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. 2024 Dec 14;53(1):gkae1204. doi: 10.1093/nar/gkae1204

When repetita no-longer iuvant: somatic instability of the CAG triplet in Huntington’s disease

Elena Cattaneo 1,2,, Davide Scalzo 3,4, Martina Zobel 5,6, Raffaele Iennaco 7,8, Camilla Maffezzini 9,10, Dario Besusso 11,12, Simone Maestri 13,14
PMCID: PMC11724284  PMID: 39673793

Abstract

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.

Graphical Abstract

Graphical Abstract.

Graphical Abstract

Introduction

The paradox of C–A–Gs: from advantage to adversity

Tandem repeats are a significant component of DNA, particularly in primates, comprising up to seven percent of the total genome. Among these, triplet repeats are blocks of three base pairs (bp) repeated many times one after the other (1), with the cytosine–adenine–guanine (C–A–G) triplet being among the most abundant in exons of the human genome (2). Throughout evolution, the size of these repeats can gradually increase within existing genes, contributing to diversification of gene expression, regulation and function, without the need for new genes. For instance, in humans, >1500 tandem repeats have been found to be specifically expanded compared to non-human primates, and this expansion has been associated with differential isoform usage in genes containing the repeats (3). This increase in repeat size is thought to facilitate faster evolutionary changes by generating a broader range of phenotypes than other types of genetic variations—such as single nucleotide polymorphisms, which produce only two variants, i.e. mutated or not mutated. This process may lead to new shades of traits or behaviors that are subject to natural selection (4–8). However, in some cases, repeats in genes encounter a paradox, shifting from being evolutionarily advantageous to becoming pathogenic in human adults.

A notable example of the paradoxical role of tandem repeats is that of the CAGs in the Huntingtin (HTT) gene, where expansion beyond a certain threshold leads to Huntington’s disease (HD). In early phylogeny, the CAG sequence first appeared in the Echinodermata HTT gene with a small number of repeats (2 CAGs). Inserted at a specific point in the gene (at the 18th triplet, as in the human gene), the CAG sequence began to lengthen throughout evolution, increasing to 4 repeats in amphibians, fishes, reptiles and birds, 7 in mice, 8 in rats and reaching 11–18 repeats in pigs and non-human primates (9). Only humans show a considerably higher number of CAG repeats, with normal range variability between 9 and 35 (10). This evidence suggests that evolution has been permissive regarding this tract and its elongation. Initially considered functionally neutral, the CAG tract in HTT was recently shown to be under purifying selection (9). A stepwise increase in CAG size—from 0 to 2, to 4, to 7—was found to influence positively neuronal parameters in cells in vitro, indicating that the tract is functionally relevant (9). The CAG repeats in the HTT gene of healthy individuals also shape brain structure, with more CAGs in the normal range correlating with an increase in grey matter (11). Furthermore, longer CAG repeats in HTT have been associated with better cognitive outcome (12). Interestingly, individuals in their teens with CAG repeats in the pathological range have been shown to exhibit improved cognitive parameters during the disease-free phase (13).

These data indicate that the CAG tract in HTT is evolutionarily advantageous, a finding that seems counterintuitive for a gene associated with disease. In fact, when >36 CAG repeats in the first of the 67 exons of the HTT gene are transmitted through the germline, HD, a genetically dominant neurodegenerative disease, will manifest (14). Each CAG triplet in the gene is translated into glutamine (Q) in the resulting HTT protein, with the mutant version therefore incorporating >36Qs (14). Symptoms typically appear in adulthood, four to five decades after birth, despite the gene’s presence since conception, marking the characteristic age of onset (AOO). The clinical presentation includes uncontrolled movements, cognitive decline and psychiatric disturbances. Within the brain, the medium spiny neurons (MSNs) and cortical neurons degenerate (14). Longer inherited CAG tracts are linked to an earlier AOO, though other genetic and environmental factors influence this timing and the progression of the disease (15). The exact relationship between pathological CAG expansion in the HTT gene, the late onset of symptoms, and the tissue- and cell-type-specific vulnerability in HD, remains unclear. This question similarly applies to other adult-onset neurodegenerative conditions, such as spinal bulbar muscular atrophy and spinocerebellar ataxias, which also stem from CAG expansions in different genes and affect various neuron types (16).

