The rs696880 Polymorphism in the Nogo-A Receptor Gene (RTN4R) Is Associated With Susceptibility to Sporadic Amyotrophic Lateral Sclerosis in the Chinese Population

Single-nucleotide polymorphisms (SNPs) in the Nogo-A receptor gene (RTN4R) have been associated with increased risk for sporadic amyotrophic lateral sclerosis (SALS) in the French population. In the present study, we investigated the associations between RTN4R tag SNPs and SALS in a large Chinese population. Four tag SNPs (rs854971, rs887765, rs696880 and rs1567871) in the RTN4R gene with an r2 threshold of 0.8 and a minor allele frequency (MAF) greater than 0.2% were selected based on Chinese population data from HapMap. A total of 499 SALS patients and 503 healthy controls were genotyped for the SNPs by SNaPshot technology. Haplotype analysis of the four SNPs was performed using the SHEsis software platform. The results showed a significant association between the rs696880 risk allele (A) and SALS in the Han Chinese population (P = 0.009, odds ratio (OR) = 1.266 [1.06–1.51]). The allele and genotype frequencies of rs854971, rs887765 and rs1567871 were not associated with SALS. The distribution of the GAAT haplotype was different between the case and control groups (P = 0.008, OR = 1.289 [1.066–1.558]). In conclusion, our study showed an association between the RTN4R SNP rs696880 and the risk of SALS in the Han Chinese population, with the A allele increasing risk.

Single-nucleotide polymorphisms (SNPs) in the Nogo-A receptor gene (RTN4R) have been associated with increased risk for sporadic amyotrophic lateral sclerosis (SALS) in the French population. In the present study, we investigated the associations between RTN4R tag SNPs and SALS in a large Chinese population. Four tag SNPs (rs854971, rs887765, rs696880 and rs1567871) in the RTN4R gene with an r 2 threshold of 0.8 and a minor allele frequency (MAF) greater than 0.2% were selected based on Chinese population data from HapMap. A total of 499 SALS patients and 503 healthy controls were genotyped for the SNPs by SNaPshot technology. Haplotype analysis of the four SNPs was performed using the SHEsis software platform. The results showed a significant association between the rs696880 risk allele (A) and SALS in the Han Chinese population (P = 0.009, odds ratio (OR) = 1.266 [1.06-1.51]). The allele and genotype frequencies of rs854971, rs887765 and rs1567871 were not associated with SALS. The distribution of the GAAT haplotype was different between the case and control groups (P = 0.008, OR = 1.289 [1.066-1.558]). In conclusion, our study showed an association between the RTN4R SNP rs696880 and the risk of SALS in the Han Chinese population, with the A allele increasing risk.
Recently, two non-coding variants (rs701427 A and rs1567871 C) in the Nogo-A receptor gene (RTN4R) were reported to confer susceptibility to SALS in the French population (Amy et al., 2015). Moreover, rs701427 showed a significant correlation with reduced gene expression. The study concluded that reduced expression of the Nogo receptor was associated with SALS. In this study, we investigated the associations between RTN4R tag singlenucleotide polymorphisms (SNPs) and SALS in a large Chinese population.

Study Population
This study consisted of 499 SALS patients and 503 healthy control subjects. All participants were of Han Chinese origin. Diagnosis of ALS was performed at Peking University Third Hospital using the E1 Escorial Word Federation criteria for definite or probable ALS (Brooks et al., 2000). This study was carried out in accordance with the recommendations of Peking University Third Hospital ethics committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Peking University Third Hospital ethics committee.

SNP Selection
Based on the Chinese population data from the HapMap database (HapMap Data Rel 27 Phase II+III, Feb 2009, on NCBI B36 assembly, dbSNP126), candidate tag SNPs in RTN4R with an r 2 threshold of 0.8 and a minor allele frequency (MAF) greater than 0.2% were selected.

SNP Analysis
Genomic DNA was extracted from leukocytes of venous blood using the phenol-chloroform method. The sense and antisense primers for SNPs are shown in Table 1. Primers were used to amplify the target SNPs, followed by agarose gel electrophoresis and product recovery. The product was primer-extended by one base and terminated. It was detected by an ABI sequencer at Tsingke Biotechnology Co. (Beijing, China) according to the ABI PRISM SNaPshot TM Multiplex Kit Protocol.

Statistical Analysis
The chi-square test was used to compare the allele frequencies and genotype differences between the case and control groups. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the risk of genetic polymorphisms. Haplotype analysis was performed with the SHEsis software platform 1 (Shi and He, 2005;Li et al., 2009). The original P value was multiplied by the 1 http://analysis.bio-x.cn  number of comparisons to obtain a Bonferroni-corrected P value. Corrected P-values less than 0.05 were considered significant.

Functional Prediction
The functions of positive loci were analyzed using MATCH software.

