Neurobiology of Aging xxx (2014) 1e4
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Screening of CHCHD10 in a French cohort confirms the involvement of this gene in frontotemporal dementia with amyotrophic lateral sclerosis patients Annabelle Chaussenot a, b, Isabelle Le Ber c, d, Samira Ait-El-Mkadem a, b, Agnès Camuzat c, Anne de Septenville c, Sylvie Bannwarth a, b, Emmanuelle C. Genin a, Valérie Serre e, Gaëlle Augé a, b, The French research network on FTD and FTD-ALS1, Alexis Brice c, Jean Pouget f, Véronique Paquis-Flucklinger a, b, * a
IRCAN, UMR CNRS 7284/INSERM U1081/UNS, School of Medicine, Nice Sophia-Antipolis University, Nice, France Department of Medical Genetics, National Centre for Mitochondrial Diseases, Nice Teaching Hospital, Nice, France c Sorbonne Université, UPMC University Paris 06, UM75, Inserm U1127, Cnrs UMR7225, Institut du Cerveau et de la Moelle épinière (ICM), Paris, France d National Reference Centre on Rare Dementias, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Paris, France e UMR7592 CNRS, Jacques Monod Institute, Paris Diderot University, Paris, France f Department of Neurology, Timone Hospital, Marseille Teaching Hospital, Marseille, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 16 July 2014 Accepted 20 July 2014
Mutations in the CHCHD10 gene have been recently identified in a large family with a complex phenotype variably associating frontotemporal dementia (FTD) with amyotrophic lateral sclerosis (ALS), cerebellar ataxia, myopathy, and hearing impairment. CHCHD10 encodes a protein located in the mitochondrial intermembrane space and is likely involved in mitochondrial genome stability and maintenance of cristae junctions. However, the exact contribution of CHCHD10 in FTD and ALS diseases spectrum remains unknown. In this study, we evaluated the frequency of CHCHD10 mutations in 115 patients with FTD and FTD-ALS phenotypes. We identified 2 heterozygous variants in 3 unrelated probands presenting FTD and ALS, characterized by early and predominant bulbar symptoms. This study demonstrates the implication of CHCHD10 in FTD and ALS spectrum. Although the frequency of mutations is low in this series (2.6%), our work suggests that CHCHD10 mutations should be searched particularly when bulbar symptoms are present at onset. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Frontotemporal lobar degeneration (FTLD) Frontotemporal dementia (FTD) Amyotrophic lateral sclerosis (ALS) CHCHD10 Mitochondrial disease
1. Introduction
* Corresponding author at: IRCAN UMR CNRS 7284 / INSERM U1081 / UNS, School of Medicine, 28 av de Valombrose, 06107 Nice Cedex 2, France. Tel.: þ33 4 93 37 77 86; fax: þ33 4 93 37 70 33. E-mail address: [email protected] (V. Paquis-Flucklinger). 1 The French research network on frontotemporal dementia and/or frontotemporal dementia with amyotrophic lateral sclerosis includes: Sophie Auriacombe (CHU Pellegrin, Bordeaux), Alexis Brice (Hôpital de la Salpêtrière, Paris), Frédéric Blanc (Hôpitaux Civils, Strasbourg), Mira Didic (CHU La Timone, Marseille), Bruno Dubois (Hôpital de la Salpêtrière, Paris), Charles Duyckaerts (Hôpital de la Salpêtrière, Paris), Marie-Odile Habert (Hôpital de la Salpêtrière, Paris), Véronique Golfier (CHU Rennes), Didier Hannequin (CHU Charles Nicolle, Rouen), Lucette Lacomblez (Hôpital de la Salpêtrière, Paris), Isabelle Le Ber (Hôpital de la Salpêtrière, Paris), Richard Levy (CHU St Antoine, Paris), Vincent Meininger (Hôpital de la Salpêtrière, Paris), Bernard-François Michel (CH SainteMarguerite, Marseille), Florence Pasquier (CHU Roger Salengro, Lille), Catherine Thomas-Anterion (CHU Bellevue, SaintEtienne), Michèle Puel (CHU Rangueil, Toulouse), François Salachas (Hôpital de la Salpêtrière, Paris), and François Sellal (CH Colmar), Martine Vercelletto (CHU Laennec, Nantes). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.07.022
