Variation in the miRNA-433 binding site of FGF20 confers risk for Parkinson disease by overexpression of alpha-synuclein

Abstract

Parkinson disease (PD) is a common neurodegenerative disorder caused by environmental and genetic factors. We have previously shown linkage of PD to chromosome 8p. Subsequently, fibroblast growth factor 20 (FGF20) at 8p21.3-22 was identified as a risk factor in several association studies. To identify the risk-conferring polymorphism in FGF20, we performed genetic and functional analysis of single-nucleotide polymorphisms within the gene. In a sample of 729 nuclear families with 1089 affected and 1165 unaffected individuals, the strongest evidence of association came from rs12720208 in the 3' untranslated region of FGF20. We show in several functional assays that the risk allele for rs12720208 disrupts a binding site for microRNA-433, increasing translation of FGF20 in vitro and in vivo. In a cell-based system and in PD brains, this increase in translation of FGF20 is correlated with increased alpha-synuclein expression, which has previously been shown to cause PD through both overexpression and point mutations. We suggest a novel mechanism of action for PD risk in which the modulation of the susceptibility gene's translation by common variations interfere with the regulation mechanisms of microRNA. We propose this is likely to be a common mechanism of genetic modulation of individual susceptibility to complex disease.

Figure 1

Gene Structure of FGF20 Exons are denoted by blocks; coding regions are shown in gray, and 3′ UTRs are shown in white. Locations of SNPs are indicated by arrows. Underlined SNPs were previously genotyped in the initial study.

Figure 2

Characterization of miR-433 and Functional Analysis of miR-433 Targeting at the 3′ UTR of FGF20 Gene (A) The predicted binding site for miR-433 at 3′ UTR of FGF20 gene. At rs12720208, allele C base paired with G in Watson-Crick mode (as shown with a solid line), whereas allele T wobble base paired with G (as shown with a dashed line). (B) The precursor molecule pre-miR-433 proceeds to matured miR-433. (C) The construct of pRL-SV40-FGF20-3′ UTR contains SV40 promoter, renilla luciferase gene, and full-length 3′ UTR of FGF20 gene with different alleles. (D) The expression of renilla luciferase in Neuro2A cells transfected by pRL-SV40-FGF20-3′ UTR was measured by relative luciferase activity. Relative luciferase activity stands for renilla luciferase activity normalized by firefly luciferase activity which is the internal control. MiR-433 (1–100 pmol) decreased the expression level of renilla luciferase significantly in allele C constructs but not in allele T constructs (^∗∗ indicates p < 0.01). Data are mean ± standard error (SE). (E) The expression of renilla luciferase is significantly higher in neuro2A cells transfected by pRL-SV40-FGF20-21bp deletion than the constructs containing either C or T alleles under miR-433 (10 pmol). Data are mean ± SE.

Figure 3

Immunoblot Analysis of Translation Levels of FGF20 and α-Synuclein in Different Genotypic Fibroblasts and Human Brains (A) MiR-433 at 1 and 10 pmol significantly suppresses FGF20 protein level in the homozygote allele C and heterozygote at rs12720208 fibroblasts. FGF20 level is much higher in heterozygote cells than in homozygote allele C cells. In the heterozygote at rs12720208 fibroblasts, treatment with miR-433 for 24 hr does not change much of monomer form FGF20 (20 KD) but dramatically decreases the oligomer form of FGF20 (100 KD). The blot was re-probed with β-actin antibody showing the same amount loading. (B) The human brain of homozygous allele T at rs12720208 has the highest level of both FGF20 and α-synuclein, comparing with the human brains of homozygous allele C or heterozygous at rs12720208. The blot was re-probed with β-actin antibody showing the same amount loading. (C) α-synuclein translation amount is dramatically increased in SH-SY5Y cells after incubation with 50ng/ml FGF20 for 24 hr. The blot was re-probed with β-actin antibody showing the same amount loading. (D) Quantification of immunoblotted α-synuclein in SH-SY5Y cells treated by FGF20. α-synuclein-band densities were normalized to their counterpart β-actin-band densities and represented the mean ± SE of three independent experiments (^∗∗ indicates p < .01).