Skip to main content
Advertisement
  • Neurology.org
  • Journals
    • Neurology
    • Clinical Practice
    • Education
    • Genetics
    • Neuroimmunology & Neuroinflammation
  • Online Sections
    • Neurology Video Journal Club
    • Diversity, Equity, & Inclusion (DEI)
    • Neurology: Clinical Practice Accelerator
    • Practice Buzz
    • Practice Current
    • Residents & Fellows
    • Without Borders
  • Collections
    • COVID-19
    • Disputes & Debates
    • Health Disparities
    • Infographics
    • Neurology Future Forecasting Series
    • Null Hypothesis
    • Patient Pages
    • Topics A-Z
    • Translations
  • Podcast
  • CME
  • About
    • About the Journals
    • Contact Us
    • Editorial Board
  • Authors
    • Submit New Manuscript
    • Submit Revised Manuscript
    • Author Center

Advanced Search

Main menu

  • Neurology.org
  • Journals
    • Neurology
    • Clinical Practice
    • Education
    • Genetics
    • Neuroimmunology & Neuroinflammation
  • Online Sections
    • Neurology Video Journal Club
    • Diversity, Equity, & Inclusion (DEI)
    • Neurology: Clinical Practice Accelerator
    • Practice Buzz
    • Practice Current
    • Residents & Fellows
    • Without Borders
  • Collections
    • COVID-19
    • Disputes & Debates
    • Health Disparities
    • Infographics
    • Neurology Future Forecasting Series
    • Null Hypothesis
    • Patient Pages
    • Topics A-Z
    • Translations
  • Podcast
  • CME
  • About
    • About the Journals
    • Contact Us
    • Editorial Board
  • Authors
    • Submit New Manuscript
    • Submit Revised Manuscript
    • Author Center
  • Home
  • Latest Articles
  • Current Issue
  • Past Issues
  • Neurology Video Journal Club
  • Residents & Fellows

User menu

  • Subscribe
  • My Alerts
  • Log in

Search

  • Advanced search
Neurology
Home
The most widely read and highly cited peer-reviewed neurology journal
  • Subscribe
  • My Alerts
  • Log in
Site Logo
  • Home
  • Latest Articles
  • Current Issue
  • Past Issues
  • Neurology Video Journal Club
  • Residents & Fellows

Share

January 23, 2018; 90 (4) ArticleOpen Access

Unaffected mosaic C9orf72 case

RNA foci, dipeptide proteins, but upregulated C9orf72 expression

Philip McGoldrick, Ming Zhang, Marka van Blitterswijk, Christine Sato, Danielle Moreno, Shangxi Xiao, Ashley B. Zhang, Paul M. McKeever, Anna Weichert, Raphael Schneider, Julia Keith, Leonard Petrucelli, Rosa Rademakers, Lorne Zinman, Janice Robertson, Ekaterina Rogaeva
First published December 27, 2017, DOI: https://doi.org/10.1212/WNL.0000000000004865
Philip McGoldrick
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ming Zhang
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marka van Blitterswijk
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christine Sato
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Danielle Moreno
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shangxi Xiao
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ashley B. Zhang
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul M. McKeever
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anna Weichert
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Raphael Schneider
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Julia Keith
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Leonard Petrucelli
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rosa Rademakers
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lorne Zinman
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Janice Robertson
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ekaterina Rogaeva
From the Tanz Centre for Research in Neurodegenerative Diseases (P.M., M.Z., C.S., D.M., S.X., A.B.Z., P.M.M., A.W., R.S., J.R., E.R.), Department of Medicine (L.Z., E.R.), Division of Neurology, and Department of Laboratory Medicine and Pathobiology (J.R.), University of Toronto, Ontario, Canada; Department of Neuroscience (M.v.B., L.P., R.R.), Mayo Clinic, Jacksonville, FL; and Sunnybrook Health Sciences Centre (J.K., L.Z.), Toronto, Ontario, Canada.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Full PDF
Citation
Unaffected mosaic C9orf72 case
RNA foci, dipeptide proteins, but upregulated C9orf72 expression
Philip McGoldrick, Ming Zhang, Marka van Blitterswijk, Christine Sato, Danielle Moreno, Shangxi Xiao, Ashley B. Zhang, Paul M. McKeever, Anna Weichert, Raphael Schneider, Julia Keith, Leonard Petrucelli, Rosa Rademakers, Lorne Zinman, Janice Robertson, Ekaterina Rogaeva
Neurology Jan 2018, 90 (4) e323-e331; DOI: 10.1212/WNL.0000000000004865

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Permissions

Make Comment

See Comments

Downloads
1443

Share

  • Article
  • Figures & Data
  • Info & Disclosures
Loading

Abstract

Objective Suggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30–90 repeats) is unknown and was investigated here.

Methods We conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS.

Results Southern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration.

Conclusion The presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

Glossary

ALS=
amyotrophic lateral sclerosis;
DPR=
dipeptide repeat;
FTLD=
frontotemporal lobar degeneration

A G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)1,–,3 with 100% penetrance of ALS in men by 86 years of age.4 The cascade leading to TDP-43 pathology in degenerating neurons of C9orf72 carriers is unknown.5 Three suggested pathomechanisms arise from the mutation6: downregulation of C9orf72 expression that could lead to C9orf72 haploinsufficiency, nuclear sense (G4C2) and antisense (C4G2) RNA foci, and dipeptide repeat (DPR) inclusions (poly-GA, -GP, -GR, -PA, -PR).

