Abstract
A new risk gene associated with amyotrophic lateral sclerosis (ALS) has recently been identified: the Tank-binding kinase 1 (TBK1) gene. Up to now, 90 TBK1 variants have been described in ALS patients with or without frontotemporal dementia (FTD), thus making TBK1 the third or fourth most frequent genetic cause of ALS and FTD. A point mutation analysis in a cohort of 69 Italian ALS patients was performed in order to analyze the frequency of TBK1 mutations and the correlation with clinical phenotypes. The analysis identified the novel variant p.Tyr424Asp in a patient with a rapid progression of the disease. Our data supports the implication of TBK1 in ALS pathogenesis in Italy.
INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease clinically, genetically, and pathologically related to frontotemporal dementia (FTD) [1]. The majority of ALS cases are sporadic (sALS) and only 5–10% of the cases are familial (fALS). The most common causative genes of fALS are Cu/Zn superoxide dismutase (SOD1) gene, TARDNA binding protein (TARDBP) gene, fused in sarcoma/translated in liposarcoma gene (FUS/TLS), and the hexanucleotide repeat expansion in the C9orf72 gene; however, more than 20 genes have been linked to the disease [2, 3]. Through a large exome screening study, Cirulli and colleagues [4] recently identified an important risk gene associated with ALS: the Tank-binding kinase1 (TBK1) gene (locus 12q14.1) encoding the Serine/threonine-protein kinase TBK1. Following this discovery, several studies analyzed the role of TBK1 in different populations [5–14], providing evidence that the gene is the third or fourth most frequent cause of ALS and FTD [15]. The high frequency of loss of function (LOF) mutations identified the TBK1 haploinsufficiency as the main disease mechanism, however, in the last year a substantial increase of TBK1 missense mutations have been reported. To our knowledge, 90 TBK1 variants, including LOF, splice site, and missense mutations have been described in ALS or FTD patients [13–15]. In this study, we performed a point mutation analysis in a cohort of Italian ALS patients in order to analyze the frequency of TBK1 mutations and their correlation with the clinical phenotype.
METHODS
Patients
Genetic screening was performed on 69 Italian ALS patients: 35 men and 34 women (age at onset 62.3±13.1 years, mean±SD), including 66 (95.6%) sALS and 6 (8.7%) patients with cognitive impairment. The presence of the C9orf72, SOD1, and TARDBP mutations was not an exclusion criteria for TBK1 genetic analysis due to the possible existence of two mutations [10]. ALS patients were clinically evaluated and fulfilled the Strong criteria [16] for ALS and all other disease processes were excluded. ALS patients were classified as familial cases following the criteria proposed by Byrne and colleagues [17]. Informed consent was obtained from each study participant and the study protocol was approved by the local ethics committee and conducted in accordance with the provisions of the Helsinki Declaration.
Genetic analysis
Total DNA was isolated from peripheral blood using standard methods. All the coding exons and the intron/exon boundaries of SOD1(RefSeq NM_000454), TARDBP (RefSeq NM_007375), and TBK1 (RefSeq NM_013254) were PCR amplified with primers designed using Primer3 software, and the analysis was performed using High Resolution Melting Analysis (99% specificity). Samples having an unusual melting profile were sequenced using BigDye TerminatorTM protocol on an automated 310 ABI PRISM Genetic Analyzer (Applied Biosystem).
The C9orf72 repeat expansion was searched using the repeat-primed PCR and sequencing [18]; the characteristic stutter amplification pattern was considered as indication of pathogenic repeat expansion (>30 repeats).
RESULTS
In our study, 69 Italian ALS cases were screened, and none of the patients were found to carry mutations in SOD1 and TARDBP genes; seven patients carried the C9orf72 repeat expansion.
TBK1 analysis identified one patient as carrying an undescribed genetic variant: the non-synonymous single nucleotide variation c.1270 T>G leading to a p.Tyr424Asp change in exon 11. The variant is found in neither ExAC [19] nor 1000G [20] data sets. The effect of the mutation on the function of the protein has been estimated using free online bioinformatics prediction tools: MutationTaster [21], MUpro [22], and PolyPhen2 [23]. In silico analysis revealed that the missense variant could be deleterious (MutationTaster-Disease causing, score 160; MUpro-Decrease stability, score –0.902; PolyPhen2-Possibly damaging, score 0.845).
