• Huntington disease
• MLH1
• MLH3
• MSH2
• MSH3
• MSH6
• PMS1
• PMS2
• mismatch repair
• repeat expansion

• Humans
• Huntington Disease/genetics
• Huntington Disease/metabolism
• DNA Mismatch Repair/genetics
• Induced Pluripotent Stem Cells/metabolism
• Trinucleotide Repeat Expansion/genetics
• Huntingtin Protein/genetics
• Huntingtin Protein/metabolism
• MutL Protein Homolog 1/genetics
• MutL Protein Homolog 1/metabolism
• MutS Homolog 2 Protein/genetics
• MutS Homolog 2 Protein/metabolism
• Genes, Modifier
• MutS Homolog 3 Protein/genetics
• MutS Homolog 3 Protein/metabolism
• DNA-Binding Proteins/genetics
• DNA-Binding Proteins/metabolism
• MutL Proteins/genetics
• MutL Proteins/metabolism
• CRISPR-Cas Systems
• Genome-Wide Association Study

• environment
• epigenetics
• gene–environment interactions
• oxidative stress
• repeat expansion disease
• repeat instability
• short tandem repeat

• R21 ES032913 NIEHS NIH HHS
• R21ES032913 and P30ES009089 NIEHS NIH HHS

• disease modeling
• drug screening
• human pluripotent stem cells
• myotonic dystrophy type 1

• R01 NS105709 NINDS NIH HHS
• R01 NS119471 NINDS NIH HHS

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in huntingtin (HTT). Given an important role for HTT in development and significant neurodegeneration at the time of clinical manifestation in HD, early treatment of allele-specific drugs represents a promising strategy. The feasibility of an allele-specific antisense oligonucleotide (ASO) targeting single-nucleotide polymorphisms (SNPs) has been demonstrated in models of HD. Here, we constructed a map of haplotype-specific insertion-deletion variations (indels) to develop alternative mutant-HTT-specific strategies. We mapped indels annotated in the 1000 Genomes Project data on common HTT haplotypes, revealing candidate indels for mutant-specific HTT targeting. Subsequent sequencing of an HD family confirmed candidate sites and revealed additional allele-specific indels. Interestingly, the most common normal HTT haplotype carries indels of big allele length differences at many sites, further uncovering promising haplotype-specific targets. When patient-derived cells carrying the most common HTT diplotype were treated with ASOs targeting the mutant alleles of candidate indels (rs772629195 or rs72239206), complete mutant specificity was observed. In summary, our map of haplotype-specific indels permits the identification of allele-specific targets in HD subjects, potentially contributing to the development of safe HTT-lowering therapeutics that are suitable for early treatment in HD.

• Huntington's disease
• SNP
• allele-specific targeting
• haplotype-specific indel
• indel

Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

Summary: Early developmental abnormalities in myotonic dystrophy type 1 are reiterated in vitro in myotubes differentiated from human embryonic stem cells that carry the mutation.

• CpG methylation
• Human embryonic stem cells
• Myogenic differentiation
• Myotonic dystrophy type 1
• RNA mis-splicing

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.

• clinical translation
• copy number variations
• differentiation
• embryonic stem cells
• genomic instability
• induced pluripotent stem cells
• mosaicism
• pluripotent stem cells

• Carcinogenesis/genetics
• Carcinogenesis/pathology
• Cell Differentiation/genetics
• Chromosome Aberrations
• DNA Copy Number Variations/genetics
• Humans
• Models, Biological
• Pluripotent Stem Cells/metabolism
• Pluripotent Stem Cells/pathology

• DNA repair
• Huntington’s disease
• mismatch repair
• myotonic dystrophy 1
• trinucleotide repeat instability

