Huntington’s disease (HD), also known as Huntington’s chorea, is a fatal neurodegenerative genetic disorder. The disease has a prevalence of 4 to 10 individuals per 100,000 people and typically affects people of 40 years old (Ross and Tabrizi 2011). From the time of diagnosis, patients succumb to the illness within 15 to 20 years. As the pathological mechanisms are not yet fully understood, there are no standardized cures for the condition. Juvenile forms of HD that affect individuals before the age of 20 are much less common, but have been known to occur. Recent studies have found that parents yet to show symptoms of the disease are at high risk of having a child with early-onset HD characteristics (Sakazume et al. 2009). These implications highlight how susceptibility of the disease is usually through inheritance. A pedigree best illustrates the complex nature of transmission in a single family (Figure 1). As an autosomal dominant disease, if a parent has HD, there is a 50% chance that the offspring will also inherit the disease. That being said, only one copy of the genetic variant that causes the disease is needed to display symptoms.
Diagnosis and detection of HD occurs in several ways. Subjective detection occurs through a checklist of psychological symptoms, but with the discovery and ongoing understanding of the gene, multiple genetic detection methods have been developed. In 1993, Dr. James Gusella and his lab group identified the gene responsible for the observed disease pathology on the 4th chromosome which he termed huntingtin (HTT) (Walker 2007). After this discovery, he also developed a patented screen (US patent US4666828 A) for a restriction fragment length polymorphism (RFLP) linked to HD (Gusella 1987). These tests have become routine, especially prenatally. Often times, these tests can determine whether an individual will suffer from HD before birth to allow for parents to screen eggs prior to conception or to plan for postnatal care. Other genetic screens have been developed using HD biomarkers in both blood (such as 8-OHdG oxidative stress marker and BDNF) and in neuroimaging (such as cortical thickness, subcortical white-matter volume, and fMRI) (Ross and Tabrizi 2011).
The HD phenotype results from an expanded CAG repeat more than 36 times (Ehrlich 2012). These pathogenic repeats can vary in number depending on the patient at hand. When the repeat is altered, the gene is referred to as disease-causing mutant huntingtin (mHTT) (Ross and Tabrizi 2011). The mutant gene confers the associated disease symptoms which include degeneration of neurons, mood swings, and discoordination of motor abilities. Despite various different single nucleotide polymorphisms (SNPs) known to occur in the gene, no other mutation has been known to as directly cause such a strong dysfunctional phenotype. Though the symptoms of HD may be attributed to a various number of diseases, the cellular impact that the expansion has on an individual drastically differs from that of other neurodegenerative diseases.
To better understand how to approach diagnosis and treatment, the molecular characters of the gene must be analyzed. The huntingtin gene can be seen in a wide variety of taxa, noting its evolutionary significance. When comparing the mouse and human HTT genes, 86% sequence similarity at the DNA level and 91% sequence similarity at the protein level acknowledge the high conservation of the character both structurally and functionally; the main difference seen between the homologs can be seen in the shorter polyglutamine tail in the mouse variant (Barnes et al. 1994). Despite this conservation, the naturally occurring CAG variant at the amino-terminus (N-terminus) of the protein only appears in humans. Mice, instead, see a variant in a CCG repeat further downstream that impacts protein function and binding affinity (Barnes et al. 1994). This difference may be due to the notable difference between the domains in each taxon. These differences, however, further complicate HD studies due to the poor representation seen through models. Though artificially introduced variants are commonplace in HD research, mice and other organisms demonstrate how the phenotype is universal but the molecular mechanisms that cause it are much more complex.
