Effects of Hydrogen Plasma Treatment on the Electrical Behavior of Solution-Processed ZnO Thin Films.

In this study, the effect of atmospheric hydrogen plasma treatment on the in-plane conductivity of solution-processed zinc oxide (ZnO) in various environments is reported. The hydrogen-plasma-treated and untreated ZnO films exhibited ohmic behavior with room-temperature in-plane conductivity in a vacuum. When the untreated ZnO film was exposed to a dry oxygen environment, the conductivity rapidly decreased, and an oscillating current was observed. In certain cases, the thin film reversibly 'switched' between the high- and low-conductivity states. In contrast, the conductivity of the hydrogen-plasma-treated ZnO film remained nearly constant under different ambient conditions. We infer that hydrogen acts as a shallow donor, increasing the carrier concentration and generating oxygen vacancies by eliminating the surface contamination layer. Hence, atmospheric hydrogen plasma treatment could play a crucial role in stabilizing the conductivity of ZnO films.

Stability of Mosaic Divergent Repeat Interruptions in X-Linked Dystonia-Parkinsonism.

BACKGROUND: X-Linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by rapidly progressive dystonia and parkinsonism. Mosaic Divergent Repeat Interruptions affecting motif Length and Sequence (mDRILS) were recently found within the TAF1 SVA repeat tract and were shown to associate with repeat stability and age at onset in XDP, specifically the AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n] mDRILS. OBJECTIVE: This study aimed to investigate the stability of mDRILS frequencies and stability of (AGAGGG)n repeat length during transmission in parent-offspring pairs. METHODS: Fifty-six families (n = 130) were investigated for generational transmission of repeat length and mDRILS. The mDRILS stability of 16 individuals was assessed at two sampling points 1 year apart. DNA was sequenced with long-read technologies after long-range polymerase chain reaction amplification of the TAF1 SVA. Repeat number and mDRILS were detected with Noise-Cancelling Repeat Finder (NCRF). RESULTS: When comparing the repeat domain, 51 of 65 children had either contractions or expansions of the repeat length. The AGGG frequency remained stable across generations at 0.074 (IQR: 0.069-0.078) (z = -0.526; P = 0.599). However, the median AGGG frequency in children with an expansion (0.072 [IQR: 0.066-0.076]) was lower compared with children with retention or contraction (0.080 [IQR: 0.073-0.083]) (z = -0.007; P = 0.003). In a logistic regression model, the AGGG frequency predicted the outcome of either expansion or retention/contraction when including repeat number and sex as covariates (ß = 80.7; z-score = 2.63; P = 0.0085). The AGGG frequency varied slightly over 1 year (0.070 [IQR: 0.063-0.080] to 0.073 [IQR: 0.069-0.078]). CONCLUSIONS: Our results show that a higher AGGG frequency may stabilize repeats across generations. This highlights the importance of further investigating mDRILS as a disease-modifying factor with generational differences. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genome-wide enhancer-associated tandem repeats are expanded in cardiomyopathy.

