Huntingtin CAG repeat expansions in induced pluripotent stem cell models of Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the huntingtin gene, HTT. The length of the CAG repeat is inversely correlated with age at motor onset, but other factors influence onset including genetic variation elsewhere in the genome. Recent genome-wide association studies have identified genetic variants in the vicinity of FAN1, a nuclease involved in DNA interstrand cross-link repair, as modifiers of age at onset of HD. It is thought that FAN1 might modify age at onset through direct modulation of the expanded HTT CAG repeat. Further expansions of the CAG repeat in post-mitotic neurons are thought to accelerate disease-onset and therefore factors that license or inhibit expansions represent therapeutic targets. This thesis demonstrates that HD-iPSC lines with expanded CAG repeat tracts of >100 CAG repeats (Q109) exhibit repeat instability in culture, with the repeat tract undergoing further expansions in pluripotent cells and upon neuronal differentiation. These cell lines therefore represent a cellular model of repeat expansion which can be utilised to characterise how DNA repair genes affect cells harbouring expanded CAG repeats. Employing CRISPR-Cas9 and a piggyBac transposon-based homologous recombination approach the expanded HTT CAG repeat tract was genetically corrected to a wild-type repeat length of 22 HTT CAGs (Q22). Corrected HD-iPSCs retained pluripotency and differentiation potential. Additionally, metabolic deficits in Q109 neural progenitor cells were rescued in Q22 neural progenitor cells. This thesis also aimed to establish the effect of FAN1 on HTT CAG repeat expansions. Using CRISPRCas9, FAN1 was knocked-out of an isogenic pair of HD-iPSCs containing either 22 or >100 HTT CAG repeats. FAN1-/- clones were significantly more susceptible to interstrand cross-linking agent MMC, indicative of defective interstrand cross-link repair. Increased CAG repeat expansion was observed in FAN1-/- iPSCs and iPSC-derived neurons, suggestive of a protective role of FAN1 against CAG repeat expansions. These novel model systems provide a platform for investigating the cellular phenotypes associated with expanded CAG repeats, and the effects of DNA-repair associated genetic modifiers

What is the pathogenic CAG expansion length in Huntington’s disease?

Huntington’s disease (HD) (OMIM 143100) is caused by an expanded CAG repeat tract in the HTT gene. The inherited CAG length is known to expand further in somatic and germline cells in HD subjects. Age at onset of the disease is inversely correlated with the inherited CAG length, but is further modulated by a series of genetic modifiers which are most likely to act on the CAG repeat in HTT that permit it to further expand. Longer repeats are more prone to expansions, and this expansion is age dependent and tissue-specific. Given that the inherited tract expands through life and most subjects develop disease in mid-life, this implies that in cells that degenerate, the CAG length is likely to be longer than the inherited length. These findings suggest two thresholds – the inherited CAG length which permits further expansion, and the intracellular pathogenic threshold, above which cells become dysfunctional and die. This two-step mechanism has been previously proposed and modelled mathematically to give an intracellular pathogenic threshold at a tract length of 115 CAG (95% confidence intervals 70-165 CAG). Empirically, the intracellular pathogenic threshold is difficult to determine. Clues from studies of people and models of HD, and from other diseases caused by expanded repeat tracts, place this threshold between 60-100 CAG, most likely towards the upper part of that range. We assess this evidence and discuss how the intracellular pathogenic threshold in manifest disease might be better determined. Knowing the cellular pathogenic threshold would be informative for both understanding the mechanism in HD and deploying treatments

Genetic modifiers of repeat expansion disorders

Repeat expansion disorders (REDs) are monogenic diseases caused by a sequence of repetitive DNA expanding above a pathogenic threshold. A common feature of the REDs is a strong genotype-phenotype correlation in which a major determinant of age at onset (AAO) and disease progression is the length of the inherited repeat tract. Over a disease-gene carrier's life, the length of the repeat can expand in somatic cells, through the process of somatic expansion which is hypothesised to drive disease progression. Despite being monogenic, individual REDs are phenotypically variable, and exploring what genetic modifying factors drive this phenotypic variability has illuminated key pathogenic mechanisms that are common to this group of diseases. Disease phenotypes are affected by the cognate gene in which the expansion is found, the location of the repeat sequence in coding or non-coding regions and by the presence of repeat sequence interruptions. Human genetic data, mouse models and in vitro models have implicated the disease-modifying effect of DNA repair pathways via the mechanisms of somatic mutation of the repeat tract. As such, developing an understanding of these pathways in the context of expanded repeats could lead to future disease-modifying therapies for REDs