Specifically in humans, the huntingtin gene is 180 kb long with 67 exons and is located on the short arm of chromosome 4 at position 16.3 (Warby et al. 2014). The housekeeping gene is regularly expressed in all areas of the body and plays a significant role in transcriptional regulation, especially during development (Figure 2). Because of the prominent regulatory role of the protein, it must be universally expressed. Higher expression can be seen in the brain, where a longer 13.7 kb transcript is expressed in place of the typical shorter 10.3 kb transcript (Lin et al. 1993). Though some argue that for epigenetic regulation, further study must identify differential methylation resulting in the transcriptomic differences. Other than epigenetic theories, upstream protein-nucleic acid interactions play a prominent role in this slight differential expression. The upstream open reading frame in the 5’ untranslated region, when transcribed and translated, results in a 21-amino acid peptide which negatively regulates huntingtin mRNA expression due to an unknown cause (Lee et al. 2012). Speculation of interaction with the gene’s promoter or ribosomal binding sites are a few of the arguments for down-regulation, but more study is required.
The genomic basis for HD varies, but modern breakthroughs in molecular analysis have determined major contributors to the phenotype. Though environment may impact the degree of the phenotype, variation in the highly conserved gene serves as the basis of disease severity. Though many genomic variants may be responsible for the HD phenotype, the most common polymorphism exists as an expanded CAG repeat in the first exon of the huntingtin gene (Ehrlich 2012). Depending on how many of the repeats exist at the beginning of the gene, a different amount of glutamine amino acids existed near the N-terminal of the resulting protein. Individuals displaying the disease phenotype typically see an excess of 40 amino acid repeats as compared to the wide range 9 to 35 seen in those normal individuals (Rubinsztein et al. 1996). The traditional model for functional huntingtin contains 17 glutamines, as seen in various models (Figure 3). The increased repeat area does not completely eliminate transcription, but results in new molecular properties of both the transcript and protein product. The domain, known as the huntingtin protein region, has unknown function and is observed only in eukaryotes (Figure 4). Predicting how the structure changes is a current focus of research. However, strides have been made to uncover how observed changes may result in drastic biochemical interactions.
As the gene encodes a regulatory protein important in binding to a variety of different transcription factors, distorting the expansion that is essential in protein-protein interaction severely impacts the effectivity of the protein. Polyglutamine expansion has been seen to cause both gain of function and loss of function in the huntingtin protein, resulting in undesired binding and a lack of required binding (Hoop et al. 2016). The severity to which this disequilibrium occurs depends largely on the amount of glutamines added; this inconsistency is problematic when determining the ramifications of the expansion prenatally. Completely throwing regulatory cascades out of whack, a variety of different molecular distortions appear as the protein sporadically interacts with a slew of different cellular components.
mHTT has pronounced effects in both the nucleus and cytoplasm. The KEGG pathway for HD (Figure 5), visualizes these effects and provides more detailed information regarding the specific protein interactions with mHTT. In the cytoplasm, the longer mutant gene can interfere with microtubule transport. This is due to the mutant’s inability to affiliate with the microtubules (Gutekunst et al. 1995). As a result, problems arise when trying to anchor the filaments to the cytoskeleton localization of many cellular components including organelles and vesicles are vastly impaired.
It can also lead to aberrant neuronal secretion and endocytosis. The normal form of HTT forms a complex with Hip1, AP2, and Clathrin, which are all involved in endocytosis (Trushina et al. 2006). The mutant form cannot form this complex, affecting endocytosis and secretion in neurons. Some forms of the variant have completely disabled cellular capabilities to both engulf and secrete compounds, highlighting the severity of the condition. In this regulatory pathway, HD primarily acts on medium spiny striatal neurons (MSN). The MSN is unique because it receives the greatest combination of glutamine and dopamine input among neuronal subtypes (Ehrlich 2012). Because of the high traffic associated with the MSN, researchers hypothesize that the area is a prime area of study due to its susceptibility to improperly function in response to interactions with mHTT.
mHTT has an effect on Ca2+ signaling by allowing increased activation of IP3R1 (located in the endoplasmic reticulum) by InsP3, leading to increased NMDAR activity and destabilizing Ca2+ handling, which is essential for normal synaptic activity and mitochondrial function (Bezprozvanny and Hayden 2004). Consequently, cascades that regulate transcription in the nucleus are impaired. In the nucleus, problems continue as mHTT interferes with gene transcription of protective elements. The transcriptional repression of these elements results in nuclear toxicity and eventually cell death. The p53-signaling pathway is also activated by mHTT, activating the apoptotic pathway resulting in cell death (Steffan et al. 2000). The well-known pathway may be one of the main motivators for many of the dysfunctional neuronal phenotypes based on its powerful cellular influence alone.
