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ObjectiveTo identify preferred neurofilament assays and clinically validate serum neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) as prognostic and potential pharmacodynamic biomarkers relevant to amyotrophic lateral sclerosis (ALS) therapy development.MethodsIn this prospective, multicenter, longitudinal observational study of patients with ALS (n = 229), primary lateral sclerosis (n = 20), and progressive muscular atrophy (n = 11), biological specimens were collected, processed, and stored according to strict standard operating procedures (SOPs). Neurofilament assays were performed in a blinded manner by independent contract research organizations.ResultsFor serum NfL and pNfH measured using the Simoa assay, there were no missing data (i.e., technical replicates below the lower limit of detection were not encountered). For the Iron Horse and Euroimmun pNfH assays, such missingness was encountered in ∼4% and ∼10% of serum samples, respectively. Mean coefficients of variation for NfL in serum and CSF were both ∼3%. Mean coefficients of variation for pNfH in serum and CSF were ∼4%–5% and ∼2%–3%, respectively, in all assays. Baseline serum NfL concentration, but not pNfH, predicted the future Revised ALS Functional Rating Scale (ALSFRS-R) slope and survival. Incorporation of baseline serum NfL into mixed effects models of ALSFRS-R slopes yields an estimated sample size saving of ∼8%. Depending on the method used to estimate effect size, use of serum NfL (and perhaps pNfH) as pharmacodynamic biomarkers, instead of the ALSFRS-R slope, yields significantly larger sample size savings.ConclusionsSerum NfL may be considered a clinically validated prognostic biomarker for ALS. Serum NfL (and perhaps pNfH), quantified using the Simoa assay, has potential utility as a pharmacodynamic biomarker of treatment effect.

ObjectiveSuggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30–90 repeats) is unknown and was investigated here.MethodsWe conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS.ResultsSouthern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration.ConclusionThe presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

Objective Suggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30–90 repeats) is unknown and was investigated here. Methods We conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS. Results Southern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration. Conclusion The presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

Item does not contain fulltext OBJECTIVE: To assess the frequency and phenotype of hexanucleotide repeat expansions in C9ORF72 in a large cohort of patients of Dutch descent with familial (fALS) and sporadic (sALS) amyotrophic lateral sclerosis (ALS), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS). METHODS: Included were 78 patients with fALS, 1,422 with sALS, 246 with PMA, and 110 with PLS, and 768 control subjects. Repeat expansions were determined by a repeat primed PCR. Familial aggregation of dementia and Parkinson disease (PD) was examined among patients with ALS who carried the repeat expansion. RESULTS: The expanded repeat was found in 33 (37%) of all patients with fALS, in 87 (6.1%) patients with sALS, in 4 (1.6%) patients with PMA, and in 1 (0.9%) patient with PLS. None of the controls carried the mutation. Patients with ALS with the repeat expansion had an earlier age at onset (median 59.3 vs 61.9 years, hazard ratio 1.55, p = 5 x 10(-5)) and shorter survival (median 2.5 vs 2.7 years, hazard ratio 1.46, p = 8 x 10(-4)). Dementia, but not PD, occurred nearly twice as often in relatives of patients with the expansion compared to all patients with ALS or controls (p = 9 x 10(-4)). CONCLUSIONS: The hexanucleotide repeat expansion in C9ORF72 is a major cause of fALS and apparently sporadic ALS in the Netherlands. Patients who carry the repeat expansion have an earlier onset, shorter survival, and familial aggregation of dementia. These results challenge the classic definition of fALS and may justify genetic testing in patients with sALS.