A Japanese SPG4 Patient with a Confirmed De Novo Mutation of the SPAST Gene.

Spastic paraplegia type 4 (SPG4) is caused by mutations of the SPAST gene and is the most common form of autosomal-dominantly inherited pure hereditary spastic paraplegia (HSP). We herein report a Japanese patient with SPG4 with a confirmed de novo mutation of SPAST. On exome sequencing and Sanger sequencing, we identified the heterozygous missense mutation p.R460L in the SPAST gene. This mutation was absent in the parents, and the paternity and maternity of the parents were both confirmed. The patient showed a pure SPG4 phenotype with an infantile onset. This study may expand the clinical and genetic findings for SPG4.

Introduction

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive weakness and spasticity in the lower limbs due to pyramidal tract dysfunction (1). Spastic paraplegia type 4 (SPG4) is due to heterozygous mutations of the SPAST gene and is the most frequent cause of both familial and sporadic HSP (2). However, sporadic SPG4 patients are generally attributed to common mechanisms like incomplete penetrance, somatic mosaicism, non-paternity, and inadequate clinical assessment of the parents (3). True de novo occurrence of a SPAST mutation, where both parents of the patient are proven not to have the mutation in lymphocytes, appears to be rare. Thus far, true de novo SPAST mutations have been reported in American, Brazilian, Canadian, Czech, Dutch, French, German, Greek, Italian, and Polish SPG4 families (3-13). However, the paternity and maternity of the parents have rarely been assessed to confirm the de novo occurrence.

We herein report a Japanese patient with a clinically pure phenotype of SPG4 with a de novo mutation of SPAST.

Case Report

A 23-year-old woman (Figure A, II-2) was the second of two siblings born to healthy, unrelated parents. Her 26-year-old brother was unaffected. She was born by vaginal delivery after an uneventful pregnancy. Her parents initially became concerned when she had not begun to walk by 12 months of age. She began to walk independently at two years old, and her gait became increasingly slow and spastic over time. However, the symptoms progressed slowly during the first two decades of her life, and she was able to run until graduation from high school. At age 20, however, she developed gait unsteadiness with frequent falling and difficulty climbing stairs.

Figure.

A: Pedigree of the HSP family. The patient is indicated (arrow). Squares indicate men; circles, women; shaded (black) symbol, individual with HSP, unshaded symbols, individuals without HSP. B: A sequence analysis revealed the c.1379 G>T mutation in exon 11 of SPAST in the patient. The red arrow indicates the position of the c.1379 nucleotide. C: A sequence analysis revealed no mutation in exon 11 of SPAST in the patient’s father. The green arrow indicates the position of the c.1379 nucleotide. D: A sequence analysis revealed no mutation in exon 11 of SPAST in the patient’s mother. The green arrow indicates the position of the c.1379 nucleotide.

On a neurological examination, she presented with increased muscle reflexes of the lower limbs, a positive Babinski's sign, contractures of the joints, and slight paresis of the extensors in the lower limbs. She was intellectually normal, and no cerebellar, sensory, or autonomic dysfunction was detected. Metabolic and routine blood investigations were unremarkable. Magnetic resonance imaging (MRI) of the brain and spine were normal.

We carried out whole-exome sequencing of genomic DNA from the patient. Genomic DNA was extracted from peripheral blood. Exome capture was performed with a SureSelect Human All Exon V6+UTR (89Mb) Kit (Agilent Technologies, Santa Clara, USA). Paired-end sequencing was carried out on a HiSeq2500 (Illumina, San Diego, USA) using a HiSeq SBS Kit V4 (Illumina), which generated 100-bp reads. The reference databases utilized included hg38 (GRCh38) (http://genome.ucsc.edu), The Human Gene Mutation Database (HGMD) (https://portal.biobase-international.com), Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org), the Genome Aggregation Database (GnomAD) (http://gnomad.broadinstitute.org), and the Single Nucleotide Polymorphism Database (dbSNP) (https://www.ncbi.nlm.nih.gov/SNP). We examined variants of 86 genes known to be responsible for HSP (Table 1). Through this analysis, we identified a heterozygous missense mutation (c.1379G＞T, p.Arg460Leu) in exon 11 of the SPAST gene in the patient and ruled out the possibility of other causative genes. We then examined exon 11 of the SPAST gene in the patient as well as the patient's father (Figure A, I-1) and mother (Figure A, I-2) via polymerase chain reaction (PCR). The genomic DNA of the patient's parents was also extracted from peripheral blood. On Sanger sequencing, we reconfirmed the p.R460L mutation in exon 11 of the SPAST gene, which was in a heterozygous state in the patient (Figure B). Arginine was replaced by leucine in an area evolutionarily conserved among the human, rhesus monkey, mouse, dog, elephant, chicken, western clawed frog, and zebrafish species. Bioinformatic analyses using the Mutation Taster (http://www.mutationtaster.org), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), Protein Variation Effect Analyzer (PROVEAN), and SIFT (http://provean.jcvi.org/genome_submit_2.php) software programs predicted that this variant was disease-causing, probably damaging, deleterious and damaging, respectively. On the other hand, the patient's parents did not exhibit the mutation on Sanger sequencing (Figure C, D). In this family, the patient harbored a mutation that was absent in her parents, and her sibling was healthy. This suggests that the mutation occurred de novo in the patient.

