Parkinsonism and Related Disorders xxx (2014) 1e5
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Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia Qian-Qian Wei a, YongPing Chen a, Zhen-Zhen Zheng a, XuePing Chen a, Rui Huang a, Yuan Yang b, JeanMarc Burgunder a, c, **, Hui-Fang Shang a, * a Department of Neurology and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China b Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu, Sichuan, China c Department of Neurology, Bern University, Bern CH3010, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Received in revised form 10 April 2014 Accepted 15 April 2014
Background: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative diseases. Mutations in the spastin (SPAST) gene are the most common cause of pure HSP. However, few data are available regarding the clinical and genetic spectrum of HSP among Chinese patients. Methods: Clinical data were collected at diagnosis and follow-up of 42 Chinese patients with pure HSP. All seventeen exons of the SPAST gene were directly sequenced. Additionally, we used a multiplex ligation dependent probe amplification (MLPA) assay targeting the SPAST gene to evaluate large exon deletion or insertion mutations in patients without SPAST point mutations. Results: The age of disease onset of our patients was 19.6 14.4 years. Six novel variations were found, including three missense mutations (p. L363P, p. D441V, and p. S595R), one insertion (c.1511dupT (p. Y505Ifs*7)), and two larger deletions (exons 5e17 and exons 10e17). Four previously reported mutations, including p. S399L, c.1215_c.1219delTATAA (p. N405Kfs*36), exon 1 deletion, and exon 16 deletion, were detected. The SPAST mutation rate was 40% (4/10) in Chinese familial patients and 33.33% (7/21) in Chinese sporadic pure HSP patients. The frequency of large deletions was high in both AD-HSP (20%, 2/ 10) and sporadic HSP (14.28%, 3/21). Conclusion: SPAST mutations are common in Chinese patients with pure HSP. Large exon deletions are an important cause of AD-HSP and sporadic pure HSP in Chinese patients. Large fragment tests should be performed to explore large SPAST mutations in familial and sporadic HSP patients without SPAST point mutations. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Hereditary spastic paraplegia SPAST Mutation MLPA
1. Introduction Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by slow progressive spasticity and weakness of the lower limbs. According to its clinical presentation, HSP can be classified into a pure form with isolated progressive spasticity and weakness of the lower limbs and a complicated form with other
* Corresponding author. Fax: þ86 028 85423550. ** Corresponding author. Department of Neurology, Bern University, Bern CH3010, Switzerland. E-mail addresses: [email protected] (J. Burgunder), [email protected], [email protected] (H.-F. Shang).
abnormalities, such as mental and cognitive changes, optic atrophy, amyotrophy, ataxia, deafness, ichthyosis, and/or peripheral neuropathy [1]. The inherited forms include the autosomal dominant (AD-HSP) form, which is observed most frequently (70e80%), as well as the autosomal recessive (AR-HSP) and X-linked forms [2]. To date, at least fifty-two chromosomal loci have been identified for HSPs, including seventeen autosomal dominant, thirty autosomal recessive and five X-linked inherited loci [3e5]. The spastin (SPAST) gene, which encodes a member of the AAA ATPase protein family and is located on chromosome 2p24-p21, has been reported to be the most common cause of HSP, accounting for 40e45% of pure ADHSP cases and approximately 10% of sporadic cases. More than 440 different mutations have been identified in the SPAST gene, including 179 missense/nonsense mutations (http://www.hgmd.cf. ac.uk/ac/all.php). Cases of HSP that are caused by SPAST mutations
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Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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Q.-Q. Wei et al. / Parkinsonism and Related Disorders xxx (2014) 1e5
tend to present as the pure form of the disease, which is often associated with a decreased sensation of vibration in the lower limbs and urinary problems [6]. In addition, HSP caused by mutation of the SPAST gene is reported more often in males than in females [7]. However, the age of onset, severity of symptoms, and progression of symptoms are highly variable, even within families [7]. Therefore, it is difficult to describe the correlation between the genotype and the phenotype of HSP. To date, most of the data regarding clinical and molecular correlations related to HSP have been generated using patients from European countries [8e10], although SPAST mutations have been reported in some Han Chinese patients [11,12]. However, the prevalence and types of SPAST mutations among Chinese patients are still largely unknown. One small study estimated that the frequency of SPAST mutations in Chinese patients is 45% (5/11) in ADHSP and 9% (1/11) in sporadic HSP [13]. Considering the limited sample size of previous studies, in the current study, we present clinical and genetic data related to the SPAST gene in a cohort of 42 patients with pure HSP from Southwest China. 2. Materials and methods 2.1. Subjects Forty-two patients, including 21 sporadic and 21 familial patients from 10 families, who presented to the Department of Neurology, West China Hospital, Sichuan University, were clinically diagnosed with pure HSP according to the Harding criteria [1]. All patients presented with a pure form of the HSP phenotype, although mild associated symptoms, such as sensory disturbances or bladder dysfunction, were allowed. Patients with the complicated form of HSP, which is associated with mental and cognitive impairment, dementia, epilepsy, aphasia, dystonia, extra-pyramidal disturbances, cerebellar abnormalities, a thin corpus callosum, optic atrophy, ataxia, deafness, and ichthyosis, were excluded [1]. The disability score of patients was evaluated on a four-point scale (1 ¼ normal, 2 ¼ able to walk but not run, 3 ¼ requires the use of a walking aid or support, 4 ¼ wheelchair bound) [14]. The patient history was obtained, and neurological examinations were performed by neurologists. 2.2. Mutation screening of the SPAST gene A total of 200 unrelated Chinese healthy controls (HC), matched with regard to gender, age, and area of residence to the HSP patients, were recruited for the study as the control group. Written informed consent was obtained from each subject, and the study was approved by the Sichuan University Ethics Committee. Genomic DNA was extracted from a 5 ml peripheral blood sample with an established method using saturated phenol-chloroform. Fifteen pairs of primers designed with the Primer Permier 5 software (Premier Biosoft International, Palo Alto, CA, USA) were used to amplify the coding region of SPAST, including seventeen exons and exon/intron boundaries (GenBank Accession No. NM_014946). The PCR products were directly sequenced with an ABI3100 automated DNA sequencing system (BGI, Shenzhen, China). The primer sequences and PCR conditions are shown in Supplementary Table 1. Moreover, for patients in whom no SPAST point mutations were found by direct sequencing, we performed a multiplex ligation-dependent probe amplification (MLPA) assay targeting the SPAST gene with the Salsa kit P165-C1 HSP (MRC-Holland, Amsterdam) according to the manufacturer’s instructions. The kit contains probes for all 17 exons in the SPAST gene. MLPA amplification products were separated by capillary electrophoresis using an ABI 3100-Avant Genetic Analyzer. MLPA data were analyzed using Coffalyser v9.4 software. Deletions were detected in exons in which the relative peak area was reduced by 35e50% of the normal control value. 2.3. Statistical analysis Statistical analysis was performed using SPSS software (v 18.0). The results of the data analysis are expressed as the mean standard deviation. Student’s t test was used to compare groups when the data were normally distributed. Categorical variables were compared using aChi-square test. Significance was set at P < 0.05.
