Article Text


Short report
Japanese amyotrophic lateral sclerosis patients with GGGGCC hexanucleotide repeat expansion in C9ORF72
  1. Takuya Konno1,
  2. Atsushi Shiga2,
  3. Akira Tsujino3,
  4. Akihiro Sugai1,
  5. Taisuke Kato1,
  6. Kazuaki Kanai4,
  7. Akio Yokoseki1,
  8. Hiroto Eguchi3,
  9. Satoshi Kuwabara4,
  10. Masatoyo Nishizawa1,
  11. Hitoshi Takahashi2,
  12. Osamu Onodera5
  1. 1Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
  2. 2Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan
  3. 3First Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Science, Nagasaki, Japan
  4. 4Department of Neurology, Chiba University School of Medicine, Chiba, Japan
  5. 5Department of Molecular Neuroscience, Brain Research Institute, Niigata University, Niigata, Japan
  1. Correspondence to Dr Osamu Onodera, Department of Molecular Neuroscience, Brain Research Institute, Niigata University, 1-757 Asahimachi-dori, Chuo-Ku, Niigata-City, Niigata 951-8585, Japan; onodera{at}


Background A GGGGCC hexanucleotide repeat expansion in C9ORF72 occurs on a chromosome 9p21 locus that is linked with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in white populations. The diseases resulting from this expansion are referred to as ‘c9FTD/ALS’. It has been suggested that c9FTD/ALS arose from a single founder. However, the existence of c9FTD/ALS in non-white populations has not been evaluated.

Results We found two index familial ALS (FALS) patients with c9FTD/ALS in the Japanese population. The frequency of c9FTD/ALS was 3.4% (2/58 cases) in FALS. No patients with sporadic ALS (n=110) or control individuals (n=180) had the expansion. Neuropathological findings of an autopsy case were indistinguishable from those of white patients. Although the frequency of risk alleles identified in white subjects is low in Japanese, one patient had all 20 risk alleles and the other had all but one. The estimated haplotype indicated that the repeat expansion in these patients was located on the chromosome with the risk haplotype identified in white subjects.

Conclusions C9ORF72 repeat expansions were present in a Japanese cohort of ALS patients, but they were rare. Intriguingly, Japanese patients appear to carry the same risk haplotype identified in white populations.

  • Amyotrophic lateral sclerosis
  • DNA repeat expansion
  • chromosome 9
  • C9ORF72
  • haplotypes

Statistics from


A chromosome 9p21 locus has been linked with both familial and sporadic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in white populations.1–4 It has been identified as a risk haplotype that is represented by a 140 kb block of linkage disequilibrium on chromosome 9p, and rs3849942 allele A is the most commonly associated single nucleotide polymorphism (SNP).3 ,5 Recently, a GGGGCC hexanucleotide repeat expansion in the non-coding region of C9ORF72 was identified in this locus.6 ,7 In normal individuals the number of repeats is no more than 23.6 The number was increased to more than 700 in four patients based on a Southern blot analysis,6 but the actual threshold and penetrance of expanded repeats of different sizes are still unclear. The diseases resulting from the expansion are now referred to as ‘c9FTD/ALS’.6 The existence of RNA foci in affected cells and a decreased amount of C9ORF72 mRNA in patients with c9FTD/ALS indicate that the expansion is the causative mutation for c9FTD/ALS.6–8

In white populations, c9FTD/ALS is the most frequent cause of familial ALS (FALS) and sporadic ALS (SALS), accounting for 23.5%–47% of FALS incidence and 4.1%–21.0% of SALS incidence.6–8 Haplotype analysis of affected individuals indicates that c9FTD/ALS arose from a single founder mutation from a common Scandinavian ancestor.3 ,5 DeJesus-Hernandez et al found that all patients with c9FTD/ALS had rs3849942 allele A, and significantly longer repeats were observed for the haplotype with rs3849942 allele A compared with the haplotype without allele A in control subjects, suggesting that de novo expansion may occur on the risk haplotype.6 However, it is still controversial whether the expansion occurs on any haplotype or only on the risk haplotype.7 Investigation of the repeat expansion on different genetic backgrounds may help solve this issue. Here we report two Japanese patients with c9FTD/ALS who appeared to have the same risk haplotype identified in white subjects.

Material and methods

Mutation screening

Repeat-primed PCR and genotyping PCR for GGGGCC repeats in C9ORF72 were performed in genomic DNA from 110 patients with SALS, 58 with FALS (unrelated to each other and having at least one first-degree relative diagnosed with ALS), 180 Japanese control subjects and two white patients with c9FTD/ALS as positive control (Coriell Cell Repositories, Camden, New Jersey, USA).6 ,7 Fluorescence fragment length analysis of PCR fragments was performed on an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, California, USA) and with Peak Scanner software v1.0 (Applied Biosystems). This study was approved by the Institutional Review Board of Niigata University, and written informed consent was obtained from all patients or their relatives.

