The age at onset of motor symptoms in Huntington disease (HD) is driven by HTT CAG repeat length but modified by other genes. We used exome sequencing of 683 HD patients with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We identified damaging coding variants in candidate modifier genes from prior 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 these variants 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 comparable to those observed with complete FAN1 knock out. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.
BackgroundThe expression of mutant HTT leads to many cellular alterations, including abnormal vesicle recycling, loss of signalling by brain-derived neurotrophic factor, excitotoxicity, perturbation of Ca2+ signalling, decreases in intracellular ATP, alterations of gene transcription, inhibition of protein clearance pathways, mitochondrial and metabolic disturbances, and ultimately cell death. While robust mammalian systems have been developed to model disease and extensive mechanistic insights have emerged, significant differences between rodent and human cells and between non-neuronal cells and neurons limit the utility of these models for accurately representing human disease. Human pluripotent stem cells can generate highly specified cell populations, including DARPP32-positive MSNs of the striatum, and provide a method for modelling HD in human neurons carrying the mutation. As it is caused by one single gene, HD is an ideal disorder for exploring the utility of modelling disease in induced pluripotent stem cells (iPSCs) through reprogramming adult cells from HD patients with known patterns of disease onset and duration.AimsGenerate iPSC lines from HD patients and controls and identify CAG-repeat expansion associated phenotypes.Methods/techniquesThrough the efforts of an international consortium effort, 14 lines were generated, differentiated into neuronal populations and assessed for CAG-repeat dependent outcome measures.Results/outcomesHD iPSC lines have reproducible CAG expansion–associated phenotypes upon differentiation, including CAG expansion-associated changes in gene expression patterns and alterations in electrophysiology, metabolism, cell adhesion, and ultimately an increased risk of cell death. While the lines with the longest repeats (HD180) showed a phenotype across all assays, those with shorter repeats (HD60) showed phenotypes in a specific sub set of assays. The most sensitive assay for establishing repeat dependent effects was found to be calcium responses to stress.ConclusionsThis HD iPSC collection represents a unique and well-characterised resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics.FundingNIH, CHDI, CIRM.
Regulation of nucleosome positioning is important for neurodevelopment, and mutation of genes mediating chromatin remodelling are strongly associated with human neurodevelopmental disorders. Unicellular organisms possess arrays of highly positioned nucleosomes within their chromatin, occupying up to 80% of their genomes. These span gene-coding and regulatory regions, and can be associated with local changes of gene transcription. In the much larger genome of human cells, the roles of nucleosome positioning are less clear, and this raises questions of how nucleosome dynamics interfaces with human neurodevelopment.We have generated genome-wide nucleosome maps from an undifferentiated human induced pluripotent stem cell (hiPSC) line and after its differentiation to the neuronal progenitor cell (NPC) stage. We found that approximately 3% of nucleosomes are highly positioned in NPC. In contrast, there are 8-fold less positioned nucleosomes in pluripotent cells, with the majority arising de novo or relocating during cell differentiation. Positioned nucleosomes do not directly correlate with active chromatin or gene transcription, such as marking Transcriptional Start Sites (TSS).Unexpectedly, we find a small population of nucleosomes that remain positioned after differentiation, occupying similar positions in pluripotent and NPC cells. They flank the binding sites of the key gene regulators NRSF/REST and CTCF, but remain in place whether or not their regulatory complexes are present. Together, these results present an alternative view in human cells, where positioned nucleosomes are sparse and dynamic, but act to alter gene expression at a distance via structural conformation at sites of chromatin regulation, not local changes in gene organisation.3 Main Nucleosome maps generated by MNase-seq show the dynamic nature of nucleosome positioning. Narrow sequence read mid-point distribution profiles are indicative of highly positioned nucleosomes present at the same position in all cells of the population, whereas broader distributions occur where there is substantial variation in nucleosome positioning between individual cells ( Figure 1A). By mapping nucleosomes in the same human induced pluripotent stem cell (hiPSC) line in both its undifferentiated, pluripotent cell state and following differentiation to the neural progenitor cell (NPC) stage, we followed the changes in nucleosome positioning during early stages of neurodevelopment. We found that only 2.7% of nucleosomes are highly positioned in human NPC (408,152 of a theoretical total of 15 million based on an assumption of 1 nucleosome per 200 bp). This is consistent with observations from other human and mammalian cells [1,2], but contrasts with the yeasts and Dictyostelium genomes where positioned nucleosomes occupy approximately 80% of the genome [3][4][5][6]. Using the same analysis on data generated from the human lymphoblastoid cell lines, K562 and GM12878 [7] we calculated that 2.4% and 1.6% of nucleosomes respectively were highly positioned in these cell line...
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