3. Results 3.1. Clinical data All patients (Patient 1 to Patient 42) presented with gait disorder due to weakness or spasticity of the lower limbs. The spasticity gradually became worse, although none of the patients were
wheelchair bound. The age of disease onset was 19.6 14.4 years (ranging from 1 to 47 years), and the disease duration was 14.3 10.9 years (ranging from 1 to 40 years). The babinski sign was present in 33 patients (78.6%). Eight patients (19.05%) had bladder disturbance and presented with urinary incontinence. Seven patients presented with pes cavus. The age of onset of patients with pes cavus did not differ from that of patients without pes cavus. In addition, an electromyography test was performed on these seven patients with pes cavus. Of these, only two patients showed mild peripheral neuropathy of the lower limbs and exhibited fibrillation waves and slowing of the sensory nerve conduction velocity. Six patients (17.7%, 6/35) showed thoracic spinal cord atrophy. 3.2. Genetic findings After direct sequencing and MLPA assay screening of all seventeen exons in each patient, 10 different mutations were found in 13 patients, including 6 familial patients (from 4 families) and 7 sporadic patients. The clinical features of the patients with these mutations are presented in Table 1. Among the ten mutations, six mutations were novel, including three missense mutations (p. L363P, p. D441V, and p. S595R), one insertion mutation (c.1511dupT (p. Y505Ifs*7)), and two large deletions (exons 5e17 and exons 10e17), whereas four known mutations (p. S399L, c.1215_c.1219delTATAA (p. N405Kfs*36), exon 1 deletion, and exon 16 deletion) were reported (Table 1) [8,10,14,15]. Direct sequencing showed that all novel mutations were absent in the 200 healthy control subjects. In addition, these mutations were not found in the single nucleotide polymorphism (SNP) database or the 1000 Genomes Project database. The p. L363P mutation in exon 7 was identified in a sporadic male patient with disease onset at 15 years of age (Patient 20, Fig. 1A). The p. D441V mutation in exon 11 was identified in a familial patient from the family 10 pedigree with disease onset at 41 years of age (Patient 17, Fig. 1B). The p. S595R mutation in exon 17 was found in a familial male patient from the family 6 pedigree with disease onset at 25 years of age (Patient 29, Fig. 1C). The same mutation was found in his son (Patient 30), who exhibited symptom onset at 15 years of age. The mutated amino acid residues of the three novel missense mutations, p. L363P, p. D441V and p. S595R, are highly conserved among species. Furthermore, these novel missense mutations are predicted to be damaging substitutions by the Sorting Intolerant from Tolerant (SIFT) (http://sift. jcvi.org/www/SIFT_enst_submit.html) and PolyPhen-2 prediction tools (http://genetics.bwh.harvard.edu/pph2/). The novel insertion mutation in exon 13, c.1511dupT (p. Y505Ifs*7), was found in a male sporadic patient with disease onset at 12 years of age (Patient 9, Fig. 1D). The exons 10e17 deletion was found in an AD-HSP family (family 4) (Patients 27 and 28, Fig. 2A and B), and the novel exons 5e17 deletion was found in a sporadic male patient with disease onset at 44 years of age (Patient 5, Fig. 2C). The previously reported mutation p. S399L was detected in Patient 8. The previously reported deletion mutation in exon 9, c.1215_c.1219delTATAA (p. N405Kfs*36), was identified in a sporadic male patient with disease onset at 8 years of age (Patient 35). The previously reported exon 1 deletions were found in a familial male patient from family 7 (Patient 23) and a sporadic female patient (Patient 15). The previously reported exon 16 deletion was found in a sporadic female patient (Patient 7). The mutation rate was 40% (4/10) in familial patients and 33.33% (7/21) in sporadic patients, and a higher mutation rate was found in male patients (39.29%, 11/28) compared to female patients (14.29%, 2/14). There were no significant differences in gender, mode of inheritance, or the age of disease onset between patients with and
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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Table 1 Clinical characteristics and identified mutations in patients with hereditary spastic paraplegia. Patient number
Family number
Sex
OA
Duration
Mode of inheritance
DS
Exon/intron
cDNA
Protein
Mutation type
Previously reported
PC
TSCA
BI
BS
5 7 8 9 15 17 20 23 27 28 29 30 35
e e e e e 10:III1 e 7:III1 4:III3 4:IV2 6:II3 6:III2 e
M F M M F M M M M M M M M
44 47 39 12 2 41 15 28 26 2 25 15 8
20 7 10 20 39 9 27 4 13 5 23 2 29
S S S S S AD S AD AD AD AD AD S
3 2 2 2 2 2 3 2 2 2 3 1 3
Ex 5e17 Ex 16 9 13 Ex 1 11 7 Ex 1 Ex 10e17 Ex 10e17 17 17 9
c.683?_1851þ?del c.1688?_1728þ?del c.1196C>T c.1511dupT c.1?_415þ?del c.1322A>T c.1088T>C c.1?_415þ?del c.1246?_1851þ?del c.1246?_1851þ?del c.1783A>C c.1783A>C c.1215_c.1219delTATAA
Unknown Unknown p. S399L p. Y505Ifs Unknown p. D441V p. L363P Unknown Unknown Unknown p. S595R p. S595R p. N405Kfs*36
Deletion Deletion Missense Duplication Deletion Missense Missense Deletion Deletion Deletion Missense Missense Deletion
N [10],a [14,15]a N [10],a N N [10],a N N N N [8],a
þ þ þ
þ ND
þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
M: male; F: female; OA: onset age; AD: autosomal dominant; S: sporadic; DS: disability score; PC: pes cavus; TSCA: thoracic spinal cord atrophy; BI: bladder involvement; BS: Babinski signs; ND, no data. a See references.
