• K01 NS126489 NINDS NIH HHS
• P30 AG066546 NIA NIH HHS
• RF1 NS112391 NINDS NIH HHS
• S10 OD010786 NIH HHS

• Huntington Disease/genetics
• Huntington Disease/diagnosis
• Humans
• Huntingtin Protein/genetics
• Trinucleotide Repeat Expansion
• Sequence Analysis, DNA/methods
• High-Throughput Nucleotide Sequencing/methods
• Computational Biology/methods
• Trinucleotide Repeats

• 742436 European Research Council, Advanced Grant
• 874758 NSC-Reconstruct Consortium, European Union's Horizon 2020 Research and Innovation Program
• JSC A11103 C.H.D.I. Foundation
• Leslie Gehry Prize for Innovation in Science from the Hereditary Disease Foundation
• GMR23T1059 Fondazione Telethon
• 2022LBENTH Ministero dell'Istruzione, dell'Università e della Ricerca

• genetic predisposition to disease
• genetics research

• Humans
• Huntington Disease/genetics
• Huntington Disease/pathology
• Huntingtin Protein/genetics
• Middle Aged
• Male
• Female
• Phenotype
• Adult
• Alleles
• Trinucleotide Repeat Expansion
• Aged
• Heterozygote
• Brain/pathology
• Brain/metabolism

• FIS PI15/02227 Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)
• Enroll-HD Departamento de Salud, Gobierno de Navarra (ref. 0011-4809-2018-000001)

• Humans
• Microsatellite Repeats/genetics
• DNA Repeat Expansion/genetics
• Genome, Human

• ATXN1
• ATXN2
• ATXN3
• ATXN7
• CAG repeat
• DNA repair
• SCA
• repeat expansion
• somatic instability
• spinocerebellar ataxia

• Humans
• Mosaicism
• Spinocerebellar Ataxias/genetics
• Trinucleotide Repeat Expansion/genetics
• Female
• Male
• Adult
• Middle Aged
• Cerebellum/metabolism
• Cerebellum/pathology
• Aged
• Brain/metabolism
• Brain/pathology
• Ataxin-1/genetics

We developed a diagnostic method for repeat expansion diseases using a long-read sequencer to improve currently available, low throughput diagnostic methods. We employed the real-time target enrichment system of the nanopore GridION sequencer using the adaptive sampling option, in which software-based target assignment is available without prior sample enrichment, and built an analysis pipeline that prioritized the disease-causing loci. Twenty-two patients with various neurological and neuromuscular diseases, including 12 with genetically diagnosed repeat expansion diseases and 10 manifesting cerebellar ataxia, but without genetic diagnosis, were analyzed. We first sequenced the 12 molecularly diagnosed patients and accurately confirmed expanded repeats in all with uniform depth of coverage across the loci. Next, we applied our method and a conventional method to 10 molecularly undiagnosed patients. Our method corrected inaccurate diagnoses of two patients by the conventional method. Our method is superior to conventional diagnostic methods in terms of speed, accuracy, and comprehensiveness.

• genetic testing
• genetics research

• amyotrophic lateral sclerosis
• motor neuron disease
• motor neurons
• single-cell sequencing
• spatial transcriptomics
• spinal cord

• Adult
• Amyotrophic Lateral Sclerosis/genetics
• Animals
• Disease Models, Animal
• Humans
• Motor Neurons/metabolism
• Spinal Cord
• Transcriptome/genetics

• R35 NS097263 NINDS NIH HHS
• T32 MH020016 NIMH NIH HHS

Repeat diseases, such as fragile X syndrome, myotonic dystrophy, Friedreich ataxia, Huntington disease, spinocerebellar ataxias, and some forms of amyotrophic lateral sclerosis, are caused by repetitive DNA sequences that are expanded in affected individuals. The age at which an individual begins to experience symptoms, and the severity of disease, are partially determined by the size of the repeat. However, the epigenetic state of the area in and around the repeat also plays an important role in determining the age of disease onset and the rate of disease progression. Many repeat diseases share a common epigenetic pattern of increased methylation at CpG islands near the repeat region. CpG islands are CG-rich sequences that are tightly regulated by methylation and are often found at gene enhancer or insulator elements in the genome. Methylation of CpG islands can inhibit binding of the transcriptional regulator CTCF, resulting in a closed chromatin state and gene down regulation. The downregulation of these genes leads to some disease-specific symptoms. Additionally, a genetic and epigenetic interplay is suggested by an effect of methylation on repeat instability, a hallmark of large repeat expansions that leads to increasing disease severity in successive generations. In this review, we will discuss the common epigenetic patterns shared across repeat diseases, how the genetics and epigenetics interact, and how this could be involved in disease manifestation. We also discuss the currently available stem cell and mouse models, which frequently do not recapitulate epigenetic patterns observed in human disease, and propose alternative strategies to study the role of epigenetics in repeat diseases.

