Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It is characterized by the progressive neuronal loss and degeneration of upper motor neurons and lower motor neurons. The death of motor neurons causes a loss of the ability of central nervous system (CNS) to control voluntary muscle movements, contributing to the development of progressive atrophy of voluntary muscles, eventually resulting in death of the patient due to respiratory failure in the later stages of the disease. Most ALS patients succumb to the disease within 3–5 years after disease onset. Although the patient may or may not present with a family history of the disease, ALS is divided into familial ALS (fALS) and sporadic ALS (sALS) (1). Several genes and/or loci associated with risk of fALS development have been identified in the recent years. Although certain genetic loci have been associated with sALS risk, the exact genetic mechanism for sALS has not been elucidated, and sALS is hypothesized to possess a more complex pathogenesis. The reasons for progressive and selective motor neuron death occurring in sALS remain elusive. The pathogenesis of sALS remains an enigma (Ludolph et al., 2012).

The recently acknowledged pathogenesis of sALS is mainly focused on environmental and genetic factors. Potential environmental factors predisposing to sALS development include viral and bacterial infections (Sher, 2017; Yu and Pamphlett, 2017; Xue et al., 2018), organophosphate, organochlorine (Su et al., 2016; Riancho et al., 2018; Lian et al., 2019), and heavy metal exposure (Callaghan et al., 2011; Garzillo et al., 2015; Peters et al., 2017; Riancho et al., 2018), intense physical activity (Tsitkanou et al., 2019), smoking, electromagnetic fields, electric shocks, cyanotoxins, and military service (Vinceti et al., 2017; Swash and Eisen, 2020). However, none of the known environmental risk factors has been conclusively determined, and no firm conclusions have been deduced thus far (Longinetti and Fang, 2019). If environmental factors are truly a causal risk factor in sALS development, the genetic susceptibility would be expected to increase the risk of sALS development due to exposure to environmental agents (Nowicka et al., 2019). Therefore, genetic factors have garnered considerable attention in the study of sALS pathogenesis since the discovery of SOD1 mutations in sALS. During the last decade, the evolution of molecular genetic technologies has rapidly advanced our knowledge concerning the genetic pathogenesis of sALS. The development of fALS has been attributed to mutations in at least 24 different genes. Certain mutations responsible for fALS development have been identified also in patients with sALS (Chia et al., 2018; Mathis et al., 2019). Recent large-scale parallel sequencing technologies have facilitated disease-gene discovery, rare variants in more than 50 genes have now been identified to be associated with sALS. Thus, sALS has been considered a complex gene-related disease (Van Doormaal et al., 2017).

Genetic testing may aid the exploration of mutations in ALS-related genes. However, the loci that are most likely to be affected in individual patients with sALS cannot be easily predicted (Yousefian-Jazi et al., 2020). Although a series of possible ALS-related genes and mutant loci have been successively reported in the literature, currently there is no single gene or mutant locus that can completely explain the pathogenesis of ALS (Morgan and Orrell, 2016; Yousefian-Jazi et al., 2020). Therefore, it has been suggested that the sALS pathogenesis is associated with multiple genes and/or mutant loci. To this end, the investigation of additional ALS-related genes and/or mutant loci is extremely important.

In this study, we selected the 27 loci that were most likely to be associated with sALS development based on the results of the present study including our GWAS study of 250 sALS and 250 controls as well as other related previous studies (Van Es et al., 2007, 2008; Laaksovirta et al., 2010; Fogh et al., 2014; Van Doormaal et al., 2014; Xie et al., 2014), and we further analyzed them in an independent cohort of the 239 individuals with sALS and the 261 controls of Han ancestry from mainland China (HACM). We discovered that the polymorphism of the rs2619566 in contactin 4 (CNTN4), the rs10260404 in dipeptidyl-peptidase 6 (DPP6), and the rs79609816 in inositol polyphosphate-5-phosphatases B (INPP5B) were markedly associated with sALS development in the subjects of HACM ethnicity. These results provide evidence to a certain extent for the further elucidation of the pathogenesis of sALS.