Huntington’s disease (HD) is a neurodegenerative disorder characterized by the progressive decline of motor, cognitive, and psychiatric functions. HD results from an autosomal dominant mutation that causes a trinucleotide CAG repeat expansion and the production of mutant Huntingtin protein (mHTT). This results in the initial selective and progressive loss of medium spiny neurons (MSNs) in the striatum before progressing to involve the whole brain. There are currently no effective treatments to prevent or delay the progression of HD as knowledge into the mechanisms driving the selective degeneration of MSNs has been hindered by a lack of access to live neurons from individuals with HD. The invention of cell reprogramming provides a revolutionary technique for the study, and potential treatment, of neurological conditions. Cell reprogramming technologies allow for the generation of live disease-affected neurons from patients with neurological conditions, becoming a primary technique for modelling these conditions in vitro. The ability to generate HD-affected neurons has widespread applications for investigating the pathogenesis of HD, the identification of new therapeutic targets, and for high-throughput drug screening. Cell reprogramming also offers a potential autologous source of cells for HD cell replacement therapy. This review provides a comprehensive analysis of the use of cell reprogramming to model HD and a discussion on recent advancements in cell reprogramming technologies that will benefit the HD field.

• Huntington’s disease
• cell reprogramming
• direct cell reprogramming
• disease modelling
• pluripotent stem cells
• striatal differentiation

• Animals
• Cellular Reprogramming
• Disease Models, Animal
• Humans
• Huntington Disease/pathology
• Models, Biological
• Phenotype
• Pluripotent Stem Cells/pathology

• DNA damage response
• DNA repair
• MUS81
• SMX nuclease
• XPF
• crosslinks
• recombination
• telomeres

• Animals
• DNA Damage
• DNA Repair
• Genomic Instability
• Humans
• MutS Homolog 3 Protein/genetics
• MutS Homolog 3 Protein/metabolism
• Neoplasms/genetics
• Neoplasms/metabolism
• Protein Interaction Maps
• Recombinases/genetics
• Recombinases/metabolism

• 11582 Cancer Research UK
• FC001212 Cancer Research UK
• Wellcome Trust
• FC001212 Wellcome Trust
• FC001212 Medical Research Council

• Cell replacement therapy
• Disease modeling
• Gene therapy
• Pluripotent stem cells
• Polyglutamine diseases

Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.

•

FECD is a common, age-related corneal dystrophy.

• CTG18.1
• FECD
• Fuchs endothelial corneal dystrophy
• RAN translation
• RNA toxicity
• Repeat-expansion
• Transcription factor 4
• Trinucleotide repeat
• Triplet repeat-mediated disease

• Fuchs' Endothelial Dystrophy/genetics
• Fuchs' Endothelial Dystrophy/pathology
• Gene Expression Regulation
• Genetic Predisposition to Disease
• Genotype
• Humans
• Polymorphism, Genetic
• Transcription Factor 4/genetics
• Trinucleotide Repeat Expansion/genetics

• MR/S031820/1 Medical Research Council
• R01 EY026490 NEI NIH HHS
• Department of Health

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients’ pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9^e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.

• Endoplasmic reticulum
• Epigenetics
• Gene set enrichment analysis
• Induced pluripotent stem cells
• Neuromuscular junctions
• Neurotransmitter
• RNA sequencing
• Spinal bulbar muscular atrophy
• Synapse
• iPSC-derived motor neurons

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease caused by an unstable cytosine thymine guanine (CTG) repeat expansion in the DMPK gene. This disease is characterized by high clinical and genetic variability, leading to some difficulties in the diagnosis and prognosis of DM1. Better understanding the origin of this variability is important for developing new challenging therapies and, in particular, for progressing on the path of personalized treatments. Here, we reviewed CTG triplet repeat instability and its modifiers as an important source of phenotypic variability in patients with DM1.

