Clinical and Genetic Analyses of SPG7 in Japanese Patients with Undiagnosed Ataxia

Abstract

Objective

Spastic paraplegia 7 (SPG7) is an autosomal recessive neurodegenerative disorder caused by biallelic pathogenic variants in SPG7. It is predominantly characterized by adult-onset slowly progressive spastic paraparesis. While SPG7 presenting with ataxia with or without spasticity is relatively common in Europe and North America, it is considered rare in Japan. This study aimed to identify SPG7 patients among those with undiagnosed ataxia within the Japanese population.

Methods

We retrospectively selected 351 patients with undiagnosed ataxia, excluding those with secondary and common spinocerebellar ataxia. Whole-exome sequence analysis was conducted, and homozygosity of the identified variants was confirmed using droplet digital polymerase chain reaction (ddPCR).

Results

Among the 351 patients, 2 were diagnosed with SPG7, and homozygosity was confirmed by ddPCR. Both patients carried homozygous pathogenic variants in SPG7: c.1948G＞A, p.Asp650Asn, and c.1192C＞T, p.Arg398Ter (NM_003119.4). Clinically, both patients presented with progressive ataxia. In addition, Patient 1 exhibited partial ophthalmoplegia and spastic paraparesis, whereas Patient 2 demonstrated cerebellar ataxia without spasticity.

Conclusion

The rarity of SPG7 in Japan may be attributed to variation in the minor allele frequency of the c.1529C＞T, p.Ala510Val variant, which is more prevalent in Europe and North America than in other areas.

Introduction

Spastic paraplegia 7 (SPG7) is an autosomal recessive neurodegenerative disorder caused by biallelic pathogenic variants in SPG7, predominantly characterized by adult-onset, slowly progressive spastic paraparesis (1). SPG7 encodes paraplegin, which, along with AFG3L2, forms a high-molecular-weight complex within the inner mitochondrial membrane essential for protein quality control (2).

SPG7 is a pure or complex form of hereditary spastic paraplegia (HSP). A complex phenotype of SPG7 includes cerebellar ataxia (3,4), optic neuropathy, and progressive external ophthalmoplegia (5). While SPG7 patients presenting with ataxia, with or without spasticity, are relatively common and constitute 5-10% of undiagnosed ataxia cases in Europe and North America (3,4), it is considered rare in Japan. As a member of the Japan Spastic Paraplegia Research Consortium (JASPAC) (6,7), the molecular epidemiology of HSP in the Japanese population has been investigated in our laboratory. A study conducted by Ishiura et al. involving a mutational analysis of 129 Japanese patients with HSP found no patients with SPG7 (6). Later, a study of 488 Japanese patients with HSP by Koh et al. identified only 1 patient with SPG7 (7), and there have been few reported cases of SPG7 presenting with spastic paraparesis or ataxia (8,9). Of note, SPG7 can present predominantly as a cerebellar phenotype without spastic paraparesis (3,4), so patients with SPG7 can be diagnosed with spinocerebellar degeneration (SCD) if they do not present with obvious spastic paraparesis. Consequently, some patients may not have been registered in the HSP cohort.

This study investigated patients with SPG7 in a series of patients referred to our department for the molecular diagnosis of ataxia.

Materials and Methods

Patients

Patients with undiagnosed ataxia were selected from a series of patients who had been followed-up at our department or referred to our department for the molecular diagnosis of neurological diseases. Among the series of patients, those with undiagnosed ataxia were selected based on the following criteria: (i) progressive ataxia; (ii) secondary ataxias caused by cerebrovascular diseases, tumors, alcohol abuse, autoimmune diseases, endocrinological disorders, or paraneoplastic syndromes were excluded; (iii) negative test results for common spinocerebellar ataxias (SCAs), namely SCA1, 2, 6, 7, 8, 12, 17, 31, 36, Machado-Joseph disease (MJD), and dentatorubral-pallidoluysian atrophy.

Genetic analyses

All patients provided their written informed consent for this study. Genomic DNA samples were prepared from peripheral blood leukocytes obtained from patients. We conducted whole-exome sequence analysis with the SureSelect Human All Exon V6+UTRs kit (Agilent Technology, Santa Clara, USA) or xGen Exome Research Panel v2 (Integrated DNA Technologies, Coralville, USA) employing HiSeq 2500 (Illumina, San Diego, USA). The sequences were aligned to GRCh37/hg19 using the Burrows-Wheeler Aligner, and variants were called using SAMtools (10,11). We searched for rare variants in SPG7 with a quality score of over 20 and minor allele frequencies less than 0.01 utilizing our in-house exome database of 1,163 Japanese controls.

Copy number analyses using droplet digital polymerase chain reaction (ddPCR)

The copy numbers of the variants were evaluated by ddPCR (12). The sequences of primers and probes used in ddPCR are listed in Supplementary material 1. Detailed protocols are documented in Supplementary material 2. The copy number ratio of SPG7 to AP3B1 was calculated from the ratio of FAM-positive droplets (detection of the pathogenic variant of the target gene SPG7) to HEX-positive droplets (detection of AP3B1 as an internal control).

