Genetic and clinical features of Chinese sporadic amyotrophic lateral sclerosis patients with TARDBP mutations

Abstract Objectives To investigate the genetic and clinical features of Chinese sporadic amyotrophic lateral sclerosis (SALS) patients with TARDBP mutations, we carried out a genetic analysis in a cohort of 391 SALS patients and explored the clinical manifestations of patients with TARDBP variants. Materials and methods The coding region of all five coding exons of TARDBP, exons 2–6, were sequenced for mutations in 391 Chinese SALS patients. The clinical features of patients with TARDBP mutations were described and compared with cases in literatures. Results Two missense mutations in TARDBP gene, c.1132A > G (p.N378D) and c.1147A > G (p.I383V), were detected in three cases, showing a low frequency (0.77%, 3/391) of TARDBP missense mutations in Chinese SALS patients. Based on a retrospective analysis of literatures, p.N378D mutation mainly presents a phenotype of early onset, whereas p.I383V mutation presents pure ALS or ALS alongside semantic variant primary progressive aphasia (svPPA), a type of frontotemporal dementia (FTD). Conclusions Our results demonstrate that TARDBP mutation is a rare cause of Chinese SALS patients and expand the spectrum of phenotype. It is implied that genetic analysis of SALS patients plays a crucial role in uncovering the cause of disease, especially for cases developing early onset or alongside FTD.

TARDBP gene encodes transactive response DNA-binding protein 43 (TDP-43), a critical protein for RNA regulation, transcription, and translation. Under pathological conditions, TDP-43 translocates to cytoplasm from nuclei and develops the ubiquitin-positive but taunegative inclusions, a pathological hallmark of ALS and FTD with ubiquitin-positive inclusions. To date, mutations in TARDBP gene associated with ALS has exceeded 60 on the Human Gene Mutation Database (HGMD). Correspondingly, clinical features of ALS patients with TARDBP mutations are heterogeneous. A review of 205 ALS patients with TARDBP variants showed that the mean age of onset was 57.3 years old, while spinal, bulbar and other onset accounted for 44.0%, 23.9%, and 32.2% of cases, respectively (Connolly et al., 2020).
A meta-analysis showed that 3.3% of FALS patients and 0.5% of SALS patients harbored TARDBP mutations (Zou et al., 2017). In Chinese ALS patients, TARDBP mutations accounted for 5.8% FALS cases and 0.3% SALS cases . Although the frequency of TARDBP mutations is much lower in SALS than in FALS, it is still an important cause of SALS that cannot be ignored. In the present study, we screened TARDBP gene in a large cohort of Chinese SALS patients to investigate the clinical features of SALS patients with TARDBP mutations. Furthermore, we reviewed and summarized SALS cases with the detected mutations in literatures to explore the correlations between genotype and phenotype.

Participants enrollment
A total of 391 Chinese patients meeting the revised El Escorial criteria (Brooks et al., 2000) for clinically definite, clinically probable or clinically possible SALS were included in the study. They were all from Chinese Han populations, as well as SOD1 and FUS gene negative. SALS is defined as without family history of ALS or FTD. If the patient had at least one first-degree or second-degree relative with ALS or FTD, the family history was considered positive. These participants were inpa-

Genetic analysis
Genomic DNA was extracted from peripheral blood leucocytes using

Genetic analysis
Sequence analysis of 2-6 exons of the TARDBP gene among 391 SALS patients identified two reported missense mutations (p.N378D and p.I383V, Figure S1) in three patients (Table 1), showing a 0.77% (3/391) frequency of TARDBP variants. In the above three patients, no pathogenic or likely pathogenic missense mutation was detected in the 80-gene panel, and the number of hexanucleotide (GGGGCC) repeat expansion in C9orf72 gene was normal. The p.N378D and p.I383V mutations in TARDBP were reported by Tsai et al. (2011) and by Rutherford et al. (2008), respectively. In this study, pathogenic analysis according to the ACMG recommendations demonstrated that p.N378D variant is pathogenic, whereas p.I383V mutation is likely pathogenic. In ExAC database and gnomAD, the MAF of p.N378D mutation is both unavailable, while the MAF of p.I383V mutation is 0.00008059 and 0.00006667, respectively. In addition, two synonymous substitutions in TARDBP gene, c.57A > G (p.P19P) and c.1098C > G (p.A366A), were also detected in this study.

