Neurobiology of Aging 35 (2014) 936.e13e936.e17

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C9orf72 repeat expansions are restricted to the ALS-FTD spectrum Nicola Ticozzi a, b, *, Cinzia Tiloca a, c, Daniela Calini a, Stella Gagliardi d, Alessandra Altieri a, Claudia Colombrita a, b, Cristina Cereda d, Antonia Ratti a, b, Gianni Pezzoli e, Barbara Borroni f, Stefano Goldwurm e, Alessandro Padovani f, Vincenzo Silani a, b a

Department of Neurology and Laboratory of Neuroscience, Istituto di Ricovero e Cura a Carattere Scientifico Istituto Auxologico Italiano, Milan, Italy Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy c Doctoral School in Molecular Medicine, Department of Sciences and Biomedical Technologies, University of Milan, Milan, Italy d Laboratory of Experimental Neurology, Istituto di Ricovero e Cura a Carattere Scientifico C. Mondino National Neurological Institute, Pavia, Italy e Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy f Centre for Neurodegenerative Disorders, University of Brescia, Brescia, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2013 Received in revised form 26 September 2013 Accepted 27 September 2013 Available online 2 October 2013

Expansion of a GGGGCC repeat (RE) in the C9orf72 gene has been recently reported as the main genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the growing evidence of genetic and clinicopathologic overlap among ALS, FTD, and other neurodegenerative diseases, we investigated the occurrence of RE in a subset of 9 patients with ALS-plus syndromes, including Parkinson’s disease (PD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and multiple system atrophy. We identified RE in 2 ALS-plus individuals (22.2%) displaying PSP and CBS features. On the basis of this finding, we extended our analysis to a cohort composed of 190 PD, 103 CBS, 107 PSP, and 177 Alzheimer’s disease cases. We did not identify any RE in these patients, indicating that C9orf72 is in all probability not involved in the pathogenesis of these disorders. However, the high frequency of C9orf72 RE in patients with ALS-plus syndromes suggests that, similar to ALS-FTD patients, individuals with combined motor neuron and extrapyramidal features should be screened for RE, independent of their family history. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Amyotrophic lateral sclerosis Frontotemporal lobar degeneration C9orf72 Progressive supranuclear palsy Corticobasal syndrome Neurogenetics

1. Introduction The recent discovery of the GGGGCC hexanucleotide repeat expansion (RE) in C9orf72 gene as the main cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represents a major landmark in the genetics of these overlapping neurodegenerative diseases (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2011; Renton et al., 2011). The repeat is highly polymorphic in the normal population (2e23 units) but is expanded both in ALS and FTD patients up to 4400 units (Beck et al., 2013). The reported mutational frequency for C9orf72 gene in different populations is 23%e50% in familial ALS cases, with a possible north-south descending gradient, and 4%e8% in individuals with sporadic ALS (Boeve et al., 2012; Byrne et al., 2012; Cooper-Knock et al., 2012; Majounie et al., 2012c; Ratti et al., 2012). Similar mutational frequencies have been described in * Corresponding author at: Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, P.le Brescia 20, Milan 20149, Italy. Tel./ fax: þ39 02 61911 2937. E-mail address: [email protected] (N. Ticozzi). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.09.037

patients with familial (15%e55%) or sporadic (2%e6%) FTD (Hsiung et al., 2012; Simon-Sanchez et al., 2012; Snowden et al., 2012). Individuals with concurrent ALS and FTD or with a family history of dementia or motor neuron disease have a higher risk of harboring C9orf72 RE (33%e86%), further indicating that the two diseases belong to the same pathogenic continuum (Byrne et al., 2012). However, clinical heterogeneous features have been observed in patients and families carrying C9orf72 RE, and the presence of additional atypical features, such as signs of parkinsonism and psychotic phenomena, have been reported (Boeve et al., 2012). RE carriers also have a higher incidence of Parkinson’s (PD) and Alzheimer’s (AD) diseases among relatives (Cooper-Knock et al., 2012; Hsiung et al., 2012; Ratti et al., 2012), and an individual with C9orf72 RE was reported to be affected with corticobasal syndrome (CBS) (Lindquist et al., 2012). All these observations suggest that C9orf72 RE may contribute to the pathogenesis of a broad spectrum of neurodegenerative diseases other than TDP-43 proteinopathies. This study aims to screen C9orf72 gene in a cohort of patients with ALS-plus syndromes, and with CBS, clinically diagnosed tauopathies (progressive supranuclear palsy, PSP), alphasynucleinopathies (PD), and amyloidopathies (AD) to further test

