A novel mutation in the senataxin gene identified in a ...

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membrane protein B (VAPB, ALS8) and TDP-43. (3Б8). Overall, 12Б23% of diagnosed FALS cases and 2Б7% of apparently SALS cases are accounted.
Amyotrophic Lateral Sclerosis. 2009; 10: 118122

ORIGINAL ARTICLE

A novel mutation in the senataxin gene identified in a Chinese patient with sporadic amyotrophic lateral sclerosis

ZHEN-HUA ZHAO1, WEN-ZU CHEN1, ZHI-YING WU1,2, NING WANG1, GUI-XIAN ZHAO2, WAN-JIN CHEN1 & SHEN-XING MURONG1 Amyotroph Lateral Scler Downloaded from informahealthcare.com by 118.175.9.242 on 05/20/14 For personal use only.

1

Department of Neurology and Institute of Neurology, First Affiliated Hospital, Centre of Neuroscience, Fujian Medical University, Fuzhou, and 2Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai, China

Abstract Our objective was to investigate the association between senataxin mutations and sporadic amyotrophic lateral sclerosis (ALS) in Chinese patients. DNA from 45 sporadic ALS patients was screened for mutations in senataxin using polymerase chain reaction (PCR) and direct sequencing. A novel variation, Thr1118Ile, was identified in a 42-year-old individual with sporadic ALS. This variation was not detected in 200 unrelated control individuals. In conclusion, the presence of this variation in a patient with sporadic ALS, and its absence in 200 controls, supports an association between senataxin and sporadic ALS. This study has broadened the mutation spectrum of senataxin and expanded the clinical phenotypes of senataxin mutations. Key words: Amyotrophic lateral sclerosis, senataxin, sporadic, Chinese

Introduction Amyotrophic lateral sclerosis (ALS) is a common adult-onset disorder of motor neurons. Individuals with ALS typically develop a combination of upper and lower motor neuron signs, with progressive muscle weakness and wasting, usually accompanied by pathologically brisk reflexes, eventually involving the limb and bulbar muscles. Although scientific progress has been made, the aetiology and pathomechanisms of the disease are still not well known (1,2). Approximately 510% cases are familial ALS (FALS), and the remaining cases are sporadic ALS (SALS) (1). FALS is caused by mutations of many genes including SOD1 (ALS1), alsin (ALS2), senataxin (SETX, ALS4), dynactin (DCTIN1), angiogenin (ANG), and synaptobrevin-associated membrane protein B (VAPB, ALS8) and TDP-43 (38). Overall, 1223% of diagnosed FALS cases and 27% of apparently SALS cases are accounted for by SOD1 mutations (35). Senataxin mutations are associated with two distinct syndromes, juvenile amyotrophic lateral

sclerosis 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2) (914). To date, only three senataxin mutations, including T3I, L389S, and R2136H, have been reported in ALS4 patients (http:// www.hgmd.cf.ac.uk) (10). However, the function of this gene remains unknown. Here, we screened for senataxin mutations in 45 SALS patients and have identified one novel mutation.

Patients and methods Patients SALS patients from the Han ethnic group in south China were enrolled in our study between 23 March 2004 and 16 June 2006. All patients had been examined by two neurologists. Neurological examinations, including electromyography (EMG) and magnetic resonance imaging (MRI) of the cervical cord, were performed. Medical history and demographic information were collected by a speciallyassigned person, and records were reviewed by two senior neurologists. All patients had ‘definite ALS’

Correspondence: Z.-Y. Wu, Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, 12 Wulumuqi Zhong Road, Shanghai 200040, China. E-mail: [email protected] or N. Wang, Department of Neurology and Institute of Neurology, First Affiliated Hospital, Center of Neuroscience, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China. (Received 31 July 2008; accepted 6 October 2008) ISSN 1748-2968 print/ISSN 1471-180X online # 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 10.1080/17482960802572673

Senataxin mutation in a Chinese SALS patient

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Table I. Primers designed for the senataxin gene and conditions of PCR. Primers

Oligonucleotide of primers

Size of PCR product (bp)

Annealing temperature(8C)

Exon1

F 5?-CTACCTCgCggCAACAgC-3? R 5?-AagCggCCCTgCAAgCAC-3? F 5?-TaggATCgATACCAggCTC-3? R 5?-TCTgTCCCTTTAggACTCTg-3? F 5?-gTgATTgATAAgCCAgCCAg-3? R 5?-TTCCCAgCTATCAAgCTCTg-3? F 5?-gAAAAggCTTTCTAggTCg-3? R 5?-AACTTgAggAACCATCAAg-3? F 5?-TCAACggATTTAgCCTgTCAg-3? R 5?-TCTTCTTTgACCCCAgAgCg-3? F 5?-AggATCAAATAAggTCCACAC-3? R 5?-CAACAgAgTgAgACTgTCTC-3? F 5?-CTTgCTTCATCTTCCTgCC-3? R 5?-CAACATACACCAATTCCTgC-3? F 5?-TagTTgggTTgCTACTCACC-3? R 5?-CCTCTTgTTAAgTAATCTAgC-3? F 5?-gCTAgATTACTTAACAAgAgg-3? R 5?-ATAgCAgTAgATTgAgAACAg-3? F 5?-CgACTTgggTgTTgAACC-3? R 5?-ATTgCTgTTCCTTTggAgC-3? F 5?-gAAgCCgTCgTCAAATgTg-3? R 5?-gAAAATgTTgggCTggAAg-3? F 5?-TggATCTgACTTCTgCATg-3? R 5?-gAgCATCATCCTTTAAAgAg-3? F 5?-gAAAggggATgTgACAgAg-3? R 5?-gAgATgATTTCTTCTCTgAAg-3? F 5?-ACAggAATTTCATgTTgATgg-3? R 5?-gTTgAAACgTgCTgCTCTg-3? F 5?-AggACAAAATATgCCTTgAg-3? R 5?-AagATgAAggCCTCACAgg-3? F 5?-CAAgACCACggAgTATTTC -3? R 5?-CTCAgCTgTTgAAgTTggC-3? F 5?-AgTTgTAgAACAACTCCTgC-3? R 5?-CATCATCAgTTgCTggAgAC-3? F 5?-gTACTTgCCAACAgTAACAg-3? R 5?-ggTTTAgATgCAggAggAg-3? F 5?-gAAgTgAAAgCAgCAgATg-3? R 5?-ACTTgAAAAgTTTggAgAgg-3? F 5?-TgTCACCTAgATTCTAgCTTC-3? R 5?-ATTgTTCCAgTAACTATCCTC-3? F 5?-AgAgTCCTTTAgTgCTgTg-3? R 5?-ACTgTATCTgACATTTCCTg-3? F 5?-CATgACCTTTACAACTAggC-3? R 5?-AAACTgTTCACCgTgAACAC-3? F 5?-ggTCACTTTggTgTCCTTg-3? R 5?-gCTAgTTAACAgCATCAgTg-3? F 5?-AgCTATgTAATAgAgCTCAC-3? R 5?-AggACTggCCTgCTCTTTCC-3? F 5?-ATAgAgACAggATTgATCAgC-3? R 5?-ACAgAgCAAgACTCTgTCTC-3? F 5?-gAggTAgTCCATTggAAgg-3? R 5?-gAgACAgAgTgAgACTCTg-3? F 5?-AggCCTgTCATAgTCAAgg-3? R 5?-AtggAgggTCATTAAggAg-3? F 5?-TgCAggAgAAgTgTgATAC-3? R 5?-CCAAgAAAgATgTCTCTCC-3? F 5?-ACCTgAACgTTgCCATATAC-3? R 5?-CACAAACAATAAggggAACTC-3? F 5?-CCAgCCCACATCACCACAC-3? R 5?-CTgTgCCCTgACACTAggC-3? F 5?-CTCAgTggAACTTAAgTTAgC-3? R 5?-CAATTTgCACAgACCACTCC-3? F 5?-ACAgAgTgAgACTCCgTCTC-3? R 5?-ACAgTgggTCTATTTCACAAC-3? F 5?-TCCACCTCTgTCAgACACg-3? R 5?-TCCTAggAAgAAgTTgCTgg-3? F 5?-ggAACTCTATgCTTCCCAAg-3? R 5?-AggTCCTggTgAACgACAg-3?

194

60

271

60

382

58

322

58

460

62

441

65

312

62

356

60

289

60

521

60

479

62

498

60

538

60

498

62

429

64

431

58

526

60

563

60

611

60

382

60

338

60

421

62

356

58

317

62

557

60

270

58

386

62

304

56

318

60

311

68

349

62

388

62

340

60

532

60

Exon2 Exon3 Exon4 Exon5 Exon6 Exon7

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Exon8 Exon9 Exon10.1 Exon10.2 Exon10.3 Exon10.4 Exon10.5 Exon10.6 Exon10.7 Exon10.8 Exon10.9 Exon10.10 Exon11 Exon12 Exon13 Exon14 Exon15 Exon16-17 Exon18 Exon19 Exon20 Exon21 Exon22 Exon23 Exon24 Exon25 Exon26.1

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Table I (Continued) Primers

Amyotroph Lateral Scler Downloaded from informahealthcare.com by 118.175.9.242 on 05/20/14 For personal use only.

Exon26.2

Oligonucleotide of primers F 5?-TTgggATCCACAACCCTCg-3? R 5?-TagATggTCCCACgAgCTg -3?

Size of PCR product (bp)

Annealing temperature(8C)

443

62

according to the Airlie House criteria (15). Fortyfive apparently SALS patients were enrolled into the study. Two hundred unrelated-age individuals ( ]65 years) with no known history of ALS were selected as a control group. This study was approved by the local ethics committee, and informed consent was obtained from the participants or their legal surrogates prior to inclusion in the study. Genomic DNA was extracted from peripheral EDTA blood with a QIAamp DNA Blood Minikit (QIAGEN, Hilden, Germany). The mean age of onset of disease in the patients was 48.2299.48 years, ranging from 32 to 69 years. The ratio of male to female patients was 3.2: 1.

Pedigree analysis of this family and mutation analysis of some relatives

Mutation scanning

The patient with the Thr1118Ile variation presented at 42 years of age with difficulty in using chopsticks and lifting his arms. He gradually progressed over the subsequent two years. Neurological examination

Mutation analysis of the SOD1 gene was performed as described previously. All 21 exons of senataxin, including the intron-exon boundaries, were analysed using primer combinations designed based on the intronic sequences of senataxin. The full length of exon10 is 4176 bp; thus, 10 pairs of primers were designed flanking this area. PCR amplification was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) with standard conditions. The sequence of the primers and the annealing temperatures are shown in Table I. Amplified products were purified and subjected to direct sequencing. Sequencing procedure is as previously reported (16). Obtained sequences were compared with the genomic DNA sequence of senataxin (NCBI Sequence Viewer NT_022184), and nucleotide changes were numbered corresponding to their position in senataxin mRNA (NCBI Sequence Viewer NM_152516).

The pedigree of the patient carrying the Thr1118Ile mutation is shown in Figure 2. His mother died at 91 years of age and his father is currently 93 years old. Three elder sisters and two brothers are over 50 years of age, and all of them are healthy. Direct sequencing was performed to screen for the Thr1118Ile mutation in the father, three sisters, and two brothers. No mutation was found in them. Samples were unavailable for other family members. Clinical features and pedigree analysis of the patient carrying the Thr1118Ile mutation

Results One novel senataxin mutation was identified in the present study All patients were screened for mutations in SOD1 and were negative. Screening for senataxin mutations revealed a heterozygous Thr1118Ile (3353C 0T) variation in one patient. This variation was not identified in 200 control subjects and it has not been reported previously. The chromatogram of this heterozygous mutation is shown in Figure 1.