3.3.1
Patient 1 Beginning at 39 years of age, patient 1 (II-1 in Figure 1a) developed weakness in the right hand and had difficulty feeding herself. Two months later, the weakness progressed to the proximal right limb, and the patient had difficulty washing and dressing herself. At the same time, atrophy of the right limb and fasciculation in the muscles both presented. One month later, she went to our clinic, and generalized hyperreflexia was found on neurological examination. EMG showed fibrillation potentials, positive sharp waves and long duration, high amplitude motor unit potentials in muscles of upper limbs. At that time, the patient was diagnosed with clinically possible ALS. Unfortunately, neuropsychological assessments were not carried out for the patient.
Genetic test of her parents, younger brother and sister were not accomplished due to loss of follow-up. However, similar symptoms or cognitive impairment in family members were denied by the patient.

Patient 2
At the age of 53 years, patient 2 (II-4 in Figure 1b) began to present dysarthria. Three months later, she exhibited obvious dysphagia and felt weakness in both hands. As the disease progressed, she also sensed fasciculation in her tongue, hands and legs. One month later, she felt mild dyspnea in a supine position and went to our clinic. The neurological examination demonstrated dysarthria, weakness, and atrophy of the lingualis and both hands, as well as generalized hyperreflexia. EMG showed fibrillation potentials, positive sharp waves, and long duration, high-amplitude motor unit potentials in muscles innervated by brainstem and all the spinal cord segments. Ultimately, she was diagnosed with clinical definite ALS. Unfortunately, patient 2 did not complete the neuropsychological assessments due to her dysarthria and weakness F I G U R E 1 Family trees and genetic analysis of three patients with TARDBP mutations Family tree of patient 1 (a), patient 2 (b), and patient 3 (c) and results of genetic analysis. The black symbol represents the proband and the crosshatched symbol represents normal family members carrying the same mutation.
of hands, but she had no cognitive impairment or abnormal behavior reported by herself or her caregivers. Twenty-three months later, she died suddenly.
Test of first-degree relatives revealed that her mother, sister, daughter, and son all had the same heterozygous mutation, while her brother did not. Her sister, brother, daughter, and son all had no any symptoms of ALS or FTD. Her mother presented cognitive impairment in her sixties and existed for more than 20 years. Although her mother did not accept a neurological examination in our clinic, manifestations of ALS was denied by her caregivers. Due to the slow progress of cognitive impairment, long duration, and absence of abnormal behavior, diagnosis of FTD for her mother was not supported.

