VAPB and C9orf72 Mutations in 1 Familial Amyotrophic Lateral Sclerosis Patient Marka Van Blitterswijka, Michael A

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Neurobiology of Aging 33 (2012) 2950.e1–2950.e4 www.elsevier.com/locate/neuaging Brief communication VAPB and C9orf72 mutations in 1 familial amyotrophic lateral sclerosis patient Marka van Blitterswijka, Michael A. van Esa, Max Koppersa, Wouter van Rheenena, Jelena Medica, Helenius J. Schelhaasb, Anneke J. van der Kooic, Marianne de Visserc, Jan H. Veldinka,1, Leonard H. van den Berga,1,* a Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, The Netherlands b Department of Neurology, and Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands c Department of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Received 4 June 2012; accepted 10 July 2012

Abstract

Previously, we have reported amyotrophic lateral sclerosis (ALS) families with multiple mutations in major ALS-associated genes. These findings provided evidence for an oligogenic basis of ALS. In our present study, we screened a cohort of 755 sporadic ALS patients, 111 familial ALS patients (97 families), and 765 control subjects of Dutch descent for mutations in vesicle-associated membrane protein B (VAPB). We have identified 1 novel VAPB mutation (p.V234I) in a familial ALS patient known to have a chromosome 9 open reading frame 72 (C9orf72) repeat expansion. This p.V234I mutation was absent in control subjects, located in a region with high evolutionary conservation, and predicted to have damaging effects. Taken together, these findings provide additional evidence for an oligogenic basis of ALS. © 2012 Elsevier Inc. Open access under the Elsevier OA license.

Keywords: Amyotrophic lateral sc lerosis; Motor neuron disease; Familial ALS; Genetics; VAPB; C9orf72

1. Introduction multiple mutations, which is higher than one might ex- pect on the basis of chance (p ϭ 1.57 ϫ 10Ϫ7). We Amyotrophic lateral sclerosis (ALS) is a complex neurode- demonstrated that C9orf72 repeat expansions were com- generative disorder of upper and lower motor neurons. Re- bined with TARDBP, FUS/TLS, and SOD1 mutations, and cently, we screened 97 families with familial ALS (FALS) for ANG TARDBP mutations in TAR DNA-binding protein (TARDBP), fused in that mutations were also combined with sarcoma/translated in liposarcoma (FUS/TLS), superox- and FUS/TLS mutations. These ﬁndings supported an ide dismutase-1 (SOD1), angiogenin (ANG), and chromo- oligogenic etiology of ALS. some 9 open reading frame 72 (C9orf72)(van Blitter- In our cohort of FALS patients, we had not yet deter- swijk et al., 2012). In 5 of these families we identiﬁed mined the mutation frequency of vesicle-associated membrane protein B (VAPB). A mutation in VAPB (p.P56S) was initially reported in Brazilian families with motor neuron

* Corresponding author at: Department of Neurology, Rudolf Magnus disease (Nishimura et al., 2004). Families with VAPB mu- Institute of Neuroscience, University Medical Center Utrecht, Heidelber- tations demonstrate a wide range of phenotypes: their age at glaan 100, 3584 CX, Utrecht, The Netherlands. Tel.: ϩ31 88 7557939; fax: onset varied from 25 to 55 years of age, their disease ϩ31 30 2542100. progression from 2 to 30 years, and they were diagnosed E-mail address: [email protected] (L.H. van den Berg). 1 Dr. J.H. Veldink and Dr. L.H. van den Berg contributed equally. with late-onset spinal muscular atrophy, atypical ALS, or