SNP genotyping and haplotype estimation

The risk haplotype consists of 20 SNPs ranged from rs1822723 (centromeric) to rs2477518 (telomeric).5 The rs3849942 SNP was genotyped using TaqMan SNP genotyping assays (Applied Biosystems).6 The other SNPs were analysed by a direct sequencing method. Haplotypes consisting of these SNPs were estimated in 30 Japanese controls using SNPAlyze V.8 (DYNACOM, Chiba, Japan).

Histopathological analysis

For histological analysis, we prepared 4 μm thick, formalin-fixed, paraffin-embedded sections of brain and spinal cord tissue. For immunostaining, we used anti-TDP-43 antibody (ProteinTech Group, Chicago, Illinois, USA), anti-phosphorylated TDP-43 (pTDP-43) antibody (S409/410) (Cosmo bio, Tokyo, Japan) and anti-p62 antibody (BD Transduction Laboratories, San Diego, California, USA) as a primary antibody. Fluorescent images were acquired with an LSM 710 NLO laser-scanning confocal microscope (Zeiss, Jena, Germany).


Genetic studies

We found two index patients with FALS (3.4%) who had the GGGGCC repeat expansion in C9ORF72 (figure 1A, table 1). Neither patients with SALS nor controls had the repeat expansion. We then investigated whether the patients with FALS shared the risk haplotype for c9FTD/ALS identified in white populations. Although we were unable to obtain DNA samples from their relatives, both patients had the same risk alleles associated with c9FTD/ALS in white subjects; one had all 20 SNPs and the other had all but one, including very rare alleles in Japanese (figure 1B). Based on haplotypes from rs1822723 to rs2477518 in Japanese controls, we estimated that each patient had the risk haplotype (figure 1B). In addition, sequence analyses for amplicons spanning the hexanucleotide repeat to rs1982915 revealed that the normal repeat and the rs1982915 A allele were located on the same chromosome, suggesting that their expanded repeats were located on the risk haplotype (figure 1C). To investigate whether the risk allele was associated with repeat length, we examined the number of repeats in Japanese controls and ALS subjects. Although there were no significant differences in the average number of repeats between groups, the individuals carrying at least one rs3849942 allele A had a significantly longer repeat than those without allele A (table 1).

Figure 1

(A) Electropherograms show capillary-based sequence traces of the repeat-primed PCR. The typical sawtooth patterns are observed in the cases carrying the GGGGCC repeat expansion and a positive control.7 The inset shows fluorescent fragment length analyses of a genotyping PCR fragment containing the GGGGCC repeat in C9ORF72. The repeat expansion carriers show one peak due to the presence of an unamplifiable repeat expansion. Numbers under the peaks indicate the number of GGGGCC repeats. (B) Schematic representation of the C9ORF72 gene and surrounding associated 20 single nucleotide polymorphisms (SNPs).5 Japanese linkage disequilibrium blocks (indicated by black bars) and risk allele frequencies (RAF) of each SNP in Japanese are presented based on HapMap-JPT ( Genotypes of Japanese c9FTD/ALS cases are shown. We estimated the haplotype consisting of these SNPs from control using SNPAlyze V.8 (DYNACOM, Chiba, Japan), and then we estimated the haplotype for each case. Case 1 has the risk haplotype (shown in red) and the most frequent non-risk haplotype (shown in black). Case 2 also has the risk haplotype except rs2814707 (shown in red) and the non-risk haplotype (shown in black). (C) Direct sequence analysis for amplicons spanning the hexanucleotide repeat to rs1982915 from case 1. Approximately 7 kbp amplicons spanning from the hexanucleotide repeat to rs1982915 are shown in the left panel (primer sequences and condition are available on request). Chromatograms for the amplicons are shown in the right panel. Because the PCR cannot amplify the expanded repeat, the amplicons only have the normal repeat. Although both cases have G/A allele for rs1982915 in genotyping, the sequence analysis of the amplicons shows that the products have only the A allele for rs1982915. The same results were obtained for case 2. (D) The pedigree of the cases carrying the repeat expansion. These families had no history of dementia or psychosis. The parents were healthy and without dementia for more than 80 years in case 1 and for more than 65 years in case 2. Arrow, the probands; squares, men; circles, women; solid symbols, affected family members. Sex of the unaffected siblings is obscured to protect privacy. (E) In the thoracic cord, myelin pallor is evident in the anterior and lateral corticospinal tracts (Klüver–Barrera stain). (F) A Bunina body (arrow) is evident in a facial nucleus motor neuron (H&E stain). (G) TDP-43-positive neuronal cytoplasmic inclusions (NCIs) are evident in a cervical anterior horn cell (TDP-43 immunostaining). (H) Double-labelling immunofluorescence staining in the neurons of the cervical anterior horn. In contrast to the colocalisation of TDP-43 and pTDP-43 in sporadic ALS (SALS) without the repeat expansion, case 1 shows that TDP-43-positive skein-like inclusions are negative for pTDP-43. (I–K) Scattered p62-positive NCIs are evident in the cerebellar granule cells (I), hippocampal CA3 neurons (J) and motor cortex (K) (p62 immunostaining). Scale bars, 20 μm. ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia.

