At present, the occurrence of RLS is believed to be related to genetic variation, abnormal iron metabolism, dopaminergic dysfunction, and pregnancy [18-20]. Among them, genetic variation plays an important role in the occurrence and development of RLS. In particular, with the widespread use of high-throughput sequencing technology in the clinic, WES technology has identified multiple genetic changes in RLS patients. For example, an early study found that five genetic susceptibility loci located at 12q13-23 [21], 14q13-21 [22], 9p24-22 [23], 2q33 [24], and 20p13 may cause RLS [25]. Recently, another 4 novel genetic susceptibility loci have been reported, namely, 6p21.2 (BTBD9), 2p14 (MEIS1), 15q23 (MAP2K5 / SKOR1 or MAP2K5 / LBXCOR1), and 9p24-922 (PTPRD) [13-17].
Among them, variants in the FAT2 gene have been reported to be associated with RLS. FAT2 is a fatty cadherin gene [26], located on the human chromosome 5q33.1, and contains 4,349 amino acids [27]. FAT2, a protein-encoding gene, is involved in regulating cell connection and movement, and is related to the occurrence and development of various human diseases [28, 29]. In normal tissues, a higher FAT2 level was observed in the cerebellum, and was also expressed in epithelial tissues [30] and blood tissues [31]. In tumor tissues, abnormal expression of FAT2 was reported in gastric cancer, ovarian cancer, esophageal cancer, and pancreatic cancer, etc. [32, 33]. Genetic variants of the FAT2 gene have also been found in head and neck squamous cell carcinoma [27, 32], 5q- myelodysplastic syndrome [31], and colorectal cancer [34]. However, the role of FAT2 in RLS is limited.
To date, one report showed that the FAT2 splice site mutation IVS17-3C> A existed in two RLS
patients from a family[30], which suggested that it may lead to abnormal splicing and protein function damage. Here, variants of the FAT2 gene were also found in an RLS family, which further supported the relationship between FAT2 and RLS occurrence. In contrast to previous studies, we identified two novel variants of the FAT2 gene, splice site mutation, and missense mutation. The frequency of these two variants was extremely rare in the normal population. Missense mutation of FAT2 was considered to be probably damaging for protein structure and function by software analysis. The pHSF3.1 software suggested that a splicing mutation occurred at the late intron position, but it may have no effect on splicing. From the perspective of protein structure and function , missense mutations in the FAT2 gene were more closely related to RLS.
Missense or nonsense mutations are the most common causes of hereditary diseases [35, 36]. Splicing mutations usually occur in spliced donors, acceptor sites, or flanking conserved sequences, which alter the splicing pattern of RNA precursors, and result in introns or deleting exon sequences in mRNA. Splicing mutations affect transcription and translation, and thus do form an incomplete protein sequence, leading to disease occurrence. Currently, the effect of splicing mutations on protein functions can be predicted using software such as GeneSplicer, Human Splicing Finder, and MaxEntScan [37], but it is still limited to distinguishing differences in genetic background in the population. In addition, it is not enough to determine or exclude it as a potential cause of disease according to the prediction results from the computer. As an example, intron 19: c.11464-8C> G mutation may not cause splicing defects via computer prediction, but it is extremely rare in the population. Hence, whether this variant is associated with the occurrence of RLS disease needs further investigation.
Although we found that there are two novel variants of the FAT2 gene in the RLS family, the molecular mechanism of FAT2 in the occurrence of RLS is still unclear. A previous report suggested that FAT2 expression is significantly higher in the cerebellum than in other tissue, and is associated with autophagy, early embryonic development, and neurogenesis, which suggests that FAT2 may directly or indirectly cause RLS by affecting the development of the nervous system or interfering with the transmission of the nervous system [32]. In addition, some studies have indicated that FAT2 mutations can lead to neurodegeneration or autophagy dysfunction [38], which in turn leads to spinal cerebellar ataxia [30] or Parkinson's disease [39, 40].
In summary, this study first reported two novel variants of the FAT2 gene in a RLS family, which provided a preliminary theoretical basis for clinical diagnosis and etiology research into RLS, but it is also necessary to verify them in a large sample and explore potential mechanisms in the future.