Glucocerebrosidase mutations in primary parkinsonism

Abstract

Introduction: Mutations in the lysosomal glucocerebrosidase (GBA) gene increase the risk of Parkinson's Disease (PD). We determined the frequency and relative risk of major GBA mutations in a large series of Italian patients with primary parkinsonism.

Methods: We studied 2766 unrelated consecutive patients with clinical diagnosis of primary degenerative parkinsonism (including 2350 PD), and 1111 controls. The entire cohort was screened for mutations in GBA exons 9 and 10, covering approximately 70% of mutations, including the two most frequent defects, p.N370S and p.L444P.

Results: Four known mutations were identified in heterozygous state: 3 missense mutations (p.N370S, p.L444P, and p.D443N), and the splicing mutation IVS10+1G>T, which results in the in-frame exon-10 skipping. Molecular characterization of 2 additional rare variants, potentially interfering with splicing, suggested a neutral effect. GBA mutations were more frequent in PD (4.5%, RR = 7.2, CI = 3.3-15.3) and in Dementia with Lewy Bodies (DLB) (13.8%, RR = 21.9, CI = 6.8-70.7) than in controls (0.63%). but not in the other forms of parkinsonism such as Progressive Supranuclear Palsy (PSP, 2%), and Corticobasal Degeneration (CBD, 0%). Considering only the PD group, GBA-carriers were younger at onset (52 ± 10 vs. 57 ± 10 years, P < 0.0001) and were more likely to have a positive family history of PD (34% vs. 20%, P < 0.001).

Conclusion: GBA dysfunction is relevant for synucleinopathies, such as PD and DLB, except for MSA, in which pathology involves oligodendrocytes, and the tauopathies PSP and CBD. The risk of developing DLB is three-fold higher than PD, suggesting a more aggressive phenotype.

Keywords: Association analysis; Functional characterization; GBA; Parkinson's disease; Parkinsonism; Splicing mutation.

Fig. 1

In-vivo analysis of the effect of the IVS10+1G>T mutation on GBA pre–mRNA splicing. a) A schematic representation of part of the GBA gene is reported: exons are represented by boxes (the thinner one corresponding to the 3′ untranslated region) and introns by lines. The gene is approximately drawn to scale. Arrows below the relevant exon indicate positions of primers used in RT-PCR experiments. The position of IVS10+1G>T is indicated by an asterisk. b) The results of RT-PCR experiments are shown. Amplified products were obtained from RNA extracted from whole blood of the PD patient carrying the IVS10+1G>T (c.1505+1G>T) mutation, as well as from a healthy donor. Products were separated on a 2% agarose gel; WM: molecular weight marker (λPstI). Two RT-PCR products were concomitantly amplified: one corresponding to the wild-type transcript, and a shorter one, of about 600 bp, possibly representing the transcript generated from the mutant allele. Direct sequencing of this aberrant product demonstrated that it is characterized by the in-frame skipping of the 117-nucleotide-long exon 10. A schematic representation of the two obtained RT-PCR products, together with their length, is also reported. c) RT-PCR experiments were also performed using, in the amplification step, a fluorescent primer. An aliquot of the fluorescent RT-PCR was run on a 3130XL Genetic Analyzer and measured with GeneMapper v4.0 software. The figure shows a GeneMapper window displaying fluorescence peaks corresponding to the 2 molecular species (wild type and mutant). The empty peaks represent the size standard (ROX-500 HD; their length is indicated below the panels), whereas the filled peaks correspond to the RT-PCR labeled products. The X-axis indicates GeneMapper data points and the Y-axis represents fluorescence units (FUs). d) Histograms indicate the quantitative analysis on fluorescence peak areas. For each sample, the sum of all the peak areas was set as 100%; the dark gray portion of the histogram corresponds to the percentage of transcripts containing transcripts lacking exon 10 (Δexon-10), whereas the light gray portion represents the percentage of wild-type isoforms. The expression of the mutant allele amounts to about one fourth of the wild-type allele.