Mutant huntingtin confers cell-autonomous phenotypes on Huntington’s disease iPSC-derived microglia

Huntington’s disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way

A Magnetohydrodynamic enhanced entry system for space transportation: MEESST

This paper outlines the initial development of a novel magnetohydrodynamic (MHD) plasma control system which aims at mitigating shock-induced heating and the radio-frequency communication blackout typically encountered during (re-)entry into planetary atmospheres. An international consortium comprising universities, SMEs, research institutions, and industry has been formed in order to develop this technology within the MEESST project. The latter is funded by the Future and Emerging Technologies (FET) program of the European Commission’s Horizon 2020 scheme (grant no. 899298). Atmospheric entry imposes one of the harshest environments which a spacecraft can experience. The combination of hypersonic velocities and the rapid compression of atmospheric particles by the spacecraft leads to high-enthalpy, partially ionised gases forming around the vehicle. This inhibits radio communications and induces high thermal loads on the spacecraft surface. For the former problem, spacecraft can sometimes rely on satellite constellations for communicating through the plasma wake and therefore preventing the blackout. On the other hand, expensive, heavy, and non-reusable thermal protection systems (TPS) are needed to dissipate the severe thermal loads. Such TPS can represent up to 30% of an entry vehicles weight, and especially for manned missions they can reduce the cost- efficiency by sacrificing payload mass. Such systems are also prone to failure, putting the lives of astronauts at risk. The use of electromagnetic fields to exploit MHD principles has long been considered as an attractive solution for tackling the problems described above. By pushing the boundary layer of the ionized gas layer away from the spacecraft, the thermal loads can be reduced, while also opening a magnetic window for radio communications and mitigating the blackout phenomenon. The application of this MHD-enabled system has previously not been demonstrated in realistic conditions due to the required large magnetic fields (on the order of Tesla or more), which for conventional technologies would demand exceptionally heavy and power-hungry electromagnets. High-temperature superconductors (HTS) have reached a level of industrial maturity sufficient for them to act as a key enabling technology for this application. Thanks to superior current densities, HTS coils can offer the necessary low weight and compactness required for space applications, with the ability to generate the strong magnetic fields needed for entry purposes. This paper provides an overview of the MEESST project, including its goals, methodology and some preliminary design considerations

A Magnetohydrodynamic enhanced entry system for space transportation: MEESST

This paper outlines the initial development of a novel magnetohydrodynamic (MHD) plasma control system which aims at mitigating shock-induced heating and the radio-frequency communication blackout typically encountered during (re-)entry into planetary atmospheres. An international consortium comprising universities, SMEs, research institutions, and industry has been formed in order to develop this technology within the MEESST project. The latter is funded by the Future and Emerging Technologies (FET) program of the European Commission’s Horizon 2020 scheme (grant no. 899298). Atmospheric entry imposes one of the harshest environments which a spacecraft can experience. The combination of hypersonic velocities and the rapid compression of atmospheric particles by the spacecraft leads to high-enthalpy, partially ionised gases forming around the vehicle. This inhibits radio communications and induces high thermal loads on the spacecraft surface. For the former problem, spacecraft can sometimes rely on satellite constellations for communicating through the plasma wake and therefore preventing the blackout. On the other hand, expensive, heavy, and non-reusable thermal protection systems (TPS) are needed to dissipate the severe thermal loads. Such TPS can represent up to 30% of an entry vehicles weight, and especially for manned missions they can reduce the cost- efficiency by sacrificing payload mass. Such systems are also prone to failure, putting the lives of astronauts at risk. The use of electromagnetic fields to exploit MHD principles has long been considered as an attractive solution for tackling the problems described above. By pushing the boundary layer of the ionized gas layer away from the spacecraft, the thermal loads can be reduced, while also opening a magnetic window for radio communications and mitigating the blackout phenomenon. The application of this MHD-enabled system has previously not been demonstrated in realistic conditions due to the required large magnetic fields (on the order of Tesla or more), which for conventional technologies would demand exceptionally heavy and power-hungry electromagnets. High-temperature superconductors (HTS) have reached a level of industrial maturity sufficient for them to act as a key enabling technology for this application. Thanks to superior current densities, HTS coils can offer the necessary low weight and compactness required for space applications, with the ability to generate the strong magnetic fields needed for entry purposes. This paper provides an overview of the MEESST project, including its goals, methodology and some preliminary design considerations

