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Molecular Carcinogenesis Apr 2009Triplet repeat expansion is the molecular basis for several human diseases. Intensive studies using systems in bacteria, yeast, flies, mammalian cells, and mice have... (Review)
Review
Triplet repeat expansion is the molecular basis for several human diseases. Intensive studies using systems in bacteria, yeast, flies, mammalian cells, and mice have provided important insights into the molecular processes that are responsible for mediating repeat instability. The age-dependent, ongoing repeat instability in somatic tissues, especially in terminally differentiated neurons, strongly suggests a robust role for pathways that are independent of DNA replication. Several genetic studies have indicated that transcription can play a critical role in repeat instability, potentially providing a basis for the instability observed in neurons. Transcription-induced repeat instability can be modulated by several DNA repair proteins, including those involved in mismatch repair (MMR) and transcription-coupled nucleotide excision repair (TC-NER). Though the mechanism is unclear, it is likely that transcription facilitates the formation of repeat-specific secondary structures, which act as intermediates to trigger DNA repair, eventually leading to changes in the length of the repeat tract. In addition, other processes associated with transcription can also modulate repeat instability, as shown in a variety of different systems. Overall, the mechanisms underlying repeat instability in humans are unexpectedly complicated. Because repeat-disease genes are widely expressed, transcription undoubtedly contributes to the repeat instability observed in many diseases, but it may be especially important in nondividing cells. Transcription-induced instability is likely to involve an extensive interplay not only of the core transcription machinery and DNA repair proteins, but also of proteins involved in chromatin remodeling, regulation of supercoiling, and removal of stalled RNA polymerases, as well as local DNA sequence effects.
Topics: Animals; Genomic Instability; Humans; Transcription, Genetic; Trinucleotide Repeats
PubMed: 18973172
DOI: 10.1002/mc.20488 -
The Journal of Molecular Diagnostics :... Aug 2021Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with...
Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 5'-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 5'-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA- and FRAXE-associated disorders.
Topics: Alleles; Electrophoresis, Capillary; Fragile X Mental Retardation Protein; Fragile X Syndrome; Genetic Association Studies; Genetic Predisposition to Disease; Genetic Testing; Humans; Multiplex Polymerase Chain Reaction; Nuclear Proteins; Reproducibility of Results; Trinucleotide Repeat Expansion
PubMed: 34111553
DOI: 10.1016/j.jmoldx.2021.04.015 -
Journal of Materials Chemistry. B Jan 2020Trinucleotide repeat (TNR) sequences introduce sequence-directed flexibility in the genomic makeup of all living species leading to unique non-canonical structure...
Trinucleotide repeat (TNR) sequences introduce sequence-directed flexibility in the genomic makeup of all living species leading to unique non-canonical structure formation. In humans, the expansions of TNR sequences are responsible for almost 24 neurodegenerative and neuromuscular diseases because their unique structures disrupt cell functions. The biophysical studies of these sequences affect their electrophoretic mobility and spectroscopic signatures. Here, we demonstrate a novel strategy to characterize and discriminate the TNR sequences by monitoring their capillary flow in the absence of an external driving force using wax-on-plastic microchannels. The wax-on-plastic microfluidic system translates the sequence-directed flexibility of TNR into differential flow dynamics. Several variables were used to characterize sequences including concentration, single- vs. double-stranded samples, type of repeat sequence, length of the repeat sequence, presence of mismatches in duplex, and presence of metal ion. All these variables were found to influence the flow velocities of TNR sequences as these factors directly affect the structural flexibility of TNR at the molecular level. An overall trend was observed as the higher flexibility in the TNR structure leads to lower capillary flow. After testing samples derived from relevant cells harboring expanded TNR sequences, it is concluded that this approach may transform into a reagent-free and pump-free biosensing platform to detect microsatellite expansion diseases.
