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For years, scientists hunted for the mutation on chromosome 9 that causes amyotrophic lateral sclerosis, and the subsequent discovery of the mysterious C9ORF72 was hardly the end of the challenge. At the 23rd annual International Symposium on ALS/MND, held 5-7 December 2012 in Chicago, Illinois, researchers discussed having to contend with a nucleotide expansion that varies in size not only from one person to the next, but also between tissues within the same person. Presented by Mariely Dejesus-Hernandez of the Mayo Clinic in Jacksonville, Florida, this finding could complicate diagnosis and some cellular disease models. Those based on lymphocytes, for example, may not exhibit the full extent of the mutation in the nervous system. This phenomenon is a familiar theme in repeat disorders, commented John Wilson of Baylor College of Medicine in Houston, Texas; for example, variable repeat size also occur in myotonic dystrophy type 1 and Fragile X syndrome.

Healthy people tote up to 30 hexanucleotide repeats in C9ORF72. People with ALS or frontotemporal dementia typically have hundreds or thousands. Since its discovery in 2011 (see ARF related news story on Renton et al., 2011, and Dejesus-Hernandez et al., 2011), there have been hints of different repeat lengths in different tissues in the same person (see ARF related news story). In Chicago, Dejesus-Hernandez described data from three people who came to autopsy. Two had died of classical ALS, and one had frontotemporal lobar degeneration and lower motor neuron disease. She examined DNA from blood, spleen, heart, muscle, liver, spinal cord, and brain tissue, the last including the frontal, temporal, parietal, and occipital cortices as well as the cerebellum. Within one person, the size of C9ORF72 repeat ranged from as little as three kilobases to 10 Kb. The person with mixed brain and spinal disease had 80-100 C9ORF72 repeats in the blood, and more than 1,000 in the cerebellum.

Even within individual tissues, there is mosaicism. Southern blots for C9ORF72 revealed smudges and smears rather than the crisp bands that would indicate a gene of fixed size. The central nervous system tends to have the longest repeats. The same is true in other repeat disorders, Wilson said. Johnathan Cooper-Knock of the University of Sheffield in the U.K. told Alzforum he has seen a similar pattern in his own C9ORF72 studies. In approximately one in 15 patients, he said, the repeat length in blood cells comes out 10-fold smaller than in brain.

“That is going to hugely complicate things, because we tend to [rely on] drawn blood [for many analyses],” commented Michael van Es of the University Medical Center Utrecht in the Netherlands. He called the finding “intriguing and horrible at the same time.” Rosa Rademakers, in whose laboratory Dejesus-Hernandez works, speculated that scientists and clinicians could miss people with C9ORF72 expansions if their blood does not reflect the lengthy repeats in the nervous system. Emphasizing that no evidence yet exists for this kind of false negative test result, Rademakers told Alzforum that it would be hard to determine if such a misdiagnosis occurred, since that would require brain biopsies. Nevertheless, missing people with C9ORF72 expansions in the brain would not only confuse genetic diagnosis in the clinic. It could also confound studies that attempt to characterize the symptoms of C9ORF72-based disease, and/or that correlate phenotype with repeat length. Cooper-Knock recommended that scientists making patient-based cell models not use blood cells from people who have only a hundred or so repeats in their blood, because that does not guarantee a sufficient number of repeats in the brain to cause pathology.

C9ORF72 is hardly the first repeat disorder to show this kind of instability, noted Wilson and Tom Cooper, also at the Baylor College of Medicine. In myotonic dystrophy type 1, which is due to a repeat expansion in dystrophia myotonica-protein kinase 1 (DMPK1), repeat shrinkage and expansion both occur in cell culture experiments, Wilson said. In animals the repeats tend toward growth. In the case of C9ORF72, Rademakers said it is unknown if people start out with one shortish repeat that grows over time, or if contraction occurs as well.

John Hardy of University College London, U.K., commented in an e-mail to Alzforum that if people with C9ORF72 expansions have different lengths in different tissues, that might explain why they have different disease phenotypes. People with the C9ORF72 repeat can exhibit symptoms of ALS, frontotemporal dementia, or both. Rademakers speculated that people with longer repeats might suffer earlier disease onset.