Table 1.

Genes Known to Be Responsible for HSP.

ATL1	SLC16A2	WDR48	GJA12	CYP7B1	KIF1A	IBA57	c12orf65
SPAST	Xp25	ARL6IP1	NT5C2	SPG7	FAM134B	MAG	CYP2U1
NIPA1	HACE1	ERLIN1	GBA2	ALDH18A1	ALS2	MTCO3	TFG
KIAA0196	LYST	AMPD2	AP4B1	SPG11	EXOSC3	MTTI	KIF1C
ALDH18A1	ALS2	ENTPD1	KIAA0415	ZFYVE26	SPOAN	MTND4	USP8
KIF5A	SACS	ARSI	TECPR2	ERLIN2	GAD1	MTATP6	FA2H
RTN2	SPPRS	PGAP1	AP4M1	SPG20	ARSACS	L1CAM	PNPLA6
HSPD1	BICD2	FLRT1	AP4E1	ACP33	MAG	PLP	c9orf12
BSCL2	CHS	RAB3GAP2	AP4S1	B4GALNT1	ARSPG75	Xq11	ZFYVE27
ATSV	IFIH1	MARS	VPS37A	DDHD1	REEP2	CPT1C	SLC33A1
REEP1	CCT5	ZFR	DDHD2	ATSV	UBAP1

Finally, to genetically confirm the paternity and maternity of the parents, we performed a paternity test. Twenty-one of the most polymorphic autosomal short tandem repeat (STR) markers commonly used for paternity testing in Japanese populations and a sex-identification marker (Amelogenin locus) were genotyped (14). The STR loci, D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338, and Amelogenin, were amplified using a GlobalfilerTM Amplification Kit (Thermo Fisher Scientific, Waltham, USA). The amplified products were detected with an ABI PRISMⓇ 310 Genetic Analyzer (Thermo Fisher Scientific). Fragment sizes were determined using the GeneScan Analysis v3.7 software program (Thermo Fisher Scientific), and the alleles were typed using the GenoTyper v3.7 software program (Thermo Fisher Scientific). The numbers of repeats in each STR marker in the family members and the bio-statistical computations are shown in Table 2. The bio-statistical calculation was performed using a spreadsheet as described previously (15). The genotypes of all 21 loci showed that the child inherited one allele from her father, and the genotypes of all 21 loci except D19S433 showed that the child inherited one allele from her mother. The DNA samples from the mother and the daughter might have shown some microsatellite instability in the D19S433 STR locus; however, the bio-statistical computations strongly supported the maternity relationship. Therefore, the paternity and maternity of the parents were both confirmed in this case.

Table 2.

Paternity and Maternity Testing by Analysis of Forensic Short Tandem Repeat (STR) Markers in the Family Members.

	Father		Daughter		Mother		Probability of Maternity	Likelihood Ratio (LR)	Probability of Paternity	Likelihood Ratio (LR)
D3S1358	15	18	17	18	12	17	0.552486187845304	1.234568	0.8855827134254	7.73993808
vWA	16	18	18	18	16	18	0.734176657587349	2.237136	0.9454008329360	2.237136465
D16S539	10	11	11	12	9	12	0.800388145990311	1.451800	0.9788351386246	2.670940171
CSF1PO	9	11	11	12	12	12	0.826078806001666	1.184553	0.9911489487954	2.421307506
TPOX	8	9	8	8	8	9	0.840105573791601	1.106195	0.9919918451746	1.10619469
D8S1179	12	14	12	14	12	13	0.914186190238607	2.027575	0.9966497467238	2.401536984
D21S11	31	31	29	31	29	30	0.915285808510090	1.014199	0.9996609381179	9.910802775
D18S51	16	19	16	19	16	17	0.953105141863687	1.881114	0.9999751728685	13.66120219
D2S441	11	14	11	14	11	14	0.985057307313079	3.243523	0.9999889840496	2.253775073
D19S433	14	15	14	null	16.2	null	0.999981797094757*	833.333333*	0.9999923845519	1.446531792
TH01	6	9	6	9	6	9	0.999989671501180	1.762410	0.9999952989702	1.619957881
FGA	21	22	21	24	24	24	0.999996723777069	3.152585	0.9999987786682	3.849114704
D22S1045	15	16	16	17	17	17	0.999998601050182	2.341920	0.9999993922649	2.009646302
D5S818	9	12	11	12	11	11	0.999999193924637	1.735509	0.9999997198340	2.169197397
D13S317	11	12	11	12	11	12	0.999999656429549	2.346173	0.9999998802570	2.339728591
D7S820	10	10	10	12	11	12	0.999999685976599	1.094092	0.9999999729141	4.42086649
SE33	18	31.2	18	25.2	16	25.2	0.999999923001444	4.078303	0.9999999965005	7.73993808
D10S1248	13	13	13	15	13	15	0.999999959106875	1.882922	0.9999999980798	1.822489521
D1S1656	13	18.3	14	18.3	14	15	0.999999986161766	2.955083	0.9999999998018	9.689922481
D12S391	18	18	18	21	21	21	0.999999996936215	4.516712	0.9999999999496	3.93236335
D2S1338	19	20	19	20	19	20	0.999999999119441	3.479363	0.9999999999838	3.107520199
Amelo.	X	Y	X	X	X	X	-	Total LR: 1135641941.9485	-	Total LR: 61666289424.4637