BACKGROUND: Cardiomyopathy is a clinically and genetically heterogeneous heart condition that can lead to heart failure and sudden cardiac death in childhood. While it has a strong genetic basis, the genetic aetiology for over 50% of cardiomyopathy cases remains unknown. METHODS: In this study, we analyse the characteristics of tandem repeats from genome sequence data of unrelated individuals diagnosed with cardiomyopathy from Canada and the United Kingdom (n = 1216) and compare them to those found in the general population. We perform burden analysis to identify genomic and epigenomic features that are impacted by rare tandem repeat expansions (TREs), and enrichment analysis to identify functional pathways that are involved in the TRE-associated genes in cardiomyopathy. We use Oxford Nanopore targeted long-read sequencing to validate repeat size and methylation status of one of the most recurrent TREs. We also compare the TRE-associated genes to those that are dysregulated in the heart tissues of individuals with cardiomyopathy. FINDINGS: We demonstrate that tandem repeats that are rarely expanded in the general population are predominantly expanded in cardiomyopathy. We find that rare TREs are disproportionately present in constrained genes near transcriptional start sites, have high GC content, and frequently overlap active enhancer H3K27ac marks, where expansion-related DNA methylation may reduce gene expression. We demonstrate the gene silencing effect of expanded CGG tandem repeats in DIP2B through promoter hypermethylation. We show that the enhancer-associated loci are found in genes that are highly expressed in human cardiomyocytes and are differentially expressed in the left ventricle of the heart in individuals with cardiomyopathy. INTERPRETATION: Our findings highlight the underrecognized contribution of rare tandem repeat expansions to the risk of cardiomyopathy and suggest that rare TREs contribute to ∼4% of cardiomyopathy risk. FUNDING: Government of Ontario (RKCY), The Canadian Institutes of Health Research PJT 175329 (RKCY), The Azrieli Foundation (RKCY), SickKids Catalyst Scholar in Genetics (RKCY), The University of Toronto McLaughlin Centre (RKCY, SM), Ted Rogers Centre for Heart Research (SM), Data Sciences Institute at the University of Toronto (SM), The Canadian Institutes of Health Research PJT 175034 (SM), The Canadian Institutes of Health Research ENP 161429 under the frame of ERA PerMed (SM, RL), Heart and Stroke Foundation of Ontario & Robert M Freedom Chair in Cardiovascular Science (SM), Bitove Family Professorship of Adult Congenital Heart Disease (EO), Canada Foundation for Innovation (SWS, JR), Canada Research Chair (PS), Genome Canada (PS, JR), The Canadian Institutes of Health Research (PS).

Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures.

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1 (replication factor C subunit 1), encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS neither is known nor is it understood why some, but not all expanded, motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, whereas (AAAAG)n•(CTTTT)n is the most common nonpathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic forms unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Nonpathogenic repeats did not form G-quadruplexes. Triple-stranded structures are formed by the pathogenic motifs but not by the nonpathogenic motifs. G- and C-richness of the pathogenic strands favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, stabilized by increased hydrogen bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the nonpathogenic motif. The ligand, TMPyP4, binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.

Autistic traits in myotonic dystrophy type 1 due to MBNL inhibition and RNA mis-splicing.

Tandem repeat expansions are enriched in autism spectrum disorder, including CTG expansion in the DMPK gene that underlines myotonic muscular dystrophy type 1. Although the clinical connection of autism to myotonic dystrophy is corroborated, the molecular links remained unknown. Here, we show a mechanistic path of autism via repeat expansion in myotonic dystrophy. We found that inhibition of muscleblind-like (MBNL) splicing factors by expanded CUG RNAs alerts the splicing of autism-risk genes during brain development especially a class of autism-relevant microexons. To provide in vivo evidence that the CTG expansion and MBNL inhibition axis leads to the presentation of autistic traits, we demonstrate that CTG expansion and MBNL-null mouse models recapitulate autism-relevant mis-splicing profiles and demonstrate social deficits. Our findings indicate that DMPK CTG expansion-associated autism arises from developmental mis-splicing. Understanding this pathomechanistic connection provides an opportunity for greater in-depth investigations of mechanistic threads in autism.

Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Remotely sensed terrestrial open water evaporation.

Terrestrial open water evaporation is difficult to measure both in situ and remotely yet is critical for understanding changes in reservoirs, lakes, and inland seas from human management and climatically altered hydrological cycling. Multiple satellite missions and data systems (e.g., ECOSTRESS, OpenET) now operationally produce evapotranspiration (ET), but the open water evaporation data produced over millions of water bodies are algorithmically produced differently than the main ET data and are often overlooked in evaluation. Here, we evaluated the open water evaporation algorithm, AquaSEBS, used by ECOSTRESS and OpenET against 19 in situ open water evaporation sites from around the world using MODIS and Landsat data, making this one of the largest open water evaporation validations to date. Overall, our remotely sensed open water evaporation retrieval captured some variability and magnitude in the in situ data when controlling for high wind events (instantaneous: r2 = 0.71; bias = 13% of mean; RMSE = 38% of mean). Much of the instantaneous uncertainty was due to high wind events (u > mean daily 7.5 m·s-1) when the open water evaporation process shifts from radiatively-controlled to atmospherically-controlled; not accounting for high wind events decreases instantaneous accuracy significantly (r2 = 0.47; bias = 36% of mean; RMSE = 62% of mean). However, this sensitivity minimizes with temporal integration (e.g., daily RMSE = 1.2-1.5 mm·day-1). To benchmark AquaSEBS, we ran a suite of 11 machine learning models, but found that they did not significantly improve on the process-based formulation of AquaSEBS suggesting that the remaining error is from a combination of the in situ evaporation measurements, forcing data, and/or scaling mismatch; the machine learning models were able to predict error well in and of itself (r2 = 0.74). Our results provide confidence in the remotely sensed open water evaporation data, though not without uncertainty, and a foundation by which current and future missions may build such operational data.

