Neurology

Biology, Pathology, and Diagnostic Testing

At a Glance

C9ORF72 disease is caused by a large GGGGCC repeat expansion that routine sequencing may miss. Repeat-primed PCR can identify the expansion, but a blood-test repeat size usually cannot reliably predict when symptoms will start, how severe they will be, or which symptoms dominate.

Understanding the biology of a C9ORF72 expansion often begins with a question: Why didn’t the first genetic tests find it? To understand the answer, we have to look at the unique sequence change in this gene and the specialized tools required to see it.

The Genetic Expansion

In a typical, healthy C9ORF72 gene, there is a short section of DNA where a specific sequence of six “letters”—GGGGCC (often called a hexanucleotide repeat)—is repeated a small number of times ^[1]^[2].

In people with this condition, this section of the gene has expanded significantly. Instead of a few repeats, the gene may have hundreds or even thousands of these GGGGCC sequences ^[3]^[4]. This massive expansion alters the gene’s function.

Why Standard Testing Often Fails

Most genetic tests, like Sanger sequencing or standard Next-Generation Sequencing (NGS), work by reading the DNA code letter by letter ^[3]. However, these tools struggle with the C9ORF72 expansion for two reasons:

Complexity: The repeat is very “GC-rich,” meaning it is made of DNA building blocks that stick together very tightly, making them hard for standard machines to “read” ^[5].
Length: Pathogenic expansions are often much longer than the “windows” standard sequencers use. It is like trying to read a long word through a magnifying glass that can only see three letters at a time ^[6].

To find this expansion, doctors must use a specialized technique called Repeat-Primed PCR (RP-PCR) ^[3]. Instead of trying to read the whole expansion, this test uses “primers” that latch onto the repeats and create a characteristic pattern on a graph. This pattern tells the lab that an expansion is present, even if they cannot count every single repeat ^[7].

How Many Repeats Are “Normal”?

The scientific community is still refining the exact “cutoff” for this disease, and laboratories use assay-specific reporting conventions ^[8]^[9]. Rather than focusing on a single number, it is important to know that repeat-primed PCR can detect an expansion pattern but generally cannot provide a reliable exact count of very large alleles. The repeat size measured in a blood test does not reliably predict your age of symptom onset, disease severity, or whether your phenotype will predominantly feature movement, ALS, or FTD symptoms ^[9]. You should review your specific laboratory report with your doctor and a genetic counselor.

The Proposed Biological Mechanisms

Once the expansion reaches a certain size, researchers propose it damages the nervous system through multiple interacting mechanisms. No single mechanism fully explains the disease:

RNA Toxicity: The cell tries to read the expanded gene and creates strands of RNA that clump together into foci (clusters) that trap important proteins, preventing them from doing their normal jobs ^[10]^[11].
Dipeptide-Repeat Proteins (DPRs): The cell mistakenly translates the repeat RNA into repetitive proteins called Dipeptide-Repeat Proteins (DPRs) ^[12]. Different types of DPRs have varying levels of toxicity and can interfere with the cell’s internal machinery ^[13].
Protein Clumping (TDP-43): Over time, a critical protein called TDP-43—which normally lives in the cell’s nucleus—begins to misfold and clump together in the wrong part of the cell ^[14]^[15]. While TDP-43 pathology is very common in C9ORF72-associated ALS and FTD, it is not a universal finding in every carrier or every tissue ^[16].
Loss of Function (Haploinsufficiency): The expansion may also reduce the normal production of the C9ORF72 protein, impairing the cell’s ability to transport materials ^[17].

By combining these proposed mechanisms, the C9ORF72 expansion slowly disrupts the health of neurons in the areas of the brain that control movement and thought ^[18].

Common questions in this guide

What is a C9ORF72 repeat expansion?

It is an unusually long stretch of the six-letter DNA sequence GGGGCC within the C9ORF72 gene. In affected people, this sequence may repeat hundreds or thousands of times, altering how the gene works and contributing to nerve-cell damage.

Why can routine genetic tests miss a C9ORF72 expansion?

Sanger sequencing and standard next-generation sequencing read DNA in small sections. The C9ORF72 repeat is unusually GC-rich and can be far longer than the region these methods can reliably read, so a specialized test may be needed.

How does repeat-primed PCR find a C9ORF72 expansion?

Repeat-primed PCR uses laboratory primers that attach to the repeated GGGGCC sequence and produces a characteristic pattern on a graph. This pattern can show that an expansion is present, but the test generally cannot provide an exact count for a very large expansion.

Can the C9ORF72 repeat count predict my symptoms or when they will start?

Not reliably. The repeat size measured in blood does not reliably predict age at symptom onset, disease severity, or whether movement, ALS, or frontotemporal dementia features will predominate. Discuss the report with your doctor and a genetic counselor.

What does an intermediate C9ORF72 repeat result mean?

There is no single cutoff used in every laboratory, and reporting conventions can differ by assay. An intermediate or uncertain result may not establish that the expansion explains your symptoms, so the laboratory report should be reviewed with a neurologist or genetic counselor.

Does a negative Huntington disease test rule out a C9ORF72 expansion?

No. A Huntington disease test and a C9ORF72 expansion test look for different repeat changes, so a previous negative Huntington result does not rule out a C9ORF72 expansion. Ask whether your new test used repeat-primed PCR.