• U.S. Department of Defense
• National Institutes of Health
• CHDI Foundation

Repeat expansion diseases are a large group of human genetic disorders caused by expansion of a specific short tandem repeat tract. Expansion in somatic cells affects age of onset and disease severity in some of these disorders. However, alleles in DNA derived from blood, a commonly used source of DNA, usually show much less expansion than disease-relevant cells in the central nervous system in both humans and mouse models. Here we examined the extent of expansion in different DNA sources from mouse models of the fragile X-related disorders, Huntington's disease, spinocerebellar ataxia type 1 and spinocerebellar ataxia type 2. We found that DNA isolated from stool is a much better indicator of somatic expansion than DNA from blood. As stool is a sensitive and noninvasive source of DNA, it can be useful for studies of factors affecting the risk of expansion, or the monitoring of treatments aimed at reducing expansion in preclinical trials, as it would allow expansions to be examined longitudinally in the same animal and allow significant changes in expansion to be observed much earlier than is possible with other DNA sources.

Summary: Stool is a readily available, noninvasive and sensitive source of DNA for monitoring repeat expansion in mouse models of four different repeat expansion diseases.

• Fragile X-related disorder
• Huntington's disease
• Peripheral DNA
• Somatic instability
• Spinocerebellar ataxia types 1 and 2
• Stool

• Animals
• Base Sequence
• Dogs
• Genomics
• Humans
• Sequence Analysis, DNA
• Tandem Repeat Sequences/genetics

• Canadian Institutes of Health Research

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

The expanded HTT CAG repeat causing Huntington’s disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans, we performed comprehensive quantitative analyses of CAG expansion in ~50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.

• DNA repair
• DNA repeat expansion
• Huntington’s disease
• spinocerebellar ataxias
• trinucleotide repeat

• DNA Repair/genetics
• Humans
• Huntington Disease/genetics
• Spinocerebellar Ataxias/genetics
• Trinucleotide Repeat Expansion/genetics

• MR/L010305/1 Medical Research Council

• CAG repeat
• DNA mismatch repair
• Somatic expansion
• anticipation
• genotype to phenotype
• mutation

• Animals
• Anticipation, Genetic/genetics
• DNA Repair/genetics
• Genetics/history
• History, 19th Century
• History, 20th Century
• History, 21st Century
• Humans
• Huntington Disease/genetics
• Huntington Disease/physiopathology
• Trinucleotide Repeat Expansion/genetics

• Huntington’s disease
• N6-furfuryladenine
• disease modifiers
• genome wide analysis studies
• somatic expansion

• DNA Repair/genetics
• Genome-Wide Association Study
• Genomic Instability/genetics
• Humans
• Huntington Disease/genetics
• Spinocerebellar Ataxias/genetics

• CIHR

We tested the hypothesis that variant repeat interruptions (RIs) within the DMPK CTG repeat tract lead to milder symptoms compared with pure repeats (PRs) in myotonic dystrophy type 1 (DM1).

We evaluated motor, neurocognitive, and behavioral outcomes in a group of 6 participants with DM1 with RI compared with a case-matched sample of 12 participants with DM1 with PR and a case-matched sample of 12 unaffected healthy comparison participants (UA).

In every measure, the RI participants were intermediate between UA and PR participants. For muscle strength, the RI group was significantly less impaired than the PR group. For measures of Full Scale IQ, depression, and sleepiness, all 3 groups were significantly different from each other with UA > RI > PR in order of impairment. The RI group was different from unaffected, but not significantly different from PR (UA > RI = PR) in apathy and working memory. Finally, in finger tapping and processing speed, RI did not differ from UA comparisons, but PR had significantly lower scores than the UA comparisons (UA = RI > PR).

Our results support the notion that patients affected by DM1 with RI demonstrate a milder phenotype with the same pattern of deficits as those with PR indicating a similar disease process.

