Prospective natural history study of C9orf72 ALS clinical characteristics and biomarkers

Abstract

Objective: To define the natural history of the C9orf72 amyotrophic lateral sclerosis (C9ALS) patient population, develop disease biomarkers, and characterize patient pathologies.

Methods: We prospectively collected clinical and demographic data from 116 symptomatic C9ALS and 12 non-amyotrophic lateral sclerosis (ALS) full expansion carriers across 7 institutions in the United States and the Netherlands. In addition, we collected blood samples for DNA repeat size assessment, CSF samples for biomarker identification, and autopsy samples for dipeptide repeat protein (DPR) size determination. Finally, we collected retrospective clinical data via chart review from 208 individuals with C9ALS and 450 individuals with singleton ALS.

Results: The mean age at onset in the symptomatic prospective cohort was 57.9 ± 8.3 years, and median duration of survival after onset was 36.9 months. The monthly change was -1.8 ± 1.7 for ALS Functional Rating Scale-Revised and -1.4% ± 3.24% of predicted for slow vital capacity. In blood DNA, we found that G4C2 repeat size correlates positively with age. In CSF, we observed that concentrations of poly(GP) negatively correlate with DNA expansion size but do not correlate with measures of disease progression. Finally, we found that size of poly(GP) dipeptides in the brain can reach large sizes similar to that of their DNA repeat derivatives.

Conclusions: We present a thorough investigation of C9ALS natural history, providing the basis for C9ALS clinical trial design. We found that clinical features of this genetic subset are less variant than in singleton ALS. In addition, we identified important correlations of C9ALS patient pathologies with clinical and demographic data.

© 2019 American Academy of Neurology.

Figures

Figure 1. Individuals with amyotrophic lateral sclerosis (ALS) with chromosome 9 open reading frame 72 expansion mutations (C9ALS) natural history: Descriptive characteristics

(A) Distribution of ages at onset for the prospective C9ALS cohort (n = 116 individuals; mean age at onset 57.9 ± 8.3 years). (B) Mortality in the prospective C9ALS cohort (n = 88 individuals reaching survival endpoint; n = 28 individuals alive or lost to follow-up; right-censored on last known date of contact; marked with tick marks; median survival 36.9 months). (C) Distribution of onset locations in the prospective C9ALS cohort. (D) Family history of ALS, dementia, or both in the prospective cohort (n = 128 individuals). (E) Distribution of ages at onset for retrospective C9ALS (n = 208 individuals; mean age at onset 59.0 ± 9.3 years) and singleton amyotrophic lateral sclerosis (SALS) (n = 450 individuals; mean age at onset 59.3 ± 12.1 years) cohorts. (F) Mortality in retrospective C9ALS (n = 195 individuals; median survival 29.9 months) and SALS cohorts (n = 214 individuals; median survival 30 months). Upper quartile survival displays significantly decreased disease duration in slow-progressing C9ALS (n = 48 individuals; median survival 55 months) as compared to SALS (n = 53 individuals; median survival 73 months) (log-rank [Mantel-Cox] test: p < 0.001).

Figure 2. Individuals with amyotrophic lateral sclerosis (ALS) with chromosome 9 open reading frame 72 expansion mutations (C9ALS) natural history: Measures of disease progression

(A) ALS Functional Rating Scale–Revised (ALSFRS-R), (B) slow vital capacity (SVC), (C) ALS Cognitive Behavioral Screen (ALS-CBS), and (D) ALS Caregiver Behavioral Questionnaire (ALSCBQ) rates of decline for individuals with C9ALS with at least 2 longitudinal data points. (ALSFRS-R: n = 88; SVC: n = 53; ALS-CBS: n = 42; ALSCBQ: n = 29.) (E–H) ALSFRS-R (n = 88) and (I–L) ALS-CBS (n = 42) subscore rates of change.

Figure 3. Relationship of G4C2 repeat size and age

G4C2 repeat size in blood, as measured by southern blot, has a significant positive correlation with (A) age at disease onset (n = 79; R = 0.254; p = 0.05) and (B) age at sample collection (n = 89; R = 0.281; p = 0.02). (C) When individuals are separated by median repeat size, age at collection is significantly higher in the top half than the bottom half (n = 89; Mann-Whitney U test: p < 0.01**). Repeat size does not correlate with (D) survival (n = 61; R = 0.031; p = 0.91) after onset or (E) ALS Functional Rating Scale–Revised (ALSFRS-R) rates of decline (n = 66; R = 0.053; p = 0.90). (F) In an unexpanded (<30 repeats) population, haplotype-carrying individuals have a significantly larger average repeat size in blood than noncarriers (n = 244 haplotype carriers, n = 430 noncarriers; Mann-Whitney U test: p < 0.0001). (G) In individuals without ALS carrying the chromosome 9 open reading frame 72 (C9orf72)–associated risk haplotype with large, yet unexpanded repeat sizes (upper quintile; 13–25 repeats), repeat size is significantly correlated with age at sample collection (n = 47; R = 0.434; p < 0.01). (H) Repeat size is significantly higher in the top half of large, unexpanded repeat carriers than the bottom half (n = 47; Mann-Whitney U test: p < 0.01**). All p values are corrected for multiple comparisons (false discovery rate).

Figure 4. Poly(GP) in individuals with amyotrophic lateral sclerosis (ALS) with chromosome 9 open reading frame 72 expansion mutations (C9ALS) CSF and size in autopsy tissue

(A) Poly(GP) in CSF is highly C9-specific, with signal only observed in full expansion carriers (n = 32 C9ALS; n = 4 singleton ALS [SALS]; n = 6 C9+ carriers without ALS [AC]). (B) Poly(GP) levels are consistent between draws for 9 out of 10 individuals with longitudinal CSF draws. (C–F) CSF poly(GP) correlations with (C) ALS age at onset (n = 30 individuals; R = 0.040; p = 0.91), (D) survival (n = 20 individuals; R = 0.023; p = 0.92), (E) ALS Functional Rating Scale–Revised (ALSFRS-R) average monthly rate of change (n = 25 individuals; R = 0.111; p = 0.90), and (F) blood DNA repeat size, as measured by southern blot (n = 34 individuals; R = 0.371; p = 0.05). (G) size exclusion chromatography (SEC) standard proteins (Bio-Rad, Hercules, CA) separated by a superdex 10/300 GL SEC column used in poly(GP) size assessments, demonstrating efficient separation of relevant protein sizes. (H) Poly(GP) is observed in large SEC-separated C9ALS autopsy CNS samples. All samples are normalized to fraction 9 (the largest SEC fraction after void volume). Dotted line represents SEC standard proteins as shown in G. Matched patient autopsies were used for cerebellum and frontal cortex analyses. All p values are corrected for multiple comparisons (false discovery rate).