      e þ þ       þ   þ þ þ þ þ      þ/ þ þ  þ þ þ/ þ þ þ þ þ þ þ þ þ þ þ þ þ e þ þ þ þ e Spinal Spinal Bulbar Bulbar Spinal Spinal Bulbar Spinal Spinal

þ þ þ þ þ þ þ þ/ þ

  þ    þ þ þ

 þ   þ    

Response to levodopa Postural instability with falls Bradykinesia Tremor LMN signs UMN signs

M F M F M F M F M 1 2 3 4 5 6 7 8 9

Site of onset

Motor neuron dysfunction Age at onset Sex Case

Table 1 Clinical features of patients with ALS-plus syndromes

Dementia

Extrapyramidal features

Genomic DNA was isolated from peripheral blood according to standard protocols. The GGGGCC hexanucleotide repeat in C9orf72 was analyzed by a 2-step protocol, including a first polymerase chain reaction (PCR) amplification using the genotyping primers previously reported (DeJesus-Hernandez et al., 2011). Only those samples presenting with a single amplification product were further analyzed in the second step by the repeat-primed PCR method on a 3500 ABI Prism Genetic Analyzer (Applied Biosystems) using primer pairs that were previously described (DeJesusHernandez et al., 2011). The presence of C9orf72 RE was assigned when the sample displayed a typical electropherogram profile with decaying stutter amplification peaks with a 6 base pair periodicity that exceeded the upper detection limit of the assay (40 hexanucleotide units) as recently reported (Ratti et al., 2012).

To validate the association of C9orf72 RE with atypical ALS phenotypes, we screened a cohort composed of 9 individuals with ALS-plus syndromes (5 ALS-PD, 2 ALS-CBS, 1 ALS-PSP, 1 ALS-MSA). Among this group, we identified RE in 2 of 9 (22.2%) individuals, of which 1 had PSP and 1 CBS features. Because of the technical limitations of the molecular approach used for C9orf72 RE analysis, we could not determine the exact number of repeat units in the expanded alleles in the 2 ALS-plus cases. The ALS-plus patient with PSP features (case 7, Table 1), an otherwise healthy 62-year-old man, developed a slowly progressive impairment of verbal fluency, associated with apathy, depression, and social withdrawal. The symptoms were initially attributed to major depressive disorder, and the patient was started on

     þ   

Autonomic dysfunction Alien limb syndrome Vertical gaze palsy

2.2. Genetic analysis

3. Results

     þ/   

Cerebellar signs

The 586 patients included in the study were recruited from 4 clinical centers experienced in the diagnosis and care of neurodegenerative diseases. The cohort consisted of 9 patients with ALSplus syndromes, displaying prominent extrapyramidal features (5 ALS-PD, 2 ALS-CBS, 1 ALS-PSP, 1 ALS-multiple system atrophy; Table 1), 103 with CBS, 107 with PSP, 190 with PD, and 177 with AD. The diagnosis was made according to the El Escorial revised criteria for ALS (Miller et al., 1999), the UK Brain Bank criteria for PD (Hughes et al., 1992), the recently proposed criteria for CBS (Armstrong et al., 2013), the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association guidelines for AD (McKhann et al., 1984), and National Institute of Neurological Disorders and the Society for Progressive Supranuclear Palsy (Litvan et al., 1996) guidelines for PSP. According to the El Escorial classification, ALS with 1 additional features (such as dementia, extrapyramidal signs, objective sensory loss, autonomic dysfunction, cerebellar degeneration) is defined as ALS-plus syndrome (Miller et al., 1999). Autoptic material for neuropathologic confirmation of the clinical diagnosis was not available for any of the analyzed cases in all cohorts. Approval for this study was requested and granted by the local ethics committees on human experimentation of each clinical center, and written informed consent was obtained from all patients participating to the study.