Topics: Humans; Materials Testing; Microfluidic Analytical Techniques; Trinucleotide Repeats; Waxes
PubMed: 31894829
DOI: 10.1039/c9tb02208b -
Disease Models & Mechanisms Jan 2018Diseases such as Huntington's disease and certain spinocerebellar ataxias are caused by the expansion of genomic cytosine-adenine-guanine (CAG) trinucleotide repeats... (Review)
Review
Diseases such as Huntington's disease and certain spinocerebellar ataxias are caused by the expansion of genomic cytosine-adenine-guanine (CAG) trinucleotide repeats beyond a specific threshold. These diseases are all characterised by neurological symptoms and central neurodegeneration, but our understanding of how expanded repeats drive neuronal loss is incomplete. Recent human genetic evidence implicates DNA repair pathways, especially mismatch repair, in modifying the onset and progression of CAG repeat diseases. Repair pathways might operate directly on repeat sequences by licensing or inhibiting repeat expansion in neurons. Alternatively, or in addition, because many of the genes containing pathogenic CAG repeats encode proteins that themselves have roles in the DNA damage response, it is possible that repeat expansions impair specific DNA repair pathways. DNA damage could then accrue in neurons, leading to further expansion at repeat loci, thus setting up a vicious cycle of pathology. In this review, we consider DNA damage and repair pathways in postmitotic neurons in the context of disease-causing CAG repeats. Investigating and understanding these pathways, which are clearly relevant in promoting and ameliorating disease in humans, is a research priority, as they are known to modify disease and therefore constitute prevalidated drug targets.
Topics: Animals; DNA Damage; DNA Repair; Genome; Humans; Models, Biological; Trinucleotide Repeat Expansion
PubMed: 29419417
DOI: 10.1242/dmm.031930 -
DNA Repair Dec 2016DNA base lesions and base excision repair (BER) within trinucleotide repeat (TNR) tracts modulate repeat instability through the coordination among the key BER enzymes...
DNA base lesions and base excision repair (BER) within trinucleotide repeat (TNR) tracts modulate repeat instability through the coordination among the key BER enzymes DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase I (LIG I). However, it remains unknown whether BER cofactors can also alter TNR stability. In this study, we discovered that proliferating cell nuclear antigen (PCNA), a cofactor of BER, promoted CAG repeat deletion and removal of a CAG repeat hairpin during BER in a duplex CAG repeat tract and CAG hairpin loop, respectively. We showed that PCNA stimulated LIG I activity on a nick across a small template loop during BER in a duplex (CAG) repeat tract promoting small repeat deletions. Surprisingly, we found that during BER in a hairpin loop, PCNA promoted reannealing of the upstream flap of a double-flap intermediate, thereby facilitating the formation of a downstream flap and stimulating FEN1 cleavage activity and hairpin removal. Our results indicate that PCNA plays a critical role in preventing CAG repeat expansions by modulating the structures of dynamic DNA via cooperation with BER enzymes. We provide the first evidence that PCNA prevents CAG repeat expansions during BER by promoting CAG repeat deletion and removal of a TNR hairpin.
Topics: Base Sequence; DNA Damage; DNA Ligase ATP; DNA Polymerase beta; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Flap Endonucleases; Gene Expression; Humans; Nucleic Acid Conformation; Proliferating Cell Nuclear Antigen; Sequence Deletion; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 27793507
DOI: 10.1016/j.dnarep.2016.10.006 -
Genome Medicine Jul 2017Microsatellite expansion, such as trinucleotide repeat expansion (TRE), is known to cause a number of genetic diseases. Sanger sequencing and next-generation short-read...
Microsatellite expansion, such as trinucleotide repeat expansion (TRE), is known to cause a number of genetic diseases. Sanger sequencing and next-generation short-read sequencing are unable to interrogate TRE reliably. We developed a novel algorithm called RepeatHMM to estimate repeat counts from long-read sequencing data. Evaluation on simulation data, real amplicon sequencing data on two repeat expansion disorders, and whole-genome sequencing data generated by PacBio and Oxford Nanopore technologies showed superior performance over competing approaches. We concluded that long-read sequencing coupled with RepeatHMM can estimate repeat counts on microsatellites and can interrogate the "unsequenceable" genomic trinucleotide repeat disorders.
