Loss of angiogenin function is related to earlier ALS onset and a paradoxical increase in ALS duration

0.5–1% of ALS (Amyotrophic Lateral Sclerosis) and Parkinson's disease (PD) are associated with mutations in the angiogenin (ANG). These mutations are thought to cause disease through a loss of ANG function, but this hypothesis has not been evaluated statistically. In addition, the potential for ANG to promote disease has not been considered. With the goal of better defining the etiology of ANG-ALS, we assembled all clinical onset and disease duration data and determined if these were correlated with biochemical properties of ANG variants. Loss of ANG stability and ribonuclease activity were found to correlate with early ALS onset, confirming an aspect of the prevailing model of ANG-ALS. Conversely, loss of ANG stability and ribonuclease activity correlated with longer survival following diagnosis, which is inconsistent with the prevailing model. These results indicate that functional ANG appears to decrease the risk of developing ALS but exacerbate ALS once in progress. These findings are rationalized in terms of studies demonstrating that distinct mechanisms contribute to ALS onset and progression and propose that ANG replacement or stabilization would benefit pre-symptomatic ANG-ALS patients. However, this study challenges the prevailing hypothesis that augmenting ANG will benefit symptomatic ANG-ALS patients. Instead, our results suggest that silencing of ANG activity may be beneficial for symptomatic ALS patients. This study will serve as a call-to-arms for neurologists to consistently publish ALS and PD patient's clinical data—if all ANG-ALS patients’ data were available our findings could be tested with considerable statistical power.


Supplementary information: Table of contents
Appendix S1: Tests for additional hypothesis Appendix S2: Correlation of ALS lifespan with ANG physicochemical properties Appendix S3: Adjustment of false discovery rate using the Benjamini-Hochberg method Figure S1    Table S1 Appendix S1. ANG-aggregation propensity did not significantly correlate with either onset (p value of 0.395, CI -0.38-0.14) (Figure S1a Relative ANG-ribonuclease activity was correlated to the onset of PD (n = 17). No significant correlation was observed between the onset of PD and %WT ribonuclease activity of ANG variants (p values range of 0.59-0.60, CI -0.61-0.38) ( Figure S2). Correlations with Parkinson's disease duration couldn't be performed as disease duration information was available for only two patients. Similarly, stability of ANG variants cannot be correlated with either PD onset or PD duration as ∆∆G data was only available for two ANG-PD variants.
Appendix S2. To illustrate the lifelong effects of ANG stability and activity, we have correlated these with the total lifespan of ALS patients by adding disease duration to ALS onset time for patients having both the data were reported (patients having only ALS onset data, but no survival data were censored). Consistent with having averaged two competing forces, no significant correlations were observed between total lifespan of ALS patients and either stability Test for Cox proportional hazards assumption (p value of 0.293) indicated there is no violation of the proportionality assumption. Likewise, using the same thresholds to categorize ribonuclease activity and stability, Kaplan-Meier analysis performed above: the median lifespans of patients with low versus high ANG stability and ribonuclease activity were 850 ± 40.7 and 876± 148, and 876 ± 115 versus 834 ± 62.6 months, respectively; and no significant difference was observed between the survival functions of stability categories with respect to total lifespan (p values range of 0.18-0.28) or ribonuclease activity (p values range of 0.62-1) (Figures S3c and S3d).
Using the same thresholds to categorize ribonuclease activity and stability, Cox proportional hazards analysis were performed: no significant difference was observed between the hazard ratio of stability categories with respect to total lifespan or ribonuclease activity categories with respect to lifespan and the data was not reported. Tests for proportionality assumption demonstrated non-parallelism between the categories indicating violation of the Cox proportionality hazard model assumption. Note, however, that patients with high ANG stability and activity survive an average of 13 and 9 years longer than the remaining ALS patients.
Appendix S3. Three covariates (ANG stability; ANG aggregation propensity; ANG ribonuclease activity) were correlated, independently, with two datasets (survival and onset). False discovery rate was therefore corrected with respect to correlating three covariates to a particular dataset.
Benjamini-Hochberg method was used to adjust for the false discovery rate. A false discovery rate of 5% was used to adjust for type I error (Table S1). A total of 25 analysis were performed on our data. All our p values found significant in our analysis were found to be significant using Benjamini-Hochberg correction except, analysis of mortality risk using Cox proportional hazards model using WT ribonuclease activity as a continuous variable. To increase stringency, we also required significance to be achieved through multiple statistical tests of each hypothesis (e.g. correlation, Kaplan Meier, and Cox; as well as the use of survival hypothesis testing with different weighing functions using Log-rank, Tarone-ware, and Breslow). c. Figure S1. ALS onset, disease duration and lifespan do not correlate with aggregation propensity. a) Scatter plot demonstrating no significant correlation between aggregation propensity and ALS onset. b) Scatter plot demonstrating no significant correlation between aggregation propensity and disease duration. c) Scatter plot demonstrating no significant correlation between aggregation propensity and ALS lifespan. The definition of the aggregation propensity calculated here is important-its components (i.e. the mathematical terms used to calculate) are the fundamental physicochemical parameters of charge, hydrophobicity, and entropy. Aggregation propensity denotes the likelihood of undergoing a phase transition from an aqueous phase to an aggregated phase (i.e. it pertains to phenomena that occur following the nucleation step that "seeds" the aggregate), and to a first approximation is independent of protein stability or activity. Our study could not address the nucleation step that initiates the aggregation cascade-which could be related to protein stability-because the data were unavailable and cannot be estimated.   demonstrating no significant correlation between ribonuclease activity and lifespan. c) Kaplan-Meier curves illustrating no significant differences in lifespan between patients with ANG variants with ∆∆G less than or equal to -1 kcal/mol and variants with ∆∆G greater than -1 kcal/mol. d) Kaplan-Meier curves illustrating no significant differences in lifespan between patients with ANG variants with %WT ribonuclease activity less than or equal to 10% and variants with %WT ribonuclease activity greater than 10%.  Figure S4: Test for Cox proportionality assumption. Test for proportionality was performed using log-log plot of disease duration and WT ribonuclease activity. Parallelism was demonstrated using log-log plots between both categories %WT ribonuclease activity less than or equal to 10% and variants with %WT ribonuclease activity greater than 10%, indicating no violation of proportionality assumption.  Table S2. Survival data of tgSOD1 G93A -ALS mice dosed with 10 μg ANG.