The role of RHOT1 and RHOT2 genetic variation on Parkinson disease risk and onset

Abstract

Genetic variation within the mitochondrial pathway contributes to the risk of Parkinson’s disease (PD). Recent genetic analyses have investigated the association between the RHOT1 and RHOT2 genes and PD etiology. Furthermore, 4 mutations in the RHOT1 gene (p.R272Q, p.R450C, p.T351A, p.T610A) have been reported to be potentially associated with disease risk. As part of the International Parkinson Disease Genomics Consortium efforts to evaluate reported PD risk factors, we assessed the role of common and low frequency variants in both RHOT1 and also RHOT2 according to the high degree of homology in their amino acid sequences. Utilizing large-scale genotyping and whole-genome sequencing data from the International Parkinson Disease Genomics Consortium and the Accelerating Medicines Partnership – Parkinson Disease initiative, our analyses did not identify evidence to support the hypothesis that RHOT1 and RHOT2 are disease causing or modifying genes for PD risk or age at onset.

1. Introduction

Mitochondrial function was the first biological process to be associated with Parkinson’s disease (PD) (Hardy et al., 2010). Overexpression of Miro was reported to cause significant loss of dopaminergic neurons in Drosophila and knockout resulted in absence of mitochondria from axons and synapses (Guo et al., 2005; Liu et al., 2012). As a consequence, RHOT1 and RHOT2, encoding Miro1 and Miro2 respectively, were initially investigated as possible candidate genes associated with PD risk. A recent genetic screening of RHOT1 identified 2 PD associated variants (p.R272Q,p.R450C) in 2 unrelated German late-onset cases (Grossmann et al., 2019). An independent study reported 2 additional RHOT1 mutations in 2 German PD patients (p.T351A, p.T610A) (Berenguer-Escuder et al., 2019). Due to the high homology between Miro1 and Miro2 (Fransson et al., 2003), we comprehensively investigate whether genetic variation within RHOT1 and RHOT2 influences PD risk and age at onset utilizing large-scale genotyping and whole-genome sequencing data from the International Parkinson Disease Genomics Consortium (IPDGC) and Accelerating Medicines Partnership – Parkinson Disease (AMP-PD) initiative.

2. Methods

We analyzed IPDGC individual-level genome-wide genotyping data consisting of 14,671 cases and 17,667 healthy controls of European ancestry (Supplementary Table 1). Additionally, we utilized AMP-PD whole-genome sequencing (WGS) data consisting of 1647 cases and 1050 healthy controls from European descent. Variants were annotated using ANNOVAR (Wang et al., 2010) and Fisher’s exact test was used to examine the differences between allele frequencies using PLINK 1.9 (Chang et al., 2015).

To assess the cumulative effect of multiple low frequency variants on the risk for PD, gene-based burden analyses were performed on both the IPDGC Genome wide association study (GWAS) data and AMP-PD WGS data by using RVTESTS (Zhan et al., 2016). Summary statistics from the latest meta-analyses on PD (Nalls et al., 2019) and age at onset (Blauwendraat et al., 2019) were further assessed.

3. Results

Leveraging the IPDGC genotyping data, 62 variants were identified within the RHOT1 gene, of which all were noncoding variants. Additionally, we detected 17 variants in the RHOT2 gene, including 4 missense variants (Supplementary Table 2). We further explored summary statistics from the latest PD risk (Nalls et al., 2019) and age at onset (Blauwendraat et al., 2019) GWAS meta-analyses and did not identify a significant association between RHOT1 and RHOT2 common genetic variation and PD risk or age at onset (Fig. 1; Supplementary Table 2). Gene-based burden analysis was performed to assess the cumulative effect of low-frequency genetic variants in RHOT1 and PD. Following this, we found no evidence for an association between RHOT1 and PD risk (N variants = 7; CMC p = 0.594, Zeggini p = 0.444, MB p = 0.381, SKAT p = 0.385, SKAT-O p = 0.388, Fp p = 0.375). Similarly, we did not identify a link between the RHOT2 gene and PD risk when the aggregated effect of low-frequency genetic variants was assessed (N variants = 3; CMC p = 0.276, Zeggini p = 0.205, MB p = 0.226, SKAT p = 0.224, SKAT-O p = 0.226, Fp = 0.226).

