Neurobiology of Aging xxx (2013) 1e4

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Genetic analysis of SS18L1 in French amyotrophic lateral sclerosis Elisa Teyssou a, Nadia Vandenberghe b, Carine Moigneu a, Séverine Boillée a, Philippe Couratier c, Vincent Meininger d, Pierre-François Pradat d, e, François Salachas d, Eric LeGuern a, f, Stéphanie Millecamps a, * a Centre de Recherche de l’Institut du Cerveau et de la Moelle Epinière, INSERM UMR_S975, CNRS UMR7225, Université Pierre et Marie Curie (UPMC)-Paris 6, Hôpital Pitié-Salpêtrière, Paris, France b Hospices Civils de Lyon, Service d’ENMG, Hôpital Neurologique Pierre Wertheimer, Bron, France c Neuroépidémiologie Tropicale, INSERM UMR1094, Université de Limoges, Limoges, France d Département des Maladies du Système Nerveux, APHP, Centre de référence maladies rares SLA, Hôpital Pitié-Salpêtrière, Paris, France e INSERM U678, UPMC UMR-S678, Laboratoire d’Imagerie Biologique, Paris, France f APHP, Unité Fonctionnelle de neurogénétique moléculaire et cellulaire, Département de Génétique et Cytogénétique, Hôpital Pitié-Salpêtrière, Paris, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 October 2013 Received in revised form 20 November 2013 Accepted 24 November 2013

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease including about 15% of genetically determined forms. A de novo mutation in the SS18L1 (also known as CREST or KIAA0693) gene encoding the calcium-responsive transactivator and/or neuronal chromatin remodeling complex subunit has recently been identified by exome sequencing of 47 sporadic ALS trios. This Q388stop mutation deleting the last 9 amino acids was shown to impair activity-dependent dendritic outgrowth. A missense mutation (c.369T>G, p.Ileu123Met) was also found in 1 of 62 ALS families previously screened for other ALS-related genes and not carrying any mutation. To confirm the contribution of SS18L1 to ALS, we sequenced the 11 coding exons and exon-intron boundaries in 87 familial ALS (FALS). We identified 2 variants: the c.660_668del, p.Gln222_Ser224del in a patient devoid of mutation in any ALS related genes and the c.790G>A, p.Ala264Thr in a patient carrying a p.Arg96Leu variant in the OPTN gene. As these variants were not found in Single Nucleotide Polymorphism databases and were absent from 180 controls they could be new SS18L1 mutations causing ALS. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Motor neuron disease Amyotrophic lateral sclerosis Familial ALS Genetic analysis CREST OPTN Double mutant

1. Introduction Amyotrophic lateral sclerosis (ALS) is an age-dependent, degenerative disorder of motor neurons that typically develops in the sixth decade and is uniformly fatal, usually within 5 years. It is caused by the progressive loss of motor neurons in the spinal cord, the cortex, and the brainstem. Most ALS cases are sporadic, whereas w10% of the cases have a positive family history of familial ALS (FALS). This prevalence increases to 17%e23% in prospective studies with active investigation of genealogies (Andersen and Al-Chalabi, 2011). In the past 20 years, the list of ALS-linked genes has constantly grown and now includes C9ORF72, SOD1, TARDBP, FUS, ANG, VAPB, DAO, OPTN, UBQLN2, VCP, SQSTM1, and PFN1, encoding proteins involved in multiple pathways including * Corresponding author at: Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière, Groupe Hospitalier Pitié-Salpêtrière, 83, Bd de l’Hôpital, 75013 Paris, France. Tel.: þ33 157274341; fax: þ33 157274784. E-mail address: [email protected] (S. Millecamps). 0197-4580/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.11.023

RNA metabolism, oxidative stress, cytoskeleton organization, vesicular trafficking, ubiquitination, and protein degradation. To date, one-third of FALS causing genes remain unidentified. All the mutated genes in FALS have also been found in sporadic ALS (Andersen and Al-Chalabi, 2011). A recent study assessed the contribution of de novo mutations in ALS by sequencing the exome of 47 trios including ALS patients with an early age at onset and their unaffected parents (Chesi et al., 2013). A de novo mutation in the synovial sarcoma translocation gene on chromosome 18-like 1 (SS18L1 also known as CREST and KIAA0693) introducing a premature termination codon in the last exon, which escaped the nonsense-mediated messenger RNA decay system and deleted the last 9 amino acids of the protein (c.1162C>T, p.Gln388*, Q388stop), was identified in 1 trio (Chesi et al., 2013). SS18L1 encodes the calcium-responsive transactivator (CREST), a chromatin regulator gene, which is involved in activity-induced dendrite outgrowth. CREST was also shown to interact with the ALS-linked protein FUS (Chesi et al., 2013). The deletion of the last 9 amino acids of the protein disrupted the histone acetylase CREB-binding protein

