Introduction

Hereditary spastic paraplegias (HSPs) are characterized by various inherited disorders in which weakness and spasticity of the lower extremities are the predominant symptoms [1]. HSPs are classified into a pure or complicated form according to the absence or presence of complications, respectively. The clinical presentations of complicated forms include ataxia, mental retardation, parkinsonism, dementia, and neuropathy. To date, the causative genes or loci of HSPs, from SPG1 to SPG79, have been reported. Furthermore, other diseases, including Charcot-Marie-Tooth disease, Parkinson disease (PD), amyotrophic lateral sclerosis, and spinocerebellar ataxia include spastic paraplegia as one of their clinical presentations, emphasizing that a lot of causative genes responsible for HSPs and other neurological diseases overlap. Therefore, gene analysis is necessary to confirm the causative genes of HSPs.

Phospholipase A2 group VI (PLA2G6) encodes a calcium-independent phospholipase. Mutations of it cause PLA2G6-associated neurodegeneration (PLAN), including infantile neuroaxonal dystrophy 1 (INAD, OMIM #256600), neurodegeneration with brain iron accumulation 2B (NBIA, OMIM #610217), and dystonia-parkinsonism [2, 3]. Very recently, PLA2G6 mutations were reported to be a cause of spastic paraplegias [4, 5]. Therefore, we screened our HSP patients to find HSPs associated with PLA2G6 mutations, and to reveal the frequencies and clinical findings.

Clinical and genetic study

In this study, 383 HSP patients were recruited through the Japan Spastic Paraplegia Research Consortium (JASPAC), which was established in 2006 to clarify the molecular epidemiology of HSPs in Japan and the pathogenesis of HSPs. The JASPAC collected Japanese patients who exhibited leg spasticity with/without other complications, including mental retardation, parkinsonism, and others. Furthermore, the JASPAC collected patients having affected parents or children, probands having an affected brother or sister, probands having parents of consanguineous marriage, and probands showing a complicated form with normal parents. This study was approved by our institutional review board, and written informed consent was obtained from all the participants.

We performed whole-exome analysis on the 383 Japanese patients to find PLA2G6 mutations, and confirmed the mutations in the probands and their families by Sanger sequencing. Known genes, SPG1-79, were excluded by whole-exome sequencing. Exon capture was performed using a Sureselect Human All Exon V4 + UTRs Kit, followed by massively parallel sequencing using an Illumina HiSeq 2000 (100 bp paired end). We aligned the exome data with a Burrows-Wheeler Aligner, and extracted single-nucleotide variations and short insertion/deletions using SAMtools [6]. We referred to the NCBI Reference Sequence (NM_003560.2) for PLA2G6.

We found six patients with PLA2G6 mutations in four Japanese families. The family trees and gene analysis results are shown in Fig. 1. Four probands (patients A-II-1, B-II-3, C-II-1, and D-II-3) had the p.Q173*/p.K545R, p.L221P/p.D331Y, p.R645*/c.1187-2 A > G, and p.R635Q/p.R635Q mutations, respectively. According to the co-segregation study, unaffected individuals A-I-1, A-I-2, B-I-2, B-II-2, C-I-1, and C-I-2 had a heterozygous mutation of p.Q173*, p.K545R, p.D331Y, p.L221P, c.1187-2 A > G, and p.R645*, respectively. These results indicate that patients A-II-1, A-II-2, B-II-3, C-II-1, and C-II-2 had compound heterozygous mutations of PLA2G6. Mutations of p.Q173* and p.L221P are not registered in ExAC [7] (accessed on 18 June 2018), Human Genetic Variation Database [8] (accessed on 18 June 2018), Integrative Japanese Genome Variation Database [9] (accessed on 18 June 2018), or 1261 in-house data. Other mutations have been reported to be causative mutations for NBIA, INAD, and PD [10,11,12,13,14]. We evaluated the functional prediction of PLA2G6 mutations by means of in silico algorithms using PolyPhen-2 [15], SIFT [16], and the PHRED-like CADD score [17] (Table 1).