Presently, the pathogenesis of sALS is not well understood, while the results of recent investigations suggest that genetic factors may play an important role. To better understand the extent by which genetic factors contribute to the risk of sALS development, it is important to find possible genes or loci that contribute to sALS susceptibility. We conducted a two-stage study in the HACM population consisting of a total of 489 sALS cases and 511 controls, excluding the rapid and slow progressing sALS cases, the early- and late-onset cases, and the sALS cases of atypical clinical manifestations. In the first stage, we performed a pooling GWAS involving 250 sALS cases and 250 controls from the HACM population to screen for possible loci associated with sALS and identified 7 SNPs that showed the most remarkable association with sALS in the HACM population (Xie et al., 2014). Furthermore, we explored previously reported candidate loci associated with the pathogenesis of sALS, and revealed that 20 SNPs were potentially associated with sALS (Table 1; Van Es et al., 2007, 2008; Laaksovirta et al., 2010; Fogh et al., 2014; Van Doormaal et al., 2014). In the second stage, we performed a sequenom massARRAY and DNA sequencing analysis using samples of an independent cohort of 239 sALS cases and 261 controls of HACM ethnicity in order to further identify the relationship with sALS risk for the above-mentioned 27 candidate loci in this study. The results revealed that the rs2619566 in CNTN4, the rs10260404 in DPP6, and the rs79609816 in INPP5B were markedly associated with sALS in the HACM population.

Furthermore, we analyzed the allele and genotype frequencies in sALS cases and controls for the 3 SNPs associated with sALS in the HACM population. All 3 SNPs were intronic polymorphisms. The MAFs of the rs2619566 in the CNTN4 gene and the rs79609816 in the INPP5B gene were significantly higher in the sALS patients than those in the controls. The minor allele frequency of the rs2619566 and the rs79609816 significantly increased the risk of sALS development in the HACM population; the subjects harboring the minor allele C (CC + CT) of rs2619566 and the minor allele T (TT + TA) of rs79609816 exhibited an increased risk of sALS development in comparison with subjects of other genotypes, which indicated that these genotypes were susceptibility factors. The minor allele of rs10260404 in the DPP6 gene significantly decreased the risk of sALS development in HACM, and the carriers with the minor C allele (CC + CT) of rs10260404 showed a decreased risk of sALS development, thus indicating that the minor allele might be a protective factor (Table 3).

Additionally, we conducted functional predictions for the 3 SNPs in the CNTN4, DPP6, and INPP5B genes. The results of this functional prediction analysis showed that the CNTN4, DPP6, and INPP5B polymorphic regions (3 SNPs) might be binding sites for transcription factors. Possible binding sites for transcription factors were identified at regions more than 700 bp upstream of the CNTN4 SNPs, more than 400 bp upstream of the DPP6 SNPs, and more than 300 bp upstream of the INPP5B SNPs. The region in the vicinity of more than 20 bp of the CNTN4 and DPP6 SNPs, and the vicinity of more than 30 bp of the INPP5B SNPs were predicted to be lncRNA non-coding regions, which might play an important role in the regulation of binding proteins (Figures 4–6 and Tables 5–7). These alterations in the CNTN4, DPP6, and INPP5B genes might change or affect their splicing, transcription, and translation, might lead to the generation of abnormal functional and/or structural proteins, and might affect the development of sALS.

CNTN4 is also known as the AXCAM or BIG-2 gene, and the gene encodes a member of the contactin family of immunoglobulins. CNTNs are axon-associated cell adhesion molecules that demonstrate certain important functions in neuronal network formation and plasticity. The encoded protein is a glycosylphosphatidylinositol-anchored neuronal membrane protein that may play a role in the formation of axon connections in the developing nervous system. The alternative splice results in the generation of multiple transcript variants (Cottrell et al., 2011; Cuoco et al., 2011; Mikulska et al., 2011; Guo et al., 2012; Kaurani et al., 2014). Additionally, the encoded protein of the gene also participates in the function of axon guidance, axonal fasciculation, axonogenesis, brain development, negative regulation of neuronal differentiation, nervous system development, neuronal cell-cell adhesion, neuronal projection development, and regulation of synaptic plasticity (Fernandez et al., 2004; Oguro-Ando et al., 2017).

