently depending on whether they are carried by a chromosome of maternal or paternal origin. Correct imprinting is established by germline-specific modifications; failure of this process underlies several inherited human syndromes. All these imprinting control defects are cis-acting, disrupting establishment or maintenance of allele-specific epigenetic modifications across one contiguous segment of the genome. In contrast, we report here an inherited global imprinting defect. This recessive maternal-effect mutation disrupts the specification of imprints at multiple, non-contiguous loci, with the result that genes normally carrying a maternal methylation imprint assume a paternal epigenetic pattern on the maternal allele. The resulting conception is phenotypically indistinguishable from an androgenetic complete hydatidiform mole, in which abnormal extra-embryonic tissue proliferates while development of the embryo is absent or nearly so. This disorder offers a genetic route to the identification of trans-acting oocyte factors that mediate maternal imprint establishment.

The fragile X-related disorders are a group of three clinical conditions resulting from the instability of a CGG-repeat tract at the 5′ end of the FMR1 transcript. Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) are disorders seen in carriers of FMR1 alleles with 55–200 repeats. Female carriers of these premutation (PM) alleles are also at risk of having a child who has an FMR1 allele with >200 repeats. Most of these full mutation (FM) alleles are epigenetically silenced resulting in a deficit of the FMR1 gene product, FMRP. This results in fragile X Syndrome (FXS), the most common heritable cause of intellectual disability and autism. The diagnosis and study of these disorders is challenging, in part because the detection of alleles with large repeat numbers has, until recently, been either time-consuming or unreliable. This problem is compounded by the mosaicism for repeat length and/or DNA methylation that is frequently seen in PM and FM carriers. Furthermore, since AGG interruptions in the repeat tract affect the risk that a FM allele will be maternally transmitted, the ability to accurately detect these interruptions in female PM carriers is an additional challenge that must be met. This review will discuss some of the pros and cons of some recently described assays for these disorders, including those that detect FMRP levels directly, as well as emerging technologies that promise to improve the diagnosis of these conditions and to be useful in both basic and translational research settings.

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLα s, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′to the paternally expressed XLα s exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLα s), and biallelically (Gsα ) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.