In this review, we will examine recent evidence showing that, in addition to germline transmission, the pathological CAG repeat expands further in somatic tissues, particularly in the neurons of patients’ brains. HD and its CAG tract in the HTT gene will serve as a case study. We will summarize the discovery of somatic instability (SI) of the CAG, outline the mechanisms that contribute to this phenomenon, and discuss its significance in pathogenesis. 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.

Neurons in the HD brain: where CAG instability spirals out of control

Like many trinucleotide repeat disorders, the CAG repeat in HTT is prone to expansion (18,19). CAG repeat expansion is a phenomenon that can occur in human germ cells and be passed on to the next generation. Although rare, this typically happens during paternal transmission within the germline. It is associated with anticipation, a phenomenon where the disease phenotype manifests earlier in offspring than in the affected parent. This earlier onset is due to a significant increase in CAG repeat length, for example, from 42 repeats in the parent to 60 or more in the child (16). Germline instability is also responsible for new mutations in families with no history of HD, where an allele in the intermediate CAG repeat range (e.g. between 27 and 35) expands into the pathological range in the offspring (20).

Notably, CAG instability also manifests within the tissues most affected by the disease. It has been discovered that the inherited pathological CAG repeat in the HTT gene is unstable in various tissues and can expand throughout an individual’s life, particularly within the affected brain (21). This phenomenon, known as SI, is pronounced in specific cell types and brain regions, particularly in MSNs of the striatum, the most affected brain area (22,23). The foundations for this pivotal discovery were laid by Kennedy and colleagues in 2003, who identified extreme somatic expansion in the striatum of HD patients using early CAG sizing technologies, after similar observations in HD mouse models (24,25). Concurrently, as instability is linked to altered DNA repair, Wheeler and colleagues made significant contributions, expanding upon work by the Messer laboratory (26), by demonstrating that knocking out the mismatch repair (MMR) gene Msh2 modifies CAG repeat instability in an HD mouse model, delaying brain pathology (27). At the time, these findings were not fully appreciated, perhaps due to technological limitations and the prevailing notion that such expansions merely increased the toxicity of inherited pathological alleles without directly causing cell death (28). In the following years, more studies confirmed the primary role of SI in AOO (29,30). Seminal work from the McMurray laboratory, for example, showed a delay in pathogenesis upon suppressing SI across multiple mouse models (31,32).

Genome-wide association studies (GWAS) provided critical insights into the relevance of SI by exploring the HD genome at the population level. These studies identified both cis-acting polymorphisms in HTT exon 1 and trans-acting polymorphisms in other loci as modifiers of AOO, while at the same time affecting SI in peripheral cells (33,34). They confirmed that CAG repeat length inherited through the germline accounts for only ∼60% of individual AOO variability (33), with the remaining variance explained by an increasing number of cis- and trans-modifiers. For instance, variations in the nucleotide composition of the HTT CAG repeat region and polymorphisms in MMR genes were found to correlate with differential SI in the blood of HD patients exhibiting accelerated clinical manifestations (34–36), supporting a link between modifiers, SI and HD onset.

A new theory, the ‘two-sequential components’ hypothesis, further links SI to AOO (37). According to this hypothesis, the inherited germline-transmitted HTT CAG repeat, once exceeding a certain instability threshold, undergoes somatic expansion at a rate modulated by trans- and cis-acting modifiers of AOO. Upon exceeding a second toxicity threshold, CAG triplets in vulnerable cell types become harmful (37). This theory challenges the longstanding hypothesis that mutant HTT (mHTT) exerts cumulative damage throughout the patient’s life, with SI merely exacerbating that damage (32,37). 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).

