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HTT Promoter Mutation has a Bi-directional Effect on HD Age of Onset

Huntington’s disease (HD) is a genetic disorder caused by a mutation in the huntingtin gene (HTT). The mutation is identified as an extended number of CAG repeats where 40 or more repeats results in mutated huntingtin protein production and Huntington’s disease. The condition is inherited in an autosomal dominant fashion— meaning that only one copy of the mutated allele, known as the “HD disease allele”, is necessary for development of HD. Consequently, if one parent has the mutated gene, then all children of that parent have a 50% chance of inheriting a mutated allele. The normal, healthy copy of the gene is known as the “wild-type allele”. More information on the genetics of HD is available here.

Determining whether an individual has the HD disease allele has become a relatively accessible process due to the development of accurate genetic testing. More information on genetic testing can be found here. However, one major factor that remains elusive is when an at-risk individual will become symptomatic, often referred to as the age of onset (AO). The AO is when an individual begins to show obvious motor, behavioral, or cognitive symptoms, and it is often predicted based on the number of CAG repeats an individual has — the more repeats, the sooner they will develop symptoms. However, this correlation only accounts for 67% of variation in age of onset(1). This means that two individuals with the same number of repeats can develop symptoms up to 20 years apart. So what about the other 33% of variation? Although the answer to this question remains unclear, the remaining variation has been found to have a heritability of about .56, suggesting additional genetic factors that modify AO(1).

An international and collaborative research effort looked to the regulatory components of gene expression in order to better understand variability in AO. The researchers hypothesized that a potential factor in the variability of AO might be the levels of HTT expression, a process in which the promoter region of a gene plays a significant role. For this reason, the researchers chose to look into the role of the promoter region of the HTT gene in HTT production. Before a gene is “read” and transcribed into mRNA in order to eventually become a protein, it must have a certain array of active regulatory elements in order to initiate the process. One such element is known as the promoter, which when targeted by transcription factors, stimulates transcription. Transcription factors are an array of proteins that bind to DNA in order to regulate gene expression and consequently, protein production. For more information on DNA structure and how it results in proteins please refer here. In this recent 2015 study, researchers looked at the sequences of the HTT promoter region from a range of HD patients(2). In order to investigate the effects of gene variation in the HTT promoter on gene expression, researchers used luciferase reporter assays. This is a biochemical test that allows for measurement of differences in transcription levels by using the promoter of a gene of interest, in this case the promoter of HTT, to drive expression of a luciferase reporter gene. The luciferase reporter gene codes for a protein that emits light when produced, which can then be directly measured. The amount of light emitted serves as a measurement for the amount of promoter activity and consequently gene of interest expression. So, the more light emitted, the higher level of promoter activity and consequently transcription. Using this technique, researchers identified a relatively common SNP (present in about 16% of HTT alleles) that resulted in a 50% reduction of transcriptional activity. A SNP is a small change in the DNA sequence due to the swapping of one nucleotide for another

Next, researchers used transcription factor binding site (TFBS) prediction tools in order to identify an overlap between the SNP and a NF-κB binding site. NF-κB, which stands for “nuclear-factor-kappa-light-chain-enhancer of activated B-cells”, is a protein that regulates gene transcription. They found that the SNP was in a location where NK-κB would possibly bind, and as such could potentially to play a role in how NF- κB interacts with the promoter.

With this information, researchers hypothesized that the SNP altered the ability of NF-κB to bind to the HTT promoter and consequently reduced transcription. Researchers investigated this hypothesis and confirmed their prediction that the identified SNP reduced the ability of NF-κB to bind to the HTT promoter. This was confirmed in multiple systems, including in vitro rat striatal cell line and human cell line (LCL), as well as in vivo mouse brain tissue.

Furthermore, researchers found higher levels of NF-κB binding in mouse striatum than in other brain regions. This is significant because the striatum is an area where HD patients experience significant neuron loss and since binding of NF-κB to the HTT promoter resulted in increased HTT expression, the authors suggest that this may be related to the increased levels of neurodegeneration seen in the striatum. In summary, the hypothesis is that increased binding of NF-κB, resulting in increased HTT expression, may be causing more mutant Huntingtin protein to be produced in striatal neurons and therefore make them more susceptible to degeneration.

So, at this point the researchers knew that NF-κB binding to the HTT promoter typically results in increased HTT transcription. They also knew that the identified SNP lowers HTT promoter activity. Putting the two together, the researchers proposed that the mechanism by which this SNP acts is by inhibiting NF-κB binding to the promoter region, therefore lowering HTT transcription. And this is exactly what the researchers found: the SNP is in fact located in the binding site of NF-κB, and can act by inhibiting the ability of NF-κB to bind to the HTT promoter, consequentially reducing HTT transcription.

In order to investigate the disease-modifying effects of this molecular mechanism, researchers turned to genetic analysis of HD patients and their ages of onset. Patient cohorts consisted of those who had either very late or very early AO. This sampling strategy of extreme AO increases the efficiency in testing for the SNP’s effect because if it is true that the SNP affects AO then it should be more common in those at the ends of the AO spectrum.

The researchers found that the identified SNP in the HTT promoter acts as a bidirectional genetic modifier in HD, meaning that it can be associated with an increase and a decrease in age of onset, depending on which allele it is located. As you might remember, HD patients have two copies of the HTT gene— typically, one is the mutant allele and the other is the normal, or “wild-type”, allele. When the SNP was found on the mutant HTT, patients developed motor symptoms on average ten years later than patients without the SNP— a “protective” effect. On the other hand, patients who had the SNP on the normal allele typically developed motor symptoms four years earlier — a “detrimental” effect. The ability of this SNP to cause both an increase and decrease in age of onset, depending on which allele it is located on, makes it a “bidirectional” genetic modifier.

It should be noted that the patient cohort in this study is not an accurate representation of the entire HD population. In addition, the variant investigated that was associated with later age of onset has not been found in high frequencies outside of the Danish HD population that was used.

Why does this matter?

Genetic modifiers such as the SNP investigated in this paper are interesting because they provide more information on AO and symptom development, giving doctors the ability to better predict when a patient may become symptomatic. In addition, genetic modifiers such as this SNP can be used as potential therapeutic targets for HD patients. In this case, a drug that could “mimic” the effects of the SNP could be used to delay symptom onset. Another important implication is the detrimental role that the SNP played when located on the WT allele. Current therapies such as antisense oligonucleotides (ASOs) are designed to reduce total Huntingtin protein. These findings suggest that reduced expression of normal HTT may cause earlier age of onset, and therefore reducing all Huntingtin protein, mutant and normal, may have undesirable effects.

It is important to keep in mind that although this is exciting information that gives us new insight into how the HTT gene is regulated and the potential relationship between HTT levels and AO, the studies were restricted to an extreme patient population and use of this knowledge for therapeutics may still be years away. Nevertheless, a better understanding of the factors that influence HD provides valuable insight for researchers as they continue to search for potential treatments and cures.

 

Citations

  • (2) Becanović, Kristina, et al. “A SNP in the HTT promoter alters NF-kB binding and is a bidirectional genetic modifier of Huntington disease.” Nature201: 5

CJE 08/15