RESULTS
A total of four SNPs of RTN4R (rs854971, rs887765, rs696880 and rs1567871) were selected for further genotyping. All four SNPs were located in the intron between exon 1 and exon 2 (Figure 1). This study included 499 SALS patients with a male-to-female ratio of 1.80 and average age of onset of 51.3 ± 11.9 years. The study also included 503 control subjects, with a male-to-female ratio of 1.30 and a mean age of 53.5 ± 12.8 years. No difference was observed between the patients and control subjects in age or sex.
The four polymorphisms (rs854971, rs887765, rs696880 and rs1567871) were in Hardy-Weinberg equilibrium in SALS patients and control subjects.
Using the method of increasing sensitivity with the lowest false-negative rate, MATCH software predicted that if allele A replaced G in rs696880, one more cooperates with myogenic proteins 1 (COMP1) transcription factor binding site would be expected.
Haplotype frequencies were calculated using the SHEsis software platform. According to the sequence of rs854971-rs887765-rs696880-rs1567871, the AGGC, GAAT, GGAC, GGAT and GGGC haplotypes, with the smallest haplotype frequency >0.03, were included in the study as common haplotypes. The GAAT haplotype and non-GGAT haplotypes differed between the case group and the control group (P = 0.008). Because the GAAT haplotype was involved in five multiple comparisons, P = 0.008 × 5 = 0.04 after Bonferroni correction; therefore, this difference was still significant ( Table 3).

DISCUSSION
The tag SNPs in the RTN4R gene are different between the Han Chinese population and the French population. Four SNPs, rs854971, rs887765, rs696880 and rs1567871, were included in our study. Three SNPs, rs701427, rs701421 and rs1567871, were included in the French study (Amy et al., 2015). The MAFs of rs701427 and rs701421 were both lower than 0.2% in the Han Chinese population; therefore, these two SNPs were not included in our study. Only the rs1567871 SNP was included in both studies. In Amy et al.'s (2015) study, the   Zou et al., 2017). The frequency of OPTN mutations was high in Japanese and Chinese populations (FALS 3.3%-3.8%, SALS 0.23%-1%), whereas these mutations occurred infrequently in Caucasian patients (Li C. et al., 2015). Nogo-A is a myelin growth inhibitor protein that exerts an inhibitory effect by binding to the Nogo-A receptor (NgR1). NgR1 is a glycosylphosphatidylinositol (GPI)-linker that is rich in leucine-rich repeats (LRRs) and is encoded by the endoplasmic reticulum-4 receptor (RTN4R) gene. NgR1 forms a receptor complex with Lingo-1, p75NTR and Troy, binding to the growth inhibitory protein Nogo-A, oligodendrocyte myelin-associated glycoprotein (OMG), and myelin-binding glycoprotein myelin-associated glycoprotein (MAG; Schmandke et al., 2014).
Rho-A, a GTP-binding protein, can activate the signal transduction that regulates NgR1 in neurons, leading to growth inhibition and congenital atrophy (Fournier et al., 2002;GrandPré et al., 2002). Motor neuron growth inhibition leads to denervation of muscles and is an important pathological mechanism of ALS (Jokic et al., 2006;Steele and Yi, 2006). However, the NgR1 receptor may also regulate the complex role of maintaining motor neuron survival in ALS patients by blocking the p75NTRmediated neuronal death induced by nerve growth factor (NGF; Dupuis et al., 2008). This shows that the Nogo-A/NgR1 signaling pathway and the complex role of NgR1 in ALS are closely related (Teng and Tang, 2008;Schmandke et al., 2014). Amy et al. (2015) concluded that expression of the Nogo receptor is positively associated with SALS.
Our study suggests that the rs696880 A allele in the RTN4R gene increases the risk of SALS in the Han Chinese population. However, the mechanism is not clear. The rs696880 SNP, located in an enhancer element of the RTN4R gene, may regulate RTN4R gene transcription by changing COMP1 transcription factor binding sites. As concluded by Amy et al. (2015), the A allele of rs696880 may reduce transcription of the RTN4R gene, but this requires further functional verification.
The above findings demonstrate that Nogo-A/NgR1 contributes to the neuropathology of ALS. Variation at the NgR locus is associated with schizophrenia (Hsu et al., 2007;Budel et al., 2008;Voineskos, 2009;Jitoku et al., 2011;Willi and Schwab, 2013). Hence, our data provide additional evidence for the view that a genetic overlap exists between ALS and schizophrenia (Byrne et al., 2013;Fahey et al., 2014).
To the best of our knowledge, this is the first analysis of RTN4R tag SNPs in Han Chinese patients with SALS. This case-control study may provide an association between the Nogo-A receptor gene SNP rs696880 and the risk of SALS in the Han Chinese population, with the A allele increasing risk. However, the results still need to be verified in a larger study. Larger studies in different ethnicities are also needed to confirm and extend our findings.

AUTHOR CONTRIBUTIONS
DF conceived this study and provided financial support and was responsible for project management. LX, JL and DT performed the experiments and analyzed the data. LC and LT conducted data management and undertook data checking. LX and DF wrote the manuscript.

FUNDING
This work was supported by the National Natural Science Foundation of China (81030019).