Figure 1.

Figure 1.

SI causes cell-type specific vulnerability. (A) Recent studies have shown that somatic expansions are not only tissue-specific, but also cell-type specific; (B) Vulnerable cell types preferentially undergo somatic expansion over the course of the patient’s lifetime, ultimately leading to transcriptional dysregulation and cell death.

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. The isolated cells were then subjected to bulk RNA-seq and HTT CAG sizing, providing matched transcriptional and instability profiles at the subpopulation level (22). In a different study, Handsaker and colleagues pushed the technological frontier further by developing a sophisticated single-cell RNA-sequencing method based on long-reads, allowing simultaneous acquisition of transcriptional profile and HTT CAG size from the same cells. This allowed the grouping of cells not only by transcriptional profile (and hence cell type) but also by acquired CAG length (28). They found that (i) SI is especially pronounced in MSNs, the most vulnerable neurons in HD; (ii) within-patient, MSNs must exceed 150 CAGs to undergo overt and cell-autonomous transcriptional dysregulation; (iii) a portion of the MSNs exhibited extreme expansions in the HTT gene, with >800 CAG repeats; and (iv) as a consequence of such extreme expansions, many cell types in the brain become transcriptionally dysregulated through non-cell autonomous mechanisms. Their data support a multi-phase model termed ‘ELongATE’, where somatic expansion rates in neurons accelerate significantly once they exceed 80 CAGs, with a threshold of about 150 CAG repeats required to trigger cell-autonomous transcriptional dysregulation in MSNs, and the consequent cell death (28). According to this model, atrophy and de-vascularization of the caudate, both typical of HD, occur only after MSN loss and despite the other cell types show modest SI (28). Future studies will determine whether the ELongATE model, which provides a plausible explanation for the central role of SI in disease pathogenesis, will stand the test of time.

DNA modifiers shaping the fate of CAG repeat expansion

Although SI at the HTT DNA locus has been known for some time, GWAS data have revitalized its study by identifying genetic variants involved in DNA repair and replication, two processes that significantly influence AOO. These findings, along with increasing evidence of somatic variability in CAG repeat expansion within the brain, suggest that DNA itself is a crucial biotype linked to disease pathogenesis. This idea is further supported by recent evidence associating variations at the HTT locus with both AOO and SI (33).

Cis-modifiers, i.e. genetic variants located near the CAG repeat, have been identified that influence HD progression (33,34,36,38). One such variant affects the penultimate triplet of the CAG repeat. In humans, the repetition of pure CAGs typically ends with a CAA–CAG segment, where the CAA, along with CAG, encodes glutamine (Q) (Figure 2, reference). This penultimate CAA is considered an interruption in the pure CAGs stretch. However, the resulting protein contains a continuous polyglutamine (polyQ) stretch encoded by the pure CAGs sequence, the penultimate CAA and the final CAG. A typical patient with 42 CAG repeats followed by the CAA–CAG produces a protein with 42 + 2Qs (Figure 2, reference).

Figure 2.

Figure 2.

HTT allele structures influence HD AOO. The upper part of the diagram represents a reference HD allele with 42 CAG repeats followed by the typically human ‘CAA–CAG’ tract, leading to a protein with 42Q + 2Q (both CAA and CAG translate to glutamine, Q). The CCG–CCA pair [representing the initial tract of the proline-rich domain (PRD)] following the CAGs is also shown. Middle and bottom sections: GWAS-identified variants in the HTT allele nucleotide sequence that alter AOO; specifically, (middle) the LOI disease haplotype, an A-to-G synonymous mutation in the polyQ tract, leads to the same protein as the reference HD allele (42Q + 2Q), but accelerates disease onset. Conversely (bottom), in the DUP disease haplotype, the inclusion of an additional ‘CAA–CAG’ tract delays disease onset despite adding two extra Qs to the protein (42Q + 4Q).