What does somatic mosaicism mean for a C9ORF72 test?

Somatic mosaicism means the repeat length can differ among cells or tissues in the same person. If your report mentions it, ask how the sample type and testing method affect interpretation, because a blood result may not represent every tissue.

Get notified when new evidence is published on Huntington disease-like syndrome due to C9ORF72 expansions.

We monitor PubMed for new peer-reviewed studies on this topic and email a short summary when something meaningful changes.

References (18)

1
The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.
Gijselinck I, Van Mossevelde S, van der Zee J, et al.
Molecular psychiatry 2016; (21(8)):1112-24 doi:10.1038/mp.2015.159.
PMID: 26481318
2
Intermediate C9orf72 alleles in neurological disorders: does size really matter?
Ng ASL, Tan EK
Journal of medical genetics 2017; (54(9)):591-597 doi:10.1136/jmedgenet-2017-104752.
PMID: 28689190
3
Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.
Cleary EM, Pal S, Azam T, et al.
Molecular and cellular probes 2016; (30(4)):218-224 doi:10.1016/j.mcp.2016.06.001.
PMID: 27288208
4
Large C9orf72 repeat expansions are seen in Chinese patients with sporadic amyotrophic lateral sclerosis.
Chen Y, Lin Z, Chen X, et al.
Neurobiology of aging 2016; (38()):217.e15-217.e22 doi:10.1016/j.neurobiolaging.2015.11.016.
PMID: 26725464
5
An update on the neurological short tandem repeat expansion disorders and the emergence of long-read sequencing diagnostics.
Chintalaphani SR, Pineda SS, Deveson IW, Kumar KR
Acta neuropathologica communications 2021; (9(1)):98 doi:10.1186/s40478-021-01201-x.
PMID: 34034831
6
Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease.
Ebbert MTW, Farrugia SL, Sens JP, et al.
Molecular neurodegeneration 2018; (13(1)):46 doi:10.1186/s13024-018-0274-4.
PMID: 30126445
7
NOTCH2NLC GGC repeats are not expanded in Italian amyotrophic lateral sclerosis patients.
Manini A, Gagliardi D, Meneri M, et al.
Scientific reports 2023; (13(1)):3187 doi:10.1038/s41598-023-30393-6.
PMID: 36823368
8
Unravelling the clinical spectrum and the role of repeat length in C9ORF72 repeat expansions.
van der Ende EL, Jackson JL, White A, et al.
Journal of neurology, neurosurgery, and psychiatry 2021; (92(5)):502-509 doi:10.1136/jnnp-2020-325377.
PMID: 33452054
9
The C9orf72 hexanucleotide repeat expansion presents a challenge for testing laboratories and genetic counseling.
Crook A, McEwen A, Fifita JA, et al.
Amyotrophic lateral sclerosis & frontotemporal degeneration 2019; (20(5-6)):310-316 doi:10.1080/21678421.2019.1588904.
PMID: 30907153
10
A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism.
Swinnen B, Bento-Abreu A, Gendron TF, et al.
Acta neuropathologica 2018; (135(3)):427-443 doi:10.1007/s00401-017-1796-5.
PMID: 29302778
11
Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy.
Cooper-Knock J, Higginbottom A, Stopford MJ, et al.
Acta neuropathologica 2015; (130(1)):63-75 doi:10.1007/s00401-015-1429-9.
PMID: 25943887
12
Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity.
Tao Z, Wang H, Xia Q, et al.
Human molecular genetics 2015; (24(9)):2426-41 doi:10.1093/hmg/ddv005.
PMID: 25575510
13
Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis.
Zhang YJ, Gendron TF, Ebbert MTW, et al.
Nature medicine 2018; (24(8)):1136-1142 doi:10.1038/s41591-018-0071-1.
PMID: 29942091
14
C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43.
Nonaka T, Masuda-Suzukake M, Hosokawa M, et al.
Human molecular genetics 2018; (27(15)):2658-2670 doi:10.1093/hmg/ddy174.
PMID: 29750243
15
Prevalence of brain and spinal cord inclusions, including dipeptide repeat proteins, in patients with the C9ORF72 hexanucleotide repeat expansion: a systematic neuropathological review.
Schipper LJ, Raaphorst J, Aronica E, et al.
Neuropathology and applied neurobiology 2016; (42(6)):547-60 doi:10.1111/nan.12284.
PMID: 26373655
16
Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia.
Vatsavayai SC, Yoon SJ, Gardner RC, et al.
Brain : a journal of neurology 2016; (139(Pt 12)):3202-3216 doi:10.1093/brain/aww250.
PMID: 27797809
17
C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.
Aoki Y, Manzano R, Lee Y, et al.
Brain : a journal of neurology 2017; (140(4)):887-897 doi:10.1093/brain/awx024.
PMID: 28334866
18
Amyotrophic lateral sclerosis caused by hexanucleotide repeat expansions in C9orf72: from genetics to therapeutics.
Mizielinska S, Hautbergue GM, Gendron TF, et al.
The Lancet. Neurology 2025; (24(3)):261-274 doi:10.1016/S1474-4422(25)00026-2.
PMID: 39986312

This page explains C9ORF72 biology and genetic testing for informational purposes only and does not constitute medical advice. Ask your neurologist or genetic counselor to interpret your report and discuss what it means for you.