A Multisite Preregistered Paradigmatic Test of the Ego-Depletion Effect

We conducted a preregistered multilaboratory project (k = 36; N = 3,531) to assess the size and robustness of ego-depletion effects using a novel replication method, termed the paradigmatic replication approach. Each laboratory implemented one of two procedures that was intended to manipulate self-control and tested performance on a subsequent measure of self-control. Confirmatory tests found a nonsignificant result (d = 0.06). Confirmatory Bayesian meta-analyses using an informed-prior hypothesis (δ = 0.30, SD = 0.15) found that the data were 4 times more likely under the null than the alternative hypothesis. Hence, preregistered analyses did not find evidence for a depletion effect. Exploratory analyses on the full sample (i.e., ignoring exclusion criteria) found a statistically significant effect (d = 0.08); Bayesian analyses showed that the data were about equally likely under the null and informed-prior hypotheses. Exploratory moderator tests suggested that the depletion effect was larger for participants who reported more fatigue but was not moderated by trait self-control, willpower beliefs, or action orientation.</p

Exome sequencing of individuals with Huntington’s disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset

The age at onset of motor symptoms in Huntington’s disease (HD) is driven by HTT CAG repeat length but modified by other genes. In this study, we used exome sequencing of 683 patients with HD with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We discovered damaging coding variants in candidate modifier genes identified in previous genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier-onset HD. Nuclease activities of purified variants in vitro correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion similar to those observed with complete FAN1 knockout. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD

The Influence of Number and Timing of Pregnancies on Breast Cancer Risk for Women With BRCA1 or BRCA2 Mutations

International audienceBACKGROUND:Full-term pregnancy (FTP) is associated with a reduced breast cancer (BC) risk over time, but women are at increased BC risk in the immediate years following an FTP. No large prospective studies, however, have examined whether the number and timing of pregnancies are associated with BC risk for BRCA1 and BRCA2 mutation carriers.METHODS:Using weighted and time-varying Cox proportional hazards models, we investigated whether reproductive events are associated with BC risk for mutation carriers using a retrospective cohort (5707 BRCA1 and 3525 BRCA2 mutation carriers) and a prospective cohort (2276 BRCA1 and 1610 BRCA2 mutation carriers), separately for each cohort and the combined prospective and retrospective cohort.RESULTS:For BRCA1 mutation carriers, there was no overall association with parity compared with nulliparity (combined hazard ratio [HRc] = 0.99, 95% confidence interval [CI] = 0.83 to 1.18). Relative to being uniparous, an increased number of FTPs was associated with decreased BC risk (HRc = 0.79, 95% CI = 0.69 to 0.91; HRc = 0.70, 95% CI = 0.59 to 0.82; HRc = 0.50, 95% CI = 0.40 to 0.63, for 2, 3, and ≥4 FTPs, respectively, P trend < .0001) and increasing duration of breastfeeding was associated with decreased BC risk (combined cohort P trend = .0003). Relative to being nulliparous, uniparous BRCA1 mutation carriers were at increased BC risk in the prospective analysis (prospective hazard ration [HRp] = 1.69, 95% CI = 1.09 to 2.62). For BRCA2 mutation carriers, being parous was associated with a 30% increase in BC risk (HRc = 1.33, 95% CI = 1.05 to 1.69), and there was no apparent decrease in risk associated with multiparity except for having at least 4 FTPs vs. 1 FTP (HRc = 0.72, 95% CI = 0.54 to 0.98).CONCLUSIONS:These findings suggest differential associations with parity between BRCA1 and BRCA2 mutation carriers with higher risk for uniparous BRCA1 carriers and parous BRCA2 carriers