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are rare neurodegenerative diseases characterized by behavioral disorders and motor neuron symptoms. FTD and ALS are considered as 2 extremes of a disease spectrum because they may be associated in the same individual or within a family. Both disorders have similar genetic background. “GGGGCC” repeats expansion in C9orf72 gene is the most frequent genetic cause of FTD-ALS spectrum (DeJesus-Hernandez et al., 2011; Renton et al., 2011); other genetic causes, involving TARDBP, SQSTM1, VCP, PFN1, UBQLN2, hnRNPA2/B1, and A1 genes, are less frequent. Despite genetic heterogeneity, FTD and ALS share similar molecular signatures with an emerging theme including dysfunction in RNA processing and protein homeostasis (Ling et al., 2013; Neumann et al., 2006). Recently, we described another mechanism responsible for FTD-ALS (Bannwarth et al., 2014). Using whole-exome sequencing, we identified a heterozygous missense mutation c.176 C > T (p.Ser59Leu)
2
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in the CHCHD10 gene in a large family with a late-onset complex phenotype associating FTD, ALS, and/or cerebellar ataxia. All patients presented a mitochondrial myopathy with the accumulation of multiple mitochondrial DNA deletions in muscle. Because the frequency of CHCHD10 mutations is not known in FTD and ALS, the aim of this study was to investigate the genetic contribution and phenotypic spectrum of CHCHD10 mutations in a large French cohort of FTD or FTD-ALS patients.
2.2. Exome sequencing
2. Methods
Genomic DNA was prepared according to Illumina TruSeq Sample Preparation v3 (Illumina, CA, USA) and sequence capture, enrichment, and elution were performed according to the manufacturer’s instructions and protocols (Illumina TruSeq Exome Enrichment). Sequencing was performed on Illumina HiSeq2000 using 100 bp paired-end reads. Sequence alignment and variant calling were performed against the reference human genome (UCSC hg19) using bwa and the Genome Analysis Toolkit.
2.1. Patients
2.3. Sequencing of CHCHD10
We have studied a cohort of 115 French unrelated probands presenting FTD or FTD-ALS, including 21 previously reported (Bannwarth et al., 2014), recruited through a national network of neurologists expert in dementia and ALS. FTD and ALS clinical diagnoses were based on international diagnosis criteria (Brooks, 1994; Rascovsky et al., 2011). This cohort included 35 familial (26 FTD-ALS, 9 FTD) and 80 sporadic cases (FTD-ALS). The mean age at onset was 62.4 7.6 years, and the mean age at examination was 65.6 7.3 years. Blood samples were collected after informed consent was obtained. The most common FTD (C9orf72, MAPT, GRN, VCP, and CHMP2B) and ALS genes (SOD1, TARDBP, FUS/TLS, ANG, PFN1, UBQLN2, hnRNPA2B1, and hnRNPA1) had been previously excluded by Sanger sequencing or by repeat-primed polymerase chain reaction (PCR) (for C9orf72) in all the probands. This study was approved by the Ethics Committee of “AP-HP de Paris.”
The 4 coding exons and flanking intronic regions of CHCHD10 (NM_213720.1) were amplified by PCR. Primer sequences and PCR conditions are available on request. PCR products were purified with Illustra ExoStar enzyme (GE Healthcare), processed with an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems), and analyzed on an ABI 3130XL automated sequencer (Applied Biosystems). Sequences were analyzed using Seqscape v2.5 software. 2.4. Homology modeling of human CHCHD10 Using the threading program PHYRE2 (Kelley and Sternberg, 2009), 142 residues of CHCHD10 (Met1 to Pro142) were modeled as previously described (Bannwarth et al., 2014).