Fig. 1. Sequencing chromatograms of novel mutations and pedigrees of the patients who carried SPAST mutations. (A) Sequencing chromatogram of the p. L363P mutation in a sporadic patient. The arrow shows the position of a T-to-C transition at nucleotide 1088 (c.1088T>C) that leads to the replacement of leucine (Leu) with proline (Pro) at codon 363. (B) Sequencing chromatogram of the p. D441V mutation and the pedigree of the patient who carried the p. D441V mutation. The arrow shows the position of an A-to-T transition at nucleotide 1322 (c.1322A>T) that leads to the replacement of aspartic acid (Asp) by valine (Val) at codon 441. The father and grandfather of the index patient suffered from HSP. Dark squares show affected patients and the arrow shows the index patient. (C) Sequencing chromatogram of the p. S595R mutation and the pedigree of the patient who carried the p. S595R mutation. The arrow shows the position of an A-to-C transition at nucleotide 1783 (c.1783A>C) that leads to the replacement of serine (Ser) by arginine (Arg) at codon 595. The son of the index patient suffered from HSP. Dark squares show affected patients and the arrow shows the index patient. (D) Sequencing chromatogram of the p. Y505Ifs*7 mutation in a sporadic patient. This mutation results in a frame-shift, which generates different codon sequences starting at position 505 and introduces a premature stop at position 512.
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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Fig. 2. Multiplex ligation-dependent probe amplification profiles of the SPAST gene. (A) No exon deletions were detected in a normal control subject. (B) Deletion of exons 10e17 was detected in familial patient 27 (III3), and the pedigree of family 4 carried exon 10e17 deletions. The son, mother, and grandmother of the index patient suffered from the disease. Dark squares show affected patients, and the arrow shows the index patient. (C) Deletion of exons 5e17 was detected in sporadic patient 5 (III1).
without SPAST gene mutations. In this study, all the patients with missense mutations were males; however, the age of disease onset was variable (ranging from 15 to 41 years). The age of disease onset of patients with truncating mutations was obviously earlier than that of patients with missense mutations. No mutations in the SPAST gene were found in patients with mild peripheral neuropathy. Three patients from the group of 7 patients with pes cavus had SPAST gene mutations. 4. Discussion In this relatively large study of Chinese patients, we found that the SPAST mutation rate is 40% in Chinese familial HSP patients and 33.33% in Chinese sporadic pure HSP patients. The frequency of mutations in the SPAST gene, which are the most common cause of AD-HSP, can vary depending on ethnicity. In Europeans, mutations in SPAST/SPG4 are observed in w40% of all AD-HSP patients [16]. In our study, we used the MLPA technique to show that two AD-HSP families without SPAST point mutations had large exon deletions. Thus, the mutation rate in our pure AD-HSP families was 40% (4/10), which is in agreement with the results of a recent SPAST mutations screening study on Chinese AD-HSP families (45%, 5/11) [13]. The mutation rate (18%, 4/22) in AD-HSP families may have been underestimated in previous Chinese studies, because assays targeting the large deletion/insertion mutations were not performed in patients without point mutations [17]. After combining our results and the results of a previous study [13], the frequency of SPAST mutations in Chinese AD-HSP families is 42.86% (9/21), which is similar to that in European HSP patients [16]. The frequency of mutation carriers in sporadic cases in our cohort was 33.33% (7/21), which was higher than the frequency observed in European HSP patients (6.5e17%) [18,19] and higher than the frequency observed in another study of Chinese patients (9% (1/11)) [13]. Multiple factors may contribute to the differences among studies, such as the sample size, ethnic differences, and the
phenotype of HSP patients. A combination of direct sequencing and the MLPA technique is useful for determining the etiology of pure HSP patients. In our study, there were 2 missense mutations and 2 large deletions in AD-HSP patients, and 2 missense mutations, 1 deletion, 1 insertion, and 3 large deletions in sporadic HSP patients, which is in agreement with previous studies showing that missense mutations are the most common type of mutation in familial patients and sporadic cases [13]. However, in our study, large deletions were found to be an important cause of AD-HSP (20%, 2/10) and sporadic HSP (14.28%, 3/21), which is comparable to the 17e20% frequency of SPAST exon deletion/insertion mutations in AD-HSP patients [10,20] but is more frequent than reported in sporadic patients (2.5e10%) [20,21]. This suggests that the detection of large deletions/insertions is pivotal for understanding the cause of HSP among patients of different ethnic origins and indicates that the frequency of mutations in HSP patients has been underestimated in previous studies [15,17]. The six novel mutations identified