High‐throughput sequencing revealed a novel SETX mutation in a Hungarian patient with amyotrophic lateral sclerosis

Abstract Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of the motor neurons. To date, 126 genes have been implicated in ALS. Therefore, the heterogenous genetic background of ALS requires comprehensive genetic investigative approaches. Methods In this study, DNA from 28 Hungarian ALS patients was subjected to targeted high‐throughput sequencing of the coding regions of three Mendelian ALS genes: FUS, SETX, and C9ORF72. Results A novel heterozygous missense mutation (c.791A>G, p.N264S) of the SETX gene was identified in a female patient presenting an atypical ALS phenotype, including adult onset and lower motor neuron impairment. No further mutations were detected in the other Mendelian ALS genes investigated. Conclusion Our study contributes to the understanding of the genetic and phenotypic diversity of motor neuron diseases (MNDs). Our results also suggest that the elucidation of the genetic background of MNDs requires a complex approach, including the screening of both Mendelian and non‐Mendelian genes.


| INTRODUCTION
Amyotrophic lateral sclerosis (ALS; ORPHA803), also known as "Lou Gehrig's disease", is a clinically heterogeneous group of neurodegenerative disorders characterized by the death of motor neurons in the brain, brainstem, and spinal cord resulting in fatal paralysis (Morrison & Harding, 1994). Approximately, 90% of ALS cases are sporadic and the remaining 10% are familial (Valdmanis & Rouleau, 2008). The lifetime risk for developing the disease is approximately 1/400 (Johnston et al., 2006). Due to the complex genetic background of the disease, the underlying disease-causing variant is rarely established for individual cases (Kenna et al., 2013). To date, 126 genes have been implicated in ALS (Abel, Powell, Andersen, & Al-Chalabi, 2012).
The most common mutants identified for the Mendelian forms are located in the superoxide dismutase 1 (SOD1) gene, which accounts for approximately 5% of the ALS forms (Andersen et al., 2007). Mutations in the senataxin (SETX) gene have been identified at a lower frequency than in the SOD1 gene in ALS. SETX encodes a helicase protein involved in DNA repair and RNA production. Homozygous or compound heterozygous SETX mutations are associated with the development of autosomal recessive ataxia with oculomotor apraxia type 2 (AOA2; Anheim et al., 2009). In addition, heterozygous SETX mutations have been associated with the autosomal dominant form of juvenile-onset ALS (ALS4; Chen et al., 2004). The fused in sarcoma (FUS) gene is also associated with the Mendelian forms of ALS. FUS encodes a nucleoprotein that functions in DNA and RNA metabolism. The chromosome 9 open reading frame 72 (C9ORF72) has also been identified as a disease-causing gene of Mendelian ALS forms (Pearson et al., 2011). A heterozygous hexanucleotide (GGGGCC) repeat expansion located between the noncoding exons 1a and 1b of C9ORF72 has been recently identified in patients with FTD and/or ALS (DeJesus-Hernandez et al., 2011;Renton et al., 2011).
Considering the onset, the symptoms, and the course of the disease, our aim was to establish the frequencies of mutations in the coding regions of the SETX, FUS, and C9ORF72 genes in Hungarian patients (n = 28) that did not carry mutations in the SOD1 gene. The investigated patients were also negative for the C9ORF72 hexanucleotide repeat expansion.

| Investigated individuals
The investigated patients (n = 28) participated in this study were recruited from the Department of Neurology, University of Szeged, Szeged, Hungary. All patients fulfilled the revised El Escorial and the Awaji-shima criteria for ALS (de Carvalho & Swash, 2009;Ludolph et al., 2015). According to the revised El Escorial criteria, the lower motor neuron disease (MND; progressive muscular atrophy) is determined as one of the "restricted phenotypes" of ALS; therefore, one patient with only lower motor neuron involvement was also diagnosed as ALS. All patients and age-and sex-matched healthy controls (n = 50) were of Hungarian ancestry. The investigation was approved by the Internal Ethical Review Board of the University of Szeged. Written informed consent was obtained from patients and healthy controls, and the study was conducted according to the Principles of the Declaration of Helsinki.

| Next-generation sequencing
Genomic DNA was isolated from blood using the DNeasy Blood and Tissue kit (QIAGEN, Godollo, Hungary). Amplicons (n = 56) were designed (range 519-704 bp; mean: 612 bp) to cover the coding regions and the flanking introns of the investigated FUS, SETX, and C9ORF72 genes, and an amplicon library was prepared according to the Amplicon Library Preparation Manual for the Roche Junior 454 nextgeneration sequencing system (Roche, Budaörs, Hungary). Amplicons were purified with the Agencourt AMPure XP kit (Beckman Coulter, Budapest, Hungary), quantified with the Quant-iT PicoGreen Assay (Life Technologies, Budapest, Hungary), diluted separately to 1 × 10 7 molecules/μl and pooled. Emulsion PCR and next-generation sequencing were performed according to the manufacturers' protocols (Roche).