One group of patients deviated from the reference HD cases by having the penultimate CAA codon replaced by CAG (loss of interruption, LOI), creating a longer uninterrupted CAG stretch, while producing the same protein. Specifically, such patients with, supposedly, 42 CAG repeats would acquire an additional CAG–CAG segment in place of CAA–CAG, thereby creating a longer stretch of pure CAGs. Despite this, the number of Qs in the resulting protein remains at 42 + 2Qs (Figure 2, LOI). Remarkably, patients with the LOI haplotype exhibited a hastening of AOO of 25 years on average. This LOI variant was also associated with a variant in the CCA codon of the PRD, also influencing AOO (34,36,39).

Another group of patients carried a duplication of the penultimate CAA-CAG tract (DUP, Duplication). In a hypothetical patient with 42 uninterrupted CAG repeats, this duplication will result in a protein with 42 + 4Qs, which could theoretically increase protein toxicity (Figure 2, DUP). However, this variation is now recognized as a beneficial cis-acting modifier, leading to an average delay of 4.2 years in AOO (34).

Importantly, both the LOI and DUP cis-variants have been associated with SI, at least in blood cells. Increased and decreased CAG instability, respectively, have been observed in these genetic conditions, pointing to a direct link between HTT locus cis-variants, CAG instability and disease phenotypes (33,34,36,38,39). Still, the full impact of this relationship within a cohesive framework of SI-driven HD pathology by cis-acting modifiers remains to be explored, and data confirming that these modifiers influence instability in human brain neurons are still needed. In addition, data on Africa-ancestry individuals contradicts the increased SI associated with the LOI variants, suggesting that other mechanisms may contribute in driving the pathogenesis (36,39).

A second set of data from GWAS points to trans-modifiers involved in DNA repair. Polymorphisms in genes such as PMS1, MLH1, MSH3, PMS2, FAN1 and LIG1, which are involved in the MMR pathway, have been implicated in DNA repair and replication events leading to instability (33). These polymorphisms, which typically induce a non-synonymous amino acid change, alter protein levels and either accelerate or delay disease onset, depending on the gene involved. Genetic perturbations in MMR genes have been shown to affect SI in mouse models of HD and other trinucleotide expansion disorders (19,30,40). The GeM-HD consortium, leveraging GWAS data, confirmed the association between a polymorphism in MSH3 and SI in blood (33,36). Newer GWAS with larger cohorts not only confirmed previous modifiers, but also identified new ones, such as POLD1 and MED15 (36). Notably, among its various activities, MED15 has been found to bind SREBP and regulate cholesterol biosynthesis (41), a process whose normalization in the HD rodent brain proved beneficial (42,43).

These findings highlight the critical role of both cis and trans variants to instability (in blood cells) and AOO, although the contribution of RNA-related mechanisms to instability cannot yet be ruled out.

From guardians to contributors: MMR in somatic instability mechanisms

During replication or transcription in post-mitotic neurons, single-stranded DNA can form secondary structures, especially when expanded repeats are present (19,35). Long CAG repeats, in particular, can give rise to complex, higher-order non-canonical structures, such as DNA triplexes, hairpins, G-quadruplexes and R-loops (18,44,45). The formation of these structures depends on various factors, including sequence motifs, the presence of interruptions (i.e. purity), length and possibly methylation status (18,44,45). Once these structures form, they often create large loops (slip-outs) that are recognized by MMR proteins, which attempt to repair the DNA damage. However, rather than repairing the damage, these proteins can contribute to the somatic expansion of pathogenic repeats (35) (Figure 3).

Figure 3.

Figure 3.