Patient 3
When he was 57 years old, patient 3 (II-2 in Figure 1c Sanger sequencing of the locus was performed for his daughter, his brother as well as daughter of his brother. Only his daughter carried the same mutation, but she had no any symptoms of ALS or FTD.
The frequency of TARDBP mutations is 2.7% in SALS patients from Italy (Lattante et al., 2012), 2.1% in those from France (Daoud et al., 2009), 0.4% in those from Japan (Iida et al., 2012), and zero in those from Korea (Kwon et al., 2012), implying TARDBP mutations may be more frequent in European SALS patients.
For patients with TARDBP missense mutations in Table 1, the average age of onset was 49.3 ± 11.4 years old. Also, an earlier age of onset was identified in French ALS patients with TARDBP mutations than in those without (Corcia et al., 2012). Although 51.3% of Caucasian patients with TARDBP mutations were upper limb onset, 58.8% of Asian patients with TARDBP mutations were bulbar onset (Corcia et al., 2012), the most frequent site of onset in the current study was still upper limb (Table 1). In addition, ALS patients with TARDBP mutations presented a longer duration (Corcia et al., 2012). In Table 1, the longest duration was more than 10 years, whereas the shortest was only 6 months. Even in SALS patients with the same missense mutation, duration also varied widely (Table 1). Last, despite one patient with p.S393L (Ju et al., 2016) and one patient with p.I383V in our study presented ALS with FTD, the majority of Chinese SALS patients with TARDBP missense mutations displayed a phenotype of pure ALS (Table 1).
The p.N378D mutation in TARDBP was first described in a male FALS patient from a Chinese Taiwan family in 2011 (Tsai et al., 2011).
His daughter was also diagnosed with ALS and died at 34 years old, 2 years after the onset of symptoms in hand muscles. In 2019, the second FALS patient with p.N378D variant was reported in a Mainland Chinese family (Deng et al., 2019). This patient developed dysphagia when she was 33 years old. Her sister died at 24 years of age due to similar symptoms. In our cohort, patient 1 also had a p.N378D mutation in TARDBP but without a family history of ALS. Unfortunately, due to loss of follow-up, patient 1 did not undergo a pedigree analysis for co-segregation. Although the male FALS patient reported by Tsai (Tsai et al., 2011) did not present weakness until the age of 60 years, his daughter, two patients from the family described by Deng (Deng et al., 2019) and patient 1 in the present study all developed symptoms of ALS before the age of 40 years. It is implied that p.N378D variant tends to present with an early-onset phenotype. Additionally, spinal onset and bulbar onset were both found in ALS patients with p.N378D variant, and none of them had symptoms of FTD (Table 2).  The p.I383V mutation in TARDBP was initially reported in a female FALS patient in 2008 (Rutherford et al., 2008). In our study, we retrospectively analyzed all reported ALS patients with p.I383V mutation and found a total of 13 cases (Table 3) (Caroppo et al., 2016;Cheng et al., 2016;González-Sánchez et al., 2018;Lattante et al., 2012;Rutherford et al., 2008;Soong et al., 2014;Ticozzi et al., 2011;van Blitterswijk et al., 2012;Wang et al., 2020). As we know, the subtypes of FTD include behavioral variant of FTD (bvFTD), semantic dementia (SD), and progressive nonfluent aphasia (PNFA). As a variant of primary progressive aphasia (PPA) (Gorno-Tempini et al., 2011), SD is also designated semantic variant primary progressive aphasia (svPPA) recently. Among 13 ALS patients with p.I383V variant in literatures, 10 cases had family histroy of ALS or FTD. Furthermore, in the above 10 ALS cases, 2 patients developed bvFTD before or after ALS (Caroppo et al., 2016;Cheng et al., 2016), one patient was reported alongside FTD but no concrete description (Soong et al., 2014), and one patient had no FTD but his older sister with p.I383V mutation pre-sented svPPA (González-Sánchez et al., 2018) (Table 3). However, in the three reported ALS patients with p.I383V mutation but without family histroy of ALS or FTD, no FTD were found before or after ALS (Table 3) (Lattante et al., 2012;van Blitterswijk et al., 2012;Wang et al., 2020).
Thus, patient 3 in the present study is the first reported SALS case with p.I383V mutation presenting ALS alongside FTD, with the svPPA type.
Among them, one patient with p.G295S mutation developed bulbaronset ALS (Benajiba et al., 2009), one patient with p.A382P mutation developed spinal-onset ALS (Caroppo et al., 2016), but one patient with p.A382T variant and four patients with p.I383V mutation had no ALS alongside (Caroppo et al., 2016;Cheng et al., 2016;Gelpi et al., 2014;González-Sánchez et al., 2018) except the family members with ALS (Cheng et al., 2016;González-Sánchez et al., 2018). Thus, patient 3 in our study is also the first svPPA case with p.I383V mutation developed ALS, which expanding the phenotype spectrum of p.I383V mutation beyond pure svPPA. Furthermore, patient 3 presented symptoms of svPPA at 57 years old, similar to the onset ages of four svPPA cases with p.I383V mutation (51 to 64 years old) in literatures (Caroppo et al., 2016;Cheng et al., 2016;Gelpi et al., 2014;González-Sánchez et al., 2018). In addition, patient 3 in this study displayed severe blade-like atrophy and hypometabolism in the temporal lobe by a PET/MR hybrid, which further supported the diagnosis of svPPA.
It is demonstrated that TDP-43 contains an N-terminal domain, two RRM RNA-binding motifs and a C-terminal region, which is essential for binding to heterogeneous nuclear ribonucleoproteins (hnRNPs) and splicing inhibition. Almost all TARDBP mutations (including p.N378D and p.I383V mutations) occur in exon 6, which encodes the highly conserved C-terminal region of TDP-43. Thus, further functional analysis of p.N378D and p.I383V mutations is important to investigate the pathogenesis, but unfortunately was not carried out at present.
In conclusion, our results demonstrate a low frequency of TARDBP mutations and expand the phenotype spectrum of TARDBP mutations in Chinese SALS cases. For early onset SALS patients and those comorbid FTD, genetic analysis is strongly recommended. Moreover, shared TARDBP mutations between ALS and FTD may aid the development of effective therapeutic strategies.

ACKNOWLEDGMENT
The authors sincerely thank all of the ALS patients for their willingness to participate in this study.

CONFLICT OF INTEREST
On behalf of all authors, the corresponding author states that there is no conflict of interest.

AVAILABILITY OF DATA AND MATERIALS
The dataset used and analyzed within this article will be made available from the corresponding author on reasonable request.