68 75 81 55 80 61 62 63 49

2.1. Patients

ALS-PD ALS-PD ALS-PD PLS-PD ALS-PD ALS-MSA ALS-PSP ALS-CBS PLS-CBS

Diagnosis

2. Methods

      þ  þ

C9orf72

the association of RE with atypical ALS phenotypes and to assess RE frequency in other neurodegenerative diseases.

936.e14 Key: ALS, amyotrophic lateral sclerosis; ALS-plus, ALS with 1 additional features (e.g., dementia, extrapyramidal signs, objective sensory loss, autonomic dysfunction, cerebellar degeneration); CBS, corticobasal syndrome; F, female; LMN, lower motor neuron; M, male; MSA, multiple system atrophy; PD, Parkinson’s disease; PLS, primary lateral sclerosis; PSP, progressive supranuclear palsy; UMN, upper motor neuron.

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antidepressant therapy with no benefit. Four years later, he developed a supranuclear vertical gaze palsy, axial rigidity, and postural instability with frequent falls. A brain magnetic resonance imaging scan performed at this time was unremarkable, showing only mild diffuse cortical atrophy. The patient was diagnosed with PSP and started on levodopa treatment with no significant improvement. During the next 2 years, he began complaining of dysarthria and dysphagia and developed a symmetrical, progressive atrophy and weakness of the distal muscles of the upper limbs. Needle electromyography showed active denervation signs in the muscles of the 4 limbs, compatible with a concurrent lower motor neuron disorder. The patient died at age 73 of respiratory insufficiency. He had a positive familial history for neurodegenerative diseases, having a sister died of ALS and dementia, and his 2 children were affected with spinal and bulbar onset ALS, respectively (eFig. 1). The ALS-plus patient with CBS features (case 9, Table 1), a man with no family history for ALS or other neurodegenerative disorders, developed spastic rigidity of the left lower limb at age 49, subsequently spreading to the other limbs (in details, to the right lower limb, left upper limb, right upper limb). In the following months, he developed dysarthria, dysphagia, and emotional lability. A neurologic examination carried out at this time showed a spastic tetraparesis with clonic deep tendon reflexes and presence of Hoffmann’s and Babinski signs bilaterally. Brain magnetic resonance imaging and needle electromyography were unremarkable. On the basis of these findings, this patient was diagnosed with primary lateral sclerosis (PLS). Two years after the onset of first symptoms, however, he began complaining of a marked worsening of the rigidity of the left arm, with failure to control the movements of the hand and the feeling that “the limb didn’t belong to him anymore,” suggesting an alien limb syndrome. A second neurologic examination revealed the presence of anosodiaphoria, partial anosognosia, ideomotor apraxia,

and of plastic rigidity with cogwheel phenomenon of the left upper limb superimposed to the preexistant spastic tetraparesis. A neuropsychological evaluation was suggestive of a cognitive impairment of frontal type, and a brain positron emission imaging scan with 18F-fludeoxyglucose revealed a diffuse frontoparietal hypometabolism. The diagnosis was further defined to PLS with suggestive CBS features, and the patient was started on levodopa and ropinirole therapy with no benefit. He died 5 years after symptom onset of aspiration pneumonia. On the basis of these findings, we decided to extend our screening to patients with possible tauopathies (103 individuals with CBS and 107 with PSP), alpha-synucleinopathies (190 PD cases), and amyloidopathies (177 AD cases), to assess the role of C9orf72 RE in the pathogenesis of neurodegenerative diseases other than ALS and FTD. In all these subgroups, however, we did not identify any RE in the C9orf72 gene. Moreover, in non-expanded samples, we did not observe any difference in allele distribution between the different subgroups (Fig. 1) or with an ethnically matched control population we recently described (Ratti et al., 2012). A single CBS individual carrying an intermediate allele (23 repeats) was observed. 4. Discussion In our mutational screening, we found that C9orf72 RE are present in >20% of patients with ALS-plus syndromes, thus broadening the phenotypic spectrum also to ALS patients with suggestive PSP and CBS features. The simultaneous presence of a motor neuron disease and extrapyramidal features in the same individual, although reported in the literature, is a rare occurrence. Most patients with ALS and parkinsonism have an upper and lower motor neuron disease; a minority has PLS. In almost all cases, the

