CTG Repeats and Their Impact on Genetic Health

Repetitive DNA sequences play a crucial role in genome stability and function, but certain repeats contribute to genetic disorders. Among these, CTG repeats are particularly significant due to their tendency to expand beyond normal lengths, leading to various health implications. Their instability is linked to several inherited diseases, making them an important subject of genetic research.

DNA Structures Formed By These Sequences

CTG repeats adopt non-canonical DNA conformations that influence their stability and biological effects. One well-documented structure is the hairpin loop, which forms when complementary C-G base pairs fold back on themselves, creating a stable secondary structure. Hairpin loops interfere with DNA replication and repair, increasing the likelihood of repeat expansion or contraction. Studies using atomic force microscopy and nuclear magnetic resonance spectroscopy confirm their presence in vitro, suggesting formation in living cells under physiological conditions.

Another key structure is the slipped-strand DNA conformation, which occurs when DNA strands misalign during replication, leading to looped-out bases that escape normal proofreading. These misalignments promote repeat instability, especially when the repeat length exceeds 50, correlating with disease-associated expansions in humans.

CTG repeats can also form triplex, or H-DNA, structures, where a third DNA strand binds to the duplex, creating a triple-helical region. These structures obstruct RNA polymerase progression, altering gene expression. Chromatin immunoprecipitation assays indicate that triplex DNA recruits specific DNA-binding proteins, further affecting genomic stability. Their presence has been detected in human cells, particularly in regions with expanded CTG repeats, reinforcing their role in disease pathology.

Genomic Regions Where They Arise

CTG repeats are concentrated in specific loci with significant biological implications. One well-known region is the DMPK gene on chromosome 19q13.3, associated with myotonic dystrophy type 1 (DM1). Repeat expansion in the 3′ untranslated region (UTR) of DMPK disrupts normal gene function, leading to pathogenic consequences. Fluorescence in situ hybridization (FISH) and long-read sequencing confirm expansions ranging from a few dozen repeats in unaffected individuals to several thousand in severely affected patients.

Beyond DMPK, CTG repeats appear in the JPH3 gene on chromosome 16q24.3, linked to Huntington’s disease-like 2 (HDL2). Unlike DMPK, where the repeats are in the UTR, JPH3 harbors them within an exon, leading to potential toxic protein effects. Research published in Brain shows that expanded CTG tracts in JPH3 cause RNA foci and mis-splicing events, similar to those in DM1.

Another notable locus is the TBP gene on chromosome 6q27, associated with spinocerebellar ataxia type 17 (SCA17). Expansions in TBP result in polyglutamine tract elongation within the protein, leading to aggregation and cellular toxicity. Structural analysis using cryo-electron microscopy reveals that expanded polyglutamine tracts form amyloid-like fibrils, contributing to neuronal dysfunction.

Mechanisms Of Repeat Instability

CTG repeat instability arises from DNA replication errors, repair pathway deficiencies, and transcriptional dynamics. During replication, secondary structures like hairpins and slipped-strand loops cause polymerase stalling and template misalignment. DNA polymerase slippage at these sites leads to repeat length variations, with expansions favored when hairpins form on the lagging strand. Studies in yeast and mammalian cell models show that instability increases when repeat length exceeds 35–50, mirroring patterns seen in affected individuals.

Mismatch repair (MMR) proteins such as MSH2 and MSH3 also promote CTG repeat instability. Research in Nature Genetics shows that these proteins bind preferentially to hairpin structures, initiating repair processes that inadvertently lead to repeat elongation. Mouse models deficient in MSH2 exhibit reduced expansion rates, highlighting MMR’s paradoxical role.

Transcription-associated instability further exacerbates repeat expansions. R-loops, three-stranded nucleic acid structures formed when RNA hybridizes with the template DNA strand, expose the non-template strand to secondary structure formation. Chromatin immunoprecipitation assays detect increased R-loop formation at expanded CTG repeats, correlating with heightened genomic instability. Recruitment of DNA damage response proteins such as BRCA1 and TOP1 underscores transcription’s role in repeat instability.

Influence On Gene Function

CTG repeats disrupt gene function depending on their location and expansion size. In untranslated regions, particularly the 3′ UTR, they interfere with RNA processing. Expanded CTG tracts sequester RNA-binding proteins like MBNL1, a key splicing regulator, leading to widespread mis-splicing. In DM1, misprocessed transcripts affect ion channels and structural proteins, contributing to muscle weakness and cardiac conduction defects. RNA immunoprecipitation assays confirm that RNA-protein aggregates accumulate in nuclear foci, worsening cellular dysfunction.

In coding regions, expansions introduce toxic polyglutamine tracts, as seen in spinocerebellar ataxia type 17. These misfolded proteins aggregate, disrupting cellular proteostasis and triggering apoptosis. Cryo-electron microscopy studies reveal that expanded polyglutamine domains form insoluble fibrils, interfering with neuronal function.

Noted Associations With Specific Disorders

CTG repeat expansions are linked to several inherited disorders, with expansion size influencing disease severity and onset. Myotonic dystrophy type 1 (DM1) is the most extensively studied, with DMPK expansions leading to multisystem dysfunction. Individuals with fewer than 50 repeats remain asymptomatic, while those with more than 1000 often experience congenital forms of the disease. Symptoms include progressive muscle weakness, myotonia, cardiac arrhythmias, and cognitive impairment, driven by RNA-mediated toxicity and splicing defects. Longitudinal studies show that larger expansions accelerate symptom onset across generations due to genetic anticipation.

Beyond DM1, CTG repeat expansions in JPH3 cause Huntington’s disease-like 2 (HDL2), with symptoms resembling Huntington’s disease, including chorea, psychiatric disturbances, and cognitive decline. Unlike Huntington’s disease, which results from a CAG repeat expansion encoding polyglutamine, HDL2 arises from untranslated CTG repeats that disrupt transcript processing. Spinocerebellar ataxia type 8 (SCA8) is also linked to CTG expansions in ATXN8OS, where bidirectional transcription produces both toxic RNA and polyglutamine-containing proteins, exacerbating neurodegeneration. Postmortem analyses reveal widespread neuronal loss and nuclear RNA foci accumulation, reinforcing the role of repeat expansions in cellular dysfunction.

Differentiation From Other Repetitive Sequences

While CTG repeats share characteristics with other trinucleotide repeats, their behavior and pathological effects distinguish them from sequences like CAG, CGG, and GAA repeats. One key difference is their ability to form stable RNA structures that disrupt cellular processes. Unlike CAG expansions, which primarily affect protein function through polyglutamine aggregation, CTG repeats exert much of their pathogenicity at the RNA level by sequestering splicing regulators and forming aberrant nuclear foci. This distinction explains why diseases like DM1 and SCA8 exhibit widespread splicing defects, while polyglutamine disorders like Huntington’s disease stem from toxic protein accumulation.

CTG repeats also differ from CGG expansions associated with fragile X syndrome. While both exhibit instability, CGG expansions often trigger methylation-mediated gene silencing, leading to transcriptional repression, whereas CTG expansions typically cause RNA toxicity rather than silencing. GAA repeats, responsible for Friedreich’s ataxia, disrupt chromatin structure and impede transcriptional elongation rather than forming toxic RNA aggregates. These distinctions highlight the unique molecular mechanisms underlying CTG repeat disorders and underscore the need for targeted therapeutic strategies.