Fig. 1.

(A) Locus zoom plot for RHOT1 variants versus PD risk. Data were obtained from the latest PD GWAS meta-analysis excluding 23andMe data, consisting of 15,056 PD cases, 18,618 UK Biobank proxy-cases, and 449,056 healthy controls (Nalls et al., 2019). (B) Locus zoom plot for RHOT2 variants versus PD risk. (C) Locus zoom plot for RHOT1 variants versus PD age at onset. Data were obtained from the most recent age at onset PD GWAS meta-analysis excluding 23andMe data, consisting of 17,996 patients (Blauwendraat et al., 2019). (D) Locus zoom plot for RHOT2 variants versus PD age at onset. p-values on the log-10 scale are plotted on the left vertical axis and the chromosomal position is plotted along the horizontal axis with the gene names and size of flanking region. The most strongly associated SNPs are indicated by a purple diamond and pairwise LD (r2) with these SNP are indicated by dotted color as described in the legend in the upper right corner. The right vertical axis indicates the regional recombination rate (cM/Mb) which is overlaid in blue. Abbreviations: LD, linkage disequilibrium; PD, Parkinson’s disease; SNP, single nucleotide polymorphism.

Finally, we identified a total of 973 variants within the RHOT1 gene in WGS data, of which 10 were coding variants, including 4 synonymous and 6 nonsynonymous variants. A total of 118 variants were detected in the RHOT2 gene, of which 48 were coding variants, including 25 synonymous and 23 nonsynonymous variants (Supplementary Table 3). Fisher’s exact test did not show a significant enrichment for any of the RHOT1 variants in PD patients versus controls after Bonferroni correction (threshold for significance = 0.05/973= 5.1 × 10⁻⁵) nor for any of the RHOT2 variants (threshold for significance = 0.05/118= 4.2 × 10⁻⁴). Additionally, we found no evidence for an association between RHOT1 low-frequency genetic variation and PD risk, when gene-based burden analyses were applied (N variants = 9; CMC p = 0.452, Zeggini p = 0.452, MB p = 0.650, SKAT p = 0.616, SKAT-O p = 0.678, Fp p = 0.330). Similarly, the RHOT2 gene did not show a consistent cumulative effect on PD risk across all the tests applied (N variants = 40; CMC p = 0.044, Zeggini p = 0.166, MB p = 0.404, SKAT p = 0.125, SKAT-O p = 0.210, Fp p = 0.659).

4. Discussion

Our study aimed at exploring the role of RHOT1 and RHOT2 in PD disease risk and onset by screening large datasets from the IPDGC and AMP-PD initiative. Following our analyses we did not identify evidence to support the hypothesis that RHOT1 and RHOT2 are disease causing or modifying genes for PD in the European population. Utilizing the largest case-control cohorts publicly available to date, we failed to detect a significant enrichment of RHOT1 and RHOT2 risk alleles in PD patients. Moreover, we did not identify a cumulative effect of multiple genetic variants within RHOT1 and RHOT2 on the risk for PD when burden analyses including low-frequency variants were performed. The latter is assuming the limitation that when using WGS data we were unpowered to prove a null effect. The RHOT1 p.T610A variant originally reported by Berenguer-Escuder et al. was the only reported variant present in our WGS data; however, the risk allele was found absent in our cohort of cases and controls (meaning that both cases and controls were carriers for the reference allele). As previously discussed (Blauwendraat et al., 2017), considering that the lifetime risk of developing PD is of 1.3%–2% (depending on sex) (Elbaz et al., 2002), one would expect ~1300 non-Finnish European individuals from the gnomAD population to develop PD. The reported RHOT1 p.T610A variant has been detected in 3 individuals out of a total of 64,336 included in the gnomAD database, which would represent around ~0.005% of all estimated PD cases. We did not identify this variant in any of the screened cases or controls. Given the low frequency of the RHOT1 p.T610A variant, we cannot completely discard a possible effect on PD risk. In summary, although genetic discoveries have lent support to the notion that mitochondrial impairment plays a major role on PD disease and onset, our results do not provide enough evidence to confirm RHOT1 and RHOT2 as major contributors to disease etiology in the European population.