Proposed model for expansion and comparison to the canonical MMR pathway. (A) In canonical MMR, MutSβ recognizes small breaks and recruits MutL to perform an excision in the strand carrying the mismatch; an exonuclease then forms a gap, and the DNA polymerase repairs the damaged strand using the intact strand as template. (B) In the proposed model for somatic expansion, MutSβ recognizes bigger loops and recruits either MutLα or MutLγ, forming a ternary complex; however, excision occurs in the strand opposite to the one carrying the mismatch and the exonuclease creates a gap. As a result, the strand carrying the damage is used as a template for re-synthesis, leading to repeat expansion.

MMR proteins form dimer complexes to address DNA damage. MutSα (MSH2–MSH6) primarily recognizes small base mismatches, while MutSβ (MSH2–MSH3) detects larger loops (2–10 base pairs), which form in pathogenic contexts. Upon recognizing a loop, MutSβ recruits either MutLα (MLH1–PMS2) or MutLγ (MLH1–MLH3), forming a ternary complex. Instead of breaking the damaged strand, MutL is believed to create a break in the opposite strand (35). An endonuclease then cuts the opposite strand, leading to the longer repeat on the damaged strand being used as template for DNA repair. This may result in the erroneous incorporation of new repeats into the gene (35).

The mechanisms by which these trans-modifiers affect AOO may not be the same across all genes, and this is still an active research area. Moreover, multiple distinguishable AOO modifiers have been associated with polymorphisms in genes from MMR pathway, such as PMS1, PMS2, MSH3 and LIG1. Also polymorphisms in FAN1 can influence AOO in both directions (33,36). Although FAN1 is not a canonical MMR factor, it binds MLH1 and competes with MSH3 for ternary complex formation, mitigating the effects of SI associated with expanded CAG repeats. Its overexpression reduces SI, contrasting the effects of other MMR proteins (46–48).

Given this mechanism of action, down-regulating MMR proteins is expected to reduce SI. This effect has been observed in multiple HD mouse models deficient in MMR genes (26,27,30,40,49). However, caution is necessary when manipulating these proteins, as deficiencies in MSH2, MSH6, MLH1 and PMS1 have been associated with cancer (35,50,51).

Targeting somatic instability to treat HD

In recent years, therapeutic efforts have largely focused on reducing mHTT levels (52), while also raising awareness and expertise in the most promising treatment approaches (53). The proposed mechanism of MMR proteins suggests that manipulating the expression of MMR genes—mimicking the effects of naturally occurring trans-modifiers—could offer promising targets to slow repeat expansion rates (19). In support of this therapeutic avenue, polymorphisms identified by GWAS are well tolerated, as they have not been negatively selected by evolutionary pressure.

The field is now poised to expand gene silencing approaches (52,53) to target molecules affecting CAG instability. Animal studies have demonstrated the therapeutic potential of compounds that reduce the expression of proteins in the MMR pathway. These studies differ in the targeted gene, the method used to modulate translation efficiency, the HD model and the phenotypic readouts, however, they all agree on the value of this approach (50,51,54).

O’Reilly and colleagues used RNA interference to reduce MSH3 protein levels, demonstrating the efficacy of their approach in both in vitro and in vivo models (54). They identified two compounds that achieved a 55%–60% reduction in MSH3 protein levels in the striatum two months post-injection. Remarkably, this level of silencing was sufficient to prevent somatic CAG expansion for up to 4 months in the striatum of HD mouse models, as confirmed by capillary electrophoresis fragment analysis (54). Notably, when they tested a siRNA targeting the HTT gene itself, despite achieving high silencing efficiency, they found no measurable impact on somatic repeat expansion (54).

Ferguson and colleagues explored the effect of targeting MMR genes identified as HD modifiers of AOO using one human HD induced pluripotent stem cell line (50). They used CRISPR interference to reduce the expression of MSH2, MSH3, MSH6, MLH1, PMS1, PMS2, MLH3 and LIG1 transcript levels by 60%–80%. The study assessed the impact of silencing each target on SI, in both proliferating cells and post-mitotic neurons. Using capillary electrophoresis fragment analysis—therefore a method with limited sensitivity for rare alleles (55)—they observed a significant reduction in SI over 2 months when targeting MSH2, MSH3 and MLH1. A moderate reduction was also observed with PMS1, PMS2 and MLH3, with PMS1 being proposed as a novel target for slowing CAG repeat expansion (50).