| Bioinformatic analysis
To improve the efficiency of the Roche pipeline, composed of the  Assessor) were used to predict the functional impact and then focused on variants with predicted deleterious consequences (nonsense SNVs, frameshift indels, essential splice variants, and complex indels). The putative effect on splicing efficiency was predicted using the Human Splicing Finder (Desmet et al., 2009). The alignments were visualized with IGV V.2.0.34. (Robinson et al., 2011). All the identified disease-causing candidate variants were confirmed by direct sequencing.

| RESULTS
Our next-generation sequencing approach did not detect mutations in the investigated coding regions of the FUS and C9ORF72 genes.   (Victor & Ropper, 2000).
In a recent work, Finsterer and Burgunder (2014) (Zhao et al., 2009). Although the p.N264S mutation is located within the N-terminal region of the protein and the p.T1118I mutation is located in a region of yet unknown function, both of these mutations result in the development of the same unusual ALS4 phenotype.
Further studies are needed to elucidate the underlying mechanism responsible for the observed unusual phenotypes associated with the heterozygous p.N264S and p.T1118I missense mutations of the SETX gene (Table 1).
Four of the 17 mutations are located within the helicase domain of the SETX protein (Chen et al., 2004;Hirano et al., 2011;Kenna et al., 2013;Saracchi et al., 2014). In AOA2, homozygous or compound heterozygous missense mutations located within the helicase domain are generally associated with less severe phenotypes than the mutations affecting other regions of the protein (Anheim et al., 2009;Moreira et al., 2004). A similar genotype-phenotype association has not been observed for the heterozygous missense mutations associated with ALS4. However, in ALS4, mutations impairing the region of the helicase domain are associated with cortical and spinal motor neuron impairment, whereas others located outside of F I G U R E 2 SETX mutations associated with ALS4. All identified disease-causing SETX mutations implicated in ALS4 are heterozygous missense. Other types of mutations have not been detected in ALS4. The identified mutations are located in coding regions and distributed nearly evenly on the encoded protein. Mutation hot spots have not been detected. Mutations are located both within the N-terminal and helicase domains and located outside these regions, suggesting that these regions have as yet unidentified pivotal biological functions  (Chen et al., 2006). However, only missense SETX mutations have been implicated in the development of ALS4, to date (Avemaria et al., 2011;Hirano et al., 2011;Kenna et al., 2013;Saracchi et al., 2014); deletions, nonsense, frameshift, or other types of mutations have not yet been detected for this disease (Table 2).
Our study further widens the geographic range for the origin of disease-causing heterozygous missense mutations of the SETX gene, which have already been implicated in ALS in patients from different countries (Avemaria et al., 2011;Hirano et al., 2011;Kenna et al., 2013;Saracchi et al., 2014). To the best of our knowledge, our study is the first demonstrating a novel SETX mutation in the Hungarian ALS population (Table 2).
In this study, we did not identify any mutations in the coding regions of the C9ORF72 gene. These results correlate well with the data reported in the literature; there are currently no reports of disease-causing mutation in the coding regions of the C9ORF72 gene (Kenna et al., 2013;Koppers et al., 2013).
No mutations in FUS, another Mendelian gene associated with domains (Shang & Huang, 2016). On the basis of our results, we hypothesize that FUS mutations in the Hungarian ALS population might be very rare. This hypothesis is supported by the literature, as FUS mutations have been reported to contribute to the development of the disease in approximately 0.5% of the patients (Kenna et al., 2013).
On the basis of our results and the results of previous studies, we also emphasize that the genetic screening of the Mendelian ALSassociated genes might not elucidate the causative genetic variant in the majority of ALS cases (Cirulli et al., 2015;Kenna et al., 2013). The genetic heterogeneity of ALS is extremely complex: rare mutations of Mendelian genes and common variants of non-Mendelian genes can also contribute to the development of the disease (Abel et al., 2012). Our comprehensive study adds novel data to the genetic and phenotypic diversity of ALS and indicates that complex approaches, high-throughput methods, and large-scale studies are needed to understand of the genetic heterogeneity of this disease.

ACKNOWLEDGMENTS
Funding of the study reported in the paper was provided by the