Some factors could modify the C9orf72 phenotype. Intermediate ATXN2 alleles (27–33 CAG repeats) render susceptibility to ALS,7 and the TMEM106B A allele of rs1990622 was associated with a later onset,8 while homozygosity for the G allele of rs3173615 protects against FTLD.9 Size of the expansions may also affect disease severity.10 Initially, >30 repeats were suggested to be pathogenic1,2; however, several 30- to 70-repeat carriers have been free from disease.11,–,13 Small expansions (<90 repeats) are unmethylated, while methylation of the large expansions is unequivocal14 and associated with ≈50% downregulation of C9orf72 expression.1,2,14,–,16 In contrast, a 70-repeat expansion increases C9orf72 expression (by 3-fold vs normal control11). This 70-repeat allele (on the typical ≈200-kb risk haplotype) jumped to pathogenic length (≈1,750 repeats) in the next generation,11 indicating that the expansions have occurred on multiple occasions.17,18

We conducted a comprehensive study of a neurologically healthy carrier of a 70-repeat allele in blood11 who recently came to autopsy at age 90. We investigated C9orf72 at the DNA, RNA, and protein levels and assessed RNA foci, DPRs, and TDP-43 pathology.

Methods

Standard protocol approvals, registrations, and patient consents

The CNS and peripheral tissues from Canadian PED25 family members (figure 1) were collected in accordance with the ethics review board. We received written informed consent for all study participants.

Figure 1
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1 Updated pedigree of Canadian family of British origin (PED25)

C9orf72 genotype is shown beneath the corresponding diamond. Age at the time of examination is shown in the top right corner. Age at death is indicated by the prefix d (the postmortem interval was within 12 hours). Onset age is indicated for individuals with disease above the identification number. The 70-repeat allele carrier (9686) died at age 90. Four of his 5 offspring had large C9orf72 repeat expansions, including 3 with amyotrophic lateral sclerosis (ALS).

Human samples

The proband (9686) carried a 70-repeat C9orf72 expansion in blood DNA,11 was free from symptoms of ALS and FTLD up to age 90 on the basis of an assessment by neurologists (L.Z. and R.S.), and died of chronic obstructive pulmonary disease. Autopsy results for 9686 were compared to his daughter (9548), diagnosed with ALS at age 57, who had a large expansion (≈1,750 repeats) in all investigated tissues.11

Genetic and epigenetic analyses

C9orf72 was genotyped by repeat-primed PCR as described previously.2 Southern blot for 9686 was conducted as reported11 to estimate the size of the expansion in heart, liver, kidney, lung, tongue, testicles, epididymis, thalamus, cervical and thoracic spinal cord, brainstem, cerebellum, and frontal, motor, temporal, and occipital cortices. To study the DNA methylation of C9orf72, we used an aliquot from the same DNA preparation as for Southern blot. DNA methylation level at the 5′ and 3′ regions flanking the G4C2 repeat was analyzed by direct bisulfite sequencing.16 Methylation status of the repeat region was investigated by a (G4C2)n methylation assay.14 ATXN2 and TMEM106B were genotyped as described previously.19

Analyses of C9orf72 expression

Reverse transcription PCR11,16 and Western blotting20 were performed as reported (details in appendix e-1, links.lww.com/WNL/A75). Relative quantification of reverse transcription PCR was calculated with the ddCt method after normalization to the reference genes. Gene expression levels were normalized to the level of C9orf72 expression in corresponding tissues of 9548. C9orf72 expression in blood was assessed for family members (9686, 9548, 9697) and normalized to expression in the wild-type allele carrier (9697). Two protein isoforms are produced from C9orf72 transcript variants: C9-L (54 kDa) and C9-S (23 kDa), with C9-L found predominantly in the urea-soluble fraction and C9-S present in the low- and high-salt Triton X-100 fractions.20 Thus, we extracted protein using the high-salt Triton X-100 buffer (to quantify C9-S), followed by high salt-sucrose buffer, and solubilized the remaining pellet in urea buffer (to quantify C9-L). For Western blotting, we used anti–C9-L or anti–C9-S rabbit polyclonal in-house primary antibodies (densitometric values normalized to β-actin).

RNA foci and DPR analyses

Details for RNA fluorescence in situ hybridization and DPR assessment are given in appendix e-1, links.lww.com/WNL/A75. For quantification, investigators were blinded, and random images were taken from each region. We used 563′ TYE-conjugated probes recognizing either the sense or antisense RNA foci. The number of nuclei positive for RNA foci was expressed as a percentage of total nuclei (all cell types). The number of RNA foci per nuclei was also quantified. Primary antibodies for each DPR were mouse anti-GA (MABN889, Millipore, Billerica, MA), rabbit anti-GP (NBP2-25018, Novus, St. Louis, MO), rat anti-GR (MABN778, Millipore), and rabbit anti-PA (Rb8604) and anti-PR (Rb8736) from Dr. Petrucelli’s laboratory.

Statistics

A nonparametric Mann-Whitney U test was used to compare intensities of the Southern blot bands (70-repeat vs 2-repeat alleles) between CNS and non-CNS tissues. Linear regression was used to evaluate the relationship between the ratio of Southern blot band intensity and the ratio of C9orf72 expression (9686 vs 9548) in multiple tissues (data meet normal distribution). Statistical analysis was conducted with SPSS (IBM, Armonk, NY), and p < 0.05 was accepted as statistically significant. For Western blot, an unpaired 2-tailed t test was used (GraphPad Software Inc, La Jolla, CA). Two-tailed t tests (GraphPad) were used to determine differences between 9686 and 9548 for burden of RNA foci or DPRs (p < 0.0125 and p < 0.01 after Bonferroni correction were deemed significant, respectively).