0197-4580 © 2012 Elsevier Inc. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.neurobiolaging.2012.07.004 2950.e2 M. van Blitterswijk et al. / Neurobiology of Aging 33 (2012) 2950.e1–2950.e4 typical ALS (Nishimura et al., 2004). Moreover, several pph2/; version 2.1.0, released May 2011). PolyPhen-2 val- patients were also described with autonomic abnormalities, ues were also used for the variable-threshold test to analyze including chronic intestinal constipation, and sexual dys- grouped mutations (Price et al., 2010). This statistical test function (Marques et al., 2006). Subsequently, this mutation increases statistical power, and uses a threshold that sepa- has been described in patients of Brazilian, Japanese, and rates mutations that are likely to be detrimental or nondet- European origin (Funke et al., 2010; Landers et al., 2008; rimental. The program R was used to perform this statistical Millecamps et al., 2010). Recently, a second missense mu- analysis (CRAN; www.r-project.org). tation in VAPB (p.T46I) was identified in a cohort of FALS patients from the United Kingdom (Chen et al., 2010). We investigated the mutation frequency of VAPB mutations in a 3. Results well-genotyped cohort of ALS patients and control subjects of Dutch descent. In our Dutch cohort we identified VAPB variants in 3.1% of the FALS families, 1.5% of the SALS patients, and 1.4% 2. Methods of the control subjects (Table 1). Only 1 of these variants, p.V234I, had not been detected in control subjects (1.0% of The study population consisted of 755 patients with FALS). The variable-threshold test displayed that there was sporadic ALS (SALS) and 111 patients with FALS (from 97 no significant difference in mutational burden between pa- different families). All patients were seen at national referral tients and control subjects, no matter whether we took centers for neuromuscular diseases (University Medical potential damaging effects of mutations into account (p ϭ Center Utrecht, Academic Medical Center, Amsterdam, and 0.49) or not (p ϭ 0.53). Radboud University Nijmegen Medical Center) and diag- We used PolyPhen-2 to predict the effects of these VAPB nosed with ALS according to the El Escorial criteria variants, and demonstrated that p.S158N and p.V234I could (Brooks et al., 2000). Their baseline characteristics are have damaging effects (Table 1). Because p.S158N was also shown in Supplementary Table 1. We also included 765 detected in 1 control subject, this variant most likely repre- control subjects of Dutch descent. Patient material was obtained with approval of the Institutional Review Board, sents a benign rare polymorphism; p.V234I, on the other and participants gave informed consent. All FALS patients, hand, was not detected in control subjects. The pathogenic- and large cohorts of SALS patients and control subjects, had ity estimate of the p.V234I mutation was 0.79 (values above been screened for mutations in TARDBP, FUS/TLS, SOD1, 0.50 indicate pathogenicity) with a sensitivity of 0.85 and ANG, and C9orf72 (van Blitterswijk et al., 2012). specificity of 0.93. Moreover, it is located in a region that is We screened coding regions of VAPB (NM_004738) for highly conserved across species, and appears to cause small mutations. Previously described primers were used for am- changes to ␣-helixes located within the VAPB protein (Sup- plification by touchdown polymerase chain reaction (Chen plementary Fig. 1). This VAPB mutation was detected in a et al., 2010). For sequencing and data analysis BigDye patient that also harbored a C9orf72 repeat expansion (Sup- Terminator 3.1 sequencing kit (Applied Biosystems, Foster plementary Fig. 1). She developed weakness in her right leg City, CA, USA), DNA Analyzer 3730XL (Applied Biosys- at 65 years of age, without signs of frontotemporal dementia tems), and PolyPhred were used (Nickerson et al., 1997). (FTD), and died after 34 months (Supplementary Table 2). We confirmed mutations on genomic DNA, and used Poly- Her mother, uncle, and grandmother had also died of ALS Phen-2 to predict the impact of these mutations on the (Supplementary Fig. 2). None of her family members had structure and function of VAPB (genetics.bwh.harvard.edu/ been diagnosed with FTD.

Table 1 Variants found in VAPB Variant Exon FALS families (n ϭ 97) SALS (n ϭ 755) CON (n ϭ 765) Prediction PolyPhen-2 p.A104T 3 0 (0.0%) 0 (0.0%) 1 (0.1%) Benign p.D130Ea 4 0 (0.0%) 1 (0.1%) 0 (0.0%) Benign p.S158N 5 1 (1.0%) 0 (0.0%) 1 (0.1%) Possibly damaging p.S160dela 5 1 (1.0%) 5 (0.7%) 4 (0.5%) Not applicable p.M170I 5 0 (0.0%) 5 (0.7%) 5 (0.7%) Benign p.V234I 6 1 (1.0%)b 0 (0.0%) 0 (0.0%) Possibly damaging Total 3 (3.1%) 11 (1.5%) 11 (1.4%) The impact of missense variants on the structure, and function of the protein was predicted with PolyPhen-2 (genetics.bwh. harvard.edu/pph2/). One of the variants, p.V234I, is likely to be pathogenic, and is denoted by the bold font. Key: CON, control subjects; FALS, familial amyotrophic lateral sclerosis; SALS, sporadic amyotrophic lateral sclerosis. a The p.S160del and p.D130E variants have been reported previously; both were detected in patients and control subjects (Conforti et al., 2006; Landers et al., 2008). b This patient also has a C9orf72 repeat expansion. M. van Blitterswijk et al. / Neurobiology of Aging 33 (2012) 2950.e1–2950.e4 2950.e3