• CTG repeat instability
• clinic variability
• myotonic dystrophy

• Animals
• Biological Variation, Population
• Cardiotocography
• Humans
• Myotonic Dystrophy/genetics
• Myotonic Dystrophy/metabolism
• Myotonin-Protein Kinase/genetics
• Myotonin-Protein Kinase/metabolism
• Phenotype
• Trinucleotide Repeats/genetics
• Trinucleotide Repeats/physiology

• DNA damage and repair
• Disease
• Gene Therapy
• Stem cells

• Differentiation
• Embryonic stem cell (ESC)
• Huntingtin (HTT)
• Huntington’s disease (HD)
• Induced pluripotent stem cell (iPSC)
• Modeling
• Striatum

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a deficiency in the fragile X mental retardation protein (FMRP) due to a CGG repeat expansion in the 5′-UTR of the X-linked FMR1 gene. When CGGs expand beyond 200 copies, they lead to epigenetic gene silencing of the gene. In addition, the greater the allele size, the more likely it will become unstable and exhibit mosaicism for expansion size between and within tissues in affected individuals. The timing and mechanisms of FMR1 epigenetic gene silencing and repeat instability are far from being understood given the lack of appropriate cellular and animal models that can fully recapitulate the molecular features characteristic of the disease pathogenesis in humans. This review summarizes the data collected to date from mutant human embryonic stem cells, induced pluripotent stem cells, and hybrid fusions, and discusses their contribution to the investigation of FXS, their key limitations, and future prospects.

• DNA methylation
• epigenetic gene silencing
• fragile X syndrome
• pluripotent stem cells
• repeat instability
• unstable repeat diseases

• CRISPR-Cas Systems
• Cells, Cultured
• Child
• Female
• Gene Editing/methods
• Humans
• Induced Pluripotent Stem Cells/metabolism
• Middle Aged
• Muscle Development/genetics
• Myoblasts/metabolism
• Myotonic Dystrophy/genetics
• Myotonic Dystrophy/metabolism
• Myotonic Dystrophy/pathology
• Trinucleotide Repeat Expansion/genetics

• DP1 GM105378 NIGMS NIH HHS

Fragile X syndrome (FXS) is caused by the maternal expansion of an unstable CGG-repeat tract located in the first exon of the FMR1 gene. Further changes in repeat number occur during embryogenesis resulting in individuals sometimes being highly mosaic. Here we show in a mouse model that, in males, expansions are already present in primary spermatocytes with no additional expansions occurring in later stages of gametogenesis. We also show that, in females, expansion occurs in the post-natal oocyte. Additional expansions and a high frequency of large contractions are seen in two-cell stage embryos. Expansion in oocytes, which are non-dividing, would be consistent with a mechanism involving aberrant DNA repair or recombination rather than a problem with chromosomal replication. Given the difficulty of replicating large CGG-repeat tracts, we speculate that very large expanded alleles may be prone to contract in the mitotically proliferating spermatagonial stem cells in men. However, expanded alleles may not be under such pressure in the non-dividing oocyte. The high degree of both expansions and contractions seen in early embryos may contribute to the high frequency of somatic mosaicism that is observed in humans. Our data thus suggest an explanation for the fact that FXS is exclusively maternally transmitted and lend support to models for repeat expansion that are based on problems arising during DNA repair.

• FX-associated primary ovarian insufficiency (FXPOI)
• FX-associated tremor and ataxia syndrome (FXTAS)
• fragile X syndrome (FXS)
• fragile X-related disorders (FXDs)
• full mutations
• gametogenesis
• premutation
• repeat expansion disease

We describe a new experimental system to study genome instability caused by fragile X (CGG)_n repeats in mammalian cells. It is based on a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)_n repeats in its 5′-UTR, which was integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)_n repeats dramatically elevate the frequency of the reporter’s inactivation making cells ganciclovir-resistant. These resistant clones have a unique mutational signature: a change in the repeat length concurrent with mutagenesis in the reporter gene. Inactivation of genes implicated in break-induced replication including POLD3, POLD4, RAD52, RAD51 and SMARCAL1, reduced the frequency of ganciclovir-resistant clones to the baseline level that was observed in the absence of (CGG)_n repeats. We propose that replication fork collapse at carrier-size (CGG)_n repeats can trigger break-induced replication, which result in simultaneous repeat length changes and mutagenesis at a distance.

