The effect of LRRK2 loss-of-function variants in humans

Abstract

Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes^1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson's disease^3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. While preclinical studies in model organisms have raised some on-target toxicity concerns^5-8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here, we systematically analyze pLoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)⁹, 49,960 exome-sequenced individuals from the UK Biobank and over 4 million participants in the 23andMe genotyped dataset. After stringent variant curation, we identify 1,455 individuals with high-confidence pLoF variants in LRRK2. Experimental validation of three variants, combined with previous work¹⁰, confirmed reduced protein levels in 82.5% of our cohort. We show that heterozygous pLoF variants in LRRK2 reduce LRRK2 protein levels but that these are not strongly associated with any specific phenotype or disease state. Our results demonstrate the value of large-scale genomic databases and phenotyping of human loss-of-function carriers for target validation in drug discovery.

Fig. 1. Annotation and curation of candidate LRRK2 pLoF variants.

a, Flow chart showing the variant filtering and curation of candidate LRRK2 LoF variants in the gnomAD, UK Biobank and 23andMe cohorts. Of the 1,103 carriers identified in 23andMe, 749 were confirmed by Sanger sequencing with the remainder untested. b, The ancestry distribution of LRRK2 pLoF variant carriers in gnomAD. AFR, African/African American; AMR, American/Latino; ASJ, Ashkenazi Jewish; EAS, East Asian; FIN, Finnish; NFE, non-Finnish European; SAS, South Asian. The pLoF variants seen more than ten times appear in color with remaining variants in gray. LRRK2 pLoF variants are mostly individually extremely rare (less than 1 in 10,000 carrier frequency), with the exception of two nonsense variants almost exclusively restricted to the admixed AMR population (Cys1313Ter and Arg1725Ter) and two largely NFE-specific variants (Leu2063Ter and Arg772Ter). All variant protein descriptions are with respect to ENSP00000298910.7. c, Schematic of the LRRK2 gene with pLoF variants marked by position, with the height of the marker corresponding to allele count in gnomAD (gray bars) and UK Biobank (blue bars). The 51 exons are shown as rectangles colored by protein domain, with the remaining exons in gray. The three variants genotyped in the 23andMe cohort are annotated with their sample count in black text.

Fig. 2. LRRK2 pLoF heterozygotes have reduced LRRK2 protein compared to cells harboring no LoF variants.

a, Immunoblot of LRRK2 and loading control GAPDH on LCLs from five individuals harboring no pLoF variants (LRRK2-WT) and three individuals harboring a heterozygous (Het) pLoF variant (Cys1313Ter; 12-40699748-T-A; Arg1483Ter; 12-40704362-C-T). Experiments were repeated ten times with similar results. b, Immunoblot of LRRK2, alpha-actinin (specific to muscle) and GAPDH on three control lines and one CRISPR/Cas9-engineered LRRK2 heterozygous line of cardiomyocytes differentiated from embryonic stem cells (ESCs) (Arg1693Ter-12-40714897-C-T). All variant protein descriptions are with respect to ENSP00000298910.7. Experiments were repeated five times with similar results. Source data

Fig. 3. LRRK2 pLoF variants are not strongly associated with either age distribution or any adverse phenotypes.

a, The age distributions of LRRK2 pLoF carriers are not significantly different from those of noncarriers in both gnomAD and 23andMe. Note that this analysis is based on age at sample collection. b, Manhattan plot of phenome-wide association study results for carriers of three LRRK2 pLoF variants against noncarriers in the 23andMe cohort. Each point represents a distinct phenotype, with these grouped into related categories (delineated by alternating black and gray points). The dotted horizontal line represents a Bonferroni-corrected P value threshold for 366 tests. Logistic regression was used for binary phenotypes and linear regression for quantitative phenotypes controlling for age, sex, genotyping platform and the first ten genetic principal components. Full association statistics are listed in Supplementary Table 8.

Fig. 4. LRRK2 pLoF carriers do not have impaired lung, liver or kidney function.

For all plots, points for individual pLoF carriers are shown in teal and noncarriers in gray. The mean and 1 × s.d. are represented by the black circle and line. a, Urine biomarkers albumin and creatinine were transformed into two clinical markers of kidney function (Methods). No pLoF carriers showed signs of severely impaired kidney function. ACR, albumin to creatinine ratio. b, Z scores of age-, sex- and height-corrected spirometry measures of lung function. FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 s. c, Blood serum biomarkers of liver function. The plots for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin and creatinine were top-truncated, removing 47, 29, 92, 8 and 27 noncarriers respectively. The violin plots and summary statistics were calculated on the full dataset. All pLoF carriers are within each plot area.

Extended Data Fig. 1. Ethnicity distribution of LRRK2 LoF carriers in the 23andMe cohort.

Bars are coloured according to the detected variant. AFR, African/African American; AMR, American/Latino; NFE, non-Finnish European; SAS, South Asian.

Extended Data Fig. 2. Details of CRISPR/Cas9-engineered embryonic stem cells and cardiomyocyte differentiation.

a, Sanger sequence of isogenic hESC engineered colony for heterozygous LRRK2 variant 10, clone 13 (GRCh37:12-40714897-C-T). The engineered cell line maintains b, a normal karyotype, c, normal colony morphology and expression of OCT4, and d, differentiates into the cardiomyocyte lineage. The bright field image of cardiomyocytes was captured at day 17 of the differentiation protocol. The cardiomyocyte differentiation was repeated 12 times and the staining for Oct4 was repeated on 30 independent colonies.

Extended Data Fig. 3. IGV visualization of the splice donor variant GRCh37:12-40626187-T-C in the GTEx LRRK2 pLoF carrier exome sequencing data and lung tissue RNA-seq data compared to a control GTEx lung RNA-seq sample.

The pLoF variant is observed on reads containing an anchoring missense variant, GRCh37:12-40626185-A-G (A green and G orange), and these reads are presenting normal splicing as seen in the control RNA-seq sample.

Extended Data Fig. 4. Sex distribution of LRRK2 pLoF carriers, G2019S risk allele carriers and non-carriers in the UK Biobank.

Males are shown in dark grey and females in light grey.

Extended Data Fig. 5. Age distribution of LRRK2 pLoF carriers, G2019S risk allele carriers and non-carriers in the UK Biobank.

Data are shown as overlapping density plots.