4. Discussion ALS-associated gene and patients with mutations in multiple ALS-associated genes (van Blitterswijk et al., 2012). Our study identified 1 novel VAPB mutation (p.V234I) The detection of mutations in multiple ALS-associated that was absent in control subjects, located in a region with genes, and their detection in families with reduced pen- high evolutionary conservation, and predicted to be patho- etrance and/or apparently sporadic patients (Chiò et al., genic. Currently, only 2 pathogenic VAPB mutations have 2012; van Blitterswijk et al., 2012), does suggest that the been reported. The first mutation, p.P56S, was identified in etiology of ALS is more complex than previously thought. motor neuron disease families with phenotypic heterogene- In our present study, we report a novel VAPB mutation in ity (Nishimura et al., 2004). The second mutation, p.T46I, a patient with a C9orf72 repeat expansion. This repeat was recently detected in 1 FALS patient from the United expansion has been reported in patients with a range of Kingdom (Chen et al., 2010). VAPB has 3 conserved do- clinical phenotypes, including FTD, ALS, ALS-FTD, and mains: an N-terminal immunoglobulin-like ␤-sheet that re- progressive muscular atrophy (Dejesus-Hernandez et al., sembles the nematode major sperm protein (amino acid 2011; Renton et al., 2011; van Rheenen et al., 2012). VAPB 1–125) and contains a double phenylalanine in an acidic mutations have been detected in patients with phenotypic tract, a central coiled–coil domain (amino acid 158–211), heterogeneity as well (Nishimura et al., 2004). We, there- and a C-terminal transmembrane domain (amino acids 220– fore, hypothesize that these phenotypic differences can be 243) (Lev et al., 2008). The p.P56S, and p.T46I mutations attributed to interactions between ALS-associated genes, are both located within the nematode major sperm protein; thus emphasizing the complex oligogenic etiology of neu- the p.V234I mutation is located in the transmembrane do- rodegenerative diseases, and of ALS in particular. main. VAPB is known to be involved in many cellular processes, including lipid metabolism, membrane traffick- Acknowledgements ing, the unfolded protein response, microtubule organiza- tion, and homeostatic- and stress-signaling systems in the This work was supported by the VSB fonds, The Thierry endoplasmic reticulum (ER) (Gkogkas et al., 2008; Latran Foundation, Prinses Beatrix Fonds, Catharijne Kanekura et al., 2009; Lev et al., 2008). Mutations in VAPB Stichting, H. Kersten and M. Kersten, J.R. van Dijk, the have already been shown to cause cytoplasmic transactive Adessium Foundation, and European Community’s Health response DNA-binding protein 43 (TDP-43) accumulations, Seventh Framework Program (FP7/2007-2013) under grant lead to ER disorganization, and increase the vulnerability of agreement number 259867. motor neuron cells to ER stress-induced death (Chen et al., 2010; Tudor et al., 2010). Disclosure statement The identified p.V234I mutation was present in 1 FALS patient, which also harbored a C9orf72 repeat expansion. The authors disclose no actual or potential conflicts of This repeat expansion has recently been identified as the interest. most common genetic cause of ALS (Dejesus-Hernandez et Patient material was obtained with approval of the Insti- al., 2011; Renton et al., 2011). We have already reported tutional Review Board, and participants gave informed con- that C9orf72 repeat expansions can be detected in approx- sent. imately 37% of the FALS families in The Netherlands (van Appendix A. Supplementary data Rheenen et al., 2012). In addition, we have shown that these repeat expansions can be identified in FALS patients with Supplementary data associated with this article can be TARDBP (p.N352S), FUS/TLS (p.Q210H), or SOD1 found, in the online version, at http://dx.doi.org/10.1016/ (p.D90A) mutations (van Blitterswijk et al., 2012). A recent j.neurobiolaging.2012.07.004. study in Sardinia also reported C9orf72 repeat expansions in TARDBP patients with (p.A382T) mutations (Chiò et al., References 2012). One of their patients had ALS and FTD, his age at onset was 43 years, his disease duration was 34 months, and Brooks, B.R., Miller, R.G., Swash, M., Munsat, T.L., 2000. El Escorial both his parents had also been diagnosed with ALS and revisited: revised criteria for the diagnosis of amyotrophic lateral scle- FTD. Furthermore, they described another patient that de- rosis. Amyotroph. Lateral Scler. Other Mot. Neuron Disord. 1, 293– veloped ALS without FTD at 35 years of age; this patient 299. Chen, H.J., Anagnostou, G., Chai, A., Withers, J., Morris, A., Adhikaree, was tracheotomized after 25 months, and none of his family J., Pennetta, G., de Belleroche, J.S., 2010. Characterization of the members was known to have ALS or FTD (reduced pen- properties of a novel mutation in VAPB in familial amyotrophic lateral etrance). They argued that the age at symptom onset was sclerosis. J. Biol. Chem. 285, 40266–40281. influenced by the genetic burden of the individual, and that Chiò, A., Restagno, G., Brunetti, M., Ossola, I., Calvo, A., Canosa, A., these patients demonstrated a relatively severe disease Moglia, C., Floris, G., Tacconi, P., Marrosu, F., Marrosu, M.G., Murru, M.R., Majounie, E., Renton, A.E., Abramzon, Y., Pugliatti, M., Sotgiu, course. We, however, could not detect significant differ- M.A., Traynor, B.J., Borghero, G., the SARDINIALS Consortium, ences in age at onset between patients with mutations in 1 2012. ALS/FTD phenotype in two Sardinian families carrying both 2950.e4 M. van Blitterswijk et al. / Neurobiology of Aging 33 (2012) 2950.e1–2950.e4

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