Novel STMN2 Variant Linked to Amyotrophic Lateral Sclerosis Risk and Clinical Phenotype

Abstract

Objective: There is a critical need to establish genetic markers that explain the complex phenotypes and pathogenicity of ALS. This study identified a polymorphism in the Stathmin-2 gene and investigated its association with sporadic ALS (sALS) disease risk, age-of onset and survival duration.

Methods: The candidate CA repeat was systematically analyzed using PCR, Sanger sequencing and high throughput capillary separation for genotyping. Stathmin-2 expression was investigated using RT-PCR in patient olfactory neurosphere-derived (ONS) cells and RNA sequencing in laser-captured spinal motor neurons.

Results: In a case-control analysis of a combined North American sALS cohort (n = 321) and population control group (n = 332), long/long CA genotypes were significantly associated with disease risk (p = 0.042), and most strongly when one allele was a 24 CA repeat (p = 0.0023). In addition, longer CA allele length was associated with earlier age-of-onset (p = 0.039), and shorter survival duration in bulbar-onset cases (p = 0.006). In an Australian longitudinal sALS cohort (n = 67), ALS functional rating scale scores were significantly lower in carriers of the long/long genotype (p = 0.034). Stathmin-2 mRNA expression was reduced in sporadic patient ONS cells. Additionally, sALS patients and controls exhibited variable expression of Stathmin-2 mRNA according to CA genotype in laser-captured spinal motor neurons.

Conclusions: We report a novel non-coding CA repeat in Stathmin-2 which is associated with sALS disease risk and has disease modifying effects. The potential value of this variant as a disease marker and tool for cohort enrichment in clinical trials warrants further investigation.

Keywords: amyotrophic lateral sclerosis; genetic association studies; genetic marker; genetic variant; motor neuron disease; stathmin-2; structural variation.

FIGURE 1

Identification and characterization of CA repeat polymorphism in STMN2. (A) Schematic of the STMN2 primary transcript and the location of the CA repeat. (B) Sanger sequencing confirming size and variability of CA alleles. (C) Representation of STMN2 CA alleles from capillary separation genotyping assay. The blue peaks depict the fluorescent signal intensity with the allele peaks to determine genotype indicated by the black star. (D) Predicted in silico pre-mRNA structure for STMN2 reference sequence (NC_000008.11) generated from RNAfold Web server. The image depicts the predicted structure spanning from base pairs (79636317–79643806) with the red box indicating the portion of the pre-mRNA containing the variable CA repeat.

FIGURE 2

Investigation of STMN2 CA genotypes and their association with sALS disease risk and survival. (A) Allele distributions of the STMN2 CA repeat for 332 healthy controls and 321 sALS cases. The dotted line indicates the cut-off point between short and long CA alleles. Those <19 CA repeats are considered a short allele and those ≥19 CA repeats are considered a long allele. Significance is indicated by the *, p < 0.05. (B) Cumulative survival for 143 sALS patients from Duke University based on those that had L/L 24 CA genotypes compared to those without. (C) Cumulative survival based on site of disease onset (bulbar and spinal) and the presence of the L/L 24 CA genotypes.

FIGURE 3

The effect of CA genotype on STMN2 mRNA expression in olfactory neurosphere derived cell lines and laser captured spinal motor neurons. (A) Immunostaining of ONS cells with Nestin, NeuN, and β-tubulin. (B) Variable expression of STMN2 in sALS ONS cell lines (lanes 5–8) compared to control ONS cell lines (lanes 1–4). (C) Relative densitometry of TARDBP and STMN2 standardized to GAPDH expression showing variable expression of STMN2 in sALS cell lines and no difference in TARDBP. (D) Trend for a stepwise decrease in STMN2 mRNA expression between control and sALS cases according to STMN2 CA genotype (S/L vs. L/L), error bars represent standard error of the mean. RNA sequencing data was analyzed by Krach et al. (2018) and STMN2 expression between cases and controls previously reported (Melamed et al., 2019). (E) Percentage of motor neurons positive for phosphorylated TDP-43 during initial sample collection done by Krach et al. (2018), according to STMN2 CA genotype. (F) Percentage of motor neuron death according to STMN2 CA genotype based on data collected by Krach et al. (2018).