Muscular dystrophies (MDs) are clinically and molecularly a highly heterogeneous group of single-gene disorders that primarily affect striated muscles. Cardiac disease is present in several MDs where it is an important contributor to morbidity and mortality. Careful monitoring of cardiac issues is necessary but current management of cardiac involvement does not effectively protect from disease progression and cardiac failure. There is a critical need to gain new knowledge on the diverse molecular underpinnings of cardiac disease in MDs in order to guide cardiac treatment development and assist in reaching a clearer consensus on cardiac disease management in the clinic. Animal models are available for the majority of MDs and have been invaluable tools in probing disease mechanisms and in pre-clinical screens. However, there are recognized genetic, physiological, and structural differences between human and animal hearts that impact disease progression, manifestation, and response to pharmacological interventions. Therefore, there is a need to develop parallel human systems to model cardiac disease in MDs. This review discusses the current status of cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC) to model cardiac disease, with a focus on Duchenne muscular dystrophy (DMD) and myotonic dystrophy (DM1). We seek to provide a balanced view of opportunities and limitations offered by this system in elucidating disease mechanisms pertinent to human cardiac physiology and as a platform for treatment development or refinement.

• Cas9/CRISPR genome editing
• Duchenne muscular dystrophy (DMD)
• Human embryonic stem cells
• cardiomyocytes
• disease modelling
• exon skipping
• gene therapy
• human induced pluripotent stem cells
• myotonic dystrophy (DM1)

• CAG repeat
• Cell models
• DNA damage response
• DNA repair
• Genetic modifiers
• Huntington’s disease
• Mouse models
• Neurodegeneration
• Somatic expansion
• Trinucleotide repeat

• DNA repair
• DNA replication
• FoSTeS
• break-induced replication
• chromothripsis
• genome instability
• microsatellite instability

The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

• PCR
• agarose
• electrophoresis
• ethidium bromide
• myotonic dystrophy
• non-B DNA
• polyacrylamide
• trinucleotide DNA repeat

• ESC
• Huntington disease
• NSC
• Neurodegenerative disease
• Neurodevelopmental disease
• Polyglutamine diseases
• Stem cells
• iPS
• iPSC
• polyQ disease

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10⁻¹²). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

• DMPK
• DNA/CpG methylation
• SIX5/DMAHP
• congenital myotonic dystrophy
• epigenetics
• maternal transmission/maternal inheritance
• myotonic dystrophy
• parent-of-origin effect(s)
• pre-natal diagnosis
• trinucleotide instability

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multi-system disease caused by expanded CTG repeats in dystrophia myotonica protein kinase (DMPK). The expanded CTG repeats are unstable and can increase the length of the gene with age, which worsens the symptoms. In order to establish a human stem cell system suitable for the investigation of repeat instability, DM1 patient-derived iPSCs were generated and differentiated into three cell types commonly affected in DM1, namely cardiomyocytes, neurons and myocytes. Then we precisely analysed the CTG repeat lengths in these cells. Our DM1-iPSCs showed a gradual lengthening of CTG repeats with unchanged repeat distribution in all cell lines depending on the passage numbers of undifferentiated cells. However, the average CTG repeat length did not change significantly after differentiation into different somatic cell types. We also evaluated the chromatin accessibility in DM1-iPSCs using ATAC-seq. The chromatin status in DM1 cardiomyocytes was closed at the DMPK locus as well as at SIX5 and its promoter region, whereas it was open in control, suggesting that the epigenetic modifications may be related to the CTG repeat expansion in DM1. These findings may help clarify the role of repeat instability in the CTG repeat expansion in DM1.

About 10% of total colorectal cancers are associated with known Mendelian inheritance, as Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). In these cancer types the clinical manifestations of disease are due to mutations in high-risk alleles, with a penetrance at least of 70%. The LS is associated with germline mutations in the DNA mismatch repair (MMR) genes. However, the mutation detection analysis of these genes does not always provide informative results for genetic counseling of LS patients. Very often, the molecular analysis reveals the presence of variants of unknown significance (VUSs) whose interpretation is not easy and requires the combination of different analytical strategies to get a proper assessment of their pathogenicity. In some cases, these VUSs may make a more substantial overall contribution to cancer risk than the well-assessed severe Mendelian variants. Moreover, it could also be possible that the simultaneous presence of these genetic variants in several MMR genes that behave as low risk alleles might contribute in a cooperative manner to increase the risk of hereditary cancer. In this paper, through a review of the recent literature, we have speculated a novel inheritance model in the Lynch syndrome; this could pave the way toward new diagnostic perspectives.