Discovery of BAY-390, a Selective CNS Penetrant Chemical Probe as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist.

Transient receptor potential ankyrin 1 (TRPA1) is a voltage-dependent, ligand-gated ion channel, and activation thereof is linked to a variety of painful conditions. Preclinical studies have demonstrated the role of TRPA1 receptors in a broad range of animal models of acute, inflammatory, and neuropathic pain. In addition, a clinical study using the TRPA1 antagonist GRC-17536 (Glenmark Pharmaceuticals) demonstrated efficacy in a subgroup of patients with painful diabetic neuropathy. Consequently, there is an increasing interest in TRPA1 inhibitors as potential analgesics. Herein, we report the identification of a fragment-like hit from a high-throughput screening (HTS) campaign and subsequent optimization to provide a novel and brain-penetrant TRPA1 inhibitor (compound 18, BAY-390), which is now being made available to the research community as an open-source in vivo probe.

Mosaic divergent repeat interruptions in XDP influence repeat stability and disease onset.

While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a SINE-VNTR-Alu(AGAGGG)n retrotransposon insertion in TAF1, with a polymorphic (AGAGGG)n repeat. Repeat length and variants in MSH3 and PMS2 explain ∼65% of the variance in age at onset (AAO) in XDP. However, additional genetic modifiers are conceivably at play in XDP, such as repeat interruptions. Long-read nanopore sequencing of PCR amplicons from XDP patients (n = 202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment confirmed the presence of identified divergent repeat motifs. In addition to the canonical pure SINE-VNTR-Alu-5'-(AGAGGG)n, we observed a mosaic of divergent repeat motifs that polarized at the beginning of the tract, where the divergent repeat interruptions varied in motif length by having one, two, or three nucleotides fewer than the hexameric motif, distinct from interruptions in other disease-associated repeats, which match the lengths of the canonical motifs. All divergent configurations occurred mosaically and in two investigated brain regions (basal ganglia, cerebellum) and in blood-derived DNA from the same patient. The most common divergent interruption was AGG [5'-SINE-VNTR-Alu(AGAGGG)2AGG(AGAGGG)n], similar to the pure tract, followed by AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n], at median frequencies of 0.425 (IQR: 0.42-0.43) and 0.128 (IQR: 0.12-0.13), respectively. The mosaic AGG motif was not associated with repeat number (estimate = -3.8342, P = 0.869). The mosaic pure tract frequency was associated with repeat number (estimate = 45.32, P = 0.0441) but not AAO (estimate = -41.486, P = 0.378). Importantly, the mosaic frequency of the AGGG negatively correlated with repeat number after adjusting for age at sampling (estimate = -161.09, P = 3.44 × 10-5). When including the XDP-relevant MSH3/PMS2 modifier single nucleotide polymorphisms into the model, the mosaic AGGG frequency was associated with AAO (estimate = 155.1063, P = 0.047); however, the association dissipated after including the repeat number (estimate = -92.46430, P = 0.079). We reveal novel mosaic divergent repeat interruptions affecting both motif length and sequence (DRILS) of the canonical motif polarized within the SINE-VNTR-Alu(AGAGGG)n repeat. Our study illustrates: (i) the importance of somatic mosaic genotypes; (ii) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; and (iii) that these variations may remain undetected without assessment of single molecules.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( ≳ 40 known), ≤ 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

De novo mutations, genetic mosaicism and human disease.