Fig. 1
figure 1

a Pedigree charts and mutations. Squares and circles indicate males and females, respectively. Filled symbols indicate affected individuals, whereas open symbols indicate unaffected individuals. The slash and double lines indicate deceased and consanguineous marriage, respectively. wt wild type. b Brain MRI of patients. Five patients (A-II-1 and 2, B-II-3, and C-II-1 and 2) showed mild cerebellar atrophy. C-II-1 and 2 showed hypointensity of the globus pallidum on T2* MRI

Full size image
Table 1 Genetic findings in four families with PLA2G6 mutations
Full size table

A summary of the clinical findings is presented in Table 2. The patients in families A, B, and C exhibited infantile/child-onset spastic paraplegia with cerebellar atrophy and mental retardation, this being similar to the clinical spectrum of atypical neuroaxonal dystrophy. Meanwhile, a female patient (D-II-3) showed adult-onset spastic paraplegia with parkinsonism. Her blood serum was positive for anti-human T-lymphotropic virus (HTLV)-1 antibody but her cerebrospinal fluid (CSF) was negative. Moreover, her elder sister (D-II-2) had spastic paraplegia similar to her (D-II-3). Brain magnetic resonance imaging (MRI) showed cerebellar atrophy in all infantile/child-onset patients, i.e., A-II-1, A-II-2, B-II-3, C-II-1, and C-II-2. T2* MRI revealed hypointensity of the globus pallidus in C-II-1 and C-II-2, this being characteristic of PLAN. However, brain MRI showed no abnormalities in the adult-onset patient (D-II-3).

Table 2 Clinical findings in four families with PLA2G6 mutations
Full size table

Discussion

In this study, we found six patients with PLA2G6 mutations in four families among 383 HSP patients on exome sequencing. HSP caused by PLA2G6 mutations was found in 1.05% (4/383) of the patients with the clinical diagnosis of HSP in Japan. Three families exhibited a uniform phenotype of infantile/child-onset complicated HSP with mental retardation, and cerebellar ataxia, this being similar to atypical neuroaxonal dystrophy. Furthermore, this study revealed six asymptomatic individuals, indicating that loss of function of PLA2G6 causes PLAN.

We found seven mutations comprising two novel ones (p.Q173* and p.L221P), two causing INAD (c.1187-2 A > G and p.R645*), two causing Parkinson disease (p.D331Y and p.R635Q), and one causing NBIA (p.K545R) [10,11,12,13,14]. These mutations were reported to be compound heterozygous ones, indicating that the INADs ones were caused by c.1187-2 A > G/p.R538C and p.R645*/p.L354P, the PD ones by p.D331Y/p.M358Ifs* and p.R635Q/p.Q452*, and the NBIA ones by p.K545R/p.M358Ifs* [10,11,12,13,14]. We found that mutations of PD and NBIA also caused a spectrum of atypical neuroaxonal dystrophy in this study.

On the other hand, we describe a late-onset spastic paraplegia patient (D-II-3) with extrapyramidal signs with a positive anti-HTLV-1 antibody result for blood serum and a negative one for CSF. Although patients with HTLV-1 infection sometimes exhibit HTLV-1-associated myelopathy (HAM) showing spasticity, they must have positive anti-HTLV-1 antibody results for both blood serum and CSF. In addition, patients with HAM do not show parkinsonism, and the elder sister also showed spastic paraplegia. Thus, we diagnosed her as having HSP. The average onset age of dystonia-parkinsonism is 20s–30s [3]. However, that for D-II-3 was 66 years. This might indicate that she has a new phenotype of late-onset PLAN as a pure form of HSP.

Brain T2* MRI was performed in one family. It showed hypointensity of the globus pallidus, which is one of the characteristic findings for PLAN. This indicates that brain T2* MRI may be useful for examining the possibility of PLAN. Thus, brain T2* MRI would be useful for differential diagnosis of HSPs when a patient shows spastic paraplegia with cerebellar atrophy and mental retardation.

PLA2G6 mutations lead to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction [18]. However, what causes the phenotype differences has not been clarified. Therefore, we should perform a phenotype-genotype correlation study to elucidate the molecular mechanism underlying PLAN.

Accession numbers

LC413511, LC413512, LC413513, LC413514, LC413515, LC413516, LC413517, LC413518, LC413519, LC413520, LC413521, LC413522, LC413523, LC413524, LC413525, LC413526, and LC413527.