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

Abstract

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.

Figure 1. Repeat lengths in the central nervous system

WT = wild-type C9ORF72 allele (web appendix p 1). On a Southern blot, frontal cortex demonstrates relatively long repeat expansions, as displayed by ten representative cases (P1–P10, A). In the cerebellum, repeat expansions are usually smaller than in the frontal cortex (P11–P20, B). The horizontal dashed lines denote 23 kb and 9·4 kb. Repeat lengths in frontal cortex (n=41), cerebellum (n=40), and blood (n=50) are displayed on a scatter plot (C). Median repeat lengths are represented by a gray line. A bar graph shows frontal cortex (black), temporal cortex (light gray) and cerebellum (dark gray) for cases for whom these tissues were all available (n=18, D). For two illustrative cases, five brain regions are shown on a Southern blot (P16, P20, E). A bar graph of patients for whom frontal cortex, spinal cord, and cerebellum were all available is presented (n=5, F).

Figure 2. Repeat lengths in other tissues

Expansion sizes determined in blood of ten representative subjects are displayed on a Southern blot, demonstrating variation in repeat length (P27–P36, A). In blood, repeat expansions can show roughly the same length as in the frontal cortex or in the cerebellum, but they can also show an unexpected length, as demonstrated by three illustrative cases (P16, P20, P19, B). Substantial variability in repeat length is also present in other tissues of a single patient (P20, C). The last lane contains a negative control. Repeat expansions in fibroblasts are smaller than in blood, and can show multiple crisp bands (P37–P41, D).

Figure 3. Differences in age, survival, and repeat length

Associations of repeat length with age at onset (n=20, A) and age at collection (n=21, B) in FTD patients are shown. A regression line is displayed for each plot of correlation to enhance visual display. Survival following disease onset according to 25^th percentile repeat length (11·1 kb [~1467 repeat units]) in the cerebellum of the overall cohort is demonstrated (n=10≤25^th percentile, n=28>25^th percentile, C). In addition, survival following disease onset according to 25^th percentile repeat length (11.1 kb) in the cerebellum of FTD patients is shown (n=5≤25^th percentile, n=14>25^th percentile, D). Repeat lengths in the frontal cortex (n=21 FTD, 11 FTD/MND, 9 MND, E), cerebellum (n=20 FTD, 12 FTD/MND, 8 MND, F), and blood (n=15 FTD, 9 FTD/MND, and 23 MND, G) are not significantly different between disease subgroups; the sample median is represented by a solid horizontal line.