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Traditional methods for triplet repeats characterization. ( A ) Southern blotting requires genomic DNA digestion with restriction enzymes, f ollo w ed b y blotting and probing with a labeled DNA fragment that specifically h ybridiz es to the repeat containing region. ( B ) Fluorescence-PCR uses at least one fluorescent primer and performs fragment analysis using a capillary electrophoresis system. ( C ) Small-pool PCR relies on serial dilutions and multiple independent PCRs across the repeat, f ollo w ed b y electrophoresis and blotting. ( D ) Sanger sequencing of PCR amplicons, after allelic separation by electrophoresis, detects fluorescence emitted by chain-terminating nucleotides.
Source publication
The accurate characterization of triplet repeats, especially the overrepresented CAG repeats, is increasingly relevant for several reasons. First, germline expansion of CAG repeats above a gene-specific threshold causes multiple neurodegenerative disorders; for instance, Huntington’s disease (HD) is triggered by >36 CAG repeats in the huntingtin (H...
Contexts in source publication
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... the 1990s, several methods have been developed to size triplet repeats ( 26 ,40-42 ). One class includes amplificationfree methods like southern blotting, where genomic DNA is digested, separated by gel electrophoresis and probed with a labeled DNA fragment that hybridizes to the repeat-containing region ( 43 ) (Figure 1 A). Southern blotting avoids PCR amplification steps -therefore bypassing amplification-related artifacts -but requires large amounts of DNA. ...
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... set of methods employs capillary electrophoresis to analyze bulk PCR products (Figure 1 B). These methods employ either one or two fluorescent primers (Fluorescence PCR) ( 48 ,49 ). ...
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... sequencing, developed in the 1970s, has been used to characterize the HTT gene's repetitive region ( 8 ) and remains widely employed in clinical practice, mainly as a confirmatory orthogonal assay ( 39 ,57-61 ) (Figure 1 D). This method uses PCR amplicons followed by a chain termination reaction, with fragments separated by capillary electrophoresis and sequenced by dye fluorescence ( 57 , 58 , 62 ). ...
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... example of this is ScaleHD, a tool developed by McAllister and colleagues, which maps reads to a reference set composed of 4000 possible HTT allele structures with variable CAG / CCG length, predicting the most likely genotypes for a sample ( 126 ). Conversely, reference-free tools may be the best solution to save computational time and resources required for reads alignment, especially in the case of extremely long triplet repeats that may cause alignment issues, particularly with short high-complexity sequences flanking the repeat ( 115 , 138 , 141 ). However, these tools often require higher sequencing coverage to obtain an accurate representation of the two alleles ( 129 ). ...
Citations
... In these studies, the precise measurement of CAG size and composition in individual brain cells, along with the corresponding transcriptional profiles, has become increasingly important. These aspects are discussed in detail in the accompanying article by some of the authors ( 17 ). Finally, we will review strategies aimed at reducing SI with the goal of fighting the disease. ...
... More recently, two pioneering studies analyzing postmortem human HD brains demonstrated that SI rates are not only organ and tissue-specific [SI is marked in the brain striatum, but only moderate in the cerebellum ( 21 )], but also cell-type specific, with MSNs-the most vulnerable cells in HD-showing the highest levels of instability ( 22 ,28 ) (Figure 1 ). These advancements have been made possible by continuous improvements in genomic and CAG sequencing technologies, as detailed in the accompanying paper by some of the authors ( 17 ). For example, Mätlik and colleagues developed a fluorescence-activated nuclear sorting (FANS) approach to isolate different cell populations from five post-mortem HD brains based on marker gene expression. ...
... Since HD phenotypes, such as SI, transcriptional dysregulation and mHTT aggregation formation ( 28 ,51 ) are often co-modulated, SI in HD-vulnerable cell types may serve as an effective early marker for potential therapies. However, to ensure consistency across studies, rigorous standards for target enrichment, sequencing methods and bioinformatics protocols will be essential ( 17 ), potentially guiding the development of clinical guidelines for SI monitoring and HD prognosis assessment. ...
Trinucleotide repeats in DNA exhibit a dual nature due to their inherent instability. While their rapid expansion can diversify gene expression during evolution, exceeding a certain threshold can lead to diseases such as Huntington’s disease (HD), a neurodegenerative condition, triggered by >36 C–A–G repeats in exon 1 of the Huntingtin gene. Notably, the discovery of somatic instability (SI) of the tract allows these mutations, inherited from an affected parent, to further expand throughout the patient’s lifetime, resulting in a mosaic brain with specific neurons exhibiting variable and often extreme CAG lengths, ultimately leading to their death. Genome-wide association studies have identified genetic variants—both cis and trans, including mismatch repair modifiers—that modulate SI, as shown in blood cells, and influence HD’s age of onset. This review will explore the evidence for SI in HD and its role in disease pathogenesis, as well as the therapeutic implications of these findings. We conclude by emphasizing the urgent need for reliable methods to quantify SI for diagnostic and prognostic purposes.
p53 plays a critical role in the pathogenesis of central nervous system (CNS) diseases, including neurodegenerative diseases, stroke, and neurodevelopmental disorders. Recent studies have shown that p53 regulates pathological processes such as apoptosis, ferroptosis, neuroinflammation, mitochondrial dysfunction, DNA damage, and synaptic dysfunction through complex molecular pathways. Preclinical studies have demonstrated that pharmacological inhibition or knockdown of p53 exerts neuroprotective effects, suggesting its potential therapeutic value. This review systematically summarizes the mechanisms of p53 in CNS diseases and the associated signaling pathways involved in these pathological processes. Additionally, p53‐related intervention strategies in CNS diseases and challenges to the development of p53‐targeted drugs are summarized, aiming to provide new insights for therapeutic treatment.