Fig. 1. Identification of the p.Pro34Ser mutation in CHCHD10. (A) CHCHD10 mutation sequences in patients 2 and 3 and in a control individual (Wild-Type). (B) Cross-species conservation of CHCHD10 flanking the amino acid p.Pro34. (C) Model of CHCHD10 based on the CHCHD5 structure (PDB ID: 2LQL). Alpha helices and the nonstructured regions are shown in red and gray, respectively. The residue Proline 34 located in the nonstructured N-terminal region is shown in green. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
A. Chaussenot et al. / Neurobiology of Aging xxx (2014) 1e4
3. Results In the large family which allowed the identification of the CHCHD10 gene, all but 1 patient had dementia although motor neuron symptoms were less frequent. Based on this observation, we studied a cohort of 115 unrelated probands with FTD or FTD-ALS. Among this cohort, 21 familial cases had been first analyzed by whole-exome sequencing. A first screening led us to identify the heterozygous c.176C > T CHCHD10 mutation (p.Ser59Leu), already known, in a 57-year-old man of Spanish origin (Bannwarth et al., 2014). The patient (P1) developed a progressive motor neuron involvement, confirmed by electromyography, with a predominant pseudobulbar syndrome. He also had a frontal lobe dysfunction and behavioral changes, associated with akineto-rigid syndrome. In addition, the patient presented bilateral sensorineural hypoacusis. His father, elder sister, and one of his brothers developed progressive ALS with prominent bulbar involvement around the age of 60 years. Secondarily, we sequenced CHCHD10 in 94 patients (14 familial and 80 sporadic cases) and found a novel heterozygous missense variant, c.100C > T (p.Pro34Ser) in 2 unrelated FTD-ALS individuals (P2 and P3) (Fig. 1A). P2 was a 61-year-old French patient with no family history, but his mother died at 49 years of age and his father was unknown. He initially presented aggressiveness, violence, disinhibition, perseverative behaviors, and grasping reflex evocative of FTD. He developed pseudobulbar syndrome with paretic dysarthria and dysphagia at age 63 years. Clinical examination revealed tongue fasciculations, pyramidal signs, progressive distal limb weakness, and amyotrophy. Electromyography confirmed diffuse motor neuron involvement. Brain MRI showed mild bilateral frontal atrophy. He died at age 65 years. P3 was a 67-year-old French man who initially presented behavior disorders then developed moderate limb weakness and predominant pseudobulbar syndrome leading to FTD-ALS diagnosis. He had no family history but without information about the age of death of his father. The p.Pro34Ser mutation changes a relatively conserved proline into a serine (Fig. 1B) and is absent in public single-nucleotide polymorphism databases (including 6.503 controls of the Exome Variant Server database) and in 200 ethnically and geographically matched control alleles. In silico analysis by Mutation Taster (http:// http://www.mutationtaster.org) predicted this variant to be disease causing with a strong probability. The modeling of CHCHD10 showed (1) a nonstructured N-terminal region; (2) a highly hydrophobic helix (Gly43 to Ala 68), which may be typically an interface of interaction with a partner protein; (3) and the CHCH domain near the C-terminal region characterized by a CX9C motif. The p.Pro34Ser mutation is located in the nonstructured N-terminal region (Fig. 1C). The biological relevance of this nonstructured N-terminal region is not already understood, but it may affect the protein’s stability or its ability to interact with other proteins. 4. Discussion Because the frequency of CHCHD10 mutations is unknown in FTD-ALS, we investigated the genetic contribution and phenotypic spectrum of CHCHD10 mutations in a large French cohort of patients with FTD-ALS. Sequencing CHCHD10 in 94 affected individuals led us to identify a novel missense variant (p.Pro34Ser) in 2 unrelated FTD-ALS patients. Muscle biopsy is usually not performed in patients with FTD-ALS phenotypes, and it has not been possible to investigate whether this variant was associated with a mitochondrial myopathy. Functional analyses will be needed to firmly establish its pathogenicity. However, its absence in public singlenucleotide polymorphism databases, in ethnically matched
3