Results

Genetic analyses

This study included 351 patients with undiagnosed ataxia. A family history of cerebellar ataxia was observed in 133 patients. Whole-exome sequence analysis revealed that two patients harbored homozygous variants in SPG7 [Patient 1 had c.1948G＞A, p.Asp650Asn, and Patient 2 had c.1192C＞T, p.Arg398Ter (NM_003119.4)]. These variants have been reported to be pathogenic in ClinVar (VCV000220101.44 and VCV000545964.24, respectively). A direct nucleotide sequence analysis of PCR products confirmed the presence of each variant (Fig. 1A, B). The minor allele frequency (MAF) of c.1948G＞A, p.Asp650Asn was 0.000009, and that of c.1192C＞T, p.Arg398Ter was 0.000018 in ToMMo 54KJPN.

Figure 1.

Genetic analyses of the present cases. A direct nucleotide sequence analysis showed c.1948 G>A, p.Asp650Asn (NM_003119.4) in Patient 1 (A) and c.1192C>T, p.Arg398Ter in Patient 2 (B). To determine whether the variant was homozygous or hemizygous, a copy number analysis was performed by droplet digital PCR (C). The copy number ratio was calculated as the ratio of FAM-positive droplets (detecting the pathogenic variant of the target gene SPG7) to HEX-positive droplets (detecting AP3B1 as an internal control). The experiment was conducted three times for biological replication (dots). The copy number ratios of SPG7 to AP3B1 were calculated as 1.04 in Patient 1 and 0.97 in Patient 2. The error bars indicate ±1 standard deviation (D).

The determination of zygosity based on ddPCR

There was no reported consanguinity in either patient. To determine whether the variant was homozygous or compound heterozygous with a structural variation, such as a gross deletion, we performed a copy number analysis using ddPCR (Fig. 1C) (13). The copy number ratio of SPG7 to AP3B1 was 1.04 in Patient 1 and 0.97 in Patient 2, confirming the homozygosity of the SPG7 variant in each patient (Fig. 1D).

Clinical presentations of the patients

•Patient 1

A 63-year-old man first noticed gait disturbance and dysarthria at 16 years old. A neurological examination at 18 years old revealed scanning speech, limb and truncal ataxia, hyperreflexia in the lower extremities, and bilateral Babinski signs. The patient was able to walk with a four-legged cane. Brain computed tomography revealed folial atrophy in the cerebellum, leading to a diagnosis of SCD. By 39 years old, slurred speech and overactive bladder became evident. Partial ophthalmoplegia is also observed. His symptoms progressed slowly, and he began to use a wheelchair at 42 years old. Neurological examination at 46 years old revealed weakness in the lower extremities, with a grade of 2 in the proximal muscles and 4 in the distal muscles on the Medical Research Council scale, while the muscle strength in the upper extremities was preserved. Brain magnetic resonance imaging (MRI) at 62 years old showed diffuse cerebellar atrophy (Fig. 2A). At 63 years old, he exhibited slurred speech, partial ophthalmoplegia, ptosis, limb and truncal ataxia, bilateral Babinski signs, weakness and atrophy in the lower extremities, overactive bladder, and hammer toes. The patellar tendon reflex was brisk, and the Achilles tendon reflex was absent. The patient had no family history of neurological disorders. His parents were reported to be non-consanguineous but came from nearby regions.

Figure 2.

Radiological analyses of the present cases. Brain MRI of Patient 1, performed at 62 years old, shows diffuse cerebellar atrophy on T1-weighted sagittal images (A, left) and T2-weighted axial images (A, right). Similarly, the brain MRI of Patient 2, performed at 52 years old, showed diffuse cerebellar atrophy on T1-weighted sagittal images (B, left) and T2-weighted axial images (B, right).

•Patient 2

A 54-year-old man noticed difficulty in maintaining balance while standing on a step ladder. By 47 years old, he had developed dysarthria, and by 50 years old, he had developed gait disturbance. Neurological examination at 52 years old showed slurred and explosive speech, limb and truncal ataxia, and bilateral Babinski signs. There was no hyperreflexia, muscle weakness, or ophthalmoplegia observed. The patient was able to walk with a cane. Brain MRI showed diffuse cerebellar atrophy (Fig. 2B). The patient had no family history of neurological disorders. His parents were reported to be non-consanguineous but came from nearby regions.

Discussion

The clinical phenotypes and variants of SPG7 in these two patients, as well as in previously reported Japanese patients, are summarized in Table (8,9). Progressive external ophthalmoplegia is a well-known feature of SPG7. Pfeffer et al. identified 9 patients with SPG7 among 68 with progressive external ophthalmoplegia of unknown cause. In their study, all nine patients showed both spasticity and ataxia (5). In the present study, Patient 1 exhibited partial ophthalmoplegia, further supporting this association.

Table.

Clinical Phenotypes of SPG7 Reported in Japan.

