This study investigated a multi-allelic intronic TTTA structural variant in NEFH as a candidate risk factor for sALS, and modifier of age and site of disease onset and survival duration. Within the Duke cohort carriage of the 9 TTTA allele was significantly enriched in bulbar onset cases. Interestingly, in the Duke cohort the 9 TTTA allele was associated with a 2x reduction in sALS risk, but only in spinal onset patients. Within the spinal onset cases from the Duke cohort, the reduction in risk remained significant after considering carriage of the 10 TTTA allele or the 9,10 genotype, confirming this effect was in fact driven by the 9 TTTA allele. Despite the 9 TTTA association not replicating in the Coriell or combined cohort, it is important to note that the trend for reduced risk in the spinal onset patients was consistent in the Coriell and combined cohorts. On the other hand, there was no association for increased disease risk for any of the NEFH TTTA alleles/genotypes in either the Duke or Coriell cohorts, or in the combined sALS cohort.
Following the association with disease risk, NEFH alleles/genotypes were investigated as potential modifiers of sALS age of disease onset using general linear modelling. The association with age at disease onset was investigated in the combined sALS cohort to minimize the effect of variability across each separate sALS cohort. Carriage of the 10 TTTA allele was found to be associated with a 2.7 year later mean age at disease onset when accounting for allele carriage, sex and site of disease onset, further supporting the notion of the 9 and 10 TTTA alleles being associated with protective traits in sALS.
Lastly, NEFH alleles were investigated as a potential modifier of survival in the Duke cohort. Due to the limited number of patients with end-point survival data, we were only able to analyze the effect of NEFH alleles and not genotypes, which we acknowledge as a limitation of this study. We analyzed the carriage of NEFH alleles, taking into account the site of disease onset. Although the survival trajectories for spinal and bulbar onset patients did not differ within this cohort, it is important to include this covariate since short structural variants have previously been shown to stratify sALS patient sub-phenotype 33–35. In the present study, no significant associations were detected between NEFH alleles and survival.
A number of possible explanations were considered for the differences in the 9 and 10 TTTA allele associations found in the two sALS cohorts. Firstly, in view of the smaller size of the Duke cohort, the possibility of a spurious association was considered, but in view of the magnitude of the associations and the follow up analyses accounting for the carriage of the 9 TTTA allele, 10 TTTA allele or the 9,10 TTTA genotype, this is considered unlikely. Differences in ethnicity were excluded on the basis of a comparison of self-reported data on ethnicity and country-of-origin from the two cohorts. However, consideration still needs to be given to differences in genetic diversity between the two cohorts, based on the sources of patient samples and geographic patterns of patient recruitment. It is pertinent in this regard that the Duke cohort were from one geographical location (Duke ALS Clinic, Durham North Carolina), whereas cases from the Coriell biobank are multicenter in origin and were recruited from geographically more diverse locations.
Currently, both light and heavy neurofilaments (pNFL and pNFH) are considered among the most promising disease biomarkers for ALS diagnosis and progression, and have been extensively reviewed 5,36,37. Levels of both CSF and serum pNFL and pNFH have been shown to distinguish patients with ALS from controls and other neurodegenerative diseases 38,39. Various studies have also reported pNFL and pNFH to be correlated with pheno-conversion, clinical measures of disease progression or the clinical subtype of ALS 39–42. Typically pNFL levels are considered a more sensitive prognostic marker, displaying a significant correlation between pNFL in the CSF and in serum 38. On the other hand, pNFH in the CSF does not correlate well with serum pNFH levels but this may be due to the masking of epitopes or post-translational modifications influencing antibody detection 38,42. Despite this, a study has reported significantly higher serum pNFH concentrations in pyramidal, bulbar and classic ALS phenotypes compared to flail arm ALS, primary muscular atrophy and primary lateral sclerosis subtypes, with a positive correlation between pNFH and progression rate, suggesting that faster neuronal degeneration may be a determinant of serum pNFH levels 43.
With evidence of NEFH playing a role at both the genetic level and as a prognostic marker for ALS, one must consider the potential impact of previously overlooked short structural variants within the NEFH gene in helping to distinguish clinical variability, particularly in sporadic ALS. A recent report has stated that 21% of sALS patients carry a genetic variant in an ALS associated gene and 13% of patients carry more than one genetic variant (including variants of unknown significance) 44. Of note, patients carrying two variants developed disease at a significantly earlier age, with variants in known ALS genes being of potential clinical importance in as many as 42% of sALS patients 44. With NEFH having a clear link to ALS disease and progression, future work should explore the impact of short structural variants in NEFH and other ALS associated genes as additional markers of disease risk and prognosis.