controls and MutationTaster predictions are in favor of a deleterious effect. Previously, the analysis of 21 FTD-ALS familial cases allowed us to find 1 family carrying the p.Ser59Leu mutation, whose deleterious effect had been proven by functional studies (Bannwarth et al., 2014). All together, these results confirm the involvement of CHCHD10 in familial and sporadic FTD-ALS. However, as this gene was involved in 3 of 115 patients in this first study (2.6%), it is likely not a common cause of FTD-ALS spectrum in French population. The 3 patients carrying a CHCHD10 mutation had onset of symptoms in their 50e60’s (Achi and Rudnicki, 2012). It is interesting to note that all 3 patients had a predominant pseudobulbar syndrome. Bulbar palsy with dysarthria and dysphagia was also found in 6 of 8 affected individuals from the initially described family that allowed the CHCHD10 identification (Bannwarth et al., 2014). Cognitive changes preceded (P2, P3) or followed (P1) ALS symptoms. Mitochondrial diseases typically affect multi-organ systems. The sensorineural hypoacusis found in P1 that was also observed in 6 of 8 affected individuals from the initial family, and the ptosis also presented by one of them were likely secondary to mitochondrial dysfunction. Such symptoms may orientate toward CHCHD10 involvement and should be searched for therefore in FTD-ALS patients. This is the first study to evaluate the role of CHCHD10 in a cohort of FTD-ALS patients. Further investigations in larger populations with different geographic origins are needed. TDP-43 protein is a major component of neuronal inclusions that are pathologic hallmark of both ALS and FTD (Neumann et al., 2006). It will be of interest to determine whether TDP-43 inclusions are observed in patients carrying a CHCHD10 mutation, and whether CHCHD10 protein may be found in neuronal inclusions. The identification of CHCHD10 mutations in FTD-ALS spectrum opens a novel field to explore the pathogenesis of these diseases and to understand the role of mitochondrial dysfunction. Although the frequency of mutations is low in this series (2.6%), our work suggests that CHCHD10 mutations should be searched for particularly when bulbar symptoms are present at onset. In addition, it will be extremely important to analyze this gene in cohorts of patients with sporadic and familial ALS to determine whether it is also involved in pure ALS and at which frequency. Disclosure statement The authors have no conflicts of interest. Acknowledgements This work was funded by grants from the Fondation pour la Recherche Médicale (FRM) (Grant number is : DPM201211255) to Véronique Paquis-Flucklinger, the Neuromics FP7 contract E12009DD to Alexis Brice, the France Alzheimer association contract R12091DD to Alexis Brice, “The Programme Hospitalier de Recherche Clinique” (PHRC) to Isabelle Le Ber and from the program “Investissements d’avenir” ANR-10-IAIHU-06. Anne De Septenville is funded by the program “Investissements d’avenir” ANR-10IAIHU-06. The authors thank the Centre national de Genotypage, Lydia Guennec, Sylvie Forlani, and Christelle Dussert (DNA and cell bank of CR-ICM, Hôpital de la Salpêtrière, Paris) for their excellent technical assistance. References Achi, E.Y., Rudnicki, S.A., 2012. ALS and frontotemporal dysfunction: a review. Neurol. Res. Int. 2012, 806306. Bannwarth, S., Ait-El-Mkadem, S., Chaussenot, A., Genin, E.C., Lacas-Gervais, S., Fragaki, K., Berg-Alonso, L., Kageyama, Y., Serre, V., Moore, D.G., Verschueren, A., Rouzier, C., Le Ber, I., Augé, G., Cochaud, C., Lespinasse, F., N’Guyen, K., de Septenville, A., Brice, A.,
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Yu-Wai-Man, P., Sesaki, H., Pouget, J., Paquis-Flucklinger, V., 2014. A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement. Brain 137, 2329e2345. Brooks, B.R., 1994. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial “Clinical limits of amyotrophic lateral sclerosis” workshop contributors. J. Neurol. Sci. 124, 96e107. DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dickson, D.W., Boylan, K.B., GraffRadford, N.R., Rademakers, R., 2011. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245e256. http://www.ncbi.nlm.nih.gov/pubmed/ 21944778. Kelley, L., Sternberg, M., 2009. Protein structure prediction on the web: a case study using the Phyre server. Nat. Protoc. 4, 363e371. Ling, S.C., Polymenidou, M., Cleveland, D.W., 2013. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79, 416e438. Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi, M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M., McCluskey, L.F., Miller, B.L., Masliah, E., Mackenzie, I.R., Feldman, H., Feiden, W., Kretzschmar, H.A., Trojanowski, J.Q., Lee, V.M., 2006. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130e133.