• CRISPR-Cas Systems
• DNA/genetics
• DNA/metabolism
• DNA Breaks, Single-Stranded
• DNA Repair
• Deoxyribonuclease I/metabolism
• Genomic Instability
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• HEK293 Cells
• Humans
• MutS Homolog 2 Protein/genetics
• MutS Homolog 2 Protein/metabolism
• Trinucleotide Repeat Expansion/genetics
• Trinucleotide Repeats/genetics

Fragile X syndrome (FXS), a common cause of intellectual disability and autism, results from the expansion of a CGG-repeat tract in the 5′ untranslated region of the FMR1 gene to >200 repeats. Such expanded alleles, known as full mutation (FM) alleles, are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP, a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial.

We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing, and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs.

We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs), some silenced alleles contract and when the repeat number drops below ~400, DNA methylation erodes, even when the repeat number remains >200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore, there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation.

Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo, (ii) large unmethylated alleles may be deleterious in stem cells, (iii) methylation can occur on alleles with >400 repeats very early in embryogenesis, and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with >200 repeats.

The online version of this article (doi:10.1186/s13229-016-0105-9) contains supplementary material, which is available to authorized users.

• Fragile X syndrome
• Repeat contractions
• Repeat expansion mutation
• Repeat-mediated gene silencing
• Stem cells

• Huntington’s disease
• Ligase I
• MSH2
• MSH3
• Mismatch repair
• MutSbeta
• PCNA
• Repeat contractions
• Repeat expansions
• Repeat instability
• Slippage
• Trinucleotide repeats

• Embryonic stem cell (ESC)
• Huntingtin (HTT)
• Huntington׳s disease (HD)
• Induced pluripotent stem cell (iPSC)
• Modeling

CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation.

•

We identify a disease-associated, differentially methylated region in DM1 hESCs

Myotonic dystrophy type 1 (DM1 or Steinert’s disease) and type 2 (DM2) are multisystem disorders of genetic origin. Progressive muscular weakness, atrophy and myotonia are the most prominent neuromuscular features of these diseases, while other clinical manifestations such as cardiomyopathy, insulin resistance and cataracts are also common. From a clinical perspective, most DM symptoms are interpreted as a result of an accelerated aging (cataracts, muscular weakness and atrophy, cognitive decline, metabolic dysfunction, etc.), including an increased risk of developing tumors. From this point of view, DM1 could be described as a progeroid syndrome since a notable age-dependent dysfunction of all systems occurs. The underlying molecular disorder in DM1 consists of the existence of a pathological (CTG) triplet expansion in the 3′ untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene, whereas (CCTG)n repeats in the first intron of the Cellular Nucleic acid Binding Protein/Zinc Finger Protein 9(CNBP/ZNF9) gene cause DM2. The expansions are transcribed into (CUG)n and (CCUG)n-containing RNA, respectively, which form secondary structures and sequester RNA-binding proteins, such as the splicing factor muscleblind-like protein (MBNL), forming nuclear aggregates known as foci. Other splicing factors, such as CUGBP, are also disrupted, leading to a spliceopathy of a large number of downstream genes linked to the clinical features of these diseases. Skeletal muscle regeneration relies on muscle progenitor cells, known as satellite cells, which are activated after muscle damage, and which proliferate and differentiate to muscle cells, thus regenerating the damaged tissue. Satellite cell dysfunction seems to be a common feature of both age-dependent muscle degeneration (sarcopenia) and muscle wasting in DM and other muscle degenerative diseases. This review aims to describe the cellular, molecular and macrostructural processes involved in the muscular degeneration seen in DM patients, highlighting the similarities found with muscle aging.