in our study expand the mutation spectrum of SPAST in pure HSP patients. Clinical and genetic variation was also found in our HSP patients. Patients who carried the same SPAST mutation, such as the exon 1 deletion, had different ages of disease onset, which was similar to what was observed in other studies [10]. Abnormalities in the paraventricular area were shown in the brain MRI of an Australian patient with a S399L mutation [14]; however, these abnormalities were not found in a European patient [15] or in our patient (Patient 8). In the current study, we also observed an anticipation phenomenon [10]. The offspring of probands carrying an S595R mutation and exon 10e17 deletions (families 4 and 6, respectively) had a significantly earlier age of disease onset than the probands. Mutations in SPAST did not contribute to the differences in age of disease onset in our study, which agreed with the finding of a recent study that included 294 HSP patients. However, in our study, similar to the finds of Loureiro et al. [22], the age of disease onset was influenced by the type of SPAST mutations. The mean age of
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disease onset of patients with SPAST frameshift mutations, which lead to the production of truncated SPAST proteins, was significantly earlier than that of patients with other types of mutations. SPAST is an ATP-dependent microtubule severing protein. In the proposed disease mechanism, the region of SPAST responsible for microtubule severing was amino acids from 228 to 616. The two frameshift mutations, p. N405Kfs*36 and Y505Ifs*7, may render SPAST unable to sever microtubules [23]. The frequency of mutation in males tended to be significantly higher than that in females, which was similar to a finding from a previous study [15]. This finding may partially explain the clinical observations that the disease is more prevalent in males compared with females. Although the exact mechanism that contributes to the difference between genders is unknown, it is known that estrogen may play an important role in neuroprotection [24]. In the current study, we did not find a correlation between pes cavus, age of disease onset, and gene mutation in HSP. However, the relatively small sample size of our study is a major limitation. 5. Conclusions SPAST mutations are common in Chinese pure HSP patients. Large exon deletions are a major cause of AD-HSP and sporadic HSP in Chinese patients. In future studies, large fragment tests should be performed to explore the role of SPAST mutations in familial and sporadic pure HSP patients without point mutations in the SPAST gene. Disclosure of conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors acknowledge all the patients and healthy subjects for their participation in this study. The authors also thank Guangzhou Kingmed Center for Clinical Laboratory Company performing the MLPA test for the HSP patients. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.parkreldis.2014.04.021. References [1] Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet 1983;1:1151e5. [2] Fink JK. Hereditary spastic paraplegia. Curr Neurol Neurosci Rep 2006;6:65e 76. [3] Slabicki M, Theis M, Krastev DB, Samsonov S, Mundwiller E, Junqueira M, et al. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 2010;8:e1000408.
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[4] Montenegro G, Rebelo AP, Connell J, Allison R, Babalini C, D’Aloia M, et al. Mutations in the ER-shaping protein reticulon 2 cause the axon-degenerative disorder hereditary spastic paraplegia type 12. J Clin Invest 2012;122:538e44. [5] Gonzalez M, Nampoothiri S, Kornblum C, Oteyza AC, Walter J, Konidari I, et al. Mutations in phospholipase DDHD2 cause autosomal recessive hereditary spastic paraplegia (SPG54). Eur J Hum Genet 2013;21:1214e8. [6] de Bot ST, van de Warrenburg BP, Kremer HP, Willemsen MA. Child neurology: hereditary spastic paraplegia in children. Neurology 2010;75:e75e 9. [7] Proukakis C, Moore D, Labrum R, Wood NW, Houlden H. Detection of novel mutations and review of published data suggests that hereditary spastic paraplegia caused by spastin (SPAST) mutations is found more often in males. J Neurol Sci 2011;306:62e5. [8] Erichsen AK, Inderhaug E, Mattingsdal M, Eiklid K, Tallaksen CM. Seven novel mutations and four exon deletions in a collection of Norwegian patients with SPG4 hereditary spastic paraplegia. Eur J Neurol 2007;14:809e14. [9] Magariello A, Muglia M, Patitucci A, Ungaro C, Mazzei R, Gabriele AL, et al. Mutation analysis of the SPG4 gene in Italian patients with pure and complicated forms of spastic paraplegia. J Neurol Sci 2010;288:96e100. [10] Depienne C, Fedirko E, Forlani S, Cazeneuve C, Ribai P, Feki I, et al. Exon deletions of SPG4 are a frequent cause of hereditary spastic paraplegia. J Med Genet 2007;44:281e4. [11] Feng Y, Ke X, Zhai M, Xin Q, Gong Y, Liu Q. Novel deletion of SPAST in a Chinese family with hereditary spastic paraplegia. Singapore Med J 2013;54: 251e4. [12] Lan MY, Fu SC, Chang YY, Wu-Chou YH, Lai SC, Chen RS, et al. Clinical and genetic analysis of four Taiwanese families with autosomal dominant