Abstract
Homozygous missense variants in PTRHD1 have been identified to cause parkinsonism by segregation analysis in families. Further replication in the Parkinson’s disease (PD) population might provide additional insight. Here, we analyzed the rare variants in a Chinese PD cohort (Ncase = 1367, Ncontrol = 3298) with whole exome sequencing. We did not identify the variants described in previous studies, and no patient carried other homozygous or compound heterozygous variants of PTRHD1. Three rare variants were identified, but rare variants of PTRHD1 were not enriched in PD. Genetic screening suggested rare variants of PTRHD1 as disease cause were rare in PD in East Asians.
INTRODUCTION
Parkinson’s disease (PD) is a common neurodegenerative disorder caused by the degeneration of dopaminergic neurons in the substantia nigra pars compacta [1]. The exact pathogenesis of PD is complex, and mounting evidence has supported the important role of genetic factors in the susceptibility of PD [2, 3]. Large genome-wide association studies have identified over 90 risk-associated variants [4]. However, the rare protein-coding variants, which might make a major contribution to the missing heritability, were still less explored [2].
Recently, homozygous mutations in PTRHD1 were suggested to cause parkinsonism and intellectual disability by co-segregation analysis in two Iranian families and one African family [5–7]. All the patients in these families presented with early-onset parkinsonism and intellectual disability or cognitive dysfunction. Though the initial links have been established, further exploration in larger cohorts was still necessary. Two recent replication studies in PD did not identify pathogenic coding variants in PTRHD1 [8, 9]. However, considering the rarity of homozygous mutations, further exploration in additional cohorts was still necessary.
In this context, we explored the rare coding variants of PTRHD1 in a large Chinese PD cohort, to examine the putative pathogenicity of PTRHD1 in PD and supplement current knowledge on PTRHD1.
METHODS
Participants
A total of 1,367 Chinese PD patients were recruited from the Department of Neurology of West China Hospital, Sichuan University. The patients were diagnosed by two neurologists specializing in movement disorders according to the Movement Disorder Society clinical diagnostic criteria for PD or the United Kingdom PD Society Brain Bank Clinical Diagnostic Criteria. Among the 1,367 patients, 950 were with early-onset PD (EOPD, age at onset < 50). The controls (n = 3,298) were from the control group of the Taiwanese Schizophrenia Trio Collection recruited from Taiwan [10]. The controls were unaffected parents of probands diagnosed with schizophrenia. West China Hospital approved this study, and all participants have signed informed consent.
Sequencing
The genomic DNA of the patients was extracted from peripheral blood mononuclear cells using standard phenol-chloroform procedures. Then whole exome sequencing was conducted on the Illumina NovaSeq 6000 system following the manufacturer’s instructions [11]. For controls from the Taiwanese Schizophrenia Trio Collection, DNA collected from blood was sequenced on Illumina HiSeq sequencers using paired-end whole-exome sequencing [10].
Variant analysis
The rare variants which met the following criteria were analyzed: 1) minor allele frequency (MAF) was lower than 0.01; 2) variants were annotated as missense, splice donor, splice acceptor, start-lost, stop-gained, stop-loss or frameshift substitution by ANNOVAR [12]; 3) the variant was either heterozygote or homozygote. Allelic association analysis was performed using standard Fisher’s exact test with default parameters, and the a p value below 0.017 (0.05/3) was considered as significant after the Bonferroni correction. The summary data of the East Asians from gnomAD v2.1.1 were also used as population controls.
Gene-based burden analysis
Gene-based rare variant burden analysis was conducted to evaluate the aggregate association of rare variants in PTRHD1 with PD using the optimized sequence kernel association test (SKAT-O, R package) under 1000 permutations. Sex and the first three principal components derived from population structure using GCTA v1.93.1 were adjusted. Principal components were calculated based on common variants (MAF > 0.01) after removing SNPs with missingness > 0.05 or out of Hardy-Weinberg Equilibrium (p < 1E-06) or in linkage disequilibrium. We categorized variants into rare variants (MAF < 0.01) and ultra-rare variants (MAF < 0.001). For each category, we tested the association for all rare variants and rare damaging variants, which were predicted as damaging or pathogenic by at least 5 of 10 in-silico tools implemented in ANNOVAR. Since homozygous mutations are usually associated with early-onset or juvenile PD [13], we further performed burden analysis focusing on EOPD with the same pipeline for all PD patients.