Patient (References)	1 (This report)	2 (This report)	(8)	(9, Patient 1)	(9, Patient 2)
Age (years)	63	54	64	74	71
Sex	M	M	M	M	M
Age at onset (years)	16	40	34	32	50
Ophthalmoplegia	+	-	+	-	+
Spastic paraparesis	+	-	+	-	+
Lower limb weakness	+	-	+	-	+
Ataxia	+	+	+	+	+
Babinski’s sign	Positive	Positive	Positive	Negative	Positive
Gait	Spastic, ataxic	Ataxic	Spastic, ataxic	Ataxic	Spastic, ataxic
Bladder dysfunction	+	-	-	+	+
Functional status	Wheelchair	Requires cane	Requires support	Wheelchair	Requires cane
Consanguinity	Reportedly -	Reportedly -	+	Reportedly -	Reportedly -
Variant in SPG7 (NM_003119.4)	c.1948G>A, p.Asp650Asn (homozygous)	c.1192C>T, p.Arg398Ter (homozygous)	c.1342dup, p.His448Profs*12 (homozygous)	c.1192C>T, p.Arg398Ter (homozygous)	c.1192C>T, p.Arg398Ter (homozygous)

Ophthalmoplegia, when observed in patients with ataxia, serves as a diagnostic clue for SPG7. In addition, cerebellar ataxia without evident spastic paraparesis was a characteristic clinical presentation in Patient 2. Although both spasticity and ataxia are usually present, ataxia without spasticity has been documented in patients with SPG7. Yahikozawa et al. reported a case of SPG7-associated cerebellar ataxia without spasticity harboring a homozygous c.1192C＞T, p.Arg398Ter variant (9). Cerebellar ataxia, ophthalmoplegia, and spasticity are key features of both SCA1 and MJD. Distinguishing these disorders within the spectrum of spastic ataxia syndromes is important, particularly due to differences in inheritance patterns and prognoses. Both SCA1 and MJD are autosomal dominant disorders caused by CAG repeat expansions, where a larger CAG repeat expansion correlates with more severe symptoms and an earlier onset. By contrast, SPG7, an autosomal recessive form of HSP, typically manifests as adult-onset spastic paraplegia. In the early stages, both SCA1 and MJD frequently present spasticity and hyperreflexia, although these features may change as the disease progresses. Eye movement abnormalities provide further diagnostic clues. SCA1 commonly shows reduced saccade speed, while MJD is characterized by large-amplitude gaze nystagmus with a preserved saccadic speed (13). In SPG7, slowed vertical saccades are characteristic (14). In advanced stages, MJD may develop ophthalmoplegia, a feature rarely seen in SCA1. Patient 1's partial ophthalmoplegia and Patient 2's absence of spasticity further exemplifies the phenotypic spectrum of SPG7. Functional analyses of the variants identified in this study have not yet been conducted. Further studies on the pathogenicity of these variants at the protein level are warranted to deepen our understanding of their role in SPG7-related phenotypes and the underlying mechanisms.

SPG7 is a relatively common cause of ataxia in Europe and North America. Pfeffer et al. found SPG7 in 18.6% (13/70) of patients with undiagnosed ataxia in the United Kingdom, all of whom carried a c.1529C＞T p.Ala510Val variant in at least one allele. Four patients were homozygous for this variant, and nine patients were compound heterozygous with other variants. Initially, 3 out of 17 patients exhibited spasticity, which later developed in 14 of them (3). Sun et al. identified SPG7 in 4.7% (8/170) of patients with undiagnosed ataxia from the United States of America and Canada. Among these, two patients were homozygous for the variant, and three were compound heterozygous. In this group, five patients displayed spasticity (15).

Conversely, SPG7 appears less common in East Asia. In a Chinese case series of familial or sporadic ataxia, SPG7 was identified in 1 out of 60 ataxia patients (16). Similarly, a study from Korea identified SPG7 in 2 out of 77 patients with a family history of ataxia or an age of onset below 50 years old (17). Notably, the c.1529C＞T, p.Ala510Val variant was detected in neither the Chinese nor Korean Studies. In all studies, patients exhibited phenotypes other than ataxia, such as spasticity. Ataxia with spasticity may be classified as SCD or HSP. Thus, the reported frequency of SPG7 in undiagnosed ataxia may not be precise. Given the above, the number of reported SPG7 cases in East Asia is still lower than that in Europe and North America.

The difference in the epidemiology of SPG7 can be attributed to variations in the MAF of the c.1529C＞T, p.Ala510Val variant. This variant is found across various populations, with a MAF of 0.007265 in Europeans and 0 in East Asians (gnomAD v4.1.0). Regional differences in the MAF of this variant may underlie the rarity of SPG7 in East Asia.

The authors state that they have no Conflict of Interest (COI).

Financial Support

This study was supported by the Japan Agency for Medical Research and Development (JP20ek0109491, JP21ek0109491, JP22ek0109491, and JP23ek0109673). This study was also supported by Grants-in-Aid from the Research Committee of Ataxia, The Ministry of Health, Labor, and Welfare of Japan, under grant number JPMH23FC1010.

Supplementary Material

Supplementary Table 1.

Primers and probes for digital droplet PCR

Supplementary Methods 1.

Protocols for digital droplet PCR