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(CBP)-binding domain and inhibited activity-dependent dendrite outgrowth. In the same study, a missense mutation (c.369T>G, p.Ileu123Met) was also found in 2 patients from 1 out of 62 families with ALS previously excluded for all the known ALS related genes (Chesi et al., 2013). We aimed to confirm the contribution of this gene in our population of French FALS patients.

individuals (360 chromosomes), they could be new SS18L1 mutations. Thus in our genetic analysis, patients with variants in SS18L1 account for 2.3% (2/87) of FALS patients devoid of mutation in main ALS related genes or 0.7% (2/293) of total FALS.

2. Methods

The aim of this study was to confirm the contribution of the SS18L1 gene to ALS in our population of French FALS patients. Mutations in this gene had previously been identified in a family with autosomal dominant transmission of the disease and in a family with a de novo mutational event. We identified 2 variants in SS18L1. Taking into account that Chesi et al. (2013) identified 1 SS18L1 mutation among 62 FALS of European descent the mutational frequency of this gene represents therefore, by now w2% (3/149) of Caucasian FALS without known pathogenic mutation after pooling their study with ours (although it should be verified that both FALS populations had similar median of disease onset). Indeed, the late onset ALS presented by our index case and his brother contrasted with the early onset disease displayed by patients reported by Chesi et al. (Chesi et al., 2013). However, the variants we found were not in the same region of the protein. The first variant we identified is supposed to introduce an in-frame deletion of 3 amino acids (Gln, Gly, Ser) in the glycine rich domain of the protein. Interestingly, these residues belong to the predicted prion-like region of SS18L1, which is also present in other ALS-linked proteins that are prone to aggregate such as TDP-43 and FUS (Chesi et al., 2013; Gitler and Shorter, 2011). The second variant is a substitution (c.790G>A, p.Ala264Thr). Two other Single Nucleotide Polymorphism are reported for the same amino acid (Ala264) in dbSNP short genetic variations database: the rs35943394 (that was found in 1 out of our 180 controls) and the rs369721048, which was not found in our control population. However, the variant we found is novel, absent from our control population, and not recorded in databases. Interestingly, the patient with the c.790G>A, p.Ala264Thr variant in SS18L1 also carried a variant in OPTN (c.287G>T, p.Arg96Leu), which is mutated in some ALS patients (Maruyama et al., 2010) and in adult onset primary open angle glaucoma (Rezaie et al., 2002). This novel R96L OPTN variant, which was not predicted to alter splicing and did not cause optineurin protein accumulation was not likely to be pathogenic and cause ALS disease (Millecamps et al., 2011). As we have now identified a variant in SS18L1 (A264T) in this patient, we cannot rule out the possibility that co-occurrence of these 2 variants could contribute to the ALS phenotype. A growing number of genetic studies have similarly reported “double mutant” ALS patients with mutations concurrently affecting different ALS genes (Lattante et al., 2012;

The 11 coding exons and exon-intron boundaries of SS18L1 (ensemble reference sequence: ENSG00000184402) were sequenced by Sanger technique in 87 French familial ALS patients devoid of mutation in C9ORF72, SOD1, TARDBP, FUS, VAPB, ANG, UBQLN2, VCP, or PFN1 (Millecamps et al., 2010b, 2012a, 2012b). Our FALS patients had also systematically been screened for DAO, OPTN, and SQSTM1 (Millecamps et al., 2010a, 2011; Teyssou et al., 2013) mutations. However, patients carrying variants in these 3 genes were included in the present study as the pathogenicity of these variants remains unclear. Mean age of onset of the analyzed series was 60 years (standard error of the mean 1, range 33e80 years) and mean disease duration was 48 months (SEM 6, range 6e260 months). The number of the initial FALS population was 293. Control samples were obtained from 180 age-matched Caucasian individuals of French background. Polymerase chain reaction conditions for amplification of these exons are described in Table 1. 3. Results We identified, at the heterozygote state, 2 variants. The c.660_668del and p.Gln222_Ser224del (in exon 6) in 1 affected man devoid of mutation in any of the previously reported ALS-related genes (Fig.1A). This patient had lower limb onset at the age of 77 years and died at 78 years after disease duration of 15 months. This patient had no cognitive impairment. The index case’s brother had a similar phenotype: he started the disease at 80 years of age and died after 13 months disease course. Their parents had died at 72 years and 83 years from other medical causes (Fig. 1C). We also identified the c.790G>A, p.Ala264Thr in exon 7 (Fig. 1B), in a patient carrying the c.287G>T, p.Arg96Leu (R96L) variant in OPTN, we previously reported (Millecamps et al., 2011). This patient had lower limb onset at 56 years of age and 38-months disease duration. His mother died from ALS disease at the age of 76 years. For both families, the affected relatives were deceased so the segregation of these new SS18L1 variants could not be established. Amino-acid residues affected by these modifications are relatively well conserved in mammals (Fig.1D). As these variants were not found in the dbSNP short genetic variations, the 1000 genomes project, and the NHLBI ESP exome variant server databases and in our analysis of 180 control Caucasian