RESULTS
To explore the putative pathogenicity of PTRHD1 in PD, we analyzed rare variants from 1,367 PD patients. The average age at onset (SD) was 46.84 (12.05) with a sex ratio of 1.05 (male/female: 701/666) (Supplementary Table 1). Three rare variants with MAF < 0.01 were identified, including 1 splice donor, 1 missense and 1 stopgain variant (Table 1, Supplementary Table 2). The previously reported pathogenic variants p.Cys52Tyr, p.His53Tyr, c.169_196del, and p.Ala57Argfs*26 (Supplementary Table 3) were not found in our cohort, and no patient carried other homozygous or compound heterozygous mutations of PTRHD1. No significant association was identified at the variant level. Gene-level burden analysis did not detect enrichment of rare variants of PTRHD1 in PD patients or EOPD patients compared with controls for both rare and ultra-rare variants (Table 2).
Rare variants of PTRHD1 identified in onset PD patients
Summary data of the East Asian population from gnomAD is utilized as population controls; MAF, minor allele frequency; Variants with MAF < 0.01 were considered rare; p and OR values were obtained using Fisher’s exact test implemented in R 3.6.2 with default parameters. RefSeq accession numbers: NM_001013663.
Gene-based burden analysis of PTRHD1 variants in Parkinson’s disease
PD, Parkinson’s disease; EOPD, early-onset Parkinson’s disease, Ctrl, Control. Damaging variants were predicted as damaging or pathogenic by at least five out of ten in-silico prediction tools. Case and Ctrl denote the number of alleles detected in patients and controls. Rare variants denote variants with minor allele frequency < 0.01, while ultra-rare variants denote variants with minor allele frequency < 0.001. OR and 95% confidence intervals (CI) were calculated using Fisher’s exact test. p values were calculated with the optimized sequence kernel association test (SKAT-O) under 1000 permutations.
DISCUSSION
Along with the wide application of next-generation sequencing, a number of causative or risk genes for PD have been discovered. However, many of these identified risk genes are still debated and awaiting further replication. Recently, homozygous missense variants in PTRHD1 were suggested to cause parkinsonism by co-segregation analysis in three families, but subsequent replication studies in large PD cohorts did not identify pathogenic variants of PTRHD1. Here, to explore the involvement of PTRHD1 in PD in the Asian population, we analyzed rare coding variants in a large Chinese PD cohort. As a result, we did not find the disease-causing variant described in the previous studies, and no patient carried other homozygous or compound heterozygous variants of PTRHD1. Meanwhile, no significant enrichment was observed for PTRHD1 variants in PD or EOPD, suggesting pathogenicity caused by PTRHD1 was rare in PD.
In the 1367 PD patients, we did not identify the disease-causing variant reported in previous studies. Failure to replicate previous results might be attributed to the confounding effect of ancestral population structure, as the variant was identified by segregation analysis in Iranian and African families. It is difficult to find rare variants from other populations in the homozygous state. Meanwhile, homozygous mutations in PTRHD1 were implicated in parkinsonism and intellectual disability by co-segregation analysis in the original studies [5–7]. Though parkinsonism might cause movement problems similar to those seen in PD such as tremor, bradykinesia and stiffness, parkinsonism also presents with other syndromes like hallucinations. Therefore, PTRHD1 variants might be associated with rare forms of atypical parkinsonism, rather than typical PD. Further replication in patients with parkinsonism was still necessary.
We did not identify other homozygous or compound heterozygous variants of PTRHD1, suggesting PTRHD1 variants as disease causes were rare in PD. In addition, the genetic analysis identified no association between variants of PTRHD1 and PD at allele and gene levels, disproving the involvement of PTRHD1 in PD to some extent. However, such a conclusion should be interpreted with causation since it’s challenging to detect significant association for rare variants in a case-control designed study, especially in the current sample size. Meanwhile, there might be bias in the burden analysis since the case and control might not be age-matched. Additionally, though at gene level rare variants were not enriched in PD patients, we could not rule out the possibility that specific variant might be pathogenic. Therefore, further exploration in larger cohorts was still necessary to better understand the role of PTRHD1 in PD.
In conclusion, current results suggested rare variants of PTRHD1 as disease cause were rare in PD. More robust independent replications are still warranted, to better clarify the role of PTRHD1 in PD.
Footnotes
ACKNOWLEDGMENTS
This research was supported by the funding of the National Key Research and Development Program of China (Grant No. 2021YFC2501200), the Sichuan Science and Technology Program (Grant No. 2022ZDZX0023 and 2021YJ0415) and the National Natural Science Foundation of China (Grant No. 81901294 and 81871000). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