hereditary spastic paraplegia. J Formos Med Assoc 2012;111:380e5. [13] Fei QZ, Tang WG, Rong TY, Tang HD, Liu JR, Guo ZL, et al. Two novel mutations in the spastin gene of Chinese patients with hereditary spastic paraplegia. Eur J Neurol 2011;18:1194e6. [14] McDermott CJ, Burness CE, Kirby J, Cox LE, Rao DG, Hewamadduma C, et al. Clinical features of hereditary spastic paraplegia due to spastin mutation. Neurology 2006;67:45e51. [15] Meijer IA, Hand CK, Cossette P, Figlewicz DA, Rouleau GA. Spectrum of SPG4 mutations in a large collection of North American families with hereditary spastic paraplegia. Arch Neurol 2002;59:281e6. [16] Fonknechten N, Mavel D, Byrne P, Davoine CS, Cruaud C, Bonsch D, et al. Spectrum of SPG4 mutations in autosomal dominant spastic paraplegia. Hum Mol Genet 2000;9:637e44. [17] Tang B, Zhao G, Xia K, Pan Q, Luo W, Shen L, et al. Three novel mutations of the spastin gene in Chinese patients with hereditary spastic paraplegia. Arch Neurol 2004;61:49e55. [18] Sauter S, Miterski B, Klimpe S, Bonsch D, Schols L, Visbeck A, et al. Mutation analysis of the spastin gene (SPG4) in patients in Germany with autosomal dominant hereditary spastic paraplegia. Hum Mutat 2002;20:127e32. [19] Shoukier M, Neesen J, Sauter SM, Argyriou L, Doerwald N, Pantakani DV, et al. Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia. Eur J Hum Genet 2009;17:187e94. [20] Sulek A, Elert E, Rajkiewicz M, Zdzienicka E, Stepniak I, Krysa W, et al. Screening for the hereditary spastic paraplaegias SPG4 and SPG3A with the multiplex ligation-dependent probe amplification technique in a large population of affected individuals. Neurol Sci 2013;34:239e42. [21] Alvarez V, Sanchez-Ferrero E, Beetz C, Diaz M, Alonso B, Corao AI, et al. Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia. BMC Neurol 2010;10:89. [22] Loureiro JL, Brandao E, Ruano L, Brandao AF, Lopes AM, Thieleke-Matos C, et al. Autosomal dominant spastic paraplegias: a review of 89 families resulting from a Portuguese survey. JAMA Neurol 2013;70:481e7. [23] White SR, Evans KJ, Lary J, Cole JL, Lauring B. Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing. J Cell Biol 2007;176:995e1005. [24] Simpkins JW, Singh M. More than a decade of estrogen neuroprotection. Alzheimers Dement 2008;4:S131e6.
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Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia Qian-Qian Wei a, YongPing Chen a, Zhen-Zhen Zheng a, XuePing Chen a, Rui Huang a, Yuan Yang b, JeanMarc Burgunder a, c, **, Hui-Fang Shang a, * a Department of Neurology and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China b Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu, Sichuan, China c Department of Neurology, Bern University, Bern CH3010, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Received in revised form 10 April 2014 Accepted 15 April 2014
Background: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative diseases. Mutations in the spastin (SPAST) gene are the most common cause of pure HSP. However, few data are available regarding the clinical and genetic spectrum of HSP among Chinese patients. Methods: Clinical data were collected at diagnosis and follow-up of 42 Chinese patients with pure HSP. All seventeen exons of the SPAST gene were directly sequenced. Additionally, we used a multiplex ligation dependent probe amplification (MLPA) assay targeting the SPAST gene to evaluate large exon deletion or insertion mutations in patients without SPAST point mutations. Results: The age of disease onset of our patients was 19.6 14.4 years. Six novel variations were found, including three missense mutations (p. L363P, p. D441V, and p. S595R), one insertion (c.1511dupT (p. Y505Ifs*7)), and two larger deletions (exons 5e17 and exons 10e17). Four previously reported mutations, including p. S399L, c.1215_c.1219delTATAA (p. N405Kfs*36), exon 1 deletion, and exon 16 deletion, were detected. The SPAST mutation rate was 40% (4/10) in Chinese familial patients and 33.33% (7/21) in Chinese sporadic pure HSP patients. The frequency of large deletions was high in both AD-HSP (20%, 2/ 10) and sporadic HSP (14.28%, 3/21). Conclusion: SPAST mutations are common in Chinese patients with pure HSP. Large exon deletions are an important cause of AD-HSP and sporadic pure HSP in Chinese patients. Large fragment tests should be performed to explore large SPAST mutations in familial and sporadic HSP patients without SPAST point mutations. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Hereditary spastic paraplegia SPAST Mutation MLPA
1. Introduction Hereditary spastic paraplegia (HSP) is a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by slow progressive spasticity and weakness of the lower limbs. According to its clinical presentation, HSP can be classified into a pure form with isolated progressive spasticity and weakness of the lower limbs and a complicated form with other
* Corresponding author. Fax: þ86 028 85423550. ** Corresponding author. Department of Neurology, Bern University, Bern CH3010, Switzerland. E-mail addresses: [email protected] (J. Burgunder), [email protected], [email protected] (H.-F. Shang).