CNTN4 alteration may contribute toward the infliction of damage of the neuronal axons, as well as affect neuron differentiation, adhesion, projections, and synaptic plasticity. In our study, we found that the subjects harboring the minor C allele (CC + CT) of the rs2619566 polymorphism in the CNTN4 gene had a significantly increased risk of developing sALS. The pathogenesis mechanisms related to sALS development may rely on the CNTN4 gene harboring the minor C allele (CC + CT) that results in rs2619566 changes or affects splicing, transcription, or translation of the CNTN4 gene, thereby generating an abnormal CNTN4 protein, which induces abnormalities in differentiation, adhesion, projection, synaptic plasticity, axonal guidance, and fasciculation of motor neurons, contributing to the development of lesions and disturbance in neuronal network formation and plasticity, subsequently resulting in the development of sALS (Figure 7A).

DPP6 also is known as VF2 DPPX and MRD33. This gene encodes a single-pass type II membrane protein that is a member of the peptidase S9B family of serine proteases. This protein has no detectable protease activity, and this is most likely attributed to the absence of the conserved serine residue normally present in the catalytic domain of serine proteases. However, it does bind specific voltage-gated potassium channels and alters their expression and biophysical properties. Variations in this gene may be associated with susceptibility to sALS (Cronin et al., 2008, 2009; Del Bo et al., 2008; Kwee et al., 2012). Only 9 published studies have reported on the association between DPP6 and sALS thus far, and of these 9 studies, only 2 independent studies were conducted in the Chinese population (Li et al., 2009; Chen et al., 2012), and both studies suggested the absence of association of the rs10260404 variant in DPP6 with sALS development in the Chinese population. However, the other 7 studies performed in European populations including Italian, Dutch, Polish, and Irish, and Americans, reported results that were dramatically different; 5 of the 7 studies suggested that DPP6 was a candidate gene for sALS, and reported several candidate loci associated with sALS (Cronin et al., 2008, 2009; Del Bo et al., 2008; Kwee et al., 2012), while 2 of the 7 studies indicated that mutations in these genes were unlikely to be associated with sALS (Chiò et al., 2009; Daoud et al., 2010). Two independent studies suggested the absence of association of the rs10260404 variant in DPP6 with sALS development in the Chinese population (Li et al., 2009; Chen et al., 2012). Actually, in our previous GWAS study, there was no correlation between the rs10260404 variant in DPP6 and sALS yet (Xie et al., 2014). But in this validation study, it showed that the rs10260404 variant in DPP6 was associated with sALS in Chinese Han populations. The contrary conclusions might be due to the differences of their ethnicity or population background or sample size. The further study need conduct in the larger sample size and different ethnicity and population background.

An alternative splicing of DPP6 results in the generation of multiple transcript variants (Lin et al., 2014). DPP6 is critical for the synaptic integration and excitation of neurons (Wolf et al., 2014). The main functions of DPP6 include the not dipeptidyl-peptidase activity, potassium channel regulator activity, serine-type peptidase activity (Yokotani et al., 1993), neuronal action potential regulation, positive regulation of potassium ion transmembrane transport, and proteolysis (Soh and Goldstein, 2008). Our results revealed that carriers of the minor allele C (CC + CT) of the rs10260404 polymorphism in the DPP6 gene had an decreased risk of developing sALS, suggesting that presence of the minor C allele might change or affect the splicing, transcription, or translation of the DPP6 gene, which might be responsible for an decreased risk of sALS development. According to the existing knowledge on the DPP6 function, the protective pathogenesis associated with sALS might be attributable to an abnormal alteration of the protein activity due to the rs10260404 polymorphism-associated changes improving the synaptic integration and excitation of motor neurons. Meanwhile, the known functions of DPP6 might be involved in the process, subsequently contributing to the decreased risk of sALS (Figure 7B).

