A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease

Abstract

In Parkinson disease, the second most common neurodegenerative disorder in humans, increased alpha-synuclein (SNCA) levels are pathogenic, as evidenced by gene copy number mutations and increased alpha-synuclein levels detected in some familial and sporadic PD cases, respectively. Gene expression can be regulated at the post-transcriptional level by elements in the 3' untranslated region (3'UTR) of mRNAs. The goal of this study was to determine whether the 3'UTR of human SNCA can affect gene expression. Comparative sequence analysis revealed very high conservation across the entire 3'UTR of human SNCA over millions of years, suggesting the presence of multiple functionally important domains. EST and RT-PCR analyses showed that four different polyadenylation events occur in the 3'UTR of human SNCA. Finally, using luciferase assays, we examined the effect of the minor allele of five naturally occurring single nucleotide polymorphisms (SNPs) in the 3'UTR of SNCA on gene expression. The minor allele of SNP rs17016074 increased luciferase expression by 32% in a transient transfection assay in SHSY5Y neuroblastoma cells. Understanding the role of the 3'UTR of human SNCA and identifying functionally important naturally occurring SNPs using reporter assays can complement disease association studies in humans, uncovering potential susceptibility or protective polymorphisms in Parkinson disease. Our findings demonstrate that the 3'UTR of human SNCA, as a whole, and rs17016074, in particular, are loci of potential clinical importance for Parkinson disease.

Fig. 1. Multiple sequence alignment of 12 alpha-synuclein orthologs

The alignment of sequences starts at the top left corner, proceeds down the left column, then continues in the column on the right. Only the first 574 nucleotides of the 3′UTR of human alpha-synuclein are shown. The most frequent (‘consensus’) nucleotide in each column is shown against a black background. Nucleotides most similar to the ‘consensus’ nucleotide in each column are indicated by a grey background. The functional polyA signals are enclosed by black rectangles.

Fig. 2. The 3′UTR sequence of SNCA

The underlined sequence in gray letters is the ORF of human alpha-synuclein. The eight predicted polyA signals are shown in bold (AATAAA), bold italic (ATTAAA), or underlined (TATAAA). The individual letters highlighted in gray indicate the nucleotide immediately upstream of cleavage sites detected by our EST analyses. These nucleotides therefore represent the 3′end (excluding the polyA tail) of all possible alpha-synuclein mRNAs in humans. The 226 nucleotides in italics, underlined and highlighted in gray, constitute the LINE element in the 3′UTR of human alpha-synuclein.

Fig. 3. Alignment of human alpha-synuclein ESTs

A diagram of the 3′UTR of human alpha-synuclein with the position of the functional polyA signals is shown above the alignment, with the 5′ end of the 3′UTR on the left and the 3′ end on the right. The numbers along the line that represents the 3′UTR of human alpha-synuclein indicate the nucleotide position of each functional polyA signal. Each colored horizontal bar represents a single EST. The tissue source of each EST is shown in the left column.

Fig. 4. All known SNPs in the first 574 nt of the 3′UTR of human alpha-synuclein and the effect of the minor allele of rs17016074 on luciferase gene expression

(a) Only the first 574 nt of the 3′UTR of human alpha-synuclein are shown. This gene segment includes the 3′UTR of up to 95% of alpha-synuclein mRNAs in humans. The minor allele of all five SNPs was introduced in luciferase reporter constructs and its effect on gene expression was examined. (b) The luciferase activity of constructs carrying the full-length (574 nt) 3′UTR of SNCA with the major (black line) and minor allele (gray line) of rs17016074 is shown. The set of data depicted represent different experiments performed under a wide range of conditions (e.g., different plasmid preparations).

Fig. 4. All known SNPs in the first 574 nt of the 3′UTR of human alpha-synuclein and the effect of the minor allele of rs17016074 on luciferase gene expression

(a) Only the first 574 nt of the 3′UTR of human alpha-synuclein are shown. This gene segment includes the 3′UTR of up to 95% of alpha-synuclein mRNAs in humans. The minor allele of all five SNPs was introduced in luciferase reporter constructs and its effect on gene expression was examined. (b) The luciferase activity of constructs carrying the full-length (574 nt) 3′UTR of SNCA with the major (black line) and minor allele (gray line) of rs17016074 is shown. The set of data depicted represent different experiments performed under a wide range of conditions (e.g., different plasmid preparations).