abnormalities, such as mental and cognitive changes, optic atrophy, amyotrophy, ataxia, deafness, ichthyosis, and/or peripheral neuropathy [1]. The inherited forms include the autosomal dominant (AD-HSP) form, which is observed most frequently (70e80%), as well as the autosomal recessive (AR-HSP) and X-linked forms [2]. To date, at least fifty-two chromosomal loci have been identified for HSPs, including seventeen autosomal dominant, thirty autosomal recessive and five X-linked inherited loci [3e5]. The spastin (SPAST) gene, which encodes a member of the AAA ATPase protein family and is located on chromosome 2p24-p21, has been reported to be the most common cause of HSP, accounting for 40e45% of pure ADHSP cases and approximately 10% of sporadic cases. More than 440 different mutations have been identified in the SPAST gene, including 179 missense/nonsense mutations (http://www.hgmd.cf. ac.uk/ac/all.php). Cases of HSP that are caused by SPAST mutations
http://dx.doi.org/10.1016/j.parkreldis.2014.04.021 1353-8020/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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tend to present as the pure form of the disease, which is often associated with a decreased sensation of vibration in the lower limbs and urinary problems [6]. In addition, HSP caused by mutation of the SPAST gene is reported more often in males than in females [7]. However, the age of onset, severity of symptoms, and progression of symptoms are highly variable, even within families [7]. Therefore, it is difficult to describe the correlation between the genotype and the phenotype of HSP. To date, most of the data regarding clinical and molecular correlations related to HSP have been generated using patients from European countries [8e10], although SPAST mutations have been reported in some Han Chinese patients [11,12]. However, the prevalence and types of SPAST mutations among Chinese patients are still largely unknown. One small study estimated that the frequency of SPAST mutations in Chinese patients is 45% (5/11) in ADHSP and 9% (1/11) in sporadic HSP [13]. Considering the limited sample size of previous studies, in the current study, we present clinical and genetic data related to the SPAST gene in a cohort of 42 patients with pure HSP from Southwest China. 2. Materials and methods 2.1. Subjects Forty-two patients, including 21 sporadic and 21 familial patients from 10 families, who presented to the Department of Neurology, West China Hospital, Sichuan University, were clinically diagnosed with pure HSP according to the Harding criteria [1]. All patients presented with a pure form of the HSP phenotype, although mild associated symptoms, such as sensory disturbances or bladder dysfunction, were allowed. Patients with the complicated form of HSP, which is associated with mental and cognitive impairment, dementia, epilepsy, aphasia, dystonia, extra-pyramidal disturbances, cerebellar abnormalities, a thin corpus callosum, optic atrophy, ataxia, deafness, and ichthyosis, were excluded [1]. The disability score of patients was evaluated on a four-point scale (1 ¼ normal, 2 ¼ able to walk but not run, 3 ¼ requires the use of a walking aid or support, 4 ¼ wheelchair bound) [14]. The patient history was obtained, and neurological examinations were performed by neurologists. 2.2. Mutation screening of the SPAST gene A total of 200 unrelated Chinese healthy controls (HC), matched with regard to gender, age, and area of residence to the HSP patients, were recruited for the study as the control group. Written informed consent was obtained from each subject, and the study was approved by the Sichuan University Ethics Committee. Genomic DNA was extracted from a 5 ml peripheral blood sample with an established method using saturated phenol-chloroform. Fifteen pairs of primers designed with the Primer Permier 5 software (Premier Biosoft International, Palo Alto, CA, USA) were used to amplify the coding region of SPAST, including seventeen exons and exon/intron boundaries (GenBank Accession No. NM_014946). The PCR products were directly sequenced with an ABI3100 automated DNA sequencing system (BGI, Shenzhen, China). The primer sequences and PCR conditions are shown in Supplementary Table 1. Moreover, for patients in whom no SPAST point mutations were found by direct sequencing, we performed a multiplex ligation-dependent probe amplification (MLPA) assay targeting the SPAST gene with the Salsa kit P165-C1 HSP (MRC-Holland, Amsterdam) according to the manufacturer’s instructions. The kit contains probes for all 17 exons in the SPAST gene. MLPA amplification products were separated by capillary electrophoresis using an ABI 3100-Avant Genetic Analyzer. MLPA data were analyzed using Coffalyser v9.4 software. Deletions were detected in exons in which the relative peak area was reduced by 35e50% of the normal control value. 2.3. Statistical analysis Statistical analysis was performed using SPSS software (v 18.0). The results of the data analysis are expressed as the mean standard deviation. Student’s t test was used to compare groups when the data were normally distributed. Categorical variables were compared using aChi-square test. Significance was set at P < 0.05.
3. Results 3.1. Clinical data All patients (Patient 1 to Patient 42) presented with gait disorder due to weakness or spasticity of the lower limbs. The spasticity gradually became worse, although none of the patients were