As the polyglutamine expansion has varying effects depending on the amount of residues present, there are possibly more interactions that may be seen that have not yet been noted. Further study taking a systems biology approach is required to determine an all-encompassing pathway to describe the diversity of mHTT regulation. Pinpointing the effects of expansion-specific phenotypes could give rise to personalized procedures and medicine to increase efficacy of treatment. Analysis of other polymorphisms, such as previously identified SNPs, should also be done to determine their role in expansion structural differences.
Currently, conventional treatments are used to reduce the negative symptoms of HD, as opposed to slowing the progression of the disease itself. There is no established cure for HD (“Learning about Huntington’s Disease” 2011). These treatments include pharmacotherapy options, antipsychotics, therapy, and lifestyle changes (Frank 2014). A mix of all different treatment combinations are used to cater to an individual’s present symptoms resulting from HD. Many pharmacotherapy options target neurotransmitter levels of dopamine (neuroleptics, dopamine agonists, lithium), glutamate (glutamate antagonists), and gamma-aminobutyric acid (anti-seizure medications, cannabinoids) (Frank 2014). The most targeted negative symptoms associated with HD for treatment include chorea and dystonia, both of which are characterized by uncontrollable muscle movements. The only FDA approved treatment to relieve chorea is tetrabenazine (TBZ) which works effectively to deplete dopamine levels with extreme negative side effects (Frank 2014). TBZ can be known to increase depression, agitation, akathisia, and hyperkinesia already associated with HD (Frank 2014; “Treating Huntington’s Disease” 2014). Other side effects include drowsiness, dizziness, fatigue, and increased suicidal tendencies. Alternatively, others have studied the effects of deutetrabenazine on chorea, a derivative of tetrabenazine. The benefits of deutetrabenazine include increased adherence by the patient due to deutetrabenazine being administered twice daily; unlike TBZ, there are very few serious negative side effects while still reducing chorea and dystonia (Paton 2017). Psychotic symptoms due to HD may be treated with antipsychotics, which can also treat chorea, dystonia, and other negative motor symptoms. Antipsychotics such as olanzapine and aripiprazole effectively reduce chorea in patients without the presence of excessive negative side effects (Frank 2014).
The presence of negative side effects remains a significant consideration when choosing treatment for HD as these side effects may affect or continue to worsen already present symptoms of HD, such as depression, mania, OCD, and emotional and cognitive decline (Frank 2014). As a result, conjunctive and hybrid treatments that combine cognitive therapy and medications are considered the best route to safely reduce the negative symptoms of HD. Speech and language therapies in conjunction with mood stabilizers and antipsychotics or antidepressants are recommended for patients with HD (“Treating Huntington’s Disease” 2011). Other therapies such as adjunctive and occupational, alternative, complementary, and behavioral can be used in combination with each other or medication to reduce negative symptoms as well (Frank 2014; “Treating Huntington’s Disease” 2011). Therapies that are focused to treat OCD, depression, and anxiety are effective in reducing these symptoms of HD. Lifestyle changes such as increased exercise, hydration, and nutrition are also considered beneficial to relieve treatments (“Treating Huntington’s Disease” 2011; “Huntington’s Disease: Hope Through Research” 1998). Some individuals with HD burn an excess number of calories by a couple thousand due to their increased anxiety, hyperexcitability, and motor movements (chorea) (“Huntington’s Disease: Hope Through Research” 1998). As a result, proper nutrition plans incorporated into a patient’s lifestyle will increase comfort for the individual.