Fig. 1. Distribution of the C9orf72 alleles within the 4 studied cohorts. Histograms showing the frequency distribution of GGGGCC hexanucleotide repeat lengths in each disease group: progressive supranuclear palsy (PSP, n ¼ 107), corticobasal syndrome (CBS, n ¼ 103), Parkinson’s disease (PD, n ¼ 190), Alzheimer’s disease (AD, n ¼ 177). Repeat size was measured using fragment-length analysis and repeat-primed polymerase chain reaction. Mann-Whitney test was applied to compare differences between groups (p > .05).

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extrapyramidal features include the classic triad of bradykinesia, rigidity, and postural instability, and response to levodopa treatment and/or dementia is present in approximately half of patients (Gilbert et al., 2010). However, we did not find RE in a large cohort of patients with CBS, PSP, AD, and PD, indicating that C9orf72 is not a main genetic cause in neurodegenerative disorders other than ALS and FTD. The presence of TDP-43-immunoreactive inclusions in the cytoplasm of surviving neurons is the main neuropathologic hallmark of TDP-43 proteinopathies such as ALS and frontotemporal lobar degeneration with tau-negative and ubiquitin-positive inclusions (FTLD-U) (Neumann et al., 2006). Interestingly, in ALS and FTLD-U autoptic tissues, TDP-43 pathology has been observed not only in motor neurons and in the frontal cortex but also in the nigrostriatal system, in hippocampal areas, and in the cerebellum, suggesting a multisystem involvement of the central nervous system (Brettschneider et al., 2013; McKee et al., 2010). In fact, a severe TDP-43 pathology in the basal ganglia, even in the absence of overt extrapyramidal symptoms, has recently been described as common in late-stage ALS cases (Brettschneider et al., 2013). Although neuropathologic information was not available for our cohorts, autoptic studies also estimated that as many as 30% of AD and 20% of PD cases show evidence of TDP-43 immunoreactivity in the central nervous system (Amador-Ortiz et al., 2007; NakashimaYasuda et al., 2007). Another important observation is that several C9orf72 RE carriers have an associated AD-type pathology with a significant load of tau-positive neurofibrillary tangles (Bieniek et al., 2013), raising the hypothesis that C9orf72 may play a role also in the pathogenesis of AD. A possible overlap with parkinsonism is also suggested by the peculiar phenotype of C9orf72 RE carriers, which often includes extrapyramidal features (Hsiung et al., 2012; Ratti et al., 2012; Simon-Sanchez et al., 2012; Snowden et al., 2012) and a positive family history for Parkinson’s disease (Hsiung et al., 2012; Ratti et al., 2012; Simon-Sanchez et al., 2012; Snowden et al., 2012). These recent observations broaden the clinical phenotype spectrum associated with C9orf72, suggesting the existence of a novel C9orf72-positive ALS-parkinsonism nosological entity (O’Dowd et al., 2012). The screening of C9orf72 RE in cohorts of patients of different geographic origins with neurodegenerative diseases other than ALS and FTD yielded conflicting results. Two patients with idiopathic PD have been found to carry RE (32 and 39 repeats, respectively), but this finding is of uncertain pathogenic significance because the 39-repeats allele does not segregate with the disease, and the pathologic role of intermediate repeat sizes needs to be further investigated (Nakashima-Yasuda et al., 2007). Similarly, a misdiagnosis of an amnestic FTD could explain the rare occurrence of C9orf72 RE in AD patients (Majounie et al., 2012b). A recent study identified RE in 5 of 1446 (0.3%) parkinsonian patients in a French cohort, although the control population was likely underpowered to detect the presence of a statistical difference (Lesage et al., 2013). Another large study found that the frequency of C9orf72 RE is significantly higher in British AD cases (1.2%) compared with healthy control subjects (0.15%) (Beck et al., 2013). However, the vast majority of studies on AD and PD cohorts of different geographic origins was negative (Akimoto et al., 2013; DejesusHernandez et al., 2012; Harms et al., 2012; Majounie et al., 2012a; Rollinson et al., 2012; Yeh et al., 2012). Even if the present study is relatively underpowered because of the cohort sizes, our mutational screening further supports these negative findings for AD and PD and shows that also in PSP and CBS cases C9orf72 RE are likely rare. In light of these conflicting results, to definitively assess the role of C9orf72 in the pathogenesis of neurodegenerative diseases other than TDP-43 proteinopathies, it will be essential to establish

