In this case series, we performed a clinical genomic study of TN with familial clustering. In this study, we found for the first time an important subset of patients with FTN in Northwest China. By using WES and Sanger sequencing, we identified multiple MARS1 mutations that may drive FTN pathogenesis, and this gene has not previously been reported to be related to FTN. Moreover, by using RNA-seq, we analysed ISR-related genes to confirm their differential gene expression, and we found that Fosl1 and NFE2 expression was significantly higher in probands (Family 1) than in nonfamilial TN subject group. Our findings suggest that MARS1 mutations may cause the chronic activation of ISR, contribute to its pathophysiological changes and cause/accelerate peripheral nerve degeneration, which may explain the early age of onset in FTN. The results presented here provide a basis for further investigation into FTN and enrich our understanding of the human molecular genetics of TN.

The occurrence of FTN is not an accidental event. From the data, it is easy to see that the age of onset in probands was more than ten years earlier than that in nonfamilial TN subject group. Notably, this finding is highly consistent with those of other studies [6, 15, 16]. This evidence suggests that genetic factors may be involved in the mechanism of TN pathophysiology and accelerate TN onset.

In all probands in 3 families, one missense mutation (c.2398C > A) was screened out so we focused on this mutation. This mutation had a high conservation score and it was classified as LP according to ACMG/AMP guidelines (Table 3), and it exhibited incomplete dominance in this family. MARS1 belongs to the aminoacyl-tRNA synthetase (AARS) gene group [17]. Aminoacyl-tRNA synthetases are important for protein synthesis [18]. He et al. also suggested that aminoacyl-tRNA synthetases sense amino acid levels, transmit amino acid signals to signalling networks, and regulate various cellular functions [19]. To our knowledge, our study is the first to identify MARS1 transmission in FTN patients.

MARS is encoded by nuclear genes, of which there are two subtypes. The gene products of MARS are vital for the translation initiation of mRNAs. MARS1 gene mutations were shown to cause Charcot-Marie-Tooth disease Type 2u (CMT2u), which is a phenotype of this autosomal incomplete dominance neurodegeneration disease according to previous studies [20]. MARS2 mutations were shown to cause autosomal recessive spastic ataxia with leukoencephalopathy (ARSAL), which is also a type of neurodegeneration disorder [21]. Mutations in MARS2 lead to elevated levels of oxidative stress, impaired mitochondrial activity, and neurodegeneration. Plum et al. found that reduced MARS2 levels in Parkinson’s disease brain tissue lead to neurodegeneration [17]. Mutations in all types of MARS are closely related to neurodegenerative diseases. A recent study suggested that AARS-related genes cause deficient myelination [22]. Notably, neurodegeneration caused by demyelination is the core assumption of the pathophysiology of TN [3, 15]. All evidence indicated a close relationship between AARS-related genes and TN under some conditions.

Recently, Burgess et al. suggested that mutant tRNA synthase can activate the ISR and lead to neurodegeneration [23]. Moreover, the transgenic overexpression of a tRNA synthase rescued ISR activation in the mouse models of tRNA synthetase mutation of this gene [24]. The ISR is the central regulator of protein homeostasis and is activated in a wide range of neurodegenerative disorders of the brain to respond to stress conditions [25,26,27]. ISR activation was demonstrated in postmortem brains of individuals and animal models of cognitive and neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, CMT, etc. In particularly, myelin cells from the central or peripheral nervous system synthesize a large number of myelin lipids and proteins and accumulate misfolded or unfolded proteins to activate the ISR in myelination disorders [28, 29]. Thus, MARS1 is a type of tRNA synthase gene, and MARS1 mutations may cause ISR activation to drive neurodegenerative diseases.

The central regulatory factor of the ISR is eukaryotic initiation factor 2 (eIF2) [28, 30]. eIF2 phosphorylation triggers the translation of specific mRNAs, including key transcription factors, such as activating transcription factor 4 (ATF4). Notably, in our study, we performed RNA-seq, and the results showed that Fosl1 and NFE2 expression was higher in probands in Family 1 than individuals in the nonfamilial TN subject group. Fos1 and NFE2 are ATF4-interacting partners [31,32,33]. A previous study suggested that a high level of NFE2 increases the expression of ATF4 and thus activates the ISR [34]. The binding of Fos1 and ATF4 as dimers plays an important role in a variety of mechanisms in transcriptional regulation [32]. Therefore, we suggest that the MARS1 mutation found in the probands may cause ISR activation which leads to TN.

We also performed RNA-seq for the probands in Family 1 and their family members in the FTN family member group. However, there was no significant difference in ISR-related genes between the 2 groups, regardless of whether FTN family members carried the MARS1 mutation. This may be related to the difference in age and the small sample size of probands and FTN family members.

CMT is a common hereditary peripheral neuropathy. To date, at least 25 genes have been identified to be associated with CMT. The core of CMT pathology is demyelination (CMT type 1) and axonal degeneration (CMT type 2) [35, 36]. Interestingly, accumulating evidence indicates a potential link between FTN and CMT. To date, at least 9 CMT families have FTN (Table 5). Several studies identified potential gene mutations in probands from FTN families [5, 6, 16]. These mutated genes are the confirmed pathogenic genes of CMT.

The MARS1 mutations screened by our research were associated with CMT2u. The mutation, c.2398C > A p.(Pro800Thr), was also reported by Hyun et. in 2014, and it was associated with CMT2u [43]. This is the first study to identify potential FTN genes related to CMT type 2. However, CMT2u is a late-onset CMT (50–60 years of age), which is different from other subtypes. The average number of probands in Family 1 was 54.33 ± 4.16 years (mean ± SD). To date, our probands do not show symptoms of CMT. We will continue to follow up with Family 1.

In the future, further case series with available genetic data are needed to confirm the relationship between CMT and FTN. Further studies, including in vitro and in vivo experiments, are necessary to confirm our findings.

In addition, the missense MARS1 mutation c.2104C > T was first reported by our study in one proband from Family 3. but according to the ACMG/AMP variant classification, it is considered a VUS. Further experiments are needed to confirm its pathogenicity.