Potential other treatments include efforts to slow progression of HD. These treatments target mitochondrial function by studying the effects of coenzyme Q10 (2CARE clinical trial) and creatine (CREST-E clinical trial) which was discontinued in 2014 due to the conclusion that creatine will have no significant effect on HD (Frank 2014; “Announcement on CREST-E Early Closure” 2015). Other treatments currently being studied include monitoring the regression of HD with polyphenol (2)-epigallocatechin-3-gallate, a green tea extract that has shown progress in reducing effects of various neurodegenerative diseases (Frank 2014; Bhullar and Rupasinghe 2013). Interventions such as medical cannabis, music and dance therapy, and video games are considered to aid in the deficit of walking abilities and balance exhibited in HD patients (Frank 2014). These unconventional methods are typically not supported by insurance companies and are at the cost of the patient if they wish to partake.
A study by the FDA in 2016 found that most “patients feel that current treatments do not adequately manage their most disabling symptoms” (“The Voice of the Patient” 2016). As the disease becomes more debilitating, the only option doctors have is to increase the range or strength of the patient’s medication. Many patients seemed to feel like their disease was out of their control since they could only mask the symptoms. One particular patient “commented that a slower progression of symptoms would mean ‘a better, more productive quality of life’” (“The Voice of the Patient” 2016). Thus, a lot of research is now being put into cutting edge treatments which look at attempting to slow the disease or even cure it.
Studies have shown promise with their preclinical evaluation of N-terminal sequences aimed at stopping the aggregation of malformed huntingtin proteins in the synapses (Burra and Thakur 2017). As huntingtin proteins clump together in the brain, they prevent proper nerve transduction and cause many of the major symptoms of the disease. The study found the best peptide inhibitors were Hum-NT17 and Xen-NT17. These inhibitors were sequence variants from humans and clawed frogs that acted upon the N-terminal side of the huntingtin protein. And, while no trials have been conducted in humans or animals, this treatment may in future be a way to slow the progression of HD.
Another possible treatment is targeting transcribed Huntingtin mRNA to inhibit its translation or trigger its degradation. Because “both RNA and proteins exert pathogenic effects”, degrading the Huntingtin mRNA transcript would slow or even prevent the progression of the disease (Urbanek et al. 2017). The study used CAG repeat-targeting reagents to specifically block translation of the Huntingtin transcript, since the pathogenic sequence is characterized by a larger number of CAG repeats. RNA degradation was initiated with RNase H as well as through the RNA interference pathway (Urbanek et al. 2017). Human cultured cells have been tested and show promise, but no trials have been conducted in complex organisms yet.
Possibly the most exciting treatment on the horizon is the use of the CRISPR-Cas9 system. CRISPR-Cas9 has a simple RNA design for precisely targeting specific sequences of DNA and cutting them out of the cell’s genome. A study published in late 2016 show-cased CRISPR-Cas9’s capabilities by using it to cut out the mutant Huntingtin allele from a human cultured cell (Shin et al. 2016). Because HD patients have only one disease causing allele, removing this sequence of DNA completely halts the production of pathogenic RNA and protein, while still allowing for the transcription of the proper protein from the normal allele, effectively permanently halting the disease’s progression. Predicted sites can be found (Figure 6). Trials have not been conducted yet in humans, but with the use of CRISPR-Cas9 on humans being recently legalized it may not be long until the progression of HD is completely halted in the first patient.
In the future, HD could be recognized early with preemptive genetic testing for individuals with family history and then, if a mutant genotype is discovered, cured with the CRISPR-Cas9 system far before the onset of clinical symptoms. For those already displaying symptoms (notably, those conceived), CRISPR-Cas9 treatment targeting all somatic cells would be much more difficult and may not completely repress the severe phenotype associated with the variant. So, while current treatments only at best cover up disease progression, there are many treatments on the horizon that may not only slow but even halt and possibly, in combination, cure HD.