wheelchair bound. The age of disease onset was 19.6 14.4 years (ranging from 1 to 47 years), and the disease duration was 14.3 10.9 years (ranging from 1 to 40 years). The babinski sign was present in 33 patients (78.6%). Eight patients (19.05%) had bladder disturbance and presented with urinary incontinence. Seven patients presented with pes cavus. The age of onset of patients with pes cavus did not differ from that of patients without pes cavus. In addition, an electromyography test was performed on these seven patients with pes cavus. Of these, only two patients showed mild peripheral neuropathy of the lower limbs and exhibited fibrillation waves and slowing of the sensory nerve conduction velocity. Six patients (17.7%, 6/35) showed thoracic spinal cord atrophy. 3.2. Genetic findings After direct sequencing and MLPA assay screening of all seventeen exons in each patient, 10 different mutations were found in 13 patients, including 6 familial patients (from 4 families) and 7 sporadic patients. The clinical features of the patients with these mutations are presented in Table 1. Among the ten mutations, six mutations were novel, including three missense mutations (p. L363P, p. D441V, and p. S595R), one insertion mutation (c.1511dupT (p. Y505Ifs*7)), and two large deletions (exons 5e17 and exons 10e17), whereas four known mutations (p. S399L, c.1215_c.1219delTATAA (p. N405Kfs*36), exon 1 deletion, and exon 16 deletion) were reported (Table 1) [8,10,14,15]. Direct sequencing showed that all novel mutations were absent in the 200 healthy control subjects. In addition, these mutations were not found in the single nucleotide polymorphism (SNP) database or the 1000 Genomes Project database. The p. L363P mutation in exon 7 was identified in a sporadic male patient with disease onset at 15 years of age (Patient 20, Fig. 1A). The p. D441V mutation in exon 11 was identified in a familial patient from the family 10 pedigree with disease onset at 41 years of age (Patient 17, Fig. 1B). The p. S595R mutation in exon 17 was found in a familial male patient from the family 6 pedigree with disease onset at 25 years of age (Patient 29, Fig. 1C). The same mutation was found in his son (Patient 30), who exhibited symptom onset at 15 years of age. The mutated amino acid residues of the three novel missense mutations, p. L363P, p. D441V and p. S595R, are highly conserved among species. Furthermore, these novel missense mutations are predicted to be damaging substitutions by the Sorting Intolerant from Tolerant (SIFT) (http://sift. jcvi.org/www/SIFT_enst_submit.html) and PolyPhen-2 prediction tools (http://genetics.bwh.harvard.edu/pph2/). The novel insertion mutation in exon 13, c.1511dupT (p. Y505Ifs*7), was found in a male sporadic patient with disease onset at 12 years of age (Patient 9, Fig. 1D). The exons 10e17 deletion was found in an AD-HSP family (family 4) (Patients 27 and 28, Fig. 2A and B), and the novel exons 5e17 deletion was found in a sporadic male patient with disease onset at 44 years of age (Patient 5, Fig. 2C). The previously reported mutation p. S399L was detected in Patient 8. The previously reported deletion mutation in exon 9, c.1215_c.1219delTATAA (p. N405Kfs*36), was identified in a sporadic male patient with disease onset at 8 years of age (Patient 35). The previously reported exon 1 deletions were found in a familial male patient from family 7 (Patient 23) and a sporadic female patient (Patient 15). The previously reported exon 16 deletion was found in a sporadic female patient (Patient 7). The mutation rate was 40% (4/10) in familial patients and 33.33% (7/21) in sporadic patients, and a higher mutation rate was found in male patients (39.29%, 11/28) compared to female patients (14.29%, 2/14). There were no significant differences in gender, mode of inheritance, or the age of disease onset between patients with and
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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Table 1 Clinical characteristics and identified mutations in patients with hereditary spastic paraplegia. Patient number
Family number
Sex
OA
Duration
Mode of inheritance
DS
Exon/intron
cDNA
Protein
Mutation type
Previously reported
PC
TSCA
BI
BS
5 7 8 9 15 17 20 23 27 28 29 30 35
e e e e e 10:III1 e 7:III1 4:III3 4:IV2 6:II3 6:III2 e
M F M M F M M M M M M M M
44 47 39 12 2 41 15 28 26 2 25 15 8
20 7 10 20 39 9 27 4 13 5 23 2 29
S S S S S AD S AD AD AD AD AD S
3 2 2 2 2 2 3 2 2 2 3 1 3
Ex 5e17 Ex 16 9 13 Ex 1 11 7 Ex 1 Ex 10e17 Ex 10e17 17 17 9
c.683?_1851þ?del c.1688?_1728þ?del c.1196C>T c.1511dupT c.1?_415þ?del c.1322A>T c.1088T>C c.1?_415þ?del c.1246?_1851þ?del c.1246?_1851þ?del c.1783A>C c.1783A>C c.1215_c.1219delTATAA
Unknown Unknown p. S399L p. Y505Ifs Unknown p. D441V p. L363P Unknown Unknown Unknown p. S595R p. S595R p. N405Kfs*36
Deletion Deletion Missense Duplication Deletion Missense Missense Deletion Deletion Deletion Missense Missense Deletion
N [10],a [14,15]a N [10],a N N [10],a N N N N [8],a
þ þ þ
þ ND
þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
M: male; F: female; OA: onset age; AD: autosomal dominant; S: sporadic; DS: disability score; PC: pes cavus; TSCA: thoracic spinal cord atrophy; BI: bladder involvement; BS: Babinski signs; ND, no data. a See references.
Fig. 1. Sequencing chromatograms of novel mutations and pedigrees of the patients who carried SPAST mutations. (A) Sequencing chromatogram of the p. L363P mutation in a sporadic patient. The arrow shows the position of a T-to-C transition at nucleotide 1088 (c.1088T>C) that leads to the replacement of leucine (Leu) with proline (Pro) at codon 363. (B) Sequencing chromatogram of the p. D441V mutation and the pedigree of the patient who carried the p. D441V mutation. The arrow shows the position of an A-to-T transition at nucleotide 1322 (c.1322A>T) that leads to the replacement of aspartic acid (Asp) by valine (Val) at codon 441. The father and grandfather of the index patient suffered from HSP. Dark squares show affected patients and the arrow shows the index patient. (C) Sequencing chromatogram of the p. S595R mutation and the pedigree of the patient who carried the p. S595R mutation. The arrow shows the position of an A-to-C transition at nucleotide 1783 (c.1783A>C) that leads to the replacement of serine (Ser) by arginine (Arg) at codon 595. The son of the index patient suffered from HSP. Dark squares show affected patients and the arrow shows the index patient. (D) Sequencing chromatogram of the p. Y505Ifs*7 mutation in a sporadic patient. This mutation results in a frame-shift, which generates different codon sequences starting at position 505 and introduces a premature stop at position 512.
