Skip to main content

Letter to the editor on a paper by Kaivola et al. (2020): carriership of two copies of C9orf72 hexanucleotide repeat intermediate-length alleles is not associated with amyotrophic lateral sclerosis or frontotemporal dementia

The Original Article was published on 09 November 2020

Sir/madam,

Pathological hexanucleotide (G4C2)n-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS), as well as frontotemporal dementia (FTD) and FTD-ALS. Since the discovery of the C9orf72 repeat expansion as cause for ALS/FTD, there have been several contradicting reports whether intermediate repeat lengths are associated with FTD and/or ALS [13]. The definition of intermediate repeat length relies on the lower limit for pathological expansions, which has not been well-established. The most studies are using the initially suggested cutoff of 30 repeats [4, 5]. Recently, Kaivola et al. added to the existing literature that carriership of two copies of intermediate-length alleles is a strong risk factor for ALS [6]. Given the prior conflicting evidence, their finding warrants replication and as there is considerable overlap of FTD and ALS, we hypothesized that two copies of the C9orf72 intermediate-length alleles might also be associated with an increased risk of FTD.

In cohorts independent from Kaivola et al., we studied the association of carriership of two intermediate-length hexanucleotide C9orf72 repeats with ALS, FTD and a range of other neurodegenerative diseases, including primary progressive aphasia (PPA), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In summary, we did not find evidence for an association of the carriership of two C9orf72 repeat intermediate-length with any of the neurodegenerative diseases.

We collected data from six different cohorts studying neurodegenerative diseases (total n = 15,021) [712]. The C9orf72 lengths in each cohort were measured using comparable PCR or whole genome sequence methods (Additional file 1: Table S1). We excluded participants with a C9orf72 repeat expansion (using a ≥ 45 repeats threshold following methods of Kaivola et al. [6], n = 295), with an unknown allele length (n = 21), with an unknown phenotype (n = 28) and the phenotypes vascular dementia, mixed dementia and psychiatric diagnoses (n = 593). The remaining 14,084 participants were included for the analysis. We compared controls (n = 9,497) with five different disease classes: (a) ALS (n = 2,054), (b) FTD (n = 1,016), (c) FTD spectrum (FTD, ALS, PPA = 208, PSP/CBS = 8), (d) PD (n = 315), and (e) AD (n = 986). Statistical power and minimal detectable effect sizes (odds ratio’s) were calculated using the Genetic Association Study (GAS) Power Calculator (e.g. the sample size for power calculations of ALS was 11,551 with a case rate of 17.8%, alpha was 0.05). Expected effect sizes were derived from Kaivola et al. [6]. Disease allele frequencies were derived from our control group (Additional File 1: Table S2, Additional File 1: Table S3). We associated the intermediate-length allele thresholds described by Kaivola et al. [6]: (1) ≥ 7/ ≥ 7 repeats, (2) ≥ 7–16/7–16, and (3) ≥ 7/ ≥ 17–45 units. We fitted separate logistic regression models to study the association of traits with each of the three different intermediate-length threshold groups, adjusting for cohort origin. In addition, we performed analysis within region of origin (North-American, United Kingdom, Northern Europe and Southern Europe) followed by a fixed-effects inverse variance meta-analysis. Statistical analyses were performed using RStudio (version 3.5.2, R Development Core team 2010, rmeta package).

Power analyses showed that our study has ~ 100% power to detect the reported association in ALS in all intermediate-length threshold groups. We found no significant association of ALS, FTD and the FTD spectrum with carriership of two copies of C9orf72 intermediate-length alleles in all three intermediate-length threshold groups (Table 1). The region of origin analysis (Additional File 1: Table S4) followed by a fixed-effects inverse variance meta-analysis showed similar negative results (Additional File 1: Table S5). We explored the association of AD and PD with carriership of two copies of C9orf72 intermediate-length alleles. No significant association was found. However, sample size was limited in these groups.

We hypothesized that the true effect is smaller than reported by Kaivola et al. Therefore, we calculated the minimum odds ratio that we have 90% power for in our sample. For the ≥ 7/17–45 intermediate-length threshold, our study has 90% power to detect odds larger than 2.12 for ALS and 2.77 for FTD.

Table 1 Individuals with two C9orf72 intermediate-length alleles in ALS, FTD, FTD spectrum, PD and AD patients, and controls after exclusion of expansion carriers

Several suggestions may explain the discrepancy between Kaivola’s strong positive findings and our negative results. First, the higher prevalence of the intermediate-length alleles in Finland [13] versus the non-Finnish Europeans and North Americans represented in our cohort, could have resulted in the Finnish study to have increased power. Second, there could be another, Finland-specific, pathological variant present on the haplotype with the intermediate length allele that associates with ALS. Likewise, there are sub-haplotypes with an increased ‘base’ repeat-length, predisposing to pathological repeat expansions [10]. Third, the genotyping in the Finnish study and in our study, was not done at one site. This may have resulted in batch or laboratory effects. In our study, we corrected for batch or laboratory effects by adjusting for cohort of origin in our logistic regression models and observed no effects. Still, we cannot fully rule out false negative findings due to cohort or technical biases. In support of the association, a Belgian study showed that lengths of ≥ 7–24 are almost exclusively present on the chromosome 9 risk haplotype tagged by the rs2814707 T-allele and that homozygous carriership of the T-allele is associated with disease (OR = 1.8, p = 0.04) [2]. Homozygous carriership of this T-allele was associated with ALS and FTD-ALS (OR = 2.08, p = 0.04) in the non-expansion group [1].

