Amyotrophic Lateral Sclerosis via the C9orf72 Gene Hexanucleotide Repeat Expansion

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Expansion Assay

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
151 C9orf72$250.00 81479 Add to Order
Targeted Testing

For ordering 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 may detect a pathogenic hexanucleotide repeat expansion in up to 40% of FALS cases and 5% of SALS cases (Byrne et al. Lancet Neurol 11(3):232-240, 2012).

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Clinical Features

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brain steam, and spinal cord (Tandan and Bradley. Ann Neurol 18(3):271-280, 1985). The dysfunction and loss of these neurons results in rapid progressive muscle weakness, atrophy and ultimately paralysis of limb, bulbar and respiratory muscles. The mean age of onset of symptoms is about 55 years of age; most cases begin between 40 and 70 years of age. The annual incidence of ALS is 1-2 per 100,000 (Cleveland and Rothstein. Nat Rev Neurosci 2(11):806-819, 2001).

The most common symptoms include twitching and cramping of muscles of the hands and feet, loss of motor control in the hands and arms, weakness and fatigue, tripping and falling. Symptoms usually begin with asymmetric involvement of the muscles. As the disease progresses, symptoms may include difficulty in talking, breathing and swallowing, shortness of breath, and paralysis.

Cognitive impairment has not been initially associated with ALS.  However, frontotemporal dementia (FTD) has been reported in several cases. Dementia has been documented in patients with ALS from different ethnic groups and affects both males and females (Wikström et al. Arch Neurol 39(11):681-683, 1982; Lipton et al. Acta Neuropathol 108(5):379-385, 2004; Mitsuyama and Inoue. Neuropathology 29(6):649-654, 2009).


About 10% of ALS cases are familial (Emery and Holloway. Adv Neurol 36:139-147, 1982). In most of these families, ALS is inherited in an autosomal dominant manner (AD-ALS) and is age-dependent with high penetrance. In rare families, the disease is transmitted in an autosomal recessive or dominant X-linked pattern.

About 90% of patients with ALS are sporadic cases (SALS) with no known affected relatives. It is unclear how many of the apparently sporadic cases are inherited with low penetrance. The clinical presentations of familial ALS (FALS) and sporadic ALS (SALS) are similar.  However, the onset of symptoms in FALS is usually earlier compared to that of SALS (Kinsley and Siddique. GeneReviews, 2012).

Autosomal Dominant ALS (AD-ALS) is a clinically and genetically heterogeneous disorder that affects all ethnic groups.  At least twelve genetic loci have been reported. Several genes have been identified and include C9orf72, SOD1, FUS, TARDBP, ANG, OPTN and VCP.

Recently, an expansion of a GGGGCC hexanucleotide repeat in a non-coding region of C9orf72 was reported in patients with ALS with or without FTD. Healthy individuals have less than 24 repeats, while affected individuals with these disorders have up to 1600 repeats (Renton et al. Neuron 72(2):257-268, 2011;  DeJesus-Hernandez et al. Neuron 72(2):245-256, 2011).  This expanded repeat appears to be a major genetic cause of ALS accounting for up to 40% of FALS cases and 5% of SALS cases (Byrne et al. Lancet Neurol 11(3):232-240, 2012). The GGGGCC hexanucleotide repeat expansion was reported in patients with ALS, ALS-FTD or FTD from various ethnic groups (Kinsley and Siddique, 2012).

Testing Strategy

The function of C9orf72 is unknown at this time.  Nonetheless, nuclear RNA foci were detected in affected tissues of patients with expanded GGGGCC repeats, suggesting defective RNA processing (Rademakers et al. Nat Rev Neurol 8(8):423-434, 2012). 

The repeat-primed PCR test is used as a screening method for the presence or absence of a pathogenic GGGGCC hexanucleotide repeat expansion located in the first intron of C9orf72.   Of note, this test is not designed to determine the number of GGGGCC repeats in alleles carrying the pathogenic expansion (Warner et al., 1996; Renton, 2011).

Indications for Test

All patients with symptoms suggestive of ALS, including AD-ALS and SALS, with or without FTD are candidates for this test.


Official Gene Symbol OMIM ID
C9orf72 614260
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Test

Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Sequencing Panel


Genetic Counselors
  • Ben Hamida, M., (1990). "Hereditary motor system diseases (chronic juvenile amyotrophic lateral sclerosis). Conditions combining a bilateral pyramidal syndrome with limb and bulbar amyotrophy." Brain 113 ( Pt 2): 347-363. PubMed ID: 2328408
  • Byrne S, Elamin M, Bede P, Shatunov A, Walsh C, Corr B, Heverin M, Jordan N, Kenna K, Lynch C, McLaughlin RL, Iyer PM, O PubMed ID: 22305801
  • Cleveland, D.W. and Rothstein, J.D. (2001). "From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS." Nat Rev Neurosci 2(11): 806-819. PubMed ID: 11715057
  • DeJesus-Hernandez M. et al. 2011. Neuron. 72: 245-56. PubMed ID: 21944778
  • Emery A.E., Holloway S. 1982. Advances in Neurology. 36: 139-47. PubMed ID: 7180680
  • Kinsley L, Siddique T. 2015 Amyotrophic Lateral Sclerosis Overview. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviewsâ„¢, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301623
  • Kress, J. A., (2005). "Novel mutation in the ALS2 gene in juvenile amyotrophic lateral sclerosis." Ann Neurol 58(5): 800-803. PubMed ID: 16240357
  • Lipton, A.M. et al. (2004). "Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration". Acta Neuropathol 108(5):379-385. PubMed ID: 15351890
  • Mitsuyama, Y. and Inoue, T. (2009). "Clinical entity of frontotemporal dementia with motor neuron disease". Neuropathology 29(6):649-654. PubMed ID: 19780984
  • Rademakers R. et al. (2012). "Advances in understanding the molecular basis of frontotemporal dementia". Nat Rev Neurol 8(8):423-434. PubMed ID: 22732773
  • Renton A.E. et al. 2011. Neuron. 72: 257-68. PubMed ID: 21944779
  • Shirakawa K. et al. (2009). "Novel compound heterozygous ALS2 mutations cause juvenile amyotrophic lateral sclerosis in Japan". Neurology 73(24):2124-2126. PubMed ID: 20018642
  • Takahashi Y. et al. (2008). "Development of a high-throughput microarray-based resequencing system for neurological disorders and its application to molecular genetics of amyotrophic lateral sclerosis". Arch Neurol. 65(10):1326-1332. PubMed ID: 18852346
  • Tandan, R. and Bradley, WG. (1985). "Amyotrophic lateral sclerosis: Part 1. Clinical features, pathology, and ethical issues in management." Ann Neurol 18(3): 271-280. PubMed ID: 4051456
  • Warner J.P. et al. 1996. Journal of medical genetics. 33: 1022-6. PubMed ID: 9004136
  • Wikström, J. et al. (1982). "Classic amyotrophic lateral sclerosis with dementia". Arch Neurol 39(11):681-683. PubMed ID: 7125994
  • Yang, Y., (2001). "The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis." Nat Genet 29(2): 160-165. PubMed ID: 11586297
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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). The PCR is performed with a fluorescently labeled 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 to size the PCR products.

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 allele that has been reported.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics. 

Genetic counseling to help to explain test results to the patients and to discuss reproductive or medical options is recommended.

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 strategy for more details.

The fluorescent fragment-length assay may or may not be designed to distinguish between individuals homozygous for a wild type allele with a small repeat number and individuals heterozygous for a pathogenic allele and a wild type allele. Please see individual gene test strategy for more 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.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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