C: Genetic modifiers最新文献

{"title":"C02 FAN1 controls cag repeat expansion in huntington’s disease by dual functions, MLH1 retention and nuclease activity","authors":"R. Goold, Joseph Hamilton, Thomas Menneteau, M. Flower, Emma L. Bunting, Sarah Aldous, Antonio Porro, J. R. Vicente, N. Allen, H. Wilkinson, G. Bates, A. Sartori, K. Thalassinos, G. Balmus, S. Tabrizi","doi":"10.1136/jnnp-2021-ehdn.26","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.26","url":null,"abstract":"Background Human genetic studies have shown that, after CAG repeat length, DNA maintenance is the main process influencing Huntington’s disease (HD) pathogenesis. This likely acts via somatic expansion - a process in which the pathogenic CAG repeat in HTT exon 1 expands throughout life. Genetic modifiers of HD in the DNA damage response include the interstrand crosslink repair nuclease FAN1 and several mismatch repair (MMR) factors, including MLH1, PMS2 and MSH3. FAN1 has shown to suppress repeat expansion, however, the mechanism of this remains unclear. Aims Explore the functional significance of the previously noted FAN1–MLH1 protein interaction in an HD context. Establish which regions or functions of FAN1 are required for CAG repeat stabilisation. Methods/Techniques In the FAN1 -/- U2OS cell system, we express FAN1 variants and assess protein interactions via co-immunoprecipitation. We measure somatic expansion in a co-expressed exogenous 118 CAG HTT exon 1 construct using fragment analysis, involving estimation of CAG repeat length by capillary electrophoresis. Functional assays such as 6-thioguanine and mitomycin C examine DNA repair integrity – mismatch repair and FAN1 nuclease activity, respectively. Results/Outcome We have elucidated FAN1’s protective function in HD and the molecular nature of its interaction with mismatch repair. In HD models (in vitro and in vivo), we confirm that FAN1 binds MLH1. We demonstrate that FAN1 directly competes with MSH3 for MLH1 through an evolutionary conserved N-terminal domain, 126SPYF129, reducing the activity of mismatch repair and also CAG repeat expansion. Additionally, FAN1’s canonical nuclease domain accounts for residual suppression of CAG repeat expansion. Conclusions The FAN1-MLH1 interaction regulates somatic expansion by attenuating mismatch repair activity, providing a novel therapeutic opportunity for HD and potentially other repeat expansion diseases.","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"64 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115211913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C01 The effect of mismatch repair proteins in a huntington’s disease cellular model","authors":"Joseph Stone, Jasmine Donaldson, Sophie Powell, N. Allen, Thomas H. Massey, L. Jones","doi":"10.1136/jnnp-2021-ehdn.25","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.25","url":null,"abstract":"Background Huntington’s disease (HD) is caused by an expanded CAG repeat in exon 1 of the Huntingtin gene (HTT). Repeats greater than 35 CAGs in length cause disease and the age at disease onset is inversely correlated with CAG repeat length. Although about 60% of the variation in age at onset of disease can be attributed to the CAG repeat length, other genetic factors have been found to modify age at disease onset. Recent genome wide association studies (GWAS) have identified several single nucleotide variants (SNVs) in genes encoding DNA damage repair factors that modify age at disease onset. There is a particular enrichment in mismatch repair genes with MLH1 and MSH3 implicated. Evidence from mouse models of HD suggests that MLH1 and MSH3 are involved in driving expansion of the CAG repeat in somatic cells. This somatic expansion is hypothesised to accelerate pathogenesis and decrease the age of disease onset. Methods To investigate the role of the mismatch repair genes MLH1 and MSH3 in HD we have used an induced pluripotent stem cell (iPSC) model of HD with 109 CAG repeats which shows expansion when cultured. We have used CRISPR Cas9 gene editing technology to knockout (KO) MLH1 and MSH3 in this cell line. Results and Conclusions MLH1 KO ablates somatic expansion of the expanded HTT CAG repeat in iPSCs when cultured for 59 days and preliminary data suggests that MSH3 KO reduces somatic expansion of the expanded HTT CAG repeat in iPSCs.","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"10 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129115996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C05 SAPAP3 scaffolding protein as a regulator of mitochondrial function in huntington’s disease","authors":"Patrícia Coelho, Lígia Fão, A. Rego","doi":"10.1136/jnnp-2021-ehdn.29","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.29","url":null,"abstract":"Background Huntington’s disease (HD) is a brain disorder characterized by motor and cognitive impairment and early psychiatric disturbances, including obsessive-compulsive disorder (OCD). HD is caused by expansion of CAG repeats at the HTT gene, resulting in expression of mutant huntingtin (mHTT), largely affecting cortico-striatal synapses that are enriched in N-methyl-D-aspartate (NMDA) receptors. Former studies demonstrated that the postsynaptic scaffold protein SAPAP3, mainly located in striatum, is an important player in OCD. Unpublished data indicate that SAPAP3 interacts with several mitochondrial proteins. Hence, striatal dysfunction linked to early mitochondrial deregulation may involve changes in SAPAP3, potentially contributing to early HD symptomatology. Aims Investigate whether altered SAPAP3 protein levels affect striatal function in HD models by focusing on mitochondrial dysfunction as a hallmark of the disease. Methods Determine SAPAP3 protein levels in mitochondria isolated from pre-symptomatic (3 m.o.) and symptomatic (6, 10-12 m.o.) YAC128 transgenic vs WT mice, primary striatal/cortical cultures from YAC128 vs WT mice and STHdhQ111/Q111 vs STHdhQ7/Q7 cells. Study the influence of modulating SAPAP3 levels on mitochondrial function and dynamics in HD cells. Results We showed reduced SAPAP3 total/mitochondrial levels in symptomatic YAC128 mice, mature primary neurons from YAC128 mice and STHdhQ111/Q111 cells, compared to respective controls. In YAC128 striatal and cortical neurons, decreased SAPAP3 levels were pronounced at distal neurites. Colocalization between SAPAP3 and both PSD-95 and GluN2B were affected in YAC128 mouse striatal neurites. Of relevance, silencing SAPAP3 impaired mitochondrial dynamics and function, whereas SAPAP3 overexpression ameliorated these mitochondrial phenotypes in HD cells. Conclusion Our data suggest that SAPAP3 levels impact on mitochondrial function, being a potential neuroprotective target in HD.","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"102 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121174515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C06 Comparison of models for estimating age at motor onset in HD","authors":"P. Holmans, Oliver Didcote","doi":"10.1136/jnnp-2021-ehdn.30","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.30","url":null,"abstract":"","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"23 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114597169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C03 FAN1 prevents crispr-CAS9 nickase-induced contractions of CAG/CTG repeats","authors":"L. Heraty, Vincent Dion","doi":"10.1136/jnnp-2021-ehdn.27","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.27","url":null,"abstract":"Background Longer CAG repeats in the HTT gene causes more severe forms of Huntington’s disease (HD), suggesting that contracting them would provide a much needed therapeutic benefit. We developed the first method to contract expanded repeats in human cell lines, whilst avoiding concomitant expansions. Unlike other systems that induce double strand breaks around the repeat tract to remove the repeat region, our method relies on the CRIPSR Cas9 nickase. The enzyme is targeted by a guide RNA against the repeat tract, resulting in nicks that are processed by the endogenous DNA repair machinery leading to contractions. The mechanism of contraction is poorly defined and identifying genetic players is important for both improving the contraction efficacy and for stratifying which patients would benefit best from this therapy. Aims Here we tested the hypothesis that loss of FAN1 impacts nickase-induced contractions. Methods/Techniques We generated a HEK293-derived cell line which contains a stably integrated CRISPR-Cas9 nickase, a sgRNA against the repeat tract and an inducible GFP-reporter system containing an ectopic CAG repeat. In this line, we also knocked out FAN1. We assayed for FAN1 loss of function using targeted DNA sequencing, western blotting, and sensitivity to mitomyocin C. We analysed repeat size using flow cytometry for GFP expression and by small-pool PCR. Results/Outcomes We found that two independent FAN1 knockout clones displayed an increase in the frequency of contraction events over 21 days. We found no effect on the frequency of expansion. Conclusions We conclude that FAN1 in our system prevents nickase-induced contractions without impacting expansion events.","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"82 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131833581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"C07 Genetic risk for psychiatric disorders is associated with psychiatric and cognitive huntington’s disease symptoms","authors":"Branduff McAllister, S. Lobanov, Thomas H. Massey, L. Jones, P. Holmans","doi":"10.1136/jnnp-2021-ehdn.31","DOIUrl":"https://doi.org/10.1136/jnnp-2021-ehdn.31","url":null,"abstract":"","PeriodicalId":447196,"journal":{"name":"C: Genetic modifiers","volume":"2 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129327214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}