Table 1

The frequency of c9FTD/ALS and average number of repeats in Japanese population

Case reports

The patients were diagnosed with ALS without dementia. Both patients had a sibling who had also been diagnosed with ALS but their parents had been healthy (figure 1D). There was no family history of dementia such as FTD or other neurological diseases in either family. Case 1 (case 4 in a previous report9) noticed hand clumsiness at age 61 years, and progressively developed dysarthria, dysphagia and limb weakness. He died 20 months after onset due to respiratory failure. Case 2 noticed dysarthria at age 63 and gradually developed weakness and muscle atrophy in her tongue and extremities. MRI revealed mild diffuse brain atrophy but the mini-mental state examination score was 29/30. She died 35 months after onset.

Histopathological studies

The brain of the case 1 patient weighed 1340 g and had no apparent abnormalities in its external appearance. Histopathological examination showed degeneration in the spinal anterolateral columns (figure 1E) as well as evidence of neuronal loss and gliosis in the spinal anterior horn, brainstem lower motor neuron nuclei (V, VII and XII) and precentral cortex. Bunina bodies were a feature in the affected lower motor neurons (figure 1F). TDP-43-immunopositive neuronal cytoplasmic inclusions (NCIs) were observed in the brainstem lower motor neuron nuclei, spinal anterior horn (figure 1G) and precentral cortex. In addition, TDP-43-immunopositive oligodendroglial cytoplasmic inclusions (GCIs) were observed in the pyramidal tract. However, pTDP-43-immunopositive NCIs and GCIs were rare. Double-labelling immunofluorescence in the neurons of the cervical anterior horn revealed that TDP-43-immunopositive skein-like inclusions were pTDP-43-immunonegative (figure 1H).

p62-Immunopositive and TDP-43-immunonegative inclusions in the cerebellum and hippocampus have been reported as pathological hallmarks of c9FTD/ALS.10 In our case, we found p62-immunoreactive NCIs in the cerebellar granule cells (figure 1I) and in the granule cells and pyramidal CA4-CA2 neurons of the hippocampus (figure 1J), which were not recognised by either TDP-43 or pTDP-43 antibodies. p62-Immunopositive inclusions were also observed in the spinal anterior horn and motor cortex (figure 1K), which were recognised by a TDP-43 antibody, but not by a pTDP-43 antibody.


Based on our study cohort, the frequency of c9FTD/ALS was 3.4% in Japanese FALS and non-existent in SALS, which is extremely lower than in white subjects. Clinical records suggest that the penetrance of this expansion is incomplete.11 Both cases had the risk alleles for c9FTD/ALS from rs1822723 to rs2477518 found in white subjects, including rare alleles in Japanese, and were estimated to have the risk haplotype described in white subjects, indicating that the expansion is closely tied to the risk haplotype.5 The low frequency of c9FTD/ALS in the Japanese could be explained by the low frequency of the risk haplotype. However, it remains unclear how the haplotype might have entered Japan from Europe. Moreover, Japanese controls and ALS subjects with rs3849942 allele A also showed significantly longer repeats than the subjects without allele A, similar to white populations.6 The result supports the hypothesis that the repeat expansion may occur on the risk haplotype.6

Neuropathological ALS findings for c9FTD/ALS are very similar to those of classical SALS; degeneration is limited to the motor neuron systems, and Bunina bodies and TDP-43-immunopositive NCIs exist.10 ,12 These features were also evident in our autopsy case. However, in this case, most of the TDP-43-immunopositive NCIs were pTDP-43-immunonegative. The decreased immunoreactivity for pTDP-43 has not been reported in c9FTD/ALS.10 ,12 Further study is necessary to clarify the significance of this finding.

Characteristic features for c9FTD/ALS in white subjects are p62-positive and pTDP-43-negative NCIs in the cerebellar cortex (including Purkinje cells) and hippocampal pyramidal layer and neuronal intranuclear inclusions in the granular cells of the cerebellum and pyramidal cells of the hippocampus.10 We observed such NCIs, mainly in the cerebellar granule cells and hippocampal CA4-CA2 pyramidal neurons, but did not find neuronal intranuclear inclusions. p62 Interacts with polyubiquitinated proteins and directs them to either the ubiquitin–proteasome system or autophagosomes, indicating that the p62-immunopositive inclusions should contain some disease-associated protein.10 ,13 The component of p62-immunopositive inclusions should be clarified in future studies.

In conclusion, based on our study cohort, c9FTD/ALS appears to be rare in the Japanese population. The clinical and neuropathological findings from Japanese patients with c9FTD/ALS were indistinguishable from those of white patients with c9FTD/ALS. Despite the risk alleles for c9FTD/ALS being very rare in the Japanese population, both of our patients had the same risk haplotype identified in white subjects, indicating that the pathogenic expansion is closely tied to the risk haplotype.


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  • Funding Supported by Grant-in-Aid for Scientific Research (A) from Japan Society for the Promotion of Science, Grant-in-Aid for the Research Committee of CNS Degenerative Diseases from Ministry of Health, Labor and Welfare, Japan and Grant-in-Aid for the Nakabayashi Trust for ALS Research and a Grant-in-Aid for JSPS Fellows from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the Niigata University Institutional Review Board.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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