Results

The 70-repeat allele is somatically unstable

Repeat-primed PCR detected an allele with >50 repeats in all examined tissues of 9686 (figure e-1, links.lww.com/WNL/A73). Southern blot of CNS tissues revealed the presence of the 2-repeat and 70-repeat alleles together with a large expansion (most evident in the cerebellum) of a size similar to that of an unrelated C9orf72-ALS case, indicative of pathologic length (figure 2, A and B). In non-CNS tissues (figure 2B), a weak signal for large expansions was detected in liver but not in kidney, heart, lung, tongue, testicles, or epididymis (similar to blood DNA11).

Figure 2
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2 Southern blots of different tissues from individual 9686

(A) Southern blot of CNS tissues including spinal cord and brain tissues. (B) Southern blot of thalamus and non-CNS tissues. (C) Bar figure showing the ratio of Southern blot band intensity between small expansion and wild-type alleles in different tissues. CNS tissues have a significantly smaller ratio than non-CNS tissues (*p < 0.05), suggesting that CNS tissues have a higher instability than non-CNS tissues. BS = brainstem; CBL = cerebellum; C-SC = cervical spinal cord; EPI = epididymis; FCX = frontal cortex; HRT = heart; KDN = kidney; LNG = lung; LVR = liver; MCX = motor cortex; NC = negative control; OCX = occipital cortex; PC = positive control; TCX = temporal cortex; TEST = testicle; THAL = thalamus; TNG = tongue; T-SC = thoracic spinal cord; WT = wild-type.

To assess the degree of instability of the small expansion, we quantified the band intensity ratio between the 70-repeat and 2-repeat alleles (a lower ratio indicates a greater number of cells without the 70-repeat allele because of its expansion). CNS tissues had a lower level of the 70-repeat allele than peripheral tissues (p = 0.001), especially in cerebellum with only ≈2% of the 70-repeat expansion remaining (figure 2C). In contrast, the 70-repeat allele remained stable in most non-CNS tissues with the exception of liver and kidney, which had an estimated loss of the 70-repeat allele by ≈74% and ≈34%, respectively (figure 2C). Hence, 9686 is a mosaic carrier of small and large C9orf72 expansions, with varying degrees of somatic instability between tissues.

Some reports suggested that ALS/FTLD could be caused21 or modified22 by inheritance of variants in several genes. Previously, we excluded additional pathogenic mutations in SOD1, FUS, and GRN in 9548.11 Here, we analyzed ATXN2 and TMEM106B (table e-1, links.lww.com/WNL/A74), which did not explain the phenotypic difference between 9686 and 9548 (e.g., we did not detect intermediate ATXN2 alleles or homozygosity for the rs3173615 G allele in TMEM106B).

Large expansions trigger DNA methylation of the G4C2 repeat

No methylation was detected at CpG islands adjacent to the G4C2 repeat for 9686 or 9548 (tables e-2 and e-3, links.lww.com/WNL/A74). In contrast, the G4C2 repeat was methylated in all 10 CNS tissues, liver, and kidney of 9686 (figure 3, A and B), where large expansions were identified by Southern blot (figure 2, A and B) or partial loss of the 70-repeat allele. Hence, the (G4C2)n methylation test revealed the presence of a large expansion even in a small fraction of the cells (it could detect methylation in a mixture of DNA standards containing >5% of highly methylated DNA14).

Figure 3
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3 Chromatograms of the (G4C2)n methylation assay in multiple tissues of individual 9686

Methylation and nonmethylation channels detect methylated (blue) or unmethylated (green) repeat alleles. The red “yes” indicates that a methylated repeat expansion was detected. (A) (G4C2)n methylation results in brain DNA. (B) (G4C2)n methylation results in non-CNS DNA. (C) (G4C2)n methylation results in spinal cord DNA. BS = brainstem; CBL = cerebellum; C-SC = cervical spinal cord; EPI = epididymis; FCX = frontal cortex; HIP = hippocampus; HRT = heart; KDN = kidney; LNG = lung; L-SC = lumbar spinal cord; LVR = liver; MCX = motor cortex; OCX = occipital cortex; TCX = temporal cortex; TEST = testicle; THAL = thalamus; TNG = tongue; T-SC = thoracic spinal cord.

Small expansion upregulates C9orf72 at the RNA and protein levels

Total C9orf72 transcript levels from heart (2/70 alleles) and 5 mosaic tissues of 9686 were compared to the same tissues from 9548 (2/1,750 genotype). The mosaic carrier had higher C9orf72 expression compared to 9548 (p < 0.05 for frontal cortex and p < 0.01 for other tissues) by 51% in cerebellum, 45% in frontal cortex, 325% in cervical spinal cord, 106% in thoracic spinal cord, 79% in liver, and 440% in heart (figure 4A). Consistent with a previous study,11 9686 had much higher C9orf72 expression in blood than 9548 or a noncarrier sibling (9697 with 2/2 genotype) by 530% and 440%, respectively (figure e-2, links.lww.com/WNL/A73). The ratio of C9orf72 expression (9686 vs 9548) and the ratio of Southern blot band intensity (70-repeat vs 2-repeat alleles) in the investigated tissues fit a linear regression model (p = 0.046, R2 = 0.67) (figure 4B), indicating that the small expansion drives C9orf72 upregulation. Next, we compared the protein levels of C9-L and C9-S in frontal cortex and cerebellar tissue of 9686 and 9548. C9-L was higher in frontal cortex (p = 0.04) and cerebellum (p = 0.04) of 9686 vs 9548 (figure 4, C–E); however, no difference was evident in C9-S levels (figure 4, D–F).

