Huntington Disease via the HTT CAG Repeat Expansion

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  • Clinical Features and Genetics
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  • Methods
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Repeat Expansion

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
2299 HTT$250 81274 Add to Order
Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

This test is designed to identify the number of CAG repeats in the two alleles for HTT. The test will determine the number of repeats within the accuracy guidelines of the ACMG (as listed below) (Bean & Bayrak-Toydemir 2014).

Number of CAG Repeats Tolerance

< 50 ±2 Repeats

50-75 ±3 Repeats

> 75 ±4 Repeats

Clinical sensitivity is nearly 100% when a patient presents with symptoms and a familial history of the disease (Saft et al. 2013). Up to 25% of HD patients may present with symptoms without familial history (Saft et al., 2013). Targeted mutation analysis, through the combination of the two gene-centered PCR methods (repeat primed and fragment assays) of the HTTx1-CAG repeat region, is predicted to have a nearly 100% detection rate for pathogenic variants (Warby et al. 2014). There is a chance that extreme expansions could be missed with the fragment analysis, but the repeat primed PCR expansion assay should identify even extreme expansions.

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Clinical Features

Huntington disease is a neurodegenerative disease characterized by atrophy of the caudate nucleus and the putamen which leads to involuntary movements (chorea), progressive dementia, and psychiatric disturbances (Hayden and Kremer 2014). The average age of onset for Huntington disease is 40 years, but can range from late teens (juvenile onset) to over 60 years. Survival after the appearance of symptoms averages 18 years (Warby et al. 2014). Development of symptoms progresses slowly, and is difficult to identify at times. The first signs to appear are a general slowing of intellectual ability, and a small personality change (Hayden and Kremer 2014). During this time, Huntington disease cannot be diagnosed based upon symptoms alone. During this period, PET scans can reveal decreased glucose metabolism in the caudate (Hayden and Kremer 2014). These symptoms eventually lead to the major signs of the disease: chorea, hyopkinesia, rigidity, and dystonia. Chorea symptoms are defining to the disease. It is characterized by involuntary muscle movements, and they increase in severity throughout the course of the disease (Hayden and Kremer 2014). Gait disturbances, global cognitive decline, and dysphagia are also common as the disease progresses (Hayden and Kremer 2014). There are currently no long term treatment options to slow disease progression. Treatment at this time is limited to palliative care, attempting to address the most serious symptoms. RNA interference studies are currently being conducted as a possible treatment option for Huntington disease (Fiszer et al. 2013).


Huntington disease is inherited in an autosomal dominant manner. It is caused by a CAG repeat expansion in the HTT gene which occurs in the first exon, and encodes a polyglutamine tract beginning at residue 18. Repeat copy numbers can be categorized into 4 different categories: < 27 repeats – normal, 27-35 – normal mutable, 36-39 – reduced penetrance, > 39 full penetrance Huntington disease (Jama et al. 2013). Typically, the more repeats in an individual, the earlier symptoms will develop. The largest repeats, ranging above 60 repeats to around 250 repeats (Bean & Bayrak-Toydemir, 2014) are causative of juvenile-onset Huntington disease (Warby et al. 2014). Huntington Disease affects 3-7 individuals per 100,000 in populations of European decent (Milunsky et al. 2003). Some regions have markedly lower rates of Huntington disease (Japan 0.38 per 100,000; African descent 0.06 per 100,000) while certain European regions have significantly higher prevalence (North Sweden 144 per 100,000; Moray Firth [Scotland] 560 per 100,000) (Hayden and Kremer 2014). Huntington disease does not necessarily follow classical Mendelian inheritance patterns. For example, larger repeat tracts are more unstable and prone to undergo expansions or contractions (Semaka et al. 2013). Anticipation, or the increase of severity of a disease over successive generations, has been widely documented (Warby et al. 2014). According to Semaka et al. (2006), 80% of juvenile onset cases (large expansions) occur on the paternally inherited allele. Mosaicism of the HTT-CAG repeat has been reported and seems to be more prominent in juvenile-onset cases. However, according to the ACMG, the degree of mosaicism is not substantial enough to affect the interpretation of results obtained from peripheral blood (Bean et al. 2014; Telenius et al. 1994). The HTT (huntingtin) gene (4p16.3) contains 67 exons and encodes the 348kDa huntingtin protein (Hayden and Kremer 2014). The exact function of the huntingtin protein is unknown (Zuccato et al. 2010). The polyglutamine region has been shown to be an essential regulator for binding partners. The conformation of this region is potentially flexible, allowing for the interactions with a multitude of other proteins (Kim et al. 2009). It has also been proposed that the polyglutamine tract plays an important role in protein aggregation, and possibly aids in escaping protein degradation (Hayden and Kremer 2014).

