Elsevier

The Lancet Neurology

Volume 12, Issue 10, October 2013, Pages 978-988
The Lancet Neurology

Articles
Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study

https://doi.org/10.1016/S1474-4422(13)70210-2Get rights and content

Summary

Background

Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are the most common known genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND). We assessed whether expansion size is associated with disease severity or phenotype.

Methods

We did a cross-sectional Southern blot characterisation study (Xpansize-72) in a cohort of individuals with FTD, MND, both these diseases, or no clinical phenotype. All participants had GGGGCC repeat expansions in C9ORF72, and high quality DNA was available from one or more of the frontal cortex, cerebellum, or blood. We used Southern blotting techniques and densitometry to estimate the repeat size of the most abundant expansion species. We compared repeat sizes between different tissues using Wilcoxon rank sum and Wilcoxon signed rank tests, and between disease subgroups using Kruskal-Wallis rank sum tests. We assessed the association of repeat size with age at onset and age at collection using a Spearman's test of correlation, and assessed the association between repeat size and survival after disease onset using Cox proportional hazards regression models.

Findings

We included 84 individuals with C9ORF72 expansions: 35 had FTD, 16 had FTD and MND, 30 had MND, and three had no clinical phenotype. We focused our analysis on three major tissue subgroups: frontal cortex (available from 41 patients [21 with FTD, 11 with FTD and MND, and nine with MND]), cerebellum (40 patients [20 with FTD, 12 with FTD and MND, and eight with MND]), and blood (47 patients [15 with FTD, nine with FTD and MND, and 23 with MND] and three carriers who had no clinical phenotype). Repeat lengths in the cerebellum were smaller (median 12·3 kb [about 1667 repeat units], IQR 11·1–14·3) than those in the frontal cortex (33·8 kb [about 5250 repeat units], 23·5–44·9; p<0·0001) and those in blood (18·6 kb [about 2717 repeat units], 13·9–28·1; p=0·0002). Within these tissues, we detected no difference in repeat length between disease subgroups (cerebellum p=0·96, frontal cortex p=0·27, blood p=0·10). In the frontal cortex of patients with FTD, repeat length correlated with age at onset (r=0·63; p=0·003) and age at sample collection (r=0·58; p=0·006); we did not detect such a correlation in samples from the cerebellum or blood. When assessing cerebellum samples from the overall cohort, survival after disease onset was 4·8 years (IQR 3·0–7·4) in the group with expansions greater than 1467 repeat units (the 25th percentile of repeat lengths) versus 7·4 years (6·3–10·9) in the group with smaller expansions (HR 3·27, 95% CI 1·34–7·95; p=0·009).

Interpretation

We detected substantial variation in repeat sizes between samples from the cerebellum, frontal cortex, and blood, and longer repeat sizes in the cerebellum seem to be associated with a survival disadvantage. Our findings indicate that expansion size does affect disease severity, which—if replicated in other cohorts—could be relevant for genetic counselling.

Funding

The ALS Therapy Alliance, the National Institute of Neurological Disorders and Stroke, the National Institute on Aging, the Arizona Department of Health Services, the Arizona Biomedical Research Commission, and the Michael J Fox Foundation for Parkinson's Research.

Introduction

Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are the most common known genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND).1, 2 These diseases have been proposed to result from the formation of toxic RNA foci when repeats are transcribed,1 from the generation of aggregating dipeptide-repeat proteins due to non-ATG translation,3, 4 or from a loss of function of the C9ORF72 protein.5, 6

Although repeat-primed PCR methods can assess the presence or absence of C9ORF72 repeat expansions, Southern blotting techniques are needed to find out the size of the GGGGCC repeat. Southern blotting, however, is time-consuming and challenging, and therefore very few studies have been done. A 2013 Southern blot study investigated a clinical cohort of 57 patients with a range of neurodegenerative diseases,7 but large-scale Southern blot studies that examine repeat sizes in the brain have not been reported. To address crucial questions, such as whether repeat length is associated with clinical phenotype, disease onset, or survival in patients with hexanucleotide repeat expansions, we did an extensive Southern blot characterisation study (Xpansize-72) of DNA samples from C9ORF72 expansion carriers.

Section snippets

Participants and study design

For patients who had post-mortem tissues available, we selected all C9ORF72 repeat expansion carriers diagnosed with FTD, MND, or both from the Mayo Clinic Florida Brain Bank (Jacksonville, FL, USA) and Banner Sun Health Research Institute (Sun City, AZ, USA) for whom sufficient tissue from the frontal cortex, cerebellum, or both was available to allow extraction of high quality DNA for Southern blotting analysis. For our clinical cohort, we selected one patient from each family carrying C9ORF72

Results

We included 84 individuals with the C9ORF72 expansion, 80 from the Mayo Clinic and four from the Banner Sun Health Research Institute (table 1; appendix); samples were collected between 1999 and 2013. Half the people were diagnosed pathologically, with most of the rest being diagnosed clinically; three had no clinical phenotype (table 1).

For all participants, frontal cortex, cerebellum, or blood samples were obtained, and these tissues comprised the primary analysis of this study. Frontal

Discussion

We confirmed the C9ORF72 repeat expansion in all participants of our study with FTD, MND, both diseases, and no clinical phenotype. In the cerebellum, the median repeat length was smaller than those in the frontal cortex and blood. We detected no association between repeat length and disease subgroups in any of these tissues. Furthermore, in patients with FTD, we found that longer repeat expansions in the frontal cortex were associated with older age at onset and age at sample collection. We

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