Mosaicism-the existence of genetically distinct populations of cells in a particular organism-is an important cause of genetic disease. Mosaicism can appear as de novo DNA mutations, epigenetic alterations of DNA, and chromosomal abnormalities. Neurodevelopmental or neuropsychiatric diseases, including autism-often arise by de novo mutations that usually not present in either of the parents. De novo mutations might occur as early as in the parental germline, during embryonic, fetal development, and/or post-natally, through ageing and life. Mutation timing could lead to mutation burden of less than heterozygosity to approaching homozygosity. Developmental timing of somatic mutation attainment will affect the mutation load and distribution throughout the body. In this review, we discuss the timing of de novo mutations, spanning from mutations in the germ lineage (all ages), to post-zygotic, embryonic, fetal, and post-natal events, through aging to death. These factors can determine the tissue specific distribution and load of de novo mutations, which can affect disease. The disease threshold burden of somatic de novo mutations of a particular gene in any tissue will be important to define.

Safety and Efficacy of a New Water Gradient Biomimetic Monthly Replacement Spherical Contact Lens Material (Lehfilcon A).

Purpose: The objective of this study was to evaluate the safety and performance of the investigational lens, lehfilcon A, when worn in a daily wear modality and replaced monthly as compared to the commercially available comfilcon A contact lens. Methods: This was a multicenter, prospective, controlled, double-masked, randomized, parallel-group clinical study with bilateral lens wear for 3 months. In all, 115 subjects completed the study (77 with test lehfilcon A and 38 with control comfilcon A contact lenses). Distance visual acuity (VA) was assessed using Snellen VA. Lens performance was assessed by examining lens fit/movement, centration, front surface wettability and front/back surface deposits using slit-lamp biomicroscopy. Results: At the 3-month follow-up visit, all eyes had a distance VA of 20/20 or better. Further, lens fit/movement was assessed as optimal in 92.9% of the eyes with lehfilcon A and 89.2% with comfilcon A. There were no ratings of unacceptably tight or loose fits for either contact lens material. Lens centration was assessed as optimal in 98.7% of eyes with lehfilcon A and 94.6% with comfilcon A. For front and back surface deposits, both materials showed minimal lens surface deposits. Front surface wettability was assessed as grade 0 or 1 for most of the study lenses in both lens groups across all attended study visits. There were no ocular adverse events related to the study lenses. Conclusion: Overall, lehfilcon A showed excellent VA, optimal lens fitting characteristics, a clean surface, high wettability, and low risk for adverse events after 3 months of lens wear.

Genome-wide tandem repeat expansions contribute to schizophrenia risk.

Tandem repeat expansions (TREs) can cause neurological diseases but their impact in schizophrenia is unclear. Here we analyzed genome sequences of adults with schizophrenia and found that they have a higher burden of TREs that are near exons and rare in the general population, compared with non-psychiatric controls. These TREs are disproportionately found at loci known to be associated with schizophrenia from genome-wide association studies, in individuals with clinically-relevant genetic variants at other schizophrenia loci, and in families where multiple individuals have schizophrenia. We showed that rare TREs in schizophrenia may impact synaptic functions by disrupting the splicing process of their associated genes in a loss-of-function manner. Our findings support the involvement of genome-wide rare TREs in the polygenic nature of schizophrenia.

Towards Intelligently Designed Evolvable Processors.

Evolution-in-Materio is a computational paradigm in which an algorithm reconfigures a material's properties to achieve a specific computational function. This article addresses the question of how successful and well performing Evolution-in-Materio processors can be designed through the selection of nanomaterials and an evolutionary algorithm for a target application. A physical model of a nanomaterial network is developed which allows for both randomness, and the possibility of Ohmic and non-Ohmic conduction, that are characteristic of such materials. These differing networks are then exploited by differential evolution, which optimises several configuration parameters (e.g., configuration voltages, weights, etc.), to solve different classification problems. We show that ideal nanomaterial choice depends upon problem complexity, with more complex problems being favoured by complex voltage dependence of conductivity and vice versa. Furthermore, we highlight how intrinsic nanomaterial electrical properties can be exploited by differing configuration parameters, clarifying the role and limitations of these techniques. These findings provide guidance for the rational design of nanomaterials and algorithms for future Evolution-in-Materio processors.

Advancing genomic technologies and clinical awareness accelerates discovery of disease-associated tandem repeat sequences.