Rascovsky, K., Hodges, J.R., Knopman, D., Mendez, M.F., Kramer, J.H., Neuhaus, J., van Swieten, J.C., Seelaar, H., Dopper, E.G., Onyike, C.U., Hillis, A.E., Josephs, K.A., Boeve, B.F., Kertesz, A., Seeley, W.W., Rankin, K.P., Johnson, J.K., GornoTempini, M.L., Rosen, H., Prioleau-Latham, C.E., Lee, A., Kipps, C.M., Lillo, P., Piguet, O., Rohrer, J.D., Rossor, M.N., Warren, J.D., Fox, N.C., Galasko, D., Salmon, D.P., Black, S.E., Mesulam, M., Weintraub, S., Dickerson, B.C., DiehlSchmid, J., Pasquier, F., Deramecourt, V., Lebert, F., Pijnenburg, Y., Chow, T.W., Manes, F., Grafman, J., Cappa, S.F., Freedman, M., Grossman, M., Miller, B.L., 2011. Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 134, 2456e2477. http://www.ncbi.nlm.nih.gov/ pubmed/21810890. Renton, A.E., Majounie, E., Waite, A., Simón-Sánchez, J., Rollinson, S., Gibbs, J.R., Schymick, J.C., Laaksovirta, H., van Swieten, J.C., Myllykangas, L., Kalimo, H., Paetau, A., Abramzon, Y., Remes, A.M., Kaganovich, A., Scholz, S.W., Duckworth, J., Ding, J., Harmer, D.W., Hernandez, D.G., Johnson, J.O., Mok, K., Ryten, M., Trabzuni, D., Guerreiro, R.J., Orrell, R.W., Neal, J., Murray, A., Pearson, J., Jansen, I.E., Sondervan, D., Seelaar, H., Blake, D., Young, K., Halliwell, N., Callister, J.B., Toulson, G., Richardson, A., Gerhard, A., Snowden, J., Mann, D., Neary, D., Nalls, M.A., Peuralinna, T., Jansson, L., Isoviita, V.M., Kaivorinne, A.L., Hölttä-Vuori, M., Ikonen, E., Sulkava, R., Benatar, M., Wuu, J., Chiò, A., Restagno, G., Borghero, G., Sabatelli, M.; ITALSGEN Consortium, Heckerman, D., Rogaeva, E., Zinman, L., Rothstein, J.D., Sendtner, M., Drepper, C., Eichler, E.E., Alkan, C., Abdullaev, Z., Pack, S.D., Dutra, A., Pak, E., Hardy, J., Singleton, A., Williams, N.M., Heutink, P., Pickering-Brown, S., Morris, H.R., Tienari, P.J., Traynor, B.J., 2011. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257e268. http:// www.ncbi.nlm.nih.gov/pubmed/21944779.
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Screening of CHCHD10 in a French cohort confirms the involvement of this gene in frontotemporal dementia with amyotrophic lateral sclerosis patients Annabelle Chaussenot a, b, Isabelle Le Ber c, d, Samira Ait-El-Mkadem a, b, Agnès Camuzat c, Anne de Septenville c, Sylvie Bannwarth a, b, Emmanuelle C. Genin a, Valérie Serre e, Gaëlle Augé a, b, The French research network on FTD and FTD-ALS1, Alexis Brice c, Jean Pouget f, Véronique Paquis-Flucklinger a, b, * a
IRCAN, UMR CNRS 7284/INSERM U1081/UNS, School of Medicine, Nice Sophia-Antipolis University, Nice, France Department of Medical Genetics, National Centre for Mitochondrial Diseases, Nice Teaching Hospital, Nice, France c Sorbonne Université, UPMC University Paris 06, UM75, Inserm U1127, Cnrs UMR7225, Institut du Cerveau et de la Moelle épinière (ICM), Paris, France d National Reference Centre on Rare Dementias, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Paris, France e UMR7592 CNRS, Jacques Monod Institute, Paris Diderot University, Paris, France f Department of Neurology, Timone Hospital, Marseille Teaching Hospital, Marseille, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 16 July 2014 Accepted 20 July 2014
Mutations in the CHCHD10 gene have been recently identified in a large family with a complex phenotype variably associating frontotemporal dementia (FTD) with amyotrophic lateral sclerosis (ALS), cerebellar ataxia, myopathy, and hearing impairment. CHCHD10 encodes a protein located in the mitochondrial intermembrane space and is likely involved in mitochondrial genome stability and maintenance of cristae junctions. However, the exact contribution of CHCHD10 in FTD and ALS diseases spectrum remains unknown. In this study, we evaluated the frequency of CHCHD10 mutations in 115 patients with FTD and FTD-ALS phenotypes. We identified 2 heterozygous variants in 3 unrelated probands presenting FTD and ALS, characterized by early and predominant bulbar symptoms. This study demonstrates the implication of CHCHD10 in FTD and ALS spectrum. Although the frequency of mutations is low in this series (2.6%), our work suggests that CHCHD10 mutations should be searched particularly when bulbar symptoms are present at onset. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Frontotemporal lobar degeneration (FTLD) Frontotemporal dementia (FTD) Amyotrophic lateral sclerosis (ALS) CHCHD10 Mitochondrial disease