INPP5B is also known as 5PTase. This gene encodes a member of a family of inositol polyphosphate-5-phosphatases (5-phosphatases) (Ross et al., 1991). The enzyme functions in the regulation of calcium signaling by inactivating inositol phosphates (IPs). The encoded protein is localized in the cytosol and mitochondria, and establishes association with membranes through an isoprenyl modification near the C-terminus. Cellular calcium signaling is controlled by the production of IPs and by phospholipase C in response to extracellular signals. The IPs signaling molecules are inactivated by activities of a family of 5-phosphatases. Several alternatively spliced transcript variants of this gene have been described, but the full-length transcript features of a few of these variants have not been determined. The main functions of INPP5B include inositol-1,4,5-trisphosphate 5-phosphatase activity, metal ion binding, phosphatidylinositol-4,5-bisphosphate 5-phosphatase activity, and protein binding (Jefferson and Majerus, 1995; Noakes et al., 2011). In the results of the present study, the MAFs for the rs79609816 polymorphism in the INPP5B gene were higher in the sALS patients than those in the controls, and subjects harboring the minor allele T (TT + TA) had a significantly higher risk of developing sALS. We hypothesize that the minor allele T of the INPP5B rs79609816 polymorphism may affect the splicing, transcription, or translation of the INPP5B gene, that may further generate an abnormal INPP5B protein, which results in an increased risk for sALS development and represents a susceptibility factor for this disease. The alteration due to the INPP5B rs79609816 variant might change the function of 5-phosphatases, affect cellular calcium signaling in the cytosol and mitochondria, induce an imbalance in the intracellular and extracellular calcium levels, contribute to calcium overload in the cytosol and mitochondria, and ultimately result in the death of motor neurons (Figure 7C). In conclusion, our study found that 3 polymorphisms, namely the CNTN4 rs2619566, the DPP6 rs10260404, and the rs79609816, were significantly associated with sALS development in the HACM population (Figure 7).

Based on the currently known functions of the CNTN4, DPP6, and INPP5B genes, it is possible to postulate that CNTN4 rs2619566 variants may inflict damage on the neuronal network formation and plasticity of motor neurons, that DPP6 rs10260404 variants may improve the synaptic integration and excitation of motor neurons, and that INPP5B rs79609816 variants may destroy cellular calcium signaling of the cytosol and mitochondria in motor neurons, ultimately influence the sALS development. In the process, the function of axon guidance, axonal fasciculation, axonogenesis, negative regulation of neuron differentiation, nervous system development, neuron cell-cell adhesion, neuron projection development, regulation of synaptic plasticity, not dipeptidyl-peptidase activity, potassium channel regulator activity, serine-type peptidase activity, neuronal action potential, positive regulation of potassium ion transmembrane transport, proteolysis, inositol-1,4,5-trisphosphate-5-phosphatase activity, metal ion binding, phosphatidylinositol-4,5-bisphosphate 5-phosphatase activity, and protein binding may all directly or indirectly participate in the development of sALS (Figure 7).

Our data are consistent with those obtained using a model of sALS pathogenesis that involves a series of complex pathophysiologic processes attributed to the presence of multiple genes and loci. These polymorphisms might change or affect splicing, transcription, and translation, resulting in the production of abnormal CNTN4, DPP6, and INPP5B proteins, which might inflict damage or disrupt or improve neuronal network formation and plasticity, the synaptic integration and excitation, and the cellular calcium signaling of the cytosol and mitochondria of motor neurons, ultimately influencing the development of sALS. Our study further suggests that multiple genes and loci participate in the susceptibility to sALS. Our results provide valuable data for conducting further studies on the elucidation of the pathogenesis of sALS.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The study was approved by the Institutional Review Board of the Hospital Human Ethics Committee of The First Affiliated Hospital of Nanchang University and was conducted in accordance with the approved guidelines and regulations. The patients/participants provided their written informed consent to participate in this study.

JZ and RX conceived, designed, and performed the experiments, analyzed the data, and wrote the manuscript. XZ, YD, and HN conceived and designed the experiments. JZ, WQ, and FH performed the experiments, analyzed the data, and were the jointed first authors. XZ, YD, HN, and RX contributed reagents, materials, tools, and services and were the co-corresponding authors. RX was the corresponding author. All authors were involved in the drafting, critical revision, read, and final approval of the manuscript for publication and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved and contributed significantly to this research and in the preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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