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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Fig. 2. Multiplex ligation-dependent probe amplification profiles of the SPAST gene. (A) No exon deletions were detected in a normal control subject. (B) Deletion of exons 10e17 was detected in familial patient 27 (III3), and the pedigree of family 4 carried exon 10e17 deletions. The son, mother, and grandmother of the index patient suffered from the disease. Dark squares show affected patients, and the arrow shows the index patient. (C) Deletion of exons 5e17 was detected in sporadic patient 5 (III1).
without SPAST gene mutations. In this study, all the patients with missense mutations were males; however, the age of disease onset was variable (ranging from 15 to 41 years). The age of disease onset of patients with truncating mutations was obviously earlier than that of patients with missense mutations. No mutations in the SPAST gene were found in patients with mild peripheral neuropathy. Three patients from the group of 7 patients with pes cavus had SPAST gene mutations. 4. Discussion In this relatively large study of Chinese patients, we found that the SPAST mutation rate is 40% in Chinese familial HSP patients and 33.33% in Chinese sporadic pure HSP patients. The frequency of mutations in the SPAST gene, which are the most common cause of AD-HSP, can vary depending on ethnicity. In Europeans, mutations in SPAST/SPG4 are observed in w40% of all AD-HSP patients [16]. In our study, we used the MLPA technique to show that two AD-HSP families without SPAST point mutations had large exon deletions. Thus, the mutation rate in our pure AD-HSP families was 40% (4/10), which is in agreement with the results of a recent SPAST mutations screening study on Chinese AD-HSP families (45%, 5/11) [13]. The mutation rate (18%, 4/22) in AD-HSP families may have been underestimated in previous Chinese studies, because assays targeting the large deletion/insertion mutations were not performed in patients without point mutations [17]. After combining our results and the results of a previous study [13], the frequency of SPAST mutations in Chinese AD-HSP families is 42.86% (9/21), which is similar to that in European HSP patients [16]. The frequency of mutation carriers in sporadic cases in our cohort was 33.33% (7/21), which was higher than the frequency observed in European HSP patients (6.5e17%) [18,19] and higher than the frequency observed in another study of Chinese patients (9% (1/11)) [13]. Multiple factors may contribute to the differences among studies, such as the sample size, ethnic differences, and the
phenotype of HSP patients. A combination of direct sequencing and the MLPA technique is useful for determining the etiology of pure HSP patients. In our study, there were 2 missense mutations and 2 large deletions in AD-HSP patients, and 2 missense mutations, 1 deletion, 1 insertion, and 3 large deletions in sporadic HSP patients, which is in agreement with previous studies showing that missense mutations are the most common type of mutation in familial patients and sporadic cases [13]. However, in our study, large deletions were found to be an important cause of AD-HSP (20%, 2/10) and sporadic HSP (14.28%, 3/21), which is comparable to the 17e20% frequency of SPAST exon deletion/insertion mutations in AD-HSP patients [10,20] but is more frequent than reported in sporadic patients (2.5e10%) [20,21]. This suggests that the detection of large deletions/insertions is pivotal for understanding the cause of HSP among patients of different ethnic origins and indicates that the frequency of mutations in HSP patients has been underestimated in previous studies [15,17]. The six novel mutations identified in our study expand the mutation spectrum of SPAST in pure HSP patients. Clinical and genetic variation was also found in our HSP patients. Patients who carried the same SPAST mutation, such as the exon 1 deletion, had different ages of disease onset, which was similar to what was observed in other studies [10]. Abnormalities in the paraventricular area were shown in the brain MRI of an Australian patient with a S399L mutation [14]; however, these abnormalities were not found in a European patient [15] or in our patient (Patient 8). In the current study, we also observed an anticipation phenomenon [10]. The offspring of probands carrying an S595R mutation and exon 10e17 deletions (families 4 and 6, respectively) had a significantly earlier age of disease onset than the probands. Mutations in SPAST did not contribute to the differences in age of disease onset in our study, which agreed with the finding of a recent study that included 294 HSP patients. However, in our study, similar to the finds of Loureiro et al. [22], the age of disease onset was influenced by the type of SPAST mutations. The mean age of
Please cite this article in press as: Wei Q-Q, et al., Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia, Parkinsonism and Related Disorders (2014), http://dx.doi.org/10.1016/j.parkreldis.2014.04.021
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disease onset of patients with SPAST frameshift mutations, which lead to the production of truncated SPAST proteins, was significantly earlier than that of patients with other types of mutations. SPAST is an ATP-dependent microtubule severing protein. In the proposed disease mechanism, the region of SPAST responsible for microtubule severing was amino acids from 228 to 616. The two frameshift mutations, p. N405Kfs*36 and Y505Ifs*7, may render SPAST unable to sever microtubules [23]. The frequency of mutation in males tended to be significantly higher than that in females, which was similar to a finding from a previous study [15]. This finding may partially explain the clinical observations that the disease is more prevalent in males compared with females. Although the exact mechanism that contributes to the difference between genders is unknown, it is known that estrogen may play an important role in neuroprotection [24]. In the current study, we did not find a correlation between pes cavus, age of disease onset, and gene mutation in HSP. However, the relatively small sample size of our study is a major limitation. 5. Conclusions SPAST mutations are common in Chinese pure HSP patients. Large exon deletions are a major cause of AD-HSP and sporadic HSP in Chinese patients. In future studies, large fragment tests should be performed to explore the role of SPAST mutations in familial and sporadic pure HSP patients without point mutations in the SPAST gene. Disclosure of conflict of interest The authors declare that they have no conflict of interest. Acknowledgments The authors acknowledge all the patients and healthy subjects for their participation in this study. The authors also thank Guangzhou Kingmed Center for Clinical Laboratory Company performing the MLPA test for the HSP patients. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.parkreldis.2014.04.021. References [1] Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet 1983;1:1151e5. [2] Fink JK. Hereditary spastic paraplegia. Curr Neurol Neurosci Rep 2006;6:65e 76. [3] Slabicki M, Theis M, Krastev DB, Samsonov S, Mundwiller E, Junqueira M, et al. A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 2010;8:e1000408.
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