Case report
The new variant p.Tyr424Asp was identified in a 68-year-old woman who was referred to our neurology unit after a five-month history of left foot dorsiflection weakness quickly progressed to four-limb muscle weakness and wasting, dysarthria, dysphagia, need of a cane for walking, and gastrostomy for feeding. Her past medical history was significant for vitiligo and hypertension. At the last evaluation, nine months from disease onset, the patient presented a marked diffuse paresis, inability to walk, signs of pyramidal tracts dysfunction, and severe dysarthria. She also had respiratory insufficiency that required non-invasive ventilatory (NIV) support, and mild anxiety that was successfully treated with citalopram. At that time, a comprehensive neuropsychological assessment was conducted (Table 1) which revealed a cognitive impairment with deficit in attention and executive functions, in particular in inhibiting automatic behavior (Stroop Test- Time Interference Effect, score 41, cut-off ≥36.92) and in organizational and planning skills (Rey Osterrieth Complex Figure Test- Copy, score 21.75, cut-off <28). The fluorodeoxyglucose PET (FDG-PET) showed a severe bilateral hypome-tabolism in the anterior cingulate and a moderate bilateral hypometabolism in the anterior prefrontal, medial frontal, and orbitofrontal cortices and moderate hypometabolism in left parieto-temporal areas (Fig. 1). Unfortunately, it was not possible to verify segregation of the variant with the disease in the family because the patient had no information about her birth family.
FDG-PET showed severe bilateral hypometabolism in frontal medial areas and moderate hypometabolism in left parieto-temporal cortex of the patient carrying the new variant Tyr424Asp at TBK1 gene.
Patient carrying the new variant Tyr424Asp at TBK1 gene: neuropsychological evaluation at 9 month from the disease onset
*score outside the normal value.
DISCUSSION
The new identified ALS causative gene TBK1 encodes a highly conserved multifunctional kinase consisting of four domains: N-terminal Kinase domain (Serine/Threonine kinase), ubiquitin-like domain, α-helical scaffold dimerization domain (SDD), and C-terminal domain [24]. The majority of TBK1 mutations have been reported in patients of Caucasian ancestry and resulted responsible for 0.4–4% of all ALS cases, based on the studied populations. Approximately half of these patients showed symptoms of FTD, however the clinical phenotype was heterogeneous (spinal onset versus bulbar onset, wide range of age at onset and duration of disease) [5]. In 2016, Borghero and colleagues analyzed the role of TBK1 in a cohort of ALS patients of Sardinian ancestry finding two missense genetic variants (p.Arg384Thr and p.Arg444Gln) and a LOF mutation (p.Met690fs) with a TBK1 mutation frequency of 1.6% [10]. However, the Sardinian cohort could be considered an isolated Italian population with a high rate of fALS and sALS cases, providing unique information on the genetics of the disease [25]. The three mutations are all linked to spinal onset but to different clinical phenotypes with regard to the duration of disease (fast course of disease for the missense genetic variants and slow course for the frame shift) and the presence of cognitive impairment. In particular, a fronto-temporal cognitive disturbance configuring the diagnosis of ALS with FTD was documented only in the patient carrying the p.Arg444Gln. In the other two cases, it was assumed that cognition was normal, however a formal assessment was not carried out and the neuropsychological profile was not provided [25].
The present work identifies and describes the first Italian patient carrying the unknown TBK1 genetic variant p.Tyr424Asp, with a frequency 1.4%, confirming an earlier report [10]. The patient presented cognitive dysfunction, in particular a frontal dysexecutive syndrome in the absence of notable posterior visuospatial dysfunction or amnestic disorder, with a pattern of predominant frontal hypometabolism [16].
The p.Tyr424Asp is localized within the SDD C-terminal region, which is important for protein dimerization, required for TBK1 trans-activation. There is little data on the pathogenetic role of TBK1 missense variants; however, a functional impairment has been displayed for some [5]. In particular, a biochemical study revealed that the variant p.Met559Arg, localized in the same domain as p.Tyr424Asp, creates severe functional damage to the protein and its activity [5].
In conclusion, although our data are based on a small cohort, we have identified the new TBK1 missense variant p. Tyr424Asp in a patient with a rapid progression of the disease and a cognitive dysfunction possibly evolving within the FTD spectrum, thus supporting the implication of TBK1 in ALS and FTD pathogenesis.