Figure 4
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4 Relative quantification of C9orf72 at the RNA and protein levels

(A) We analyzed C9orf72 expression levels in brain tissues (frontal cortex [FCX] and cerebellum [CBL]), cervical (C-SC) and thoracic spinal cord (T-SC), and non-CNS tissues (liver [LIV] and heart [HRT]) from 9686 (C9orf72 genotype: 2/70/expansion) and 9548 (C9orf72 genotype: 2/expansion). Tissues carrying the small expansion had higher C9orf72 expression than tissues with large expansions (*p < 0.05, **p < 0.01). The relative quantification of each sample of 9686 was adjusted to that of 9548. Error bars represent the standard error of the triplicates. (B) A correlation analysis to understand the relationship between repeat expansion size and C9orf72 expression. The ratio of Southern blot (SB) band intensity (70-repeat allele vs wildtype [WT]) in 9686 is significantly correlated with the ratio of C9orf72 expression (9686 vs 9548) (p = 0.046). (C) C9-L levels were examined in the urea fraction of frontal cortex tissue of 9686 and 9548. Three separate samples of tissue were used from each case. C9-L levels were normalized to β-actin and revealed to be significantly higher in 9686 (*p = 0.044). (D) C9-S levels were examined from the high-salt Triton X-100 (HST) fraction. No difference was seen between 9686 and 9548. (E) C9-L was significantly increased in the urea fraction of cerebellum of 9686 compared to 9548 (*p = 0.042). C9-L was normalized to β-actin, and 3 separate samples were used. (F) C9-S levels in the HST fraction of cerebellum did not differ between 9686 and 9548. Data are mean ± SEM.

Brain pathology in PED25 family

We quantified the burden of RNA foci in brain regions (figure 5, A–Q) and spinal cord of 9686 and 9548. There was no difference between 9686 and 9548 in the number of nuclei positive for sense RNA foci in frontal cortex (6.63 ± 2.24% vs 6.64 ± 0.42%), motor cortex (10.28 ± 0.39% vs 9.27 ± 1.26%), or hippocampus (21.55 ± 2.06% vs 20.13 ± 1.07%). Remarkably, in cerebellum of 9686, there were twice as many nuclei positive for sense RNA foci compared to ALS-affected 9548 (21.20 ± 1.42% vs 11.38 ± 0.76%; p = 0.0037) (figure 5I). The burden of antisense RNA foci was similar between 9686 and 9548 (figure 5R). In addition, we assessed the number of RNA foci per nucleus, which suggested similar dynamics of RNA foci production between 9686 and 9548 (figure e-3, links.lww.com/WNL/A73). Quantification of sense or antisense RNA foci in motor neurons of the cervical and lumbar spinal cord did not reveal any differences between 9686 and 9548 (figure e-4).

Figure 5
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5 RNA foci analyses in brains of individuals 9686 and 9548

RNA fluorescence in situ hybridization with a probe complementary to the sense-direction sequence (GGGGCC) was used to detect RNA foci (red) in nuclei (blue) of the cerebellum (CBL), frontal cortex (FCX), motor cortex (MCX), and hippocampus (HIP) of 9686 (A–D, respectively) and 9548 (E–H, respectively). Quantification of the total number of nuclei positive for sense RNA foci (I) revealed more sense RNA foci–positive cells in the cerebellum of 9686 compared to 9548 (n = 3, **p < 0.001); however, there were no differences in other regions. Antisense RNA foci (red, J–Q), detected with a probe complementary to the antisense sequence (CCCCGG), were also seen in the cerebellum, frontal cortex, motor cortex, and hippocampus of 9686 (J–M) and 9548 (N–Q). Quantification of the number of antisense RNA foci–positive nuclei did not reveal any significant differences between 9686 and 9548 (R). n = 3 slides per region. Data are mean ± SEM. PKC = Purkinje and molecular cell layer.

All 5 types of DPR inclusions were detectable in brains of 9686 and 9548 (figure e-5, links.lww.com/WNL/A73). Our result is consistent with the reported relative abundance of DPRs in C9orf72 cases (GA > GP > GR > PA/PR), with PA and PR inclusions being very rare.23 To determine whether DPR burden is altered in 9686 compared to 9548, we scored the average number of DPR inclusions per field from different brain regions (figure e-5L). In agreement with a prior report,23 DPR inclusions in the spinal cord were too scarce for quantification. Strikingly, in granule cells of the cerebellum, DPR burden was increased (up to 7 times) in neurologically normal 9686 compared to 9548: GA (205.80 ± 11.62 vs 30.33 ± 4.13, p < 0.001), GP (49.60 ± 6.64 vs 30.50 ± 2.40, p < 0.01), and GR (7.48 ± 0.66 vs 1.00 ± 0.22, p < 0.001, respectively) (figure e-5L). There was no difference in DPR burden in frontal cortex (figure e-5M) or hippocampus (figure e-5N), and although the number of GA inclusions was lower in the motor cortex of 9686 compared to 9548 (3.70 ± 1.77 vs 5.53 ± 2.36, p < 0.001) (figure e-5O), other DPRs did not show differences in this region. Our findings do not rule out DPRs as mediators of toxicity; rather, we consider that alone they are insufficient for neurodegeneration.