Testing Strategy

This test consists of a combination of two complementary analyses: (1) a repeat-primed PCR assay, and (2) a fluorescent fragment-length assay. Repeat-primed PCR Assay The repeat-primed PCR assay is used to determine the presence or absence of a nucleotide repeat expansion, as previously described (Warner et al. 1996; Kalman et al. 2007; Jama et al. 2013). PCR is performed with a fluorescently labeled forward primer specific to the HTT locus. The reverse primer anneals to multiple locations within the HTT repeat region. Due to the multiple annealing sites, amplicons will vary in size according to the number of repeats. PCR products are then analyzed on an ABI3730xl sequencer. Fluorescent Fragment-length Assay PCR analysis using primers that flank the HTT-CAG repeat allows full length amplification of both normal and expanded alleles. The purpose of this assay is to confirm the results obtained from the repeat primed PCR assay, as well as to help distinguish between individuals homozygous for an allele versus those with one normal sized allele and a second allele containing a large expansion. This procedure also helps to confirm allele sizes in individuals who are heterozygous for two alleles that are very close in repeat number. Expanded repeats have been known to fail to amplify due to their large size. At this time, expansions of up to ~250 repeats (Bean & Bayrak-Toydemir) have been reported in affected individuals. Therefore in affected individuals carrying such large repeats, PCR amplification using specific primers that flank the CAG repeat sequence may result in the amplification of only the smaller sized allele. Of note, we have successfully visualized a sample with an allele of approximately 180 repeats using this method.

Indications for Test

Testing should follow the guidelines as proposed by the ACMG. All patients with symptoms suggestive of Huntington disease are candidates for this test. This test is also recommended for patients with a familial history of Huntington disease. Predictive testing is not recommended for patients under the age of 18 (to allow for a patient’s own, informed decision) due to the debilitating nature of the disease, lack of treatment options (Bean & Bayrak-Toydemir 2014), and the potential psychological issues that may result from either a positive or negative test result (Semaka et al 2006).

While prenatal testing is generally not recommended for adult-onset conditions, we are able to offer prenatal testing for Huntington disease on a case-by-case basis due to unique clinical circumstances. Please contact us in advance to discuss the possibility of prenatal Huntington testing. Please also note that for such testing we require that parental samples, particularly from the affected or at-risk parent, be sent along with a fetal sample. We do not, however, issue parental reports. If approved, testing can be ordered using test code #990. Acceptable specimen types are cultured cells or direct samples from an amniocentesis or CVS procedure.


Official Gene Symbol OMIM ID
HTT 613004
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Huntington Disease AD 143100


Genetic Counselors
  • Bean L., Bayrak-Toydemir P. 2014. Genetics in medicine : official journal of the American College of Medical Genetics. 16: e2. PubMed ID: 25356969
  • Fiszer A. et al. 2013. Nucleic acids research. 41: 10426-37. PubMed ID: 24038471
  • Hayden M.R., Kremer B. 2014. Huntington Disease. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
  • Jama M. et al. 2013. The Journal of molecular diagnostics : JMD. 15: 255-62. PubMed ID: 23414820
  • Kalman L., Johnson MA. 2007. Genetics in medicine. 9: 719-23. PubMed ID: 18073586 PubMed ID: 18073586
  • Kim MW., Chelliah Y. 2009. Structure (London, England : 1993). 17: 1205-12. PubMed ID: 19748341
  • Milunsky JM. et al. 2003. Clinical genetics. 64: 70-3. PubMed ID: 12791042
  • Saft C. et al. 2014. European journal of human genetics : EJHG. 22: N/A. PubMed ID: 24105375
  • Semaka A. et al. 2006. Clinical genetics. 70: 283-94. PubMed ID: 16965319
  • Semaka A. et al. 2013. Journal of medical genetics. 50: 696-703. PubMed ID: 23896435
  • Telenius H. et al. 1994. Nature Genetics. 6: 409-14. PubMed ID: 8054984
  • Warby SC. et al. 2014. Huntington Disease. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle.
    PubMed ID: 20301482
  • Warner J.P. et al. 1996. Journal of medical genetics. 33: 1022-6. PubMed ID: 9004136
  • Zuccato C. et al. 2010. Physiological reviews. 90: 905-81. PubMed ID: 20664076
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Combination Of Repeat-Primed PCR and Fluorescent Fragment-Length Assay

Test Procedure

As required, DNA is extracted from the patient specimen.

Repeat-primed PCR

The repeat-primed PCR assay is used to determine the presence or absence of a nucleotide repeat expansion, as previously described (Warner et al. 1996; Renton et al. 2011; DeJesus-Hernandez et al. 2011; Jama et al. 2013). PCR is performed with a fluorescently labeled unique forward primer specific to the gene of interest. The reverse primer is specific to the repeat region allowing for annealing at multiple locations within the repeat region. Due to the multiple annealing sites, amplicons will vary in size according to the number of nucleotides in the repeat. A tail anchored primer may also be used in conjunction with the reverse primer to minimize progressive shortening of amplicons.

PCR products are then analyzed on an ABI3730xl sequencer.

Fluorescent Fragment-length Assay

The purpose of this assay is to confirm results obtained from the repeat-primed PCR, using flanking primers specific to the gene repeat of interest. The PCR is carried out using a fluorescently labeled forward primer and a reverse primer which flank the repeat of interest as described (DeJesus-Hernandez et al. 2011; Jama et al. 2013). PCR products are then analyzed on an ABI3730xl sequencer. Two peaks will be observed in patients with two different sized alleles. In patients with a very large expansion, it is possible that only one allele will be observed. The repeat-primed PCR will determine if the patient is a true homozygote or if they have a large expansion.

Analytical Validity

A variety of positive and negative control DNA samples from affected and unaffected individuals were used to validate this test. Controls spanned the full range from the smallest normal alleles to near the largest expansion that has been reported.

Analytical Limitations

The repeat-primed PCR assay may or may not be designed to determine the number of repeats in alleles carrying the pathogenic expansion. Please see individual gene test descriptions for details.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

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