• aging
• muscle wasting
• myotonic dystrophy
• sarcopenia
• satellite cells

Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neuronal and neuromuscular diseases. All result from expanded tracts of repetitive DNA sequences (mostly microsatellites) that become unstable beyond a critical length when transmitted across generations. Nearly all are inherited as autosomal dominant conditions and are typically associated with anticipation. Pathologic unstable repeat expansions can be classified according to their length, repeat sequence, gene location and underlying pathologic mechanisms. This review summarizes the current contribution of mutant pluripotent stem cells (diseased human embryonic stem cells and patient-derived induced pluripotent stem cells) to the research of unstable repeat pathologies by focusing on particularly large unstable noncoding expansions. Among this class of disorders are Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome, myotonic dystrophy type 1 and myotonic dystrophy type 2, Friedreich ataxia and C9 related amyotrophic lateral sclerosis and/or frontotemporal dementia, Facioscapulohumeral Muscular Dystrophy and potentially more. Common features that are typical to this subclass of conditions are RNA toxic gain-of-function, epigenetic loss-of-function, toxic repeat-associated non-ATG translation and somatic instability. For each mechanism we summarize the currently available stem cell based models, highlight how they contributed to better understanding of the related mechanism, and discuss how they may be utilized in future investigations.

• Unstable repeat associated disorders
• Human embryonic stem cells
• Patient-derived induced pluripotent stem cells
• Disease modeling
• Epigenetics
• repeat-associated non-ATG translation
• RNA toxicity
• Repeat somatic instability

• Cell Differentiation/genetics
• Cell Line
• Human Embryonic Stem Cells/metabolism
• Humans
• Huntington Disease/pathology
• Mutation/genetics
• Myocytes, Cardiac/cytology
• Pluripotent Stem Cells/cytology
• Trinucleotide Repeat Expansion/genetics

• G0701172 Medical Research Council
• G0801061 Medical Research Council
• RG/14/1/30588 British Heart Foundation
• G1000035/1 National Centre for the Replacement, Refinement and Reduction of Animals in Research
• MR/L012618/1 Medical Research Council

• Chromosome fragility
• DNA damage checkpoint
• DNA structure
• recombination
• repair
• replication
• structure-specific helicases
• trinucleotide repeat expansion

• DNA mismatch repair
• hereditary neurological diseases
• non-B-DNA structures
• repeat expansion diseases
• triplet repeats

• human embryonic stem cells
• implantation failure
• preimplantation genetic diagnosis
• trophoblast differentiation
• unbalanced translocation

• disease modeling
• embryonic stem cells (ESCs)
• induced pluripotent stem cells (iPSCs)

Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.

In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson’s disease and Alzheimer’s disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.

• Animals
• Congenital Abnormalities/epidemiology
• Disease Models, Animal
• Embryonic Stem Cells
• Epigenesis, Genetic
• Europe
• Female
• Fertilization in Vitro/methods
• Genetic Counseling
• Genetic Testing
• Genetic Variation
• Genetics, Medical
• Guidelines as Topic
• Humans
• Infertility/genetics
• Infertility/therapy
• Male
• Medical Tourism
• Policy
• Pregnancy
• Preimplantation Diagnosis
• Regenerative Medicine
• Reproduction/genetics
• Reproductive Techniques, Assisted/adverse effects
• Reproductive Techniques, Assisted/ethics
• Reproductive Techniques, Assisted/legislation & jurisprudence
• Reproductive Techniques, Assisted/trends
• Societies, Scientific

More than fifteen genetic diseases, including Huntington’s disease, myotonic dystrophy 1, fragile X syndrome and Friedreich ataxia, are caused by the aberrant expansion of a trinucleotide repeat. The mutation is unstable and further expands in specific cells or tissues with time, which can accelerate disease progression. DNA damage and base excision repair (BER) are involved in repeat instability and might contribute to the tissue selectivity of the process. In this review, we will discuss the mechanisms of trinucleotide repeat instability, focusing more specifically on the role of BER.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG₃₇ and CAG₅₁ typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG₅₁ neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

• GABAergic neurons
• Huntington's disease
• human embryonic stem cells
• neuronal differentiation