• ALS
• Alzheimer's disease
• DNA damage
• Huntington's disease
• Mosaicism
• Multiple system atrophy
• Neurodegeneration
• Parkinson's disease
• Single cell sequencing
• Somatic mutation
• Synucleinopathy

DNA trinucleotide repeats (TRs) can exhibit dynamic expansions by integer numbers of trinucleotides that lead to neurodegenerative disorders. Strand slipped hairpins during DNA replication, repair and/or recombination may contribute to TR expansion. Here, we combine single-molecule FRET experiments and molecular dynamics studies to elucidate slipping dynamics and conformations of (CAG)_n TR hairpins. We directly resolve slipping by predominantly two CAG units. The slipping kinetics depends on the even/odd repeat parity. The populated states suggest greater stability for 5′-AGCA-3′ tetraloops, compared with alternative 5′-CAG-3′ triloops. To accommodate the tetraloop, even(odd)-numbered repeats have an even(odd) number of hanging bases in the hairpin stem. In particular, a paired-end tetraloop (no hanging TR) is stable in (CAG)_{n = even}, but such situation cannot occur in (CAG)_{n = odd}, where the hairpin is “frustrated’’ and slips back and forth between states with one TR hanging at the 5′ or 3′ end. Trinucleotide interrupts in the repeating CAG pattern associated with altered disease phenotypes select for specific conformers with favorable loop sequences. Molecular dynamics provide atomic-level insight into the loop configurations. Reducing strand slipping in TR hairpins by sequence interruptions at the loop suggests disease-associated variations impact expansion mechanisms at the level of slipped hairpins.

• Atlas-based analysis
• CERES
• FreeSurfer
• SCA1
• Spinocerebellar ataxia type 1

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.

• CAG repeat
• DNA damage
• DNA repair
• Huntington's disease
• Spinocerebellar ataxia

• Animals
• DNA Damage/genetics
• DNA Repair/genetics
• Genome
• Humans
• Models, Biological
• Trinucleotide Repeat Expansion/genetics

• MR/L010305/1 Medical Research Council
• MR/P001629/1 Medical Research Council

• Ataxia
• Biomarker
• MRI
• Polyglutamine disorders
• Spinal cord
• Spinocerebellar ataxia type 1

• homorepeats
• intrinsically disordered protein
• intrinsically disordered protein region
• protein aggregation
• protein repeat expansion
• proteinopathies

• Cell Line
• Humans
• Intrinsically Disordered Proteins/genetics
• Intrinsically Disordered Proteins/metabolism
• Microsatellite Repeats
• Peptides/genetics
• Peptides/metabolism
• Protein Aggregates
• Protein Aggregation, Pathological/genetics
• Protein Aggregation, Pathological/metabolism
• Protein Conformation
• RNA, Messenger/genetics
• RNA, Messenger/metabolism

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

Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant cerebellar ataxia disorder, is caused by a non-coding ATTCT microsatellite repeat expansion in the ataxin 10 gene. In a subset of SCA10 families, the 5’-end of the repeat expansion contains a complex sequence of penta- and heptanucleotide interruption motifs which is followed by a pure tract of tandem ATCCT repeats of unknown length at its 3’-end. Intriguingly, expansions that carry these interruption motifs correlate with an epileptic seizure phenotype and are unstable despite the theory that interruptions are expected to stabilize expanded repeats. To examine the apparent contradiction of unstable, interruption-positive SCA10 expansion alleles and to determine whether the instability originates outside of the interrupted region, we sequenced approximately 1 kb of the 5’-end of SCA10 expansions using the ATCCT-PCR product in individuals across multiple generations from four SCA10 families. We found that the greatest instability within this region occurred in paternal transmissions of the allele in stretches of pure ATTCT motifs while the intervening interrupted sequences were stable. Overall, the ATCCT interruption changes by only one to three repeat units and therefore cannot account for the instability across the length of the disease allele. We conclude that the AT-rich interruptions locally stabilize the SCA10 expansion at the 5’-end but do not completely abolish instability across the entire span of the expansion. In addition, analysis of the interruption alleles across these families support a parsimonious single origin of the mutation with a shared distant ancestor.