We also made an interesting observation when reviewing the clinical records of carriers of two copies of repeat intermediate-length in one cohort. These patients showed a bvFTD phenotype with noteworthy co-symptoms of PSP and ALS. A co-existence that, based on clinicopathology, is not to be expected and as far as we are aware, has not previously been associated with C9orf72 repeat intermediate-length [14].

Altogether, in this multinational cohort we could not confirm an association of carriership of two copies of C9orf72 repeat intermediate-length alleles with ALS or FTD.

References

  1. Gijselinck I, et al (2016) The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychaitry 21:8:1112–1124

    CAS  Article  Google Scholar 

  2. van der Zee J, et al (2013) A pan-European study of the C9orf72 repeat associated with FTLD: geographic prevalence, genomic instability, and intermediate repeats. Hum Mutat 34(2):363–373

    Article  Google Scholar 

  3. Rutherford NJ, Heckman MG, Dejesus-Hernandez M, Baker MC, Soto-Ortolaza AI, Rayaprolu S, Rademakers R (2012) Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype. Neurobiol Aging 33(12):2950.e2955-2957

    Article  Google Scholar 

  4. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Rademakers R (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72(2):245–256

    CAS  Article  Google Scholar 

  5. Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Traynor BJ (2011) A hexanucleotide repeat expansion in C9ORF72 Is the cause of chromosome 9p21-Linked ALS-FTD. Neuron 72(2):257–268

    CAS  Article  Google Scholar 

  6. Kaivola K, Salmi SJ, Jansson L, Launes J, Hokkanen L, Niemi A-K, Tienari PJ (2020) Carriership of two copies of C9orf72 hexanucleotide repeat intermediate-length alleles is a risk factor for ALS in the Finnish population. Acta Neuropathol Commun 8:187

    CAS  Article  Google Scholar 

  7. Huisman MHB et al (2011) Population based epidemiology of amyotrophic lateral sclerosis using capture-recapture methodology. J Neurol Neurosurg and Psychiat 82(10):1165–1170

    Article  Google Scholar 

  8. Beck J, Poulter M, Hensman D, Rohrer JD, Mahoney CJ, Adamson G, Mead S (2013) Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population. Am J Hum Genet 92(3):345–353

    CAS  Article  Google Scholar 

  9. Mol MO, van Rooij JGJ, Wong TH, Melhem S, Verkerk A, Kievit AJA, van Minkelen R, Rademakers R, Pottier C, Kaat LD, Seelaar H, van Swieten JC, Dopper EGP (2021) Underlying genetic variation in familial frontotemporal dementia: sequencing of 198 patients. Neurobiol Aging 97:148.e149-148.e116

    Article  Google Scholar 

  10. Reus LM, Jansen IE, Mol MO, van Ruissen F, van Rooij J, van Schoor NM, van der Lee SJ (2021) Genome-wide association study of frontotemporal dementia identifies a C9ORF72 haplotype with a median of 12–G4C2 repeats that predisposes to pathological repeat expansions. Transl Psychiatry 11(1):451–451

    CAS  Article  Google Scholar 

  11. Serpente M, Fenoglio C, Arighi A, Fumagalli GG, Arcaro M, Sorrentino F, Galimberti D (2021) Analysis of C9orf72 intermediate alleles in a retrospective cohort of neurological patients: risk factors for alzheimer’s disease? J Alzheimers Dis 81:1445–1451. https://doi.org/10.3233/JAD-210249

    CAS  Article  PubMed  Google Scholar 

  12. Xi Z, Zinman L, Grinberg Y, Moreno D, Sato C, Bilbao JM, Rogaeva E (2012) Investigation of c9orf72 in 4 neurodegenerative disorders. Arch Neurol 69(12):1583–1590. https://doi.org/10.1001/archneurol.2012.2016

    Article  PubMed  PubMed Central  Google Scholar 

  13. Laaksovirta H, Launes J, Jansson L, Traynor BJ, Kaivola K, Tienari PJ (2022) ALS in Finland. Neurol Genet 8(2):e665

    Article  Google Scholar 

  14. Ng ASL, Tan EK (2017) Intermediate C9orf72 alleles in neurological disorders: does size really matter? J Med Genet 54(9):591–597

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Contributions

All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sterre C. M. de Boer.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de Boer, S.C.M., Woolley, L., Mol, M.O. et al. Letter to the editor on a paper by Kaivola et al. (2020): carriership of two copies of C9orf72 hexanucleotide repeat intermediate-length alleles is not associated with amyotrophic lateral sclerosis or frontotemporal dementia. acta neuropathol commun 10, 141 (2022). https://doi.org/10.1186/s40478-022-01438-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40478-022-01438-0