Expansions of gene-specific DNA tandem repeats (TRs), first described in 1991 as a disease-causing mutation in humans, are now known to cause >60 phenotypes, not just disease, and not only in humans. TRs are a common form of genetic variation with biological consequences, observed, so far, in humans, dogs, plants, oysters, and yeast. Repeat diseases show atypical clinical features, genetic anticipation, and multiple and partially penetrant phenotypes among family members. Discovery of disease-causing repeat expansion loci accelerated through technological advances in DNA sequencing and computational analyses. Between 2019 and 2021, 17 new disease-causing TR expansions were reported, totaling 63 TR loci (>69 diseases), with a likelihood of more discoveries, and in more organisms. Recent and historical lessons reveal that properly assessed clinical presentations, coupled with genetic and biological awareness, can guide discovery of disease-causing unstable TRs. We highlight critical but underrecognized aspects of TR mutations. Repeat motifs may not be present in current reference genomes but will be in forthcoming gapless long-read references. Repeat motif size can be a single nucleotide to kilobases/unit. At a given locus, repeat motif sequence purity can vary with consequence. Pathogenic repeats can be "insertions" within nonpathogenic TRs. Expansions, contractions, and somatic length variations of TRs can have clinical/biological consequences. TR instabilities occur in humans and other organisms. TRs can be epigenetically modified and/or chromosomal fragile sites. We discuss the expanding field of disease-associated TR instabilities, highlighting prospects, clinical and genetic clues, tools, and challenges for further discoveries of disease-causing TR instabilities and understanding their biological and pathological impacts-a vista that is about to expand.

CAG repeat-binding small molecule improves motor coordination impairment in a mouse model of Dentatorubral-pallidoluysian atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a devastating genetic disease presenting myoclonus, epilepsy, ataxia, and dementia. DRPLA is caused by the expansion of a CAG repeat in the ATN1 gene. Aggregation of the polyglutamine-expanded ATN1 protein causes neuro-degeneration of the dentatorubral and pallidoluysian systems. The expanded CAG repeats are unstable, and ongoing repeat expansions contribute to disease onset, progression, and severity. Inducing contractions of expanded repeats can be a means to treat DRPLA, for which no disease-modifying or curative therapies exist at present. Previously, we characterized a small molecule, naphthyridine-azaquinolone (NA), which binds to CAG slip-out structures and induces repeat contraction in Huntington's disease mice. Here, we demonstrate that long-term intracerebroventricular infusion of NA leads to repeat contraction, reductions in mutant ATN1 aggregation, and improved motor phenotype in a murine model of DRPLA. Furthermore, NA-induced contraction resulted in the modification of repeat-length-dependent dysregulation of gene expression profiles in DRPLA mice. Our study reveals the therapeutic potential of repeat contracting small molecules for repeat expansion disorders, such as DRPLA.

FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability.

Ongoing inchworm-like CAG and CGG repeat expansions in brains, arising by aberrant processing of slipped DNAs, may drive Huntington's disease, fragile X syndrome, and autism. FAN1 nuclease modifies hyper-expansion rates by unknown means. We show that FAN1, through iterative cycles, binds, dimerizes, and cleaves slipped DNAs, yielding striking exo-nuclease pauses along slip-outs: 5'-C↓A↓GC↓A↓G-3' and 5'-C↓T↓G↓C↓T↓G-3'. CAG excision is slower than CTG and requires intra-strand A·A and T·T mismatches. Fully paired hairpins arrested excision, whereas disease-delaying CAA interruptions further slowed excision. Endo-nucleolytic cleavage is insensitive to slip-outs. Rare FAN1 variants are found in individuals with autism with CGG/CCG expansions, and CGG/CCG slip-outs show exo-nuclease pauses. The slip-out-specific ligand, naphthyridine-azaquinolone, which induces contractions of expanded repeats in vivo, requires FAN1 for its effect, and protects slip-outs from FAN1 exo-, but not endo-, nucleolytic digestion. FAN1's inchworm pausing of slip-out excision rates is well suited to modify inchworm expansion rates, which modify disease onset and progression.

ATRX proximal protein associations boast roles beyond histone deposition.

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions.