PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of actamyolLink to Publisher's site
 
Acta Myol. Oct 2012; 31(2): 134–138.
PMCID: PMC3476858
TARDBP mutations are not a frequent cause of ALS in Finnish patients
HANNA-KAISA MENTULA,1* LAURA TUOVINEN,1* SINI PENTTILÄ,2 TIINA SUOMINEN,2 BJARNE UDD,2,3,4 and JOHANNA PALMIO1,2
1 School of Medicine, University of Tampere, Tampere, Finland;
2 Neuromuscular Research Unit, Department of Neurology, Tampere University Hospital and University of Tampere, Tampere, Finland;
3 Folkhälsan Institute of Genetics, Department of Medical Genetics and Haartman Institute, University of Helsinki;
4 Department of Neurology, Vaasa Central Hospital, Vaasa, Finland
*Equal contribution
Address for correspondence: Johanna Palmio, Neuromuscular Research Unit, Department of Neurology, Tampere University Hospital and University of Tampere. E-mail: johanna.palmio/at/uta.fi
In previous studies 1-3 % of ALS patients have TARDBP mutations as the cause of the disease. TARDBP mutations have been reported in ALS patients in different populations but so far there are no studies on the frequency of TARDBP mutations in Finnish ALS patients. A cohort of 50 Finnish patients, 44 SALS and 6 FALS patients, were included in the study. Genomic DNA was extracted from venous blood or muscle tissue and a mutation analysis of TARDBP was performed. No definitely pathogenic mutations could be identified in TARDBP in our patient cohort. However, two previously unknown variations were found: one silent mutation in exon 2 and one relatively deep intronic single nucleotide insertion in intron 5. In addition, two previously known non-pathogenic polymorphisms in intron 5 were detected. The size of our cohort is obviously not large enough to conclusively exclude TARDBP mutations as a very rare cause of ALS in Finland. However, based on our results TARDBP mutations do not appear to be a frequent cause of familial or sporadic ALS in Finland.
Key words: Amyotrophic lateral sclerosis, mutation screening, TARDBP
Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neuromuscular disease characterized by progressive loss of motor neurons in the brain and spinal cord, leading to paralysis and death from respiratory failure typically within 3-5 years after symptom onset (1). The incidence is 2-3 per 100 000 person-years and prevalence 4-6 per 100 000 (2-4). Most cases are sporadic (SALS), but approximately 10 % are familial (FALS). Mutations in SOD1 gene account for 15-20 % of FALS cases (5, 6), and to an even higher degree in Finnish ALS patients (7). Mutations in several other genes, including TARDBP, ALS2, SETX, FUS, VAPB, ANG, DCTN1, and UBQLN2, are described as rare causes of FALS (6, 8-16). Recently, Renton et al. (2011) reported a hexanucleotide repeat expansion within C9orf72 gene as the cause of chromosome 9p21-linked ALS (17). In their data this expansion mutation accounted for 46 % of familial and 21 % of sporadic ALS in the Finnish population, and in one-third of FALS cases of wider European ancestry making it the most common genetic cause of ALS identified to date.
Transactive response DNA-binding protein 43 (TDP- 43) is a nuclear protein composed of 414 amino acids, encoded by TAR-DNA-binding protein-gene (TARDBP) on chromosome 1p36.22 (18). TARDBP contains 5 coding and 2 non-coding exons (19). Mutations in TARDBP gene are found in 1-3% of ALS cases (20). All identified mutations seem to cluster in exon 6, except for one in exon 4. Missense mutations have been found in sporadic and familial SOD1-negative ALS (8, 21-24), and a frame-shift mutation that creates a premature stop codon (Y374X) has also been reported (25). TDP-43 is a protein expressed ubiquitously in the tissues of the human body. It has been identified as taking part in numerous cellular processes including regulating transcription and alternative splicing, as well as transport, translation and metabolism of RNA (26). Besides being primarily mutated, TDP-43 has been recognized in the inclusions of motor neurons also in other kinds of ALS, and is therefore considered a major disease protein in ALS (27, 28). Even though the neurotoxicity of TDP-43 aggregates has been established, the exact molecular mechanism of neurodegeneration remains elusive. Phosphorylation, truncation, mislocalization (26) and ubiquitination of the protein have been reported to contribute to the disease pathogenesis (16, 27, 28).
TARDBP mutations have been reported in ALS patients in different populations but so far no studies on the frequency of TARDBP mutations in Finnish ALS patients have been performed.
Subjects
A cohort of 50 ALS patients was included in the study. DNA was extracted from a blood sample in 19 patients and from a muscle tissue sample in 31 patients.
Of the patients, 24 were male and 26 female with a mean age at onset of symptoms of 62.4 years. The diagnosis of ALS, based on El Escorial criteria (29), was made between 2004 and 2011. Upper motor neuron (UMN) signs were evaluated clinically and based on findings divided in to probable and definite UMN signs (30) by neurologist. Neuropsychological assessment was performed when a patient was suspected to have cognitive decline. Six patients had FALS and 44 SALS. In all FALS cases and 17 SALS cases SOD1 D90A mutation was excluded before their inclusion in the present study. A muscle biopsy was obtained from all patients as part of the routine diagnostic evaluation. Medical history and demographic data were collected from patient documents.
All the patients gave their written informed consent. The study was approved by the Institutional Review Board of Tampere University Hospital and performed in accord with the Helsinki declaration.
Mutation screening
Genomic DNA was extracted from venous blood or muscle tissue using Archive Pure DNA Blood Kit (5 Prime, Hamburg, Germany). The coding region of TARDBP, i.e. exons 2-6 were amplified by polymerase chain reaction (PCR Master Mix, Fermentas, St. Leon-Rot, Germany). Primer sequences were designed to include the entire exons and exon-intron borders with Primer3 version 0.4.0 (31). PCR products were sequenced using Big- Dye Terminator v3.1 kit on ABI3130xl automatic DNA sequencer system (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using Chromas 1.45 (Griffith University, Southport, Queensland, Australia) and MACAW 2.0.5 (National Center for Biotechnology Information, Bethesda, MD, USA) software.
DNA extracted from venous blood or from muscle tissue was used to screen for mutations in TARDBP gene in the 50 ALS patients. Clinical information from the 44 SALS and 6 FALS patients is summarized in Table 1. At the time of the analysis of the data, 12 patients were alive, 35 deceased, and of three patients there was no current information available. One of the patients was mechanically ventilated. The age of symptom onset ranged from 41.0 to 78.75 years and the age at the point of ALS-diagnosis from 42.25 to 79.25 years. The disease duration ranged from 0.75 to16.5 years for all patients and for the 35 deceased patients from 0.75 to 6.5 years. Nine patients had probable and 41 definite UMN signs at the time of diagnosis. Frontotemporal dementia was diagnosed in three patients, nonspecific dementia in one and mild cognitive decline in three patients.
Table 1.
Table 1.
Clinical characteristics and TARDBP mutations in the Finnish ALS patients.
No definitely pathogenic mutations could be identified in the genetic analysis of TARDBP. We identified a previously unknown silent mutation in exon 2 (c.163C > A R55R), and a deep intronic single nucleotide insertion in intron 5 (c.715-75_715-74insT). Neither of these is recorded in the international polymorphism database, dbSNP (32). According to the prediction program Human Splicing Finder version 2.4.1 the effect of these two new mutations on splicing cannot be excluded (33). To estimate the pathogenicity of these two previously unknown variations we screened 101 Finnish normal population controls for them. None of the control samples harboured the mutation c.163C > A (p.R55R) whereas, two control samples were heterozygous for intronic c.715- 75_715-74insT. Two previously known common polymorphisms (c.714+68_714+69insG and c.715-126delG) in intron 5 were detected as well. The majority of patients 41/50 had both of these polymorphisms. Forty-six patients showed the common insertion polymorphism, c.714+68_714+69insG. Thirty-seven of these were homozygous and 9 heterozygous. The common deletion polymorphism, c.715-126delG, was found in 42 patients (33 homozygous and 9 heterozygous). Thus, these two known polymorphisms in intron 5 constitute the major allele in the Finnish population (Table 1).
In this study we screened a cohort of 50 SALS and FALS patients for mutations in TARDBP gene, but we did not find any definitely pathogenic mutations. A previously unknown heterozygous silent mutation in exon 2 was identified in one patient. Silent mutations are likely to be insignificant. However, activation of cryptic splice sites is possible with silent mutations in coding sequences. Such an event cannot be excluded in our patient as the mutation in exon 2 was not detected in any of the 101 control samples. Two patients showed a heterozygous single nucleotide insertion mutation in intron 5. This mutation was another one not found in the database. However, the nucleotide insertion lies relatively deep in the intron and because it was found in two control samples it is unlikely to be pathogenic.
The incidence and prevalence of ALS in Finland are among the highest in the world outside Western Pacific (1, 34). Increasing incidence of ALS in Sweden and Norway has also been reported in the last few decades. These Nordic countries appear to show higher incidence than most other European countries. Explanations for this observed increase in incidence have ranged from aging population to improved diagnostics and better neurologic services. However, these factors alone could not account for the entire increase in incidence, and the real cause remains unclear (35, 36). Many studies have addressed the role of environment as contributory factor in ALS disease process and also as an explanation for increased incidence but none of the risk factors have been reported consistently (35-37). Genetic background could be more important aspect in Finland since the frequency of the new C9orf72 gene mutation was reported to be higher in a Finnish cohort than in other similar European studies (17, 38).
The D90A allele of SOD1 occurs with increased frequency in Finland and northern Sweden but it accounts for only a proportion of the high incidence of ALS in Finland (7, 39). A second and even more important cause of ALS in the Finnish population has been associated with chromosome 9p21 (40). Recently a hexanucleotide (GGGGCC) repeat expansion within C9orf72 gene was identified as the cause of choromosome 9p21-linked ALS (17). This repeat expansion mutation is common in Finland: it was identified in 46.0% of FALS and 21.1% of SALS cases in Finnish population. In populations of wider European ancestry 38.1 % of FALS patients carried the same hexanucleotide mutation on a common haplotype background. Together with the SOD1 D90A mutation, this repeat expansion explains 87% of familial ALS in Finland (17). Apart from these two causes of ALS, no other gene mutations have yet been identified in Finnish ALS patients.
A subgroup of frontotemporal dementias (FTD) is characterized by prominent TDP-43 pathology presenting another TDP-43 proteinopathy (41). Evidence shows that frontal executive deficits are found in half of ALS patients and obvious FTD in smaller group of patients (42). Our study was not designed to evaluate possible cognitive decline or behavioral symptoms in our patients, therefore, all patients were not routinely assessed by neuropsychologist. However, three patients were diagnosed with FTD and three patients with milder symptoms. Patients with ALS and FTD should be further screened for C9orf72 gene (43).
In previous studies, the frequency of TARDBP mutations in ALS patients has been reported at 1-3 % (20). Studies on different populations show some variation of frequencies of TARDBP mutations in ALS patients. The occurrence was 0.5% in SALS and 0.6% in non-SOD1 FALS patients in an English population (8) and 6.5% in non-SOD1 FALS cases in a German cohort (44). Therefore, the size of our cohort may not be large enough to exclude TARDBP mutations as a very rare cause of ALS in Finland. We did not identify any definitely pathogenic mutations; however, pathogenicity of the silent mutation is not excluded, although unlikely. In any case, based on our results TARDBP mutations do not appear to be a frequent cause of familial or sporadic ALS in Finland.
1. Majoor-Krakauer D, Willems PJ, Hofman A. Genetic epidemiology of amyotrophic lateral sclerosis. Clin Genet. 2003;63:83–101. [PubMed]
2. Chiò A, Mora G, Calvo A, et al. Epidemiology of ALS in Italy: a 10-year prospective population-based study. Neurology. 2009;72:725–731. [PubMed]
3. O'Toole O, Traynor BJ, Brennan P, et al. Epidemiology and clinical features of amyotrophic lateral sclerosis in Ireland between 1995 and 2004. J Neurol Neurosurg Psychiatry. 2008;79:30–32. [PubMed]
4. Forbes RB, Colville S, Parratt J, et al. The incidence of motor neuron disease in Scotland. J Neurol. 2007;254:866–869. [PubMed]
5. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59–62. [PubMed]
6. Dion PA, Daoud H, Rouleau GA. Genetics of motor neuron disorders: new insights into pathogenic mechanisms. Nat Rev Genet. 2009;10:769–782. [PubMed]
7. Andersen PM, Forsgren L, Binzer M, et al. Autosomal recessive adult-onset amyotrophic lateral sclerosis associated with homozygosity for Asp90Ala CuZn-superoxide dismutase mutation. A clinical and genealogical study of 36 patients. Brain. 1996;119:1153–1172. [PubMed]
8. Sreedharan J, Blair IP, Tripathi VB, et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008;319:1668–1672. [PubMed]
9. Hadano S, Hand CK, Osuga H, et al. A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat Genet. 2001;29:166–173. [PubMed]
10. Chen YZ, Bennett CL, Huynh HM, et al. DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4) Am J Hum Genet. 2004;74:1128–1135. [PubMed]
11. Kwiatkowski TJ, Jr, Bosco DA, Leclerc AL, et al. Mutations in the FUS/TLS Gene on Chromosome 16 Cause Familial Amyotrophic Lateral Sclerosis. Science. 2009;323:1205–1208. [PubMed]
12. Vance C, Rogelj B, Hortobágyi T, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;323:1208–1211. [PubMed]
13. Nishimura AL, Mitne-Neto M, Silva HCA, et al. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004;75:822–831. [PubMed]
14. Greenway MJ, Andersen PM, Russ C, et al. ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis. Nat Genet. 2006;38:411–413. [PubMed]
15. Puls I, Jonnakuty C, Lamonte BH, et al. Mutant dynactin in motor neuron disease. Nat Genet. 2003;33:455–456. [PubMed]
16. Deng HX, Chen W, Hong ST, et al. Mutation in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature. 2011;477:211–215. [PMC free article] [PubMed]
17. Renton AE, Majounie E, Waite A, et al. A Hexanucleotide Repeat Expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72:257–268. [PMC free article] [PubMed]
18. Ou SH, Wu F, Harrich D, et al. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. J Virol. 1995;69:3584–3596. [PMC free article] [PubMed]
19. Rutherford NJ, Zhang YJ, Baker M, et al. Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis. PLoS Genet. 2008;4:e1000193–e1000193. [PMC free article] [PubMed]
20. Valdmanis PN, Daoud H, Dion PA, et al. Recent advances in the genetics of amyotrophic lateral sclerosis. Curr Neurol Neurosci Rep. 2009;9:198–205. [PubMed]
21. Yokoseki A, Shiga A, Tan CF, et al. TDP-43 mutation in familial amyotrophic lateral sclerosis. Ann Neurol. 2008;63:538–542. [PubMed]
22. Gitcho MA, Baloh RH, Chakraverty S, et al. TDP-43 A315T mutation in familial motor neuron disease. Ann Neurol. 2008;63:535–538. [PMC free article] [PubMed]
23. Kabashi E, Valdmanis PN, Dion P, et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008;40:572–574. [PubMed]
24. Deerlin VM, Leverenz JB, Bekris LM, et al. TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. Lancet Neurol. 2008;7:409–416. [PMC free article] [PubMed]
25. Daoud H, Valdmanis PN, Kabashi E, et al. Contribution of TARDBP mutations to sporadic amyotrophic lateral sclerosis. J Med Genet. 2009;46:112–114. [PubMed]
26. Gendron TF, Josephs KA, Petrucelli L. Review: transactive response DNA-binding protein 43 (TDP-43): mechanism of neurodegeneration. Neuropathol Appl Neurobiol. 2010;36:97–112. [PMC free article] [PubMed]
27. Neumann M, Sampathu DM, Kwong LK, et al. Ubiquitinated TDP- 43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science. 2006;314:130–133. [PubMed]
28. Arai T, Hasegawa M, Akiyama H, et al. TDP-43 is a component of ubiquitin-positivi tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun. 2006;351:602–611. [PubMed]
29. Brooks BR, Miller RG, Swash M, et al. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000;1:293–299. [PubMed]
30. Kaufmann P, Pullman SL, Shungu DC, et al. Objective tests for upper motor neuron involvement in amyotrophic lateral sclerosis (ALS) Neurology. 2004;62:1753–1757. [PubMed]
31. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–386. [PubMed]
32. National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/SNP/ (accessed 09/12/2011)
33. Desmet FO, Hamroun D, Lalande M, et al. Human splicing finder: An online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67–e67. [PMC free article] [PubMed]
34. Cronin S, Hardiman O, Traynor BJ. Ethnic variation in the incidence of ALS: a systematic review. Neurology. 2007;68:1002–1007. [PubMed]
35. Seljeseth YM, Vollset SE, Tysnes OB, et al. Increasing mortality from amyotrophic lateral sclerosis in Norway? Neurology. 2000;55:1262–1266. [PubMed]
36. Fang F, Valdimarsdóttir U, Bellocco R, et al. Amyotrophic Lateral Sclerosis in Sweden, 1991-2005. Arch Neurol. 2009;66:515–519. [PubMed]
37. Ahmed A, Wicklund MP. Amyotrophic lateral sclerosis: what role does environment play? Neurol Clin. 2011;29:689–711. [PubMed]
38. García-Redondo A, Dols-Icardo O, Rojas R, et al. Analysis of the C9orf72 gene in patients with amyotrophic lateral sclerosis in Spain and different populations worldwide. Hum Mutat. 2012 Aug 30; [Epub ahead of print] [PubMed]
39. Andersen PM, Nilsson P, Ala-Hurula V, et al. Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase. Nat Genet. 1995;10:61–66. [PubMed]
40. Laaksovirta H, Peuralinna T, Schymick JC, et al. Chromosome 9p21 in amyotrophic lateral sclerosis in Finland: a genome wide association study. Lancet Neurol. 2010;9:978–985. [PMC free article] [PubMed]
41. Lee EB, Lee VM, Trojanowski JQ, et al. Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration. Nat Rev Neurosci. 2011;13:38–50. [PMC free article] [PubMed]
42. Lomen-Hoerth C, Murphy J, Langmore S, et al. Are amyotrophic lateral sclerosis patients cognitively normal? Neurology. 2003;60:1094–1097. [PubMed]
43. Daoud H, Suhail H, Sabbagh M, et al. C9orf72 hexanucleotide repeat expansions as the causative mutation for chromosome 9p21- linked amyotrophic lateral sclerosis and frontotemporal dementia. Arch Neurol. 2012;69:1159–1163. [PubMed]
44. Kühnlein P, Sperfeld AD, Vanmassenhove B, et al. Two german kindreds with familial amyotrophic lateral sclerosis due to TARDBP mutations. Arch Neurol. 2008;65:1185–1189. [PMC free article] [PubMed]
Articles from Acta Myologica are provided here courtesy of
Pacini Editore