1. Introduction
* Corresponding author at: IRCAN UMR CNRS 7284 / INSERM U1081 / UNS, School of Medicine, 28 av de Valombrose, 06107 Nice Cedex 2, France. Tel.: þ33 4 93 37 77 86; fax: þ33 4 93 37 70 33. E-mail address: [email protected] (V. Paquis-Flucklinger). 1 The French research network on frontotemporal dementia and/or frontotemporal dementia with amyotrophic lateral sclerosis includes: Sophie Auriacombe (CHU Pellegrin, Bordeaux), Alexis Brice (Hôpital de la Salpêtrière, Paris), Frédéric Blanc (Hôpitaux Civils, Strasbourg), Mira Didic (CHU La Timone, Marseille), Bruno Dubois (Hôpital de la Salpêtrière, Paris), Charles Duyckaerts (Hôpital de la Salpêtrière, Paris), Marie-Odile Habert (Hôpital de la Salpêtrière, Paris), Véronique Golfier (CHU Rennes), Didier Hannequin (CHU Charles Nicolle, Rouen), Lucette Lacomblez (Hôpital de la Salpêtrière, Paris), Isabelle Le Ber (Hôpital de la Salpêtrière, Paris), Richard Levy (CHU St Antoine, Paris), Vincent Meininger (Hôpital de la Salpêtrière, Paris), Bernard-François Michel (CH SainteMarguerite, Marseille), Florence Pasquier (CHU Roger Salengro, Lille), Catherine Thomas-Anterion (CHU Bellevue, SaintEtienne), Michèle Puel (CHU Rangueil, Toulouse), François Salachas (Hôpital de la Salpêtrière, Paris), and François Sellal (CH Colmar), Martine Vercelletto (CHU Laennec, Nantes). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.07.022
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are rare neurodegenerative diseases characterized by behavioral disorders and motor neuron symptoms. FTD and ALS are considered as 2 extremes of a disease spectrum because they may be associated in the same individual or within a family. Both disorders have similar genetic background. “GGGGCC” repeats expansion in C9orf72 gene is the most frequent genetic cause of FTD-ALS spectrum (DeJesus-Hernandez et al., 2011; Renton et al., 2011); other genetic causes, involving TARDBP, SQSTM1, VCP, PFN1, UBQLN2, hnRNPA2/B1, and A1 genes, are less frequent. Despite genetic heterogeneity, FTD and ALS share similar molecular signatures with an emerging theme including dysfunction in RNA processing and protein homeostasis (Ling et al., 2013; Neumann et al., 2006). Recently, we described another mechanism responsible for FTD-ALS (Bannwarth et al., 2014). Using whole-exome sequencing, we identified a heterozygous missense mutation c.176 C > T (p.Ser59Leu)
2
A. Chaussenot et al. / Neurobiology of Aging xxx (2014) 1e4
in the CHCHD10 gene in a large family with a late-onset complex phenotype associating FTD, ALS, and/or cerebellar ataxia. All patients presented a mitochondrial myopathy with the accumulation of multiple mitochondrial DNA deletions in muscle. Because the frequency of CHCHD10 mutations is not known in FTD and ALS, the aim of this study was to investigate the genetic contribution and phenotypic spectrum of CHCHD10 mutations in a large French cohort of FTD or FTD-ALS patients.
2.2. Exome sequencing
2. Methods
Genomic DNA was prepared according to Illumina TruSeq Sample Preparation v3 (Illumina, CA, USA) and sequence capture, enrichment, and elution were performed according to the manufacturer’s instructions and protocols (Illumina TruSeq Exome Enrichment). Sequencing was performed on Illumina HiSeq2000 using 100 bp paired-end reads. Sequence alignment and variant calling were performed against the reference human genome (UCSC hg19) using bwa and the Genome Analysis Toolkit.
2.1. Patients
2.3. Sequencing of CHCHD10
We have studied a cohort of 115 French unrelated probands presenting FTD or FTD-ALS, including 21 previously reported (Bannwarth et al., 2014), recruited through a national network of neurologists expert in dementia and ALS. FTD and ALS clinical diagnoses were based on international diagnosis criteria (Brooks, 1994; Rascovsky et al., 2011). This cohort included 35 familial (26 FTD-ALS, 9 FTD) and 80 sporadic cases (FTD-ALS). The mean age at onset was 62.4 7.6 years, and the mean age at examination was 65.6 7.3 years. Blood samples were collected after informed consent was obtained. The most common FTD (C9orf72, MAPT, GRN, VCP, and CHMP2B) and ALS genes (SOD1, TARDBP, FUS/TLS, ANG, PFN1, UBQLN2, hnRNPA2B1, and hnRNPA1) had been previously excluded by Sanger sequencing or by repeat-primed polymerase chain reaction (PCR) (for C9orf72) in all the probands. This study was approved by the Ethics Committee of “AP-HP de Paris.”
