African ancestry neurodegeneration risk variant disrupts an intronic branchpoint in GBA1

Abstract

Recently, a novel African ancestry specific Parkinson's disease (PD) risk signal was identified at the gene encoding glucocerebrosidase (GBA1). This variant (rs3115534-G) is carried by ~50% of West African PD cases and imparts a dose-dependent increase in risk for disease. The risk variant has varied frequencies across African ancestry groups, but is almost absent in European and Asian ancestry populations. GBA1 is a gene of high clinical and therapeutic interest. Damaging bi-allelic protein-coding variants cause Gaucher disease and mono-allelic variants confer risk for PD and Dementia with Lewy Bodies, likely by reducing the function of glucocerebrosidase. Interestingly, the novel African ancestry specific GBA1 risk variant is a non-coding variant, suggesting a different mechanism of action. Using full length RNA transcript sequencing, we identified intron 8 expression in risk variant carriers (G) but not in non-variant carriers (T). Antibodies targeting the N-terminus of glucocerebrosidase showed that this intron-retained isoform is likely not protein coding and subsequent proteomics did not identify a shorter protein isoform, suggesting the disease mechanism is RNA-based. CRISPR editing of the reported index variant (rs3115534) revealed that this is the responsible sequence alteration driving production of these intron 8 containing transcripts. Follow-up analysis of this variant showed that it is in a key intronic branchpoint sequence and therefore has important implications in splicing and disease. In addition, when measuring glucocerebrosidase activity we identified a dose-dependent reduction in risk variant carriers (G). Overall, we report the functional effect of a GBA1 non-coding risk variant, which acts by interfering with the splicing of functional GBA1 transcripts, resulting in reduced protein levels and reduced glucocerebrosidase activity. This understanding reveals a novel therapeutic target in an underserved and underrepresented population.

Figure 1:. Overview of the African ancestry Parkinson’s disease GBA1 GWAS locus.

A) LocusZoom plot showing rs3115534 as index variant (purple diamond) and located in intron 8 of GBA1. B) rs3115534 as eQTL for GBA1 RNA expression from Kachuri et al 2023 showing increased GBA1 expression with G risk genotypes. C) rs3115534 as pQTL for GBA1 protein expression from Surapaneni et al 2022 showing decreased protein levels with G risk genotypes. For all boxplots, the center line represents the median, edges of box represent Q1 and Q3, and ends of bars represent the maximum and minimum not including outliers.

Figure 2:. GBA1 intron 8 expression is correlated with rs3115534 genotype.

A) Oxford Nanopore Technologies long-read RNA sequencing of 8 lymphoblastoid cell lines (LCL) shows a consistent pattern where the rs3115534-G risk allele is associated with intron 8 expression and absent in the homozygous T (non-risk allele) individuals generated using Integrative Genomics Viewer. B) Quantification of intron 8 expression is significantly associated with the G allele in both the 40bp region prior to exon 9 and C) the full intron 8 (linear regression, p < 0.05). D,E) No significant differences were identified in the two neighboring exons 8 and 9. Coverage for all panels normalized by dividing mean depth by total number of mapped reads per million as detailed in methods. Error bars represent standard deviation for all panels.

Figure 3:. Increased intron 8 expression across datasets in G allele carriers.

A) Intron 8 coverage from human frontal cortex sequenced with Oxford Nanopore (n=8). Intron 8 expression is only present in G allele carriers but does not reach statistical significance (p=0.281) likely due to a smaller sample size. B) Intron 8 coverage from LCLs (n-18) sequenced with Illumina. Expression is significantly associated with G allele (p=3.88E-5). C) Intron 8 expression from human frontal cortex sequenced with Illumina (n=92). Expression is significantly associated with G allele (p=2.76E-15). D) Intron 8 coverage from LCLs in 1000 Genomes cohort (n=88). Expression is significantly associated with G allele (p=4.08E-31). E) Intron 8 coverage from blood in AMP-PD cohort (n=148). Expression is significantly associated with G allele (p=8.05E-7). E) CRISPR editing of two lymphoblastoid cell lines shows that the rs3115534-G risk allele is significantly associated with intron 8 expression (p=0.036). Coverage for all panels normalized by dividing mean regional depth by total number of mapped reads per million as detailed in methods. Error bars represent standard deviation for all panels.

Figure 4:. The GBA1 intronic rs3115534 variant acts as a splicing branchpoint.

The causal variant rs3115534 (highlighted in gray on the top and in red dashed box on the bottom) in intron 8 is located in the key splicing branchpoint according to Branchpointer. When rs3115534 in non-risk state (T), on the antisense strand, the A allele functions as a branch site for the spliceosome, while in the risk state (G), on the antisense strand, the C allele disrupts this branch site resulting in abnormal splicing.

Figure 5:. Measuring GCase activity across genotypes.

A) GCase activity was measured across rs3115534 GBA1 genotypes showing a significant dose G-allele dependent reduction across genotypes. B) When measuring GCase activity across multiple GBA1 genotypes it appears that rs3115534-GT and rs3115534-GG reduce GCase activity to similar levels as PD risk variants (E326K and T369M) but remain higher than GBA1 Gaucher causing variants (GBA1_mild eg. N370S and GBA1_severe eg. L444P).

Figure 6:. Suggested variant to function hypothesis of rs3115534.

rs3115534-T is transcribed to pre-mRNA as an A–a highly conserved branchpoint nucleotide. However, rs3115534-G, which confers elevated PD risk, is instead transcribed to a C, causing the observed branchpoint disruption (rs3115534 denoted as red star). This single nucleotide change in intron 8 impacts splicing by disrupting the normal binding from the adenosine branchpoint nucleotide and the 5’ splice site (5’ss) (GU). Subsequently, an alternative branchpoint is utilized, uncovering an alternative 3’ splice site (3’ss) upstream of the normal splice site immediately proximal to exon 9 and resulting in partial and complete intron retention and fewer functional GBA1 mRNA transcripts. Downstream, abnormal splicing of GBA1 leads to reduced GCase protein and subsequent lower GCase activity which is a known pathomechanism of PD/DLB. 3’ and 5’ss refers to splice sites. Generated with biorender.com.