• Alleles
• Ataxin-10/genetics
• Base Sequence
• Epilepsy/genetics
• Epilepsy/pathology
• Female
• Gene Expression
• Genetic Variation
• Humans
• Inheritance Patterns
• Male
• Microsatellite Repeats
• Mutation
• Pedigree
• Phenotype
• Spinocerebellar Ataxias/genetics
• Spinocerebellar Ataxias/pathology

• National Institute of Neurological Disorders and Stroke

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder that primarily affects the cerebellum and brainstem. The genetic mutation is an expansion of CAG trinucleotide repeats within the coding region of the ataxin-1 gene, characterizing SCA1 as a polyglutamine expansion disease like Huntington’s. As with most polyglutamine expansion diseases, SCA1 follows the rules of genetic anticipation: the larger the expansion, the earlier and more rapid the symptoms. Unlike the majority of polyglutamine expansion diseases, the presence of histidine interruptions within the polyglutamine tract of ataxin-1 protein can prevent or mitigate disease.

The present review aims to synthesize three decades of research on the ataxin-1 polyglutamine expansion mutation that causes SCA1. Data from genetic population studies and case studies is gathered along with data from manipulation studies in animal models. Specifically, we examine the molecular mechanisms that cause tract expansions and contractions, the molecular pathways that confer instability of tract length in gametic and somatic cells resulting in gametic and somatic mosaicism, the influence of maternal or paternal factors in inheritance of the expanded allele, and the effects of CAT/histidine interruptions to the ataxin-1 allele and protein product.

Our review of existing data supports the following conclusions. First, polyCAG expansion of gametic alleles occur due to the failure of gap repair mechanisms for single or double strand breaks during the transition from an immature haploid spermatid to a mature haploid sperm cell. Equivalent failures were not detected in female gametic cells. Second, polyCAG expansion of somatic alleles occur due to hairpins formed on Okazaki fragments and slipped strand structures due to failures in mismatch repair and transcription-coupled nucleotide excision repair mechanisms. Third, CAT trinucleotide interruptions, which code for histidines in the translated protein, attenuate the formation of slipped strand structures which may protect the allele from the occurrence of large expansions. Many of the mechanisms of expansion identified in this review differ from those noted in Huntington’s disease indicating that gene -or sequence-specific factors may affect the behavior of the polyCAG/glutamine tract. Therefore, synthesis and review of research from the SCA1 field is valuable for future clinical and diagnostic work in the treatment and prevention of SCA1.

Spinocerebellar ataxia-3 (SCA3) is the most common dominant inherited ataxia worldwide and is caused by an unstable CAG trinucleotide expansion mutation within the ATXN3 gene, resulting in an expanded polyglutamine tract within the ATXN3 protein. Many in vitro studies have examined the role of autophagy in neurodegenerative disorders, including SCA3, using transfection models with expression of pathogenic proteins in normal cells. In the current study, we aimed to develop an improved model for studying SCA3 in vitro using patient-derived cells. The patient-derived iPS cells presented a phenotype similar to that of human embryonic stem cells and could be differentiated into neurons. Additionally, these cells expressed abnormal ATXN3 protein without changes in the CAG repeat length during culture for at least 35 passages as iPS cells, up to 3 passages as neural stem cells, and after 4 weeks of neural differentiation. Furthermore, we demonstrated that neural differentiation in these iPS cells was accompanied by autophagy and that rapamycin promoted autophagy through degradation of mutant ATXN3 proteins in neurally differentiated spinocerebellar ataxia-3 human induced pluripotent stem cells (p < 0.05). In conclusion, patient-derived iPS cells are a good model for studying the mechanisms of SCA3 and may provide a tool for drug discovery in vitro.

Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by a coding polyglutamine expansion in the Ataxin-1 gene (ATXN1), which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

• 14-3-3
• Ataxin-1
• aggregation
• autophagy
• mTOR
• nuclear inclusions
• spinocerebellar ataxia type 1
• ubiquitin proteasome system

• DMPK
• High resolution melting
• Methylation
• Myotonic dystrophy type 1
• SIX5
• Variant expansions

• Age of Onset
• Aged
• Amyotrophic Lateral Sclerosis/blood
• Amyotrophic Lateral Sclerosis/genetics
• Amyotrophic Lateral Sclerosis/physiopathology
• Blotting, Southern
• C9orf72 Protein
• Cohort Studies
• Cross-Sectional Studies
• DNA Repeat Expansion
• Female
• Frontotemporal Lobar Degeneration/blood
• Frontotemporal Lobar Degeneration/genetics
• Frontotemporal Lobar Degeneration/physiopathology
• Genotyping Techniques
• Haplotypes
• Humans
• Linear Models
• Male
• Middle Aged
• Multivariate Analysis
• Pattern Recognition, Automated
• Phenotype
• Polymorphism, Single Nucleotide
• Proteins/genetics
• Proteins/metabolism
• Time Factors