The 4 coding exons and flanking intronic regions of CHCHD10 (NM_213720.1) were amplified by PCR. Primer sequences and PCR conditions are available on request. PCR products were purified with Illustra ExoStar enzyme (GE Healthcare), processed with an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems), and analyzed on an ABI 3130XL automated sequencer (Applied Biosystems). Sequences were analyzed using Seqscape v2.5 software. 2.4. Homology modeling of human CHCHD10 Using the threading program PHYRE2 (Kelley and Sternberg, 2009), 142 residues of CHCHD10 (Met1 to Pro142) were modeled as previously described (Bannwarth et al., 2014).
Fig. 1. Identification of the p.Pro34Ser mutation in CHCHD10. (A) CHCHD10 mutation sequences in patients 2 and 3 and in a control individual (Wild-Type). (B) Cross-species conservation of CHCHD10 flanking the amino acid p.Pro34. (C) Model of CHCHD10 based on the CHCHD5 structure (PDB ID: 2LQL). Alpha helices and the nonstructured regions are shown in red and gray, respectively. The residue Proline 34 located in the nonstructured N-terminal region is shown in green. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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3. Results In the large family which allowed the identification of the CHCHD10 gene, all but 1 patient had dementia although motor neuron symptoms were less frequent. Based on this observation, we studied a cohort of 115 unrelated probands with FTD or FTD-ALS. Among this cohort, 21 familial cases had been first analyzed by whole-exome sequencing. A first screening led us to identify the heterozygous c.176C > T CHCHD10 mutation (p.Ser59Leu), already known, in a 57-year-old man of Spanish origin (Bannwarth et al., 2014). The patient (P1) developed a progressive motor neuron involvement, confirmed by electromyography, with a predominant pseudobulbar syndrome. He also had a frontal lobe dysfunction and behavioral changes, associated with akineto-rigid syndrome. In addition, the patient presented bilateral sensorineural hypoacusis. His father, elder sister, and one of his brothers developed progressive ALS with prominent bulbar involvement around the age of 60 years. Secondarily, we sequenced CHCHD10 in 94 patients (14 familial and 80 sporadic cases) and found a novel heterozygous missense variant, c.100C > T (p.Pro34Ser) in 2 unrelated FTD-ALS individuals (P2 and P3) (Fig. 1A). P2 was a 61-year-old French patient with no family history, but his mother died at 49 years of age and his father was unknown. He initially presented aggressiveness, violence, disinhibition, perseverative behaviors, and grasping reflex evocative of FTD. He developed pseudobulbar syndrome with paretic dysarthria and dysphagia at age 63 years. Clinical examination revealed tongue fasciculations, pyramidal signs, progressive distal limb weakness, and amyotrophy. Electromyography confirmed diffuse motor neuron involvement. Brain MRI showed mild bilateral frontal atrophy. He died at age 65 years. P3 was a 67-year-old French man who initially presented behavior disorders then developed moderate limb weakness and predominant pseudobulbar syndrome leading to FTD-ALS diagnosis. He had no family history but without information about the age of death of his father. The p.Pro34Ser mutation changes a relatively conserved proline into a serine (Fig. 1B) and is absent in public single-nucleotide polymorphism databases (including 6.503 controls of the Exome Variant Server database) and in 200 ethnically and geographically matched control alleles. In silico analysis by Mutation Taster (http:// http://www.mutationtaster.org) predicted this variant to be disease causing with a strong probability. The modeling of CHCHD10 showed (1) a nonstructured N-terminal region; (2) a highly hydrophobic helix (Gly43 to Ala 68), which may be typically an interface of interaction with a partner protein; (3) and the CHCH domain near the C-terminal region characterized by a CX9C motif. The p.Pro34Ser mutation is located in the nonstructured N-terminal region (Fig. 1C). The biological relevance of this nonstructured N-terminal region is not already understood, but it may affect the protein’s stability or its ability to interact with other proteins. 4. Discussion Because the frequency of CHCHD10 mutations is unknown in FTD-ALS, we investigated the genetic contribution and phenotypic spectrum of CHCHD10 mutations in a large French cohort of patients with FTD-ALS. Sequencing CHCHD10 in 94 affected individuals led us to identify a novel missense variant (p.Pro34Ser) in 2 unrelated FTD-ALS patients. Muscle biopsy is usually not performed in patients with FTD-ALS phenotypes, and it has not been possible to investigate whether this variant was associated with a mitochondrial myopathy. Functional analyses will be needed to firmly establish its pathogenicity. However, its absence in public singlenucleotide polymorphism databases, in ethnically matched
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controls and MutationTaster predictions are in favor of a deleterious effect. Previously, the analysis of 21 FTD-ALS familial cases allowed us to find 1 family carrying the p.Ser59Leu mutation, whose deleterious effect had been proven by functional studies (Bannwarth et al., 2014). All together, these results confirm the involvement of CHCHD10 in familial and sporadic FTD-ALS. However, as this gene was involved in 3 of 115 patients in this first study (2.6%), it is likely not a common cause of FTD-ALS spectrum in French population. The 3 patients carrying a CHCHD10 mutation had onset of symptoms in their 50e60’s (Achi and Rudnicki, 2012). It is interesting to note that all 3 patients had a predominant pseudobulbar syndrome. Bulbar palsy with dysarthria and dysphagia was also found in 6 of 8 affected individuals from the initially described family that allowed the CHCHD10 identification (Bannwarth et al., 2014). Cognitive changes preceded (P2, P3) or followed (P1) ALS symptoms. Mitochondrial diseases typically affect multi-organ systems. The sensorineural hypoacusis found in P1 that was also observed in 6 of 8 affected individuals from the initial family, and the ptosis also presented by one of them were likely secondary to mitochondrial dysfunction. Such symptoms may orientate toward CHCHD10 involvement and should be searched for therefore in FTD-ALS patients. This is the first study to evaluate the role of CHCHD10 in a cohort of FTD-ALS patients. Further investigations in larger populations with different geographic origins are needed. TDP-43 protein is a major component of neuronal inclusions that are pathologic hallmark of both ALS and FTD (Neumann et al., 2006). It will be of interest to determine whether TDP-43 inclusions are observed in patients carrying a CHCHD10 mutation, and whether CHCHD10 protein may be found in neuronal inclusions. The identification of CHCHD10 mutations in FTD-ALS spectrum opens a novel field to explore the pathogenesis of these diseases and to understand the role of mitochondrial dysfunction. Although the frequency of mutations is low in this series (2.6%), our work suggests that CHCHD10 mutations should be searched for particularly when bulbar symptoms are present at onset. In addition, it will be extremely important to analyze this gene in cohorts of patients with sporadic and familial ALS to determine whether it is also involved in pure ALS and at which frequency. Disclosure statement The authors have no conflicts of interest. Acknowledgements This work was funded by grants from the Fondation pour la Recherche Médicale (FRM) (Grant number is : DPM201211255) to Véronique Paquis-Flucklinger, the Neuromics FP7 contract E12009DD to Alexis Brice, the France Alzheimer association contract R12091DD to Alexis Brice, “The Programme Hospitalier de Recherche Clinique” (PHRC) to Isabelle Le Ber and from the program “Investissements d’avenir” ANR-10-IAIHU-06. Anne De Septenville is funded by the program “Investissements d’avenir” ANR-10IAIHU-06. The authors thank the Centre national de Genotypage, Lydia Guennec, Sylvie Forlani, and Christelle Dussert (DNA and cell bank of CR-ICM, Hôpital de la Salpêtrière, Paris) for their excellent technical assistance. References Achi, E.Y., Rudnicki, S.A., 2012. ALS and frontotemporal dysfunction: a review. Neurol. Res. Int. 2012, 806306. Bannwarth, S., Ait-El-Mkadem, S., Chaussenot, A., Genin, E.C., Lacas-Gervais, S., Fragaki, K., Berg-Alonso, L., Kageyama, Y., Serre, V., Moore, D.G., Verschueren, A., Rouzier, C., Le Ber, I., Augé, G., Cochaud, C., Lespinasse, F., N’Guyen, K., de Septenville, A., Brice, A.,
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