Recently, Wang and colleagues studied the impact of knocking out different MMR genes in mice (51). They generated homozygous and heterozygous KO alleles for 9 MMR genes in a HD knock-in mouse model with extremely long CAG tract, including Msh3, Mlh1, Pms1, Pms2, Ccdc82, Tcerg1, Msh2, Msh6 and Polq. After 6 months, capillary electrophoresis fragment analysis revealed in knock-in mice a significant linear increase in SI in the brain regions most affected by HD, along with transcriptional alterations and mHTT aggregation, a typical HD hallmark (56,57). Notably, KO for Msh3, Msh2 and Pms1 showed a gene-dosage-dependent rescue of HD phenotypes (51). Specifically, Msh3 KO not only reduced SI compared to unperturbed knock-in mice, but also led to a transcriptional rescue in MSNs, with the effect being more pronounced when both gene copies were knocked out. To further assess cell-type-specific mosaicism of the CAG repeats, Wang and colleagues performed fragment analysis on MSNs purified using FANS. Compared to the bulk striatal tissue, they observed a much narrower distribution of CAG sizes, with unperturbed knock-in mice almost doubling the initial germline CAG number over 16 months (51).

Recent studies emphasize MSH3 and PMS1, key components of MutSβ and MutLβ, as safe and effective targets for reducing SI and ameliorating HD phenotypes. Targeting both genes simultaneously may also yield synergistic effects (50). Future strategies will likely focus on enhancing the potency, stability and duration of gene knockdowns in in vivo models, alongside developing high-throughput, long-term screening systems (58). Neuronal organoid cultures could serve as a valuable tool, enabling co-culturing and perturbation screening of various cis and trans-modifiers (59). 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.

The two sides of the coin: heads - HD is driven by SI

Transcriptional profiling and matched CAG sizing in postmortem HD brains has highlighted a primary role of SI in HD pathogenesis (28). The proposed ‘ELongATE’ model—enabled by advancements in cutting-edge technology (17)—provides a plausible explanation to some unresolved questions. Firstly, the progressive nature of MSN loss during HD neurodegeneration may be attributed to the time required for the accumulation of extreme expansion events in these neurons over a lifetime, with up to 840 CAGs detected in post-mortem HD brains (28). Relevant work from the Heintz lab reviewed SI in cell types within the striatum and found SI in MSNs but also in cell types that do not degenerate (22,60). They conclude that although SI may be important, it alone is not sufficient for cell death. However, these studies used short-read sequencing methods (technically limited to approximately 110 CAG) and may have overlooked extreme expansion events above 150 CAGs, which seem to be the key driver of pathogenesis (28). Secondly, the prolonged period of degeneration observed in HD patients—typically 10–20 years from diagnosis to death (53,61)—seems compatible with the fast but asynchronous neuronal degeneration proposed in the ‘ELongATE’ model. According to this model, despite all cells in the patient’s brain express mHTT, only mHTT with >150 CAG is indeed toxic. As such, at any given time, only a few MSNs may produce this extremely expanded and toxic version of the mHTT protein, thereby contributing to the slow, progressive degeneration. Thirdly, the limited efficacy of the initial ASO strategies targeting HTT—despite persistent, dose-dependent decreases in mHTT levels in cerebrospinal fluid (53)—may be due to the small number of MSNs producing the extremely expanded and toxic mHTT protein at any given time, which are not preferentially targeted by the ASO (28). Most of the mHTT present may actually be harmless, thus providing minimal clinical benefit from its depletion (28). Conversely, the small fraction of toxic mHTT should probably be depleted at higher efficiency, to see a consistent therapeutic benefit.