Figure 1. The chromatogram of the Thr1118Ile mutation. The normal sequence is shown in the upper half and the corresponding mutation is shown below.

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Figure 2. The pedigree of the patient carrying the Thr1118Ile mutation.

showed evident amyotrophy of the lingual muscle and distal muscles of the upper limbs. Muscle weakness was noted in all four extremities. There were no pyramidal signs, and the deep-tendon reflexes were reduced. EMG testing showed reduced amplitudes of compound muscle-action potentials, positive sharp waves, fibrillations, and normal motor and sensory NCV. He was treated with coenzyme Q10 120 mg daily and followed with follow-up examinations every three months. He continued to progress and developed bulbar symptoms three years after the onset of symptoms, and type II respiratory failure two years later.

Discussion Senataxin encodes a 302.8 kD protein and is associated with ALS4 and AOA2. Only three missense mutations have been detected in patients with ALS4, while many mutations have been identified in patients with AOA2 (1013). Northern blot analysis identified two prominent transcripts of 11.5 and 9.0 kb in all tissues examined, including the brain and spinal cord (10). It has been suggested that senataxin may have both RNA and DNA helicase activities and acts in the DNA repair pathway, like several other proteins defective in autosomal recessive cerebellar ataxias (10). ALS4 is a rare autosomal dominant form of juvenile ALS. It is characterized by distal muscle weakness and atrophy, normal sensation, pyramidal signs, and relentless slow disease progression. Patients with ALS4 usually have a juvenile onset of symptoms (ages B25 years), and the condition does not usually affect respiratory muscles or life expectancy (17). ALS4 and AOA2 show little clinical overlap, establishing a distinctive genotype-phenotype correlation associated with senataxin mutations. Our study indicates that senataxin mutations might also occur in sporadic ALS. None of the patients in our study had family history or mutation of the SOD1 gene. The patient with the Thr1118Ile variation had the onset of symptoms at the age of 42 years, which is considerably older than that generally associated with ALS4. Furthermore, he had ‘classical ALS’. His parents remain unaffected in their nineties, and no relatives were found to have a similar condition. The Thr1118Ile variation was

not detected in his father, his three sisters, his two brothers, or 200 unrelated control individuals. On this basis, we propose that Thr1118Ile is a novel mutation in this SALS patient, and that senataxin might be associated with sporadic ALS. Although the significance of this variation remains unknown, our study shows that possible clinical phenotypes of senataxin mutations may be wider than originally considered. Further studies are required to confirm this finding. Acknowledgements The authors thank the ALS patients and their family members. The authors also thank the anonymous reviewers for improving the manuscript. This project was supported by grant FMU-RT002 of the program for Innovative Research Team in Science and Technology in Fujian Province University and grant JS6037 from Fujian Medical College for professorship (Fuzhou), and a grant from Huashan Hospital for special professorship of Fudan University (Shanghai), China. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Mitchell JD, Borasio GD. Amyotrophic lateral sclerosis. Lancet. 2007;369:203141. 2. Shaw PJ. Molecular and cellular pathways of neurodegeneration in motor neuron disease. J Neurol Neurosurg Psychiatry. 2005;76:104657. 3. del Aguila MA, Longstreth WT Jr, McGuire V, Koepsell TD, van Belle G. Prognosis in amyotrophic lateral sclerosis: a population-based study. Neurology. 2003;60:8139. 4. Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci. 2006;7:71023. 5. Gros-Louis F, Gaspar C, Rouleau GA. Genetics of familial and sporadic amyotrophic lateral sclerosis. Biochim Biophys Acta. 2006;1762:95672. 6. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008;319:166872. 7. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, van der Velde C, et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008;40:5724. 8. van Deerlin VM, Leverenz JB, Bekris LM, Bird TD, Yuan W, Elman LB, et al. TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. Lancet Neurol. 2008;7:40916.

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