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the frequency of C9orf72 RE in the general population, as well as the repeat upper limits for healthy control subjects. In fact, frequencies ranging from 0.15% to 0.4% have been reported in mutational screenings of healthy control subjects of different ethnicities (Beck et al., 2013; Ratti et al., 2012; Renton et al., 2011). Our mutational screening of the Italian population confirms that C9orf72 does not represent a significant genetic determinant of AD and PD, either in familial and sporadic cases, and suggests that the same may be true also for tauopathies such as PSP and CBS. However, given that the presence of features of motor neuron dysfunction are highly unusual in CBS and PSP, our finding that >20% of patients with ALS-plus syndromes carry C9orf72 RE is of interest. For comparison, in a large cohort of ALS patients of similar geographic origin previously screened by us, the mutational frequency was about the same in ALS-FTD cases, and half in individuals with pure motor neuron disease (Ratti et al., 2012). In conclusion, our findings confirm that C9orf72 RE are mostly specific for the FTD/ALS spectrum and also suggest that all patients presenting with a combination of extrapyramidal features and signs of motor neuron dysfunction should be screened for C9orf72. This recommendation is further justified by the recent evidence that C9orf72 RE carriers display the same distribution of TDP-43 pathology as non-mutated ALS cases, although with a greater regional burden of lesions (Brettschneider et al., 2013). An increased lesion load in extramotor areas, including the nigrostriatal system, may cross a critical threshold, thus causing in C9orf72 RE carriers the early appearance of extrapyramidal features otherwise extremely uncommon in non-mutated ALS patients. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements Research support was provided by the Italian Ministry of Health (RF 2009-1473856), and by Agenzia Italiana per la Ricerca sulla SLA (grant NOVALS 2012 cofinanced with the contribution of 5x1000, Healthcare Research support of the Ministry of Health). The PD samples were obtained from the Parkinson Institute Biobank (http://www.parkinsonbiobank.com), member of the Telethon Network of Genetic Biobank (project n. GTB12001) funded by TELETHON Italy, and supported by Fondazione Grigioni per il Morbo di Parkinson. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2013.09.037. References Akimoto, C., Forsgren, L., Linder, J., Birve, A., Backlund, I., Andersson, J., Nilsson, A.C., Alstermark, H., Andersen, P.M., 2013. No GGGGCC-hexanucleotide repeat expansion in C9ORF72 in parkinsonism patients in Sweden. Amyotroph. Lateral. Scler. Frontotemporal. Degener. 14, 26e29. Amador-Ortiz, C., Lin, W.L., Ahmed, Z., Personett, D., Davies, P., Duara, R., GraffRadford, N.R., Hutton, M.L., Dickson, D.W., 2007. TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer’s disease. Ann. Neurol. 61, 435e445. Armstrong, M.J., Litvan, I., Lang, A.E., Bak, T.H., Bhatia, K.P., Borroni, B., Boxer, A.L., Dickson, D.W., Grossman, M., Hallett, M., Josephs, K.A., Kertesz, A., Lee, S.E., Miller, B.L., Reich, S.G., Riley, D.E., Tolosa, E., Troster, A.I., Vidailhet, M., Weiner, W.J., 2013. Criteria for the diagnosis of corticobasal degeneration. Neurology 80, 496e503. Beck, J., Poulter, M., Hensman, D., Rohrer, J.D., Mahoney, C.J., Adamson, G., Campbell, T., Uphill, J., Borg, A., Fratta, P., Orrell, R.W., Malaspina, A., Rowe, J., Brown, J., Hodges, J., Sidle, K., Polke, J.M., Houlden, H., Schott, J.M., Fox, N.C.,

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