Effect of the 50 bp deletion polymorphism in the SOD1 promoter on SOD1 mRNA levels in Italian ALS patients

Abstract

The genetic association between homozygosity for a 50 bp deletion polymorphism in the SOD1 promoter, 1684 bp upstream of the ATG, and an increased age of symptom onset was observed in various populations of ALS patients. Moreover, it has been demonstrated that this deletion reduces SOD1 expression in vitro. The objective of the present study was to test whether the observed association is replicated in patients from an Italian population and to check whether the deletion correlates with reduced SOD1 mRNA expression in vivo. Genomic DNA from 235 Italian SALS cases and 245 age- and sex-matched donors from the same ethnic background was screened for the 50 bp SOD1 promoter deletion by real time PCR assays. No differences were observed between ALS patients and controls for the frequency of both the alleles (D = deleted, N = non-deleted; p = 0.95) and genotypes (p = 0.90). Furthermore, stratification of the ALS samples showed that this variation was not associated with increased age of onset in ND and DD patients in comparison to NN patients (p = 0.48). Finally, we performed real-time RT-PCR to quantify SOD1 mRNA levels in 48 patients and we did not find a relevant difference among the three sub-groups of genotypes (p = 0.30). Our data suggest that the studied polymorphism does not modulate SOD1 mRNA level and disease phenotype in an Italian population.

Introduction

Amyotrophic Lateral Sclerosis is a late-onset neurodegenerative disease clinically characterized by progressive muscular paralysis reflecting the degeneration of both upper and lower motor neurons. Although the majority (90%) of cases is sporadic (SALS), the remainder presents a family history (FALS). Mutations in the gene coding for Cu/Zn superoxide dismutase (SOD1) represent the most common form of familial ALS (ALS1); moreover, it has been estimated that approximately 1% of SALS cases are caused by mutations in the coding region of this gene [1], [2]. Mutations in other genes, such as ALS2, SETX, VAPB, ANG, DCTN1, TARDBP, FUS/TLS, FIG4, OPTN have also been linked to FALS and SALS [3], [4], [5], [6].

Interestingly, a recent finding suggested that the increase of SOD1 mRNA expression could be a specific biomarker for ALS pathology [7]. SOD1 mRNA level is elevated both in nervous areas typically affected by ALS disease (i.e., brain stem and spinal cord) and in peripheral blood mononuclear cells (PBMCs) from SALS patients compared to control population. The authors also demonstrated the disease specificity of this phenomenon, showing that SOD1 mRNA levels in patients with a different neurodegenerative disease (i.e. AD subjects) do not vary from age-matched controls. Therefore, it is important to evaluate whether polymorphisms in the regulatory region of the SOD1 promoter could be associated with altered SOD1 mRNA levels and/or ALS phenotype variability. In fact, the promoter region is of great importance for the fine-tuned modulation of SOD1 mRNA levels since it contains the binding sites for several transcription factors [8] and sequence differences in these cis-acting responsive elements may be causative for varied mRNA expression. Niemann and co-workers [9] identified a heterozygous A to G transition located at position − 109 from initiation codon ATG, corresponding to the second residue of the highly conserved SOD1 TATA-box (TATAAA → TGTAAA). This variant was identified in one FALS and two SALS patients of 301 cases and functional studied demonstrated that this sequence change caused a reduction in SOD1 gene expression in vitro. Nevertheless it was not supposed to be disease-causing or associated with increased risk of ALS. Another A to G variant at position − 278 from the ATG was described [10]. Bioinformatic analysis showed that this change abolished the responsive elements for the transcription factor Hmx2/Nkx5-2 and potentially created the binding sites for three other transcription factors (NUDR, Elk-1, c-Rel). However, no molecular studies have yet been performed to validate the in silico results and to experimentally determine the functional relevance of this mutation for ALS pathology.

Moreover, a 50 bp deletion polymorphism in SOD1 promoter (1684 bp upstream of the ATG start codon) was described and in vitro analysis demonstrated that this deletion reduced promoter activity in cells, because of the loss of two Sp1 binding sites, thus highlighting a functional effect of such polymorphism [11], [12]. Population studies demonstrated a positive association for the deletion homozygosity with an increased age of symptoms in UK and Irish patients, raising the possibility that altered expression of SOD1 mRNA may modulate the phenotype in SALS.

The aim of the present study was to verify whether the 50 bp deletion polymorphism correlated, in vivo, with varied SOD1 mRNA expression and with consequent phenotypic heterogeneity in an Italian SALS population. Indeed, epidemiological researches reported phenotype variations in age and site of onset, disease duration and also symptomatology in both familial and sporadic ALS, which may reflect the effect of mRNA modulation caused by inter-individual genetic differences [13], [14], [15].