Large C9orf72 repeat expansions are not a common cause of Parkinson’s disease

Abstract

The concept of a pathological overlap between neurodegenerative disorders is gaining momentum. We sought to determine the contribution of C9orf72 repeat expansions, recently discovered as a cause of frontotemporal dementia and amyotrophic lateral sclerosis, in a large number of Parkinson’s disease patients. No large expansions were identified in our cohort.

1. Introduction

Parkinson’s disease (PD; OMIM 168600) is 1 of the most common neurodegenerative disorders, affecting 1%–2% of the population older than 65 years old. The rare monogenic forms of PD have offered insight into the genetic etiology of this disease (Lesage and Brice, 2012), and the use of genome-wide association has revealed a large number of risk loci for PD (IPDGC-WTCCC2, 2011; Nalls et al., 2011).

We recently identified a hexanucleotide repeat expansion within C9orf72 that accounts for a significant proportion of familial amyotrophic lateral sclerosis (ALS; OMIM #105400105400) and frontotemporal dementia (FTD; OMIM #600274600274; Renton et al., 2011; Majounie et al., 2012b). Several lines of evidence support the concept of a pathological overlap between neurodegenerative disorders. First, predominantly FTD/ALS families harboring concomitant symptoms of parkinsonism have been described (Chio et al., 2011; Gilbert et al., 2010). Second, recent evidence suggests that classic FTD/ALS genes may give rise to rare cases of clinical PD (Kacem et al., 2012; Quadri et al., 2011). Finally, studies of C9orf72expansion carriers reported both clinical features of parkinsonism in the probands and increased incidence of PD in their relatives (Cooper-Knock et al., 2012; Hsiung et al., 2012). Consequently, we investigated a large cohort of PD cases under the hypothesis that the C9orf72 expansion may also contribute to idiopathic PD.

2. Methods

The PD cohort consisted of sporadic and familial cases from the United States obtained from the Coriell Institute for Biomedical Research. All subjects were Caucasian, non-Hispanic individuals. The average age of symptom onset was 60 years old (range, 12–91 years). The number of (GGGGCC) n repeat within C9orf72was assayed using a fluorescent repeat-primed (RP)-polymerase chain reaction approach, as previously described (Renton et al., 2011). Additional statistical analysis of repeat length correlation to age of symptom onset, family history, and Mini Mental State Examination scores was conducted using a generalized linear model.

3. Results

We screened a total of 781 PD cases, including 245 individuals with a familial history of the disease, for the hexanucleotide expansion in the C9orf72 gene. The repeat-primed polymerase chain reaction assays can resolve up to approximately 60 repeats, allowing for rapid discrimination of wild type (< 20 repeats) and expanded (> 30 repeats) alleles (Renton et al., 2011). We did not observe any large (GGGGCC) n expansions in our PD cohort. Four samples harbored marginally larger-than-normal alleles (at 21, 23, 24, and 38 repeats) but did not show a classic large expansion pattern ( Supplementary Figs. 1 and 2). The contribution of such alleles to disease is unclear (Traynor BJ, Renton AE and Majounie E, unreported observations). These 4 cases showed classic signs of PD (activation tremor, asymmetric onset, bradykinesis, gait difficulties, and postural instability). The 38-repeat allele did not segregate with disease in the proband’s sibling, also affected with PD. On average, the number of repeats detected were 3 (range, 0–38) and the allelic distribution is represented in Supplementary Fig. 3. These results are similar to that observed in a previously reported outbred control Caucasian population ( Supplementary Fig. 4; Renton et al., 2011; Majounie et al., 2012b). There was no correlation between repeat length and family history or age of onset.

4. Discussion

To our knowledge, we report here the first investigation of the C9orf72 hexanucleotide repeat in a large cohort of PD patients. Although it is possible that our cohort was underpowered to detect the C9orf72 expansion, our study demonstrates that such expansions are likely to be a rare occurrence in PD; certainly rarer than that seen in Alzheimer’s disease, where approximately 1% of clinically diagnosed patients harbor the mutation (Majounie et al., 2012a). The pathogenic mechanism underlying C9orf72 expansion is largely unknown, and changes in C9orf72 isoform expression or formation of toxic RNA inclusions may contribute to disease (Renton et al., 2011). We did not screen the length of the C9orf72 gene for single nucleotide variants that may influence expression and contribute to disease risk.

Cases with concomitant dementia represent approximately a quarter of PD (Aarsland et al., 2005) and may be of particular interest in future studies. Limited phenotypic data were available in our cohort. However, Mini Mental State Examination scores for 217 PD patients did not correlate with allelic length. In conclusion, our data show that the C9orf72 expansion is not commonly associated with PD.

Supplementary Material

suppl material

Supplementary figure 1. Sequence traces of the repeat-primed PCR in a PD case with intermediate repeats alleles compared to a published positive control (Renton et al., 2011). Fluorescence intensity is recorded on the vertical axis. Size marker is represented by orange peaks. PD cases (A) and (B) departed from the typical saw tooth, decreasing tail pattern seen in the positive control (C) and were called as rare non-pathogenic intermediate alleles by an experience operator. Arrows indicate the cut-off peaks for allele calling. The presence of intermediate alleles was further confirmed by an independent fluorescent sizing assay across the C9orf72 locus (supplementary figure 4). This suggests the PD cases do not carry a large expansion despite a number of repeats (n=24, n=38) that is markedly larger than the average seen in controls (n=3).

Supplementary figure 2. Sequence trace from the FAM-fluorescent PCR in PD case B (38 repeats). The assay amplifies across the C9orf72 repeat locus and confirms the presence of two alleles at 254bp (wild-type) and ~423bp (38 repeats), thus validating the repeat-primed PCR results. The “stutter” artifact seen in the expanded allele is a common phenomenon in PCR amplification of repeat sequences. While this artifact can hinder accurate calling of the true allele size, the aim of this assay was primarily to confirm the absence of a large expansion in sample B.

Supplementary figure 3. Distribution of repeat lengths obtained from the repeat-primed PCR assay in Caucasian PD cases (n = 781) and published controls (n=1605; Renton et al., 2011; Majounie et al., 2012b).

Supplementary figure 4. Density plot of C9orf72 repeat number in PD cases compared to previously published controls (Renton et al., 2011; Majounie et al., 2012b).