• P01 AG032953 NIA NIH HHS
• P30 AG010124 NIA NIH HHS
• T32 MH065218 NIMH NIH HHS
• P01 AG017586 NIA NIH HHS
• K08 AG039510 NIA NIH HHS
• 2014100 Doris Duke Charitable Foundation
• T32MH065218 NIMH NIH HHS
• K01 AG043503 NIA NIH HHS
• K23 NS088341 NINDS NIH HHS

• Base excision repair
• Global genome repair
• Mismatch repair
• Oxidative damage
• Repeat Expansion Diseases
• Transcription coupled repair

• Animals
• Ataxin-1
• Ataxins
• Base Sequence
• Canada
• DNA Repeat Expansion
• Humans
• Huntingtin Protein
• Models, Molecular
• Nerve Tissue Proteins/chemistry
• Nerve Tissue Proteins/genetics
• Nerve Tissue Proteins/metabolism
• Neurodegenerative Diseases/genetics
• Neurodegenerative Diseases/pathology
• Neurodegenerative Diseases/physiopathology
• Neurodegenerative Diseases/therapy
• Nuclear Proteins/chemistry
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Nucleic Acid Conformation
• Peptides/genetics
• Proteasome Endopeptidase Complex/metabolism
• Protein Conformation
• Receptors, Androgen/metabolism
• Signal Transduction/physiology

• DNA repair
• DNA replication
• DNA structure
• Genetic instability
• Transcription

• DNA Damage/genetics
• DNA Repair/genetics
• DNA Replication/genetics
• DNA, B-Form/chemistry
• DNA, B-Form/genetics
• Genomic Instability
• Nucleic Acid Conformation
• Repetitive Sequences, Nucleic Acid/genetics
• Transcription, Genetic

• R01 CA093729 NCI NIH HHS
• CA093729 NCI NIH HHS

Trinucleotide repeat disorders are a heterogeneous group of diseases caused by the expansion, beyond a pathogenic threshold, of unstable DNA tracts in different genes. Sequence interruptions in the repeats have been described in the majority of these disorders and may influence disease phenotype and heritability. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by a CAG trinucleotide expansion in the androgen receptor (AR) gene. Diagnostic testing and previous research have relied on fragment analysis polymerase chain reaction to determine the AR CAG repeat size, and have therefore not been able to assess the presence of interruptions. We here report a sequencing study of the AR CAG repeat in a cohort of SBMA patients and control subjects in the United Kingdom. We found no repeat interruptions to be present, and we describe differences between sequencing and traditional sizing methods.

• CAG
• Interruptions
• Kennedy's disease
• Spinal bulbar muscular atrophy
• Trinucleotide

• 2-D gel electrophoresis
• DNA structure
• Genetic instability
• Mutation
• Replication
• Search engine

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.

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder resulting in loss of coordination and balance. It is caused by an expanded repeated DNA sequence (CAG) in the gene ATXN1. The CAG repeat region is normally interrupted by the DNA sequence CAT. Loss of this interruption is believed to cause instability whereby the CAG repeat expands beyond a key threshold resulting, ultimately, in polyglutamine protein aggregation and cell death. Here we examine how interruptions influence pathology in patients and establish a cellular model to support our findings. We distinguish our patients into two sub-groups based on whether or not their expanded CAG repeat stretches contained an interruption. This is not possible with conventional diagnostic techniques. Differentiating the sub-group with no interruptions led to improved accuracy in predicting their age at onset. The other sub-group, with interruptions, reveals a delay in age at onset that shows greater alignment with the longest stretch of CAG repeats. These findings are significant for genetic counselling and prognosis. Our cellular model and in vitro studies confirmed the relationship between disease severity and uninterrupted repeat length and showed that interruptions do not significantly affect the polyglutamine protein aggregation, but do slow down the aggregation rate.