The neuropathologic diagnosis of the daughter (9548) was ALS with findings typical for C9orf72 carriers. This included gliosis and loss of motor neurons from the anterior horn, TDP-43 and p62 inclusions in remaining motor neurons, and degeneration of corticospinal tracts. Neuronal p62 inclusions, some with a starburst morphology, were scattered throughout all cortical regions, the Ammon horn, the entorhinal cortex, and the cerebellar granular layer. There was a normal population of Betz cells, with some containing p62- and TDP-43–positive inclusions. In contrast, the father (9686) did not have any evidence of upper or lower motor neuron degeneration, and there was no TDP-43 pathology in the motor pathway. There was no evidence of FTLD TDP-43; the frontal and temporal cortices were not atrophic and did not contain TDP-43 inclusions. The most significant finding was the typical starburst-shaped p62 inclusions encountered in C9orf72 carriers known to reflect DPRs. These inclusions were observed in the cerebellar granular layer, pyramidal neurons of CA1, and all sampled areas of the cerebral cortex. Other minor coexisting neurodegenerative phenomena included Alzheimer-type neuropathologic changes and scant TDP-43 pathology restricted to the mesial temporal regions (short neurites, neuronal cytoplasmic inclusions in the superficial layers of the entorhinal cortex, and dot-like inclusions of the dentate granular layer).

Discussion

We have conducted a study of a neurologically normal individual (9686) at age 90 carrying a 70-repeat C9orf72 allele in blood that expanded to a pathogenic range in 4 offspring.11 We were unable to collect a sperm sample from 9686, but the 70-repeat allele likely expanded to ≈1,750 repeats in the germ cells of 9686, rather than arising 4 times separately in each child. Therefore, on the background of the permissive risk haplotype, small expansions (premutations) may jump to pathogenic lengths in the parental generation, which could explain the high frequency of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS1,2). Such a frequency is unusually high for an autosomal dominant disorder, even if family history in some sporadic patients is masked by the early death of parents or complexity of C9orf72 subphenotypes (e.g., neuropsychiatric symptoms).

Southern blot revealed the mosaicism of small and large expansions in 9686 with a weak signal for large expansions in spinal cord and frontal, temporal, and motor cortices, whereas cerebellum showed mainly large expansion. Somatic instability was described for other small expansions (e.g., 61–92 repeats17,18). However, detailed investigation of C9orf72 expression and brain pathology (RNA foci, DPRs, TDP-43) in these cases was unreported (table e-4, links.lww.com/WNL/A74). A study of a 30-repeat carrier, unaffected at age 84, did not show repeat instability in the investigated frontal cortex and cerebellum. However, it is unclear if he carried the risk haplotype.12 Like 9686, this individual had RNA foci and DPRs but lacked TDP-43 pathology. Similar to 9686, non-CNS tissues of reported mosaic cases rarely have large expansions, which may reflect differences in efficiency of DNA repair and cell turnover rate between CNS (lifetime) and non-CNS (days) cells. In contrast to 9686, some mosaic C9orf72 cases had strong signals for large expansions in ALS/FTLD–relevant regions, leading to a typical disease course.10,17,18,24 Thus, the degree of mosaicism in disease-relevant regions may be a key determinant for clinical manifestation.

Toxic effects of RNA foci and DPRs have been demonstrated in model systems; however, the neurodegeneration in C9orf72 cases does not correlate with the distribution of RNA foci25 or DPRs23,26 (only 1 report suggests an association between cerebellar GP inclusions and cognitive score in a small FTLD cohort27). Similarly, downregulation of C9orf72 orthologs caused mobility defects in Caenorhabditis elegans28 and motor neuron degeneration in zebrafsh29; however, knockout mice lack signs of neurodegeneration and show only immune system dysfunction,30 suggesting that C9orf72 downregulation alone is insufficient to cause disease. However, a recent report of a patient with ALS with a loss-of-function C9orf72 mutation (p.I201fsX235) supports a role of C9orf72 haploinsufficiency in ALS.31

We have previously shown that the 70-repeat expansion in blood DNA of 9686 is associated with upregulation of C9orf72.11 A similar phenomenon was reported for FMR1 in which unaffected carriers of a premutation (55–200 CGG repeats) have upregulation of FMR1, whereas larger expansions cause fragile X mental retardation syndrome by silencing FMR1 through hypermethylation.32 Consistent with prior findings,11 tissues from 9686 revealed higher C9orf72 expression at RNA and protein levels compared to tissues from the ALS-affected daughter (9548). The extent of C9orf72 upregulation correlated with the level of the 70-repeat allele, suggesting that small expansions drive upregulation, which may offset haploinsufficiency caused by large expansions. Because C9orf72 plays important roles in nucleocytoplasmic transport, autophagy, intercellular trafficking, and TDP-43 aggregation,20,33,–,38 modulating C9orf72 expression might be a therapeutic option. Indeed, a recent report found that increased expression of C9orf72 variant 1 conferred a survival advantage in expansion carriers.39 However, it is also possible that unknown genetic or environmental factors protected 9686 from disease. For instance, DNA methylation analysis of the 353 age-linked CpGs from the genome-wide 450k BeadChip revealed that acceleration of the epigenetic clock in C9orf72 carriers is significantly correlated with a shorter disease duration and earlier age at onset.19 Notably, 9686 was aging more slowly than other PED25 family members (his epigenetic age was ≈9 years younger than his chronologic age).19 However, it currently is not clear whether the epigenetic clock reacts to aging or causes aging.