Topics: Algorithms; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA; Trinucleotide Repeats
PubMed: 28720120
DOI: 10.1186/s13073-017-0456-7 -
The Journal of Molecular Diagnostics :... Aug 2022Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in...
Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in intron 1 of the FXN gene. It is common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles <200 GAA repeats (±1 to 5 repeats) and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles >200 GAA repeats (±50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described herein is a diagnostic genotyping assay that reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles, as indicated by the TR-PCR assay. The use of this two-tier method offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline, and expanded alleles.
Topics: Adenine; Friedreich Ataxia; Genotype; Guanine; Humans; Iron-Binding Proteins; Polymerase Chain Reaction; Sepharose; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 35595154
DOI: 10.1016/j.jmoldx.2022.04.008 -
Turkish Journal of Medical Sciences Dec 2022: Infertility is a global problem that brings about serious sexual and social consequences that strain the health sector and society. The expansion of CAG and GGC...
BACKGROUND
: Infertility is a global problem that brings about serious sexual and social consequences that strain the health sector and society. The expansion of CAG and GGC repeats in androgen receptor (AR) gene (Ensembl number ENSG00000169083) may lead to reduced fertility. Our objective was to determine the association of CAG and GGC repeats with altered sperm parameters in male infertile subjects.
METHODS
This was a cross-sectional study conducted at Aga Khan University, Karachi, Pakistan. A total of 376 males were recruited, out of which group A (N = 208) and group B (N = 168) were comprised of subjects with normal and altered sperm parameters, respectively, from 18 to 60 years. The numbers of CAG and GGC repeats were determined by using PCR amplification and sequence analysis using the Molecular Evolutionary Genetic Analysis (MEGA) software version 6.0. Statistical analysis was performed using the SPSS version 20 and the P-value of <0.05 was considered significant.
RESULTS
The mean androgen receptor gene CAG repeats were significantly longer in males with altered sperm parameters as compared to male subjects with normal sperm parameters (P < 0.001). There was no significant difference found for GGC repeats for subjects with altered sperm parameters.
DISCUSSION
Longer CAG length corresponded to greater severity of spermatogenic defect and may lead to subfertility recommendations.
Topics: Humans; Male; Receptors, Androgen; Cross-Sectional Studies; Semen; Infertility, Male; Exons; Trinucleotide Repeats
PubMed: 36945970
DOI: 10.55730/1300-0144.5525 -
Genes Oct 2021(FMRP translational regulator 1) variants other than repeat expansion are known to cause disease phenotypes but can be overlooked if they are not accounted for in...
(FMRP translational regulator 1) variants other than repeat expansion are known to cause disease phenotypes but can be overlooked if they are not accounted for in genetic testing strategies. We collected and reanalyzed the evidence for pathogenicity of coding, noncoding, and copy number variants published to date. There is a spectrum of disease-causing variation, with clinical and functional evidence supporting pathogenicity of five splicing, five missense, one in-frame deletion, one nonsense, and four frameshift variants. In addition, deletions occur in both mosaic full mutation patients and as constitutional pathogenic alleles. De novo deletions arise not only from full mutation alleles but also alleles with normal-sized CGG repeats in several patients, suggesting that the CGG repeat region may be prone to genomic instability even in the absence of repeat expansion. We conclude that clinical tests for potentially -related indications such as intellectual disability should include methods capable of detecting small coding, noncoding, and copy number variants.
Topics: 5' Untranslated Regions; Adult; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Frequency; Genetic Association Studies; Humans; Infant; Infant, Newborn; Male; Mutation; Open Reading Frames; Pregnancy; RNA, Messenger; Sequence Deletion; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 34828275
DOI: 10.3390/genes12111669 -
Trends in Genetics : TIG Apr 2015Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the... (Review)
Review
Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.
Topics: Animals; Endonucleases; Genetic Therapy; Genomic Instability; Humans; Protein Engineering; Substrate Specificity; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 25743488
DOI: 10.1016/j.tig.2015.02.003