The two sides of the coin: tails - HD is not driven by SI

The ‘ELongATE’ model must also address potentially conflicting evidence gathered over the past 15 years. Firstly, recent GWA studies have identified certain disease haplotypes that accelerate AOO without increasing SI in blood and, potentially, in the HD brain (36,39). If SI in MSNs is not involved in these cases, alternative SI-independent pathogenic mechanisms may be at play, such as those associated with the production of truncated HTT exon1 transcript or protein (53). Secondly, extreme SI in HD patients has been described in peripheral tissues, such as the liver, which do not exhibit HD pathology (21,55,62). Although this may be associated with the liver’s regenerative potential or its clearing capacity which may prevent mHTT accumulation, this aspect requires further investigation. Thirdly, some evidence suggests that CAG length in HTT influences brain development (13,63), with mHTT potentially providing an early advantage that is followed by an accelerated aging process (64). Since these phenotypes are likely associated with transcriptional changes occurring very early during neurodevelopment, they are unlikely to be driven by SI. Accordingly, it can be concluded that the CAG tract in HTT appears to influence phenotype even before reaching the proposed toxicity threshold of 150 CAGs. Lastly, numerous studies have shown transcriptional changes in differentiated neurons derived from human pluripotent stem cell lines with <150 CAGs (59,65–69). According to the ‘ELongATE’ model, transcriptional changes at lower CAGs—at least in the HD striatum—are considered part of a neurodegeneration process resulting from the death of the co-existing MSNs carrying extreme expansions (28). If this holds true, no transcriptional changes are expected in HD neurons, if none of those neurons had enough time to reach the proposed toxicity threshold. This hypothesis may be tested in telencephalic organoids, that recapitulate the microarchitecture of the brain. Data from our lab showed transcriptional differences in 56Q versus 20Q cell lines after only 45 days of in vitro differentiation (59), presumably a period insufficient to reach the proposed CAG toxicity threshold. Further studies are needed to verify the impact of extreme SI in HD and to explore the mechanisms by which neurons below the toxicity threshold degenerate.

Conclusions

In conclusion, while many aspects remain to be fully elucidated, SI is emerging as a critical pathogenic mechanism in HD, with implications for other neurodegenerative disorders involving triplet repeats (33). Harnessing advanced technologies to monitor SI is becoming essential for both diagnostic and therapeutic approaches in HD. As we further investigate the mechanisms driving CAG repeat expansion in specific brain regions, particularly in neurons, it is evident that SI not only influences AOO but also shapes disease progression. By identifying and targeting key cis- and trans- modifiers, new therapeutic strategies can be developed to slow or even halt the expansion process. This innovative direction underscores the need for precise diagnostic tools that go beyond simple CAG counts, capturing the complexities of genetic variability to offer more tailored and effective treatments. With advancements in gene silencing and genome editing technologies, the potential to directly modify SI could pave the way for transformative interventions, offering hope to patients, scientists and physicians confronting this devastating condition.

Contributor Information

Elena Cattaneo, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Davide Scalzo, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Martina Zobel, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Raffaele Iennaco, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Camilla Maffezzini, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Dario Besusso, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Simone Maestri, Department of Biosciences, University of Milan, street Giovanni Celoria, 26, 20133, Milan, Italy; INGM, Fondazione Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, street Francesco Sforza, 35, 20122, Milan, Italy.

Data availability

No new data were generated or analysed in support of this research.

Funding

European Research Council, Advanced Grant [742436]; NSC-Reconstruct Consortium, European Union’s Horizon 2020 Research and Innovation Program [874758]; C.H.D.I. Foundation, New York, U.S.A. [JSC A11103]; Leslie Gehry Prize for Innovation in Science from the Hereditary Disease Foundation (New York, U.S.A.); Fondazione Telethon [GMR23T1059 and GMR23T1216]; Ministero dell’Istruzione, dell’Università e della Ricerca [2022LBENTH]. Funding for open access charge: H2020 European Research Council Grant [742436].

Conflict of interest statement. None declared.

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