4. Discussion

Table 1 SS18L1 amplification conditions Exons

Forward primer

Reverse primer

Annealing temperature

Fragment size (pb)

Specific notification

1

CGCCCAGCGCAGCCGGAGTATC

CAGGCGTGGGACGCGGGGAA

70

297

Solution Q (Qiagen) glycerol 5%

2 3 4 5 6 7 8 9 10 11

TAGATGTGTAGCGATGGCTG TCAGGCTGTCTTGTTCACACTTT TGGCTGCTGATTACGAACATTGA ACTTGAAGGGAAATTGGGGTGT GGGCGCAGGAGGTAGTTGGGT CCCAGTGAGCGAGCAGGTCCTCT GCATGGTTTTGGGGAACACGG CAATGGATTTGGTTTTGGGA AAGAAAGAGGTGTCCGTTTTG TGGACGTCTGTCTTCCTTTCA

GACTCTTGGGACGTATCTGG CTACCTGAGATGACATGCCCAC CTGTGGCTACCCCAGGAGGA AACGTGCATGTGTCAAGGGTCT AGGCTCCCCGAAGAGAAGCACA CCACACCCTCCTTGCCCTCCTT AGGGACTCACTGAAGGCAGGCAC GGTACTGAGCACGTACCCATGT GAAATCTTCATTATGTCTCGACAG ACCAAAAGTATCACACCACCAA

65 65 65 60 65 65 65 60 60 60

239 271 424 337 334 261 345 309 246 300

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Fig. 1. Detection of 2 novel variants in the SS18L1 gene in 2 FALS. Part of the chromatogram of SS18L1 showing the normal (control) and the c. 660_668del, p.Gln222_Ser224del variant (patient) sequence (A). The position of the deletion is pointed by an arrow. Part of the chromatogram of SS18L1 showing the normal (control) and the c.790G>A, p.Ala264Thr missense variant (patient) sequence (B). The position of the substitution is pointed by an arrow. Pedigree of the patient carrying the c.660_668del, p.Gln222_Ser224del variant (C). Index case is indicated by an arrow. To keep confidentiality of the family, some patients were symbolized by rhomb, and number of siblings was indicated by “n”. The age and cause of death are indicated in brackets. Sequence alignment (from HomoloGene) of part of the CREST amino acids (200e270) from diverse species (D). The position of the in-frame deletion and the missense variants (boxed) are in red. Sequences used include Homo sapiens (NP_945173.1), Macaca mulatta (XP_001089944.2), Rattus norvegicus (NP_620273.1), Mus musculus (NP_848865.4) and Gallus gallus (XP_417402.3). Abbreviations: CREST, calcium responsive transactivator; FALS, familial amyotrophic lateral sclerosis.

Millecamps et al., 2010b, 2012b; van Blitterswijk et al., 2013). These results therefore underline the interest of analyzing ALS patients with identified mutations for any novel discovered genes. To conclude, in this study, we sequenced SS18L1 in 87 FALS and found 2 variants that may cause ALS disease.

Disclosure statement The authors declare no actual or potential conflicts of interest.

Acknowledgements The authors are grateful to the patients and their families. They thank the Généthon cell and DNA bank (Evry, France) and the CRicm DNA and cell bank (Paris, France) for patients’ DNA. This work was financed by the Association pour la Recherche sur la Sclérose latérale amyotrophique et autres maladies du motoneurone (ARSla, France), the Association française contre les myopathies (AFM, France) and the Fondation NRJ-Institut de France (France). Protocols were approved by the Medical Research

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Ethics Committee of “Assistance Publique-Hôpitaux de Paris” and all participants signed a consent form for the research.

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Genetic analysis of SS18L1 in French amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease including about 15% of genetically determined forms. A de novo mutation in t...
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