The striking difference between the mosaic case (9686) and the large expansion carrier (9548) is the absence of ALS/FTLD symptoms and TDP-43 pathology in 9686. Burdens of RNA foci and DPRs were similar between 9686 and 9548, except for the cerebellum, which surprisingly had more DPR inclusions and sense RNA foci in the neurologically normal father compared to the daughter with ALS. Our results suggest that RNA foci and DPRs alone are insufficient to drive neurodegeneration or require additional stressors such as downregulation of C9orf72 expression (figure e-6, links.lww.com/WNL/A73). Although a recent study reported that small and large DPRs have differential effects in vitro,40 because DPRs are not associated with regions of neurodegeneration in carriers of large expansions,23,26 it is possible that the absence of disease in 9686 is due to upregulated C9orf72 expression rather than fewer long DPRs. This is in agreement with the observation that the highest C9orf72 expression among CNS tissues of unaffected individuals was detected in the cerebellum,1 a region with high abundance of DPRs in C9orf72 cases but no degeneration.23,26

Currently, there are only limited data on the properties of small expansions, yet they may provide important knowledge on C9orf72-related disease: of the 3 potential pathogenic mechanisms, only downregulation of C9orf72 expression was absent in the unaffected father. This individual lacked signs of ALS or FTLD, but the burden of RNA foci and DPRs was similar to that of an ALS-affected daughter with large expansion. Because the current study is limited to a single family, it is important to investigate other small expansion carriers who could be identified among unaffected parents of sporadic C9orf72 cases, especially frequent in the Finnish population.1,2 We suggest that caution should be taken when consulting carriers of small C9orf72 expansions in blood because disease manifestation may be dependent on the extent of the somatic instability in disease-relevant CNS tissues.

Author contributions

Dr. McGoldrick was responsible for the tissue collection and processing, Western blotting, immunohistochemistry and RNA fluorescence in situ hybridization experiments, and subsequent analysis. Dr. Zhang was responsible for DNA methylation and RNA quantification of C9orf72 and Southern blot analysis. Dr. van Blitterswijk conducted Southern blotting. Ms Sato and Ms Moreno participated in DNA/RNA preparation. Ms Zhang assisted in immunohistochemistry, and Dr. Xiao assisted in Western blotting and C9orf72 antibody generation. Mr McKeever, Dr. Weichert, and Dr. Schneider participated in tissue collection and processing. Dr. Keith carried out pathologic analysis. Dr. Petrucelli provided reagents. Dr. Zinman was responsible for obtaining clinical findings and editing the manuscript. Dr. Rademakers contributed to Southern blotting experiments and editing the manuscript. Dr. Robertson and Dr. Rogaeva supervised the project and wrote the manuscript with Dr. McGoldrick and Dr. Zhang.

Study funding

No targeted funding reported.

Disclosure

P. McGoldrick was supported by a Milton Safenowitz Postdoctoral Fellowship from the American ALS Association and currently holds the Christopher Chiu Fellowship from ALS Double Play. M. Zhang receives funding from the Canadian Consortium on Neurodegeneration in Aging. M. van Blitterswijk receives NIH grants R35 NS097261 and P01 NS084974 and is supported by the Clinical Research in ALS and Related Disorders for Therapeutic Development (CReATe) Clinical Research Fellowship. CReATe (U54 NS092091) is part of the Rare Diseases Clinical Research Network, an initiative of the Office of Rare Diseases Research, National Center for Advancing Translational Sciences (NCATS). CReATe is funded through collaboration between NCATS and the National Institute of Neurologic Disorders and Stroke. C. Sato, D. Moreno, S. Xiao, and A. Zhang report no disclosures relevant to the manuscript. P. McKeever holds a Doctoral Award from the Alzheimer Society of Canada. A. Weichert reports no disclosures relevant to the manuscript. R. Schneider receives the Clinical Research Fellowship from ALS Canada. J. Keith reports no disclosures relevant to the manuscript. L. Petrucelli receives NIH grant P01 NS084974. R. Rademakers receives NIH grants R01 NS080882, R35 NS097261, and P01 NS084974. L. Zinman and J. Robertson receive funds from the ALS Canada-Brain Canada Hudson Grant and James Hunter ALS Initiative and the Temerty Family Foundation. E. Rogaeva receives funds from the Canadian Consortium on Neurodegeneration in Aging and the ALS Canada-Brain Canada Hudson Grant. Go to Neurology.org/N for full disclosures.

Acknowledgment

The authors are thankful to the family members for the donation of tissues for this study, as well as for the assistance of Denise Miletic (Tanz Centre for Research in Neurodegenerative Diseases), Shawn Hobbs, Shirley Pham, and Olive Grozelle (Sunnybrook Health Sciences Centre).

Footnotes

  • Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. The Article Processing Charge was funded by the ALS Canada Brain Canada Hudson Grant.

  • ↵* These authors contributed equally to this work.

  • Received July 4, 2017.
  • Accepted in final form October 16, 2017.
  • Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

