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Parkinson's disease (PD, OMIM no. 168600) is the second most common neurodegenerative disorder next to Alzheimer's disease. Although the cause remains unclear, PD is thought to be a heterogeneous disease caused by the interaction of multiple genetic factors and environmental factors associated with aging. Indeed, case–control studies identified some genetic risk factors for PD, such as SNCA,1, 2, 3, 4 LRRK23, 4, 5, 6, 7, 8 and GBA variants.9, 10, 11 To elucidate the exact etiology of PD, identifying the effect of each of the multiple factors and their combined effects is important.

Recently, PLA2G6 was reported to be the causative gene for PARK14 in patients with autosomal recessive early-onset dystonia-parkinsonism.12 PLA2G6 is also the causative gene for infantile neuroaxonal dystrophy, neurodegeneration associated with brain iron accumulation and Karak syndrome.13, 14, 15 Some patients with neurodegeneration associated with brain iron accumulation show very early-onset and rapid psychomotor regression, early cerebellar signs, pyramidal signs and visual disturbances. Patients with PLA2G6 mutations frequently exhibit brain iron accumulation, which is a feature of neurodegeneration associated with brain iron accumulation. In our recent study, we revealed two novel compound heterozygous PLA2G6 mutations in Japanese patients who had levodopa-responsive parkinsonism with or without brain iron accumulation.16 Although there are few PLA2G6 mutation analyses in parkinsonism so far, its role in parkinsonism or PD and the mechanism of neurodegeneration and iron accumulation have not been clarified.

Very recently, Tan et al.17 in Singapore reported the results of PLA2G6 analysis in 96 PD patients with young-age onset/dystonia. One of the 96 patients, who had a novel heterozygous p.P806R (c.2417C>G) mutation in exon 17, had typical features of late-onset PD with levodopa responsiveness and dystonic spasms. Although they could not conduct a segregation analysis, this mutation was not found in 100 healthy controls. Their result emphasized the potential role of this mutation and the PLA2G6 mutation as pathogenic mutations or risk factors for PD in Chinese or other races. To confirm this intriguing finding of PLA2G6, we conducted an extended mutation analysis and association study in Japanese patients with sporadic PD and normal controls.

The study was approved by the Institutional Review Board of Juntendo University, and all subjects provided an informed consent. We collected blood samples from each participant and extracted genomic DNA by using standard methods. Sequences of the primers/probes and conditions of PCR/sequencing are available upon request to the corresponding author or the first author. We directly sequenced the exon 17 of PLA2G6 from 379 Japanese patients with sporadic PD and 310 normal Japanese subjects as controls (Table 1).

Table 1 Profile of analyzed subjects and allele frequency of PLA2G6 p.P806R (c.2417C>G) in Japanese patients with sporadic Parkinson's disease (SPD) and control subjects from the general population
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We identified a heterozygous p.P806R mutation in 12 patients with PD and in 10 controls (χ2=0.0019, P=0.96; odds ratio (genotype)=1.02, 95% confidence interval: 0.44–2.37, Table 1). We found no homozygous p.P806R mutations. The allele frequency was 1.58% in sporadic PD and 1.61% in controls. We also found heterozygous synonymous p.T787T (c.2355C>T) variant in two patients and one control. No other variants were found in exon 17. Moreover, we performed direct sequencing of all exons and exon–intron boundaries of PLA2G6 in 116 Japanese patients with sporadic PD (males 60, females 56; age range, 12–92 years; mean age, 60.7±18.1 years; mean disease duration, 6.3±5.8 years). Among them, we found two novel single heterozygous non-synonymous variants (p.R301C, p.D331N). Both frequencies of the two variants were 1/379=0.26% in patients and 0/310=0% in Japanese normal controls. The roles of their rare variants found in Japanese patients with sporadic PD remained unclear (Table 2).

Table 2 PLA2G6 variants (excluding p.P806R) found in patients with sporadic PD and the allele frequency
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The reported clinical features of neurodegeneration associated with mutations in the PLA2G6 gene (PLAN) are axonal dystrophy, dystonia, dementia, visual disturbances, cerebellar signs and brain atrophy with or without iron accumulation.12, 13, 14, 15, 18, 19 Showing clinical heterogeneity, patients with PARK14-linked parkinsonism have levodopa responsiveness, levodopa-induced dyskinesia and dementia with an older-age onset and a longer disease duration than those with infantile neuroaxonal dystrophy.12, 16 These studies have suggested that patients with PLA2G6 mutation can show heterogeneous phenotype.

Although the precise function of PLA2G6 in neurodegeneration and iron accumulation remains obscure, defective phospholipid metabolism is implicated in neurodegenerative diseases featuring brain iron dyshomeostasis.14 PLA2G6 is thought to be responsible for the development of autosomal recessive disorders through its loss of function; hence, the role of a single heterozygous PLA2G6 mutation is intriguing. Indeed, two of the 10 infantile neuroaxonal dystrophy patients were previously reported to have one-allele mutations, suggesting that single heterozygous mutation in PLA2G6 could be pathogenic.19

The aim of this study was to clarify the role of the PLA2G6 mutation in PD. Although patients with PLA2G6 mutations have been reported to show atypical parkinsonism, the heterozygous PLA2G6 p.P806R mutation was found in late-onset PD patients with typical parkinsonism.17 In our extended case–controlled study of a large sample size, no association of PLA2G6 p.P806R was identified in Japanese PD patients and controls. Thus, our data suggest that PLA2G6 p.P806R is a non-PD-associated polymorphism at least in Japanese PD patients. This result should help clinicians in genetic counseling for PD patients.

Furthermore, in the previous report, there were no other possible PD-associated variants in any of the 17 exons in the 96 PD patients.17 Therefore, combined with the data from Singapore,17 our findings emphasize that PLA2G6 mutations are unlikely to be the major causes or risk factors of PD at least in Asian populations.

However, because there have been no adequate PLA2G6 mutation analyses in parkinsonism, disease-associated variants in PLA2G6 could exist in patients with atypical/typical parkinsonism, or PD in specific races. In parkinsonism-dystonia patients, PLA2G6 mutations have thus far been reported in only certain populations, such as Indians, Pakistanis and Iranians.12, 15, 19 In heterogeneous clinical setting of patients with PLA2G6 mutations, the roles of PLA2G6 should be clarified including the effect of heterozygous mutation. As brain iron accumulation is frequently observed in common diseases, such as PD and Alzheimer's disease, the role of PLA2G6 in iron accumulation is elusive in neurodegenerative disorders.

Thus, further large studies in various populations and functional studies for PLA2G6 are needed in neurodegenerative disorders with or without brain iron accumulation.