Association of a structural variant within the SQSTM1 gene with amyotrophic lateral sclerosis

Abstract

Objective: As structural variations may underpin susceptibility to complex neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the objective of this study was to investigate a structural variant (SV) within sequestosome 1 (SQSTM1).

Methods: A candidate insertion/deletion variant within intron 5 of the SQSTM1 gene was identified using a previously established SV evaluation algorithm and chosen according to its subsequent theoretical effect on gene expression. The variant was systematically assessed through PCR, polyacrylamide gel fractionation, Sanger sequencing, and reverse transcriptase PCR.

Results: A reliable and robust assay confirmed the polymorphic nature of this variant and that the variant may influence SQSTM1 transcript levels. In a North American cohort of patients with familial ALS (fALS) and sporadic ALS (sALS) (n = 403) and age-matched healthy controls (n = 562), we subsequently showed that the SQSTM1 variant is associated with fALS (p = 0.0036), particularly in familial superoxide dismutase 1 mutation positive patients (p = 0.0005), but not with patients with sALS (p = 0.97).

Conclusions: This disease association highlights the importance and implications of further investigation into SVs that may provide new targets for cohort stratification and therapeutic development.

Figure 1. Characterization of the SQSTM1 variant

(A) PCR and native polyacrylamide gel electrophoresis across a random selection of control DNA samples and compared against a 100bp ladder. (B) Sanger sequencing of the I allele and D allele. (C) SQSTM1 transcript levels of SQSTM1 exon 4–7 analyzed using RT-PCR on RNA from a panel of control ONS cells. Relative densitometry was calculated with SQSTM1 transcript signal standardized to each respective GAPDH signal. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; I/D = insertion/deletion; ONS = olfactory neurosphere derived cells; RT-PCR = Reverse transcriptase PCR; SQSTM1 = sequestosome 1.

Figure 2. Association of the SQSTM1 variant with age at onset of disease and survival

(A) The median and distribution of age at onset (years) of SOD1 mutation-positive patients with ALS (n = 167) grouped by each SQSTM1 variant genotype. (B) Kaplan-Meier survival curves of SOD1 mutation-positive patients, comparing the SQSTM1 genotypes assuming independent measurements. A robust log-rank test accounting for familial correlation was performed to assess any association between the groups. Survival was measured in months from ALS diagnosis until death. ALS = amyotrophic lateral sclerosis; SOD1 = superoxide dismutase 1; SQSTM1 = sequestosome 1.