References

  1. 1.↵
    1. Renton AE,
    2. Majounie E,
    3. Waite A, et al
    . A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72:257–268.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. DeJesus-Hernandez M,
    2. Mackenzie IR,
    3. Boeve BF, et al
    . Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011;72:245–256.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Majounie E,
    2. Renton AE,
    3. Mok K, et al
    . Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol 2012;11:323–330.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Williams KL,
    2. Fifita JA,
    3. Vucic S, et al
    . Pathophysiological insights into ALS with C9ORF72 expansions. J Neurol Neurosurg Psychiatry 2013;84:931–935.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Ling SC,
    2. Polymenidou M,
    3. Cleveland DW
    . Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 2013;79:416–438.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Hardy J,
    2. Rogaeva E
    . Motor neuron disease and frontotemporal dementia: sometimes related, sometimes not. Exp Neurol 2014;262:75–83.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. van Blitterswijk M,
    2. Mullen B,
    3. Heckman MG, et al
    . Ataxin-2 as potential disease modifier in C9ORF72 expansion carriers. Neurobiol Aging 2014;35:e2413–2427.
    OpenUrl
  8. 8.↵
    1. Gallagher MD,
    2. Suh E,
    3. Grossman M, et al
    . TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions. Acta Neuropathol 2014;127:407–418.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. van Blitterswijk M,
    2. Mullen B,
    3. Nicholson AM, et al
    . TMEM106B protects C9ORF72 expansion carriers against frontotemporal dementia. Acta Neuropathol 2014;127:397–406.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. van Blitterswijk M,
    2. DeJesus-Hernandez M,
    3. Niemantsverdriet E, et al
    . Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study. Lancet Neurol 2013;12:978–988.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Xi Z,
    2. van Blitterswijk M,
    3. Zhang M, et al
    . Jump from pre-mutation to pathologic expansion in C9orf72. Am J Hum Genet 2015;96:962–970.
    OpenUrl
  12. 12.↵
    1. Gami P,
    2. Murray C,
    3. Schottlaender L, et al
    . A 30-unit hexanucleotide repeat expansion in C9orf72 induces pathological lesions with dipeptide-repeat proteins and RNA foci, but not TDP-43 inclusions and clinical disease. Acta Neuropathol 2015;130:599–601.
    OpenUrl
  13. 13.↵
    1. Xi Z,
    2. Zinman L,
    3. Grinberg Y, et al
    . Investigation of c9orf72 in 4 neurodegenerative disorders. Arch Neurol 2012;69:1583–1590.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Xi Z,
    2. Zhang M,
    3. Bruni AC, et al
    . The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients. Acta Neuropathol 2015;129:715–727.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Xi Z,
    2. Rainero I,
    3. Rubino E, et al
    . Hypermethylation of the CpG-island near the C9orf72 G(4)C(2)-repeat expansion in FTLD patients. Hum Mol Genet 2014;23:5630–5637.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Xi Z,
    2. Zinman L,
    3. Moreno D, et al
    . Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion. Am J Hum Genet 2013;92:981–989.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Fratta P,
    2. Polke JM,
    3. Newcombe J, et al
    . Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion. Neurobiol Aging 2015;36:e541–e547.
    OpenUrl
  18. 18.↵
    1. Nordin A,
    2. Akimoto C,
    3. Wuolikainen A, et al
    . Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD. Hum Mol Genet 2015;24:3133–3142.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Zhang M,
    2. Tartaglia MC,
    3. Moreno D, et al
    . DNA methylation age-acceleration is associated with disease duration and age at onset in C9orf72 patients. Acta Neuropathol 2017;134:271–279.
    OpenUrl
  20. 20.↵
    1. Xiao S,
    2. MacNair L,
    3. McGoldrick P, et al
    . Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis. Ann Neurol 2015;78:568–583.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. van Blitterswijk M,
    2. van Es MA,
    3. Hennekam EA, et al
    . Evidence for an oligogenic basis of amyotrophic lateral sclerosis. Hum Mol Genet 2012;21:3776–3784.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Zhang M,
    2. Xi Z,
    3. Misquitta K, et al
    . C9orf72 and ATXN2 repeat expansions coexist in a family with ataxia, dementia, and parkinsonism. Mov Disord 2017;32:158–162.
    OpenUrl
  23. 23.↵
    1. Mackenzie IR,
    2. Frick P,
    3. Grasser FA, et al
    . Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol 2015;130:845–861.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Gijselinck I,
    2. Van Mossevelde S,
    3. van der Zee J, et al
    . The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychiatry 2016;21:1112–1124.
    OpenUrl
  25. 25.↵
    1. DeJesus-Hernandez M,
    2. Finch NA,
    3. Wang X, et al
    . In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers. Acta Neuropathol 2017;134:255–269.
    OpenUrl
  26. 26.↵
    1. Davidson Y,
    2. Robinson AC,
    3. Liu X, et al
    . Neurodegeneration in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9orf72 is linked to TDP-43 pathology and not associated with aggregated forms of dipeptide repeat proteins. Neuropathol Appl Neurobiol 2016;42:242–254.
    OpenUrl
  27. 27.↵
    1. Gendron TF,
    2. van Blitterswijk M,
    3. Bieniek KF, et al
    . Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol 2015;130:559–573.
    OpenUrlCrossRefPubMed
  28. 28.↵
    1. Therrien M,
    2. Rouleau GA,
    3. Dion PA,
    4. Parker JA
    . Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS One 2013;8:e83450.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Ciura S,
    2. Lattante S,
    3. Le Ber I, et al
    . Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann Neurol 2013;74:180–187.
    OpenUrlPubMed
  30. 30.↵
    1. O'Rourke JG,
    2. Bogdanik L,
    3. Yanez A, et al
    . C9orf72 is required for proper macrophage and microglial function in mice. Science 2016;351:1324–1329.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Liu F,
    2. Liu Q,
    3. Lu CX, et al
    . Identification of a novel loss-of-function C9orf72 splice site mutation in a patient with amyotrophic lateral sclerosis. Neurobiol Aging 2016;47:219.e211–219.e215.
    OpenUrl
  32. 32.↵
    1. Tassone F,
    2. Hagerman RJ,
    3. Taylor AK,
    4. Gane LW,
    5. Godfrey TE,
    6. Hagerman PJ
    . Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet 2000;66:6–15.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Amick J,
    2. Roczniak-Ferguson A,
    3. Ferguson SM
    . C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling. Mol Biol Cell 2016;27:3040–3051.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Maharjan N,
    2. Kunzli C,
    3. Buthey K,
    4. Saxena S
    . C9ORF72 regulates stress granule formation and its deficiency impairs stress granule assembly, hypersensitizing cells to stress. Mol Neurobiol 2017;54:3062–3077.
    OpenUrl
  35. 35.↵
    1. Sellier C,
    2. Campanari ML,
    3. Julie Corbier C, et al
    . Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death. EMBO J 2016;35:1276–1297.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Sivadasan R,
    2. Hornburg D,
    3. Drepper C, et al
    . C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons. Nat Neurosci 2016;19:1610–1618.
    OpenUrlCrossRef
  37. 37.↵
    1. Webster CP,
    2. Smith EF,
    3. Bauer CS, et al
    . The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. EMBO J 2016;35:1656–1676.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Yang M,
    2. Liang C,
    3. Swaminathan K, et al
    . A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy. Sci Adv 2016;2:e1601167.
    OpenUrlFREE Full Text
  39. 39.↵
    1. van Blitterswijk M,
    2. Gendron TF,
    3. Baker MC, et al
    . Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72. Acta Neuropathol 2015;130:863–876.
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Bennion Callister J,
    2. Ryan S,
    3. Sim J,
    4. Rollinson S,
    5. Pickering-Brown SM
    . Modelling C9orf72 dipeptide repeat proteins of a physiologically relevant size. Hum Mol Genet 2016;25:5069–5082.
    OpenUrl