• Age of Onset
• Alleles
• Cell Adhesion Molecules, Neuronal/genetics
• Cell Adhesion Molecules, Neuronal/metabolism
• Female
• Humans
• Male
• Middle Aged
• Mutation
• Pedigree
• Peptides/genetics
• Peptides/metabolism
• Spinocerebellar Ataxias/genetics
• Spinocerebellar Ataxias/pathology
• Spinocerebellar Degenerations/genetics
• Spinocerebellar Degenerations/pathology
• Trinucleotide Repeat Expansion/genetics

• MC_PC_13054 Medical Research Council
• MC_U117584256 Medical Research Council

AIM: To investigate mitochondrial ATP 6 and 8 polymorphisms in the colon and ileum of patients with irritable bowel syndrome with diarrhea (IBS-D).

METHODS: Twenty-eight patients fulfilling the Rome III criteria for IBS-D and 28 healthy subjects were investigated. All study participants underwent screening colonoscopy and mucosal biopsies were obtained from the colon and/or terminal ileum. Genomic DNA was extracted from specimens based on standard protocols. Mitochondrial ATP (MT-ATP) 6 and 8 genes in specimens were polymerase chain reaction amplified and sequenced. Sequencing data were analyzed via Variant Reporter™ Software and compared with the reference sequence from Genbank (accession No. NC_012920) to indicate possible polymorphisms. The protocol was registered at www.clinicaltrials.gov as NCT01028898.

RESULTS: Twenty-five polymorphic sites of MT-ATP 6 and 8 genes were detected and 12 of them were missense mutations. A median of two polymorphic sites in MT-ATP genes was found in colon specimens of controls while a median of three polymorphic sites was noted in patients with IBS-D (Mann-Whitney test, P = 0.012). The variants of the colon and ileum specimens from the same subjects were identical in all but one case. Symptom duration in IBS was not found to be a significant factor associated with the mtDNA polymorphism (Spearman correlation, P = 0.592). The mitochondrial DNA change at 8860 was present in all cases of both groups. The frequency of the 8701 polymorphism was found to be the second most frequent; however, no statistical difference was noted between the groups (χ² test, P = 0.584).

CONCLUSION: Patients with IBS-D have a higher incidence of MT-ATP 6 and 8 polymorphisms than healthy subjects, implying that the mtDNA polymorphism may play a role in IBS-D.

• Irritable bowel syndrome
• Diarrhea
• Mitochondrial ATP 6 gene
• Mitochondrial ATP 8 gene
• Polymorphism

• Adolescent
• Adult
• Biopsy
• Case-Control Studies
• Colon/pathology
• Colonoscopy
• Comorbidity
• Diarrhea/epidemiology
• Diarrhea/genetics
• Female
• Humans
• Ileum/pathology
• Incidence
• Irritable Bowel Syndrome/epidemiology
• Irritable Bowel Syndrome/genetics
• Male
• Middle Aged
• Mitochondrial Proton-Translocating ATPases/genetics
• Mutation, Missense/genetics
• Polymorphism, Single Nucleotide/genetics
• Young Adult

Alpha-synuclein (SNCA) is crucial in the pathogenesis of Parkinson's disease (PD), yet mutations in the SNCA gene are rare. Evidence for somatic genetic variation in normal humans, also involving the brain, is increasing, but its role in disease is unknown. Somatic SNCA mutations, arising in early development and leading to mosaicism, could contribute to PD pathogenesis and yet be absent or undetectable in DNA derived from peripheral lymphocytes. Such mutations could underlie the widespread pathology in PD, with the precise clinical outcome dependent on their type and the timing and location of their occurrence. We recently reported a novel SNCA mutation (c.150T>G, p.H50Q) in PD brain-derived DNA. To determine if there was mosaicism for this, a PCR and cloning strategy was used to take advantage of a nearby heterozygous intronic polymorphism. No evidence of mosaicism was found. High-resolution melting curve analysis of SNCA coding exons, which was shown to be sensitive enough to detect low proportions of 2 known mutations, did not reveal any further mutations in DNA from 28 PD brain-derived samples. We outline the grounds that make the somatic SNCA mutation hypothesis consistent with genetic, embryological, and pathological data. Further studies of brain-derived DNA are warranted and should include DNA from multiple regions and methods for detecting other types of genomic variation. © 2013 Movement Disorder Society

• Brain/pathology
• Humans
• Spinocerebellar Ataxias/classification
• Spinocerebellar Ataxias/pathology
• Spinocerebellar Ataxias/physiopathology