Frameshift Mutations in Huntington’s Disease

Understanding Frameshift Mutations: How It Can Change Lives

Exploring the genetics of neurodegenerative disorders means understanding diseases and developing better treatments. In Huntington’s disease, CAG repeats, and frameshift mutations have already been studied by using antibodies to detect mutant polyalanines. Read on to learn how frameshifts affect toxic inclusions and how our protein arrays can help you in your own research of neurodegenerative disease and beyond.

Huntington’s Disease: Genetic Basis and Clinical Consequences

Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by a defective huntingtin protein. A mutation in the HTT gene results in an abnormally long protein, prone to cleavage and aggregation.
In HD patients, there is an expansion of the cysteine-adenosine-guanine (CAG) repeat. This, in turn, leads to a longer glutamine repeat at the N-terminus of the protein product. (Schilling et al., 2007)

Caspases cleave the mutant huntingtin, resulting in N-terminal peptide fragments, which are likely to misfold, aggregate and form inclusion bodies. Over time, the toxic aggregates interfere with normal neuronal function, bringing about the classic signs of the disease.

The Huntingtin pathophenotype consists of:

  • Motor dysfunction, including progressive chorea, rigidity, psychomotor impairment.
  • Cognitive disturbances, declining into dementia.
  • Psychiatric symptoms, beginning as subtle personality changes and potentially progressing into anxiety, depression, emotional blunting, etc.

Frameshift Mutation and Neurodegenerative Disease

Huntington’s disease is the most prevalent and best-known member of the polyglutamine disease group. But you will also see a poly-G expansion due to CAG repeats in:

  • Spinocerebellar ataxias (SCA) type 1, 2, 3, 6, 7 and 17
  • Spinobulbar muscular atrophy
  • Dentatorubal-pallidoluysianatrophy

In these disorders, the mutant gene encodes aggregate-prone proteins with long polyglutamine tracts.

However, a dinucleotide deletion or single nucleotide insertion within the CAG tract shifts the coding frame by +1 to GCA. GCA encodes alanine, leading to polyalanine expansion in polyglutamine diseases.
A study by Davies and Rubinsztein confirmed both +1 and +2 frameshift-mutated protein in Huntington’s disease patients. The authors suggested this can be relevant to disease pathogenesis, considering previous findings that polyserine and polyalanine-containing protein modify mutant huntingtin toxicity. (Berger et al., 2006)

How did they go about this? By searching for antibodies rather than directly looking for the mutation.

Finding the Frameshift Mutation: How Antisera Made It Happen

To find the mutated protein, Davies and Rubinsztein started by modelling possible frameshift products of the HTT exon 1. Then, they predicted the mutant epitopes and raised polyclonal antibodies against them.

These antibodies were a fast and easy way to find +1 and +2 frameshift products. The antisera were used directly on postmortem patient brain and transgenic mouse brain samples – and here is what they found:

Polyalanine Mutant Protein in Polyglutamine Diseases: Findings & Consequences

Davies and Rubinsztein only found frameshifts 4% of protein aggregated . This supports the theory of low-level, time-dependent frameshift product occurrence.

Why Polyalanines Appear Along With Long Polyglutamines

The frameshift that causes polyalanines to appear happens more frequently in expanded CAG tracts. The longer the repeat sequence is, the more likely it is for a frameshift mutation to happen – and for polyalanines to appear.
Toulouse et al. have hypothesised that this happens because of ribosomal slippage, which occurs with long CAG tracts. (Toulouse et al., 2005)

They also state that polyalanine products increase poly-G toxicity, contributing to the pathogenesis of neurodegenerative disease. If we administer anisomycin, which interacts with ribosomes, the toxicity drops – and this seems to confirm the theory.

Drugs that act on the ribosomes could be a promising new therapy for Huntington’s patients. There is still more to explore. Anisomycin reduces protein synthesis as a whole. Thus, the protective effect might be due to a lowering of the poly-G peptide and not because the drug prevents frameshifting.

But there is more:
The polyalanine frameshift products have actually been shown to protect against polyglutamine toxicity (Davies et al., 2006). While high amounts of long polyalanines are toxic, these proteins have a positive effect at a lower level.

How Polyalanines Reduce Toxicity

One of the hypotheses tested was through sequestration of the polyglutamine protein. However, tests failed to show any direct interaction between the mutant repeats. Instead, the protective effect of polyalanines was reminiscent of heat-shock protein interaction with ataxin-1. Polyalanine repeats would reduce the proportion of cells with inclusions, but they’d increase the number of inclusions per cell.

Could it be that long polyalanines induced a heat-shock response, even at subtoxic doses? It appears so. In particular, polyalanines were found to:

  • Be the specific reason behind heat-shock response induction and polyglutamate toxicity protection – other aggregate-prone protein did not have the same property.
  • Induce hsp70 (70 kilodalton heat shock protein), which is known to protect from apoptosis.
  • Cause the induction through HSF-1 driven transcription.

Frameshift Mutations, engine Protein Arrays, and What This Means For You

The research into frameshift mutations and neurodegenerative diseases is exciting, but we still have much to learn. Asking more questions and testing more hypotheses will help us understand disease processes better and develop treatment and management options for Huntington’s disease patients.

This is where our protein arrays come in:

Our protein arrays don’t just cover full-length proteins but include neoantigens and frameshift peptides, also called as out-of-frame peptides, as well. The hEXselect array contains over 57 000 spots, 41.8% of them being neoantigens and frameshift peptides.

And, we’re here to support you with full-service analysis. All we need is a 50 µl sample to run a one-shot analysis of over 10,000 antigens and deliver the report within two weeks. engine shortens your time to discover biomarkers and antibody specificity, but you will still have complete control over data, results and samples.

You can use the engine protein arrays for:

  • Epitope mapping of potential new drugs that frameshift products,
  • Investigating the interactome of frameshift products and elucidating an interaction network,
  • Specific biomarker screening for frameshift-mutation-linked diseases
  • Researching the immune response to frameshift expression

Are you ready to expand your knowledge on Huntington’s disease and beyond? Whether you have some sera from Huntington’s patients you want to test or want to explore neoantigens, we are here to help – reach out and let’s talk! Send us your question.


References

  • Schilling, G., Klevytska, A., Tebbenkamp, A. T., Juenemann, K., Cooper, J., Gonzales, V., Slunt, H., Poirer, M., Ross, C. A., & Borchelt, D. R. (2007). Characterisation of huntingtin pathologic fragments in human Huntington disease, transgenic mice, and cell models. Journal of neuropathology and experimental neurology, 66(4), 313–320. https://doi.org/10.1097/nen.0b013e318040b2c8
  • Berger, Z., Davies, J. E., Luo, S., Pasco, M. Y., Majoul, I., O’Kane, C. J., & Rubinsztein, D. C. (2006). Deleterious and protective properties of an aggregate-prone protein with a polyalanine expansion. Human Molecular Genetics, 15(3), 453–465. https://doi.org/10.1093/hmg/ddi460
  • Toulouse, A., Au-Yeung, F., Gaspar, C., Roussel, J., Dion, P., & Rouleau, G. A. (2005). Ribosomal frameshifting on MJD-1 transcripts with long CAG tracts. Human Molecular Genetics, 14(18), 2649–2660. https://doi.org/10.1093/hmg/ddi299
  • Davies, J. E., & Rubinsztein, D. C. (2006). Polyalanine and polyserine frameshift products in Huntington’s disease. Journal of Medical Genetics, 43(11), 893–896. https://doi.org/10.1136/jmg.2006.044222

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