Letters: Rapid online correspondence

No comments have been published for this article.
Comment

REQUIREMENTS

You must ensure that your Disclosures have been updated within the previous six months. Please go to our Submission Site to add or update your Disclosure information.

Your co-authors must send a completed Publishing Agreement Form to Neurology Staff (not necessary for the lead/corresponding author as the form below will suffice) before you upload your comment.

If you are responding to a comment that was written about an article you originally authored:
You (and co-authors) do not need to fill out forms or check disclosures as author forms are still valid
and apply to letter.

Submission specifications:

  • Submissions must be < 200 words with < 5 references. Reference 1 must be the article on which you are commenting.
  • Submissions should not have more than 5 authors. (Exception: original author replies can include all original authors of the article)
  • Submit only on articles published within 6 months of issue date.
  • Do not be redundant. Read any comments already posted on the article prior to submission.
  • Submitted comments are subject to editing and editor review prior to posting.

More guidelines and information on Disputes & Debates

Compose Comment

More information about text formats

Plain text

  • No HTML tags allowed.
  • Web page addresses and e-mail addresses turn into links automatically.
  • Lines and paragraphs break automatically.
Author Information
NOTE: The first author must also be the corresponding author of the comment.
First or given name, e.g. 'Peter'.
Your last, or family, name, e.g. 'MacMoody'.
Your email address, e.g. higgs-boson@gmail.com
Your role and/or occupation, e.g. 'Orthopedic Surgeon'.
Your organization or institution (if applicable), e.g. 'Royal Free Hospital'.
Publishing Agreement
NOTE: All authors, besides the first/corresponding author, must complete a separate Publishing Agreement Form and provide via email to the editorial office before comments can be posted.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.

Vertical Tabs

You May Also be Interested in

Back to top
  • Article
    • Abstract
    • Glossary
    • Methods
    • Results
    • Discussion
    • Author contributions
    • Study funding
    • Disclosure
    • Acknowledgment
    • Footnotes
    • References
  • Figures & Data
  • Info & Disclosures
Advertisement

Brain Metabolism Related to Mild Cognitive Impairment and Phenoconversion in Patients With Isolated REM Sleep Behavior Disorder

Dr. David E. Vaillancourt and Dr. Shannon Y. Chiu

► Watch

Related Articles

  • No related articles found.

Topics Discussed

  • All Genetics
  • Amyotrophic lateral sclerosis

Alert Me

  • Alert me when eletters are published

Recommended articles

  • Article
    Multiparametric MRI study of ALS stratified for the C9orf72 genotype
    Peter Bede, Arun L.W. Bokde, Susan Byrne et al.
    Neurology, June 14, 2013
  • Views & Reviews
    C9orf72 and the Care of the Patient With ALS or FTD
    Progress and Recommendations After 10 Years
    Jennifer Roggenbuck et al.
    Neurology: Genetics, December 21, 2020
  • Article
    C9orf72 promoter hypermethylation is neuroprotective
    Neuroimaging and neuropathologic evidence
    Corey T. McMillan, Jenny Russ, Elisabeth M. Wood et al.
    Neurology, March 20, 2015
  • Articles
    Hexanucleotide repeat expansions in C9ORF72 in the spectrum of motor neuron diseases
    Wouter van Rheenen, Marka van Blitterswijk, Mark H.B. Huisman et al.
    Neurology, July 25, 2012
Neurology: 100 (22)

Articles

  • Ahead of Print
  • Current Issue
  • Past Issues
  • Popular Articles
  • Translations

About

  • About the Journals
  • Ethics Policies
  • Editors & Editorial Board
  • Contact Us
  • Advertise

Submit

  • Author Center
  • Submit a Manuscript
  • Information for Reviewers
  • AAN Guidelines
  • Permissions

Subscribers

  • Subscribe
  • Activate a Subscription
  • Sign up for eAlerts
  • RSS Feed
Site Logo
  • Visit neurology Template on Facebook
  • Follow neurology Template on Twitter
  • Visit Neurology on YouTube
  • Neurology
  • Neurology: Clinical Practice
  • Neurology: Education
  • Neurology: Genetics
  • Neurology: Neuroimmunology & Neuroinflammation
  • AAN.com
  • AANnews
  • Continuum
  • Brain & Life
  • Neurology Today

Wolters Kluwer Logo

Neurology | Print ISSN:0028-3878
Online ISSN:1526-632X

© 2023 American Academy of Neurology

  • Privacy Policy
  • Feedback
  • Advertise