*The frequency of allele "null" was set as the lowest allele frequency, "0.0003", in the database we used (14).

Since realistically, the allele "null" has not been found in the database, its frequency is expected to be less than 0.0003. Therefore, both the probability of maternity and the likelihood ratio are expected to be greater than those calculated at the lowest frequency.

Discussion

The p.R460L mutation of the SPAST gene was first reported as a disease-causing mutation in a European family with autosomal dominant pure HSP. This mutation is located in the AAA ATPase cassette of spastin (from amino acid 342 to 616), which is crucial for microtubule-severing activity (16). This mutation was not present in the patients who were reported to have true de novo SPAST mutations in the literature (3-13). Since the causative mutation of the SPG4 gene in Japanese was first confirmed in 2001 (17), true de novo SPAST mutations in cases of Japanese or Asian ethnicity have rarely been reported. After we obtained DNA samples from the patient's father (54 years old) and mother (51 years old), who are both currently unaffected, we were able to establish that the p.R460L mutation was a de novo event, as both parents exhibited normal sequencing.

True de novo occurrence of SPAST mutations was the topic of focus for the first time in the report by Schieving et al. in 2019 (3). They reported that most of the SPAST mutations that occur de novo are also present in families with multiple generations with pure HSP. Furthermore, they suggested that the majority of patients (81%) with de novo mutations have an extremely early onset of the disease. This finding fits our patient. However, it is possible that this is because patients with early-onset disease simply tend to undergo a trio analysis. The relationship between the age of onset and the de novo occurrence of the mutation in SPAST may need further study.

It has been reported that 5.7% of SPG4 cases occur sporadically (16). However, it is very difficult to identify true de novo occurrence from incomplete penetrance or non-paternity because both parents need to be examined and genetically tested. Therefore, the frequency of de novo variants causing SPG4 is unknown. We reported a proven case of a de novo mutation in the SPAST gene in a Japanese patient. We were unable to rule out the possibility of gonadal mosaicism in either of the unaffected parents, even though it would still represent a de novo event. We suggest also including genes exhibiting an autosomal dominant mode of inheritance in patients with apparently sporadic HSP if a genetic analysis is performed.

Of the previously reported 27 patients with a de novo SPAST mutation identified, 9 (33%) harbored the common c.1496G＞A mutation (3-13). Although the low number of cases did not allow for any conclusions to be drawn, more clinical cases should be evaluated in order to determine if there are any mutational hot spots for the de novo occurrence of SPAST.

There are many kinds of mutations in SPAST, and all of them arose de novo at some point in the past. It has been suggested that some mutations in SPAST identified in certain populations had a founder effect (18), while some pathogenic variants of genetic disorders arose only once in human history (19). Our study indicates that a de novo mutation of SPAST can arise in an Asian population independently, thus contradicting the possibility of sharing a common ancestral origin with European populations.

In conclusion, we encountered a case of a pure SPG4 phenotype with an infantile onset caused by a de novo SPAST mutation in a Japanese patient. The paternity and maternity of the parents were both confirmed in this case. This study may expand the clinical and genetic findings for SPG4.

The present clinical and genetic study was approved by the institutional review board of Yamanashi University, and written informed consent was obtained from all participating individuals.

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

Financial Support

This work was supported by Grants-in-Aid from the Research Committee for Ataxic Disease (Y.T.), the Ministry of Health, Labor and Welfare, Japan, and JSPS KAKENHI Grant Number JP18K07495 (Y.T.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.