Murky Mechanism
How might the expansions grow and contract? Scientists agree that the repeat sequences could cause replication or repair enzymes to stutter, adding or deleting sections as they go; however, the precise mechanism remains controversial. Some researchers who spoke with Alzforum believe the size change must occur early in development, while others argue that the gene could keep stretching and contracting in adults.

The resizing likely happens during DNA replication, reasoned Michael Baughn of the University of California, San Diego (UCSD). “The polymerases that copy DNA have a hard time with repetitive sequences,” he said. Since neurons do not divide, neuronal repeats must have arisen during early development, when precursors are still reproducing. Why, then, would non-dividing cells—the neurons—have longer repeats than regularly mitotic types such as blood cells?

At what point in development the expansions occur might explain their tissue distribution. Dejesus-Hernandez presented preliminary evidence that the length of repeats correlated, loosely, with the embryonic origin of each tissue. In one patient, repeats were shortest in mesoderm-derived cells such as spleen and blood, medium length in endoderm-based tissues such as liver, and longest in nerve tissues derived from the ectoderm. One would expect this pattern if the C9ORF72 size was set early in embryonic development, Baughn said. “It is just an observation at this point,” Rademakers cautioned. Her laboratory will gather as many tissue types from as many subjects as possible to better characterize the repeats’ size, range, and the cutoff between health and disease.

Cooper suggested an alternative explanation to when expansions might change. “While DNA replication does not happen in neurons, what does happen in all cells is DNA repair,” he said. Repeats could confuse repair enzymes, making them add to the expansion. In myotonic dystrophy type 1, Cooper noted, muscle biopsies in adults show that the DMPK expansion grows over time. Similarly, huntingtin repeats grow in Huntington’s disease model mice as they age (Kennedy et al., 2003).

Two cellular processes recruit repair enzymes to repeat sequences, Wilson said. One is transcription, which exposes and separates DNA strands. This allows the repeats to form structures such as hairpins, which the DNA repair machinery recognizes as abnormal and tries to repair (reviewed in Lin and Wilson, 2011). The formation of hairpins by CAG repeats, such as found in Huntington’s disease, and CGG repeats as in Fragile X, has long been considered a factor in expansion (Gacy et al., 1995). Supporting Wilson’s theory, knocking out some DNA repair proteins stabilizes the length of trinucleotide repeats (reviewed in Lin et al., 2009). The second possibility Wilson noted is that the high oxygen concentration in the brain damages DNA. That, too, would recruit repair enzymes and prime the system to alter the number of repeats (see ARF related news story on Kovtun et al., 2007). Rademakers suggested that multiple mechanisms might destabilize repeats.

A final answer on the process is still a way off. “One of the most fascinating aspects of all these repeat diseases is the pattern of expansions in different tissues. But it is incredibly complicated,” Wilson said. For now, the heterogeneity of the repeats is an important factor for researchers studying C9ORF72 to keep in mind, Dejesus-Hernandez concluded.—Amber Dance


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News Citations

  1. Corrupt Code: DNA Repeats Are Common Cause for ALS and FTD
  2. C9ORF72 Update: ALS Gene Is a Variable, and Global, Phenomenon
  3. A Growing Problem: Oxidative Damage Drives Trinucleotide Expansions in Aging Brain

Paper Citations

  1. . A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011 Oct 20;72(2):257-68. Epub 2011 Sep 21 PubMed.
  2. . Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011 Oct 20;72(2):245-56. Epub 2011 Sep 21 PubMed.
  3. . Dramatic tissue-specific mutation length increases are an early molecular event in Huntington disease pathogenesis. Hum Mol Genet. 2003 Dec 15;12(24):3359-67. PubMed.
  4. . Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell. 1995 May 19;81(4):533-40. PubMed.
  5. . Transcription destabilizes triplet repeats. Mol Carcinog. 2009 Apr;48(4):350-61. PubMed.
  6. . OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature. 2007 May 24;447(7143):447-52. PubMed.

Other Citations

  1. Read a PDF of the entire series.

Further Reading


  1. . C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing. J Alzheimers Dis. 2013 Jan 1;34(2):485-99. PubMed.
  2. . How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and frontotemporal dementia: can we learn from other noncoding repeat expansion disorders?. Curr Opin Neurol. 2012 Dec;25(6):689-700. PubMed.
  3. . Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype. Neurobiol Aging. 2012 Dec;33(12):2950.e5-7. PubMed.