PTPN9 Genes Polymorphism as a Protect Factor in Lumbar Disc Herniation Development

Background: Lumbar disc herniation (LDH) is a high incidence spinal disease caused by disc degeneration, nuclear pulse displacement or ber ring degeneration. This study aimed to explore the correlation between PTPN9 susceptibility and lumbar disc herniation in Chinese Han population. Methods: The subjects were 504 patients with lumbar disc hernia and 503 controls. PTPN9 polymorphism (rs76107647, rs10851882, rs753992, rs11072552) are genotype using Agena MassArray. In addition, Logistic regression was used for odds ratios (ORs) and 95% condence intervals (CIs) to assess the impact of genetic polymorphisms on LDH occurrence. Results: In the allele model, rs76107647 A (p = 0.023) is associated with a reduced risk of LDH . In the analysis of genetic models, we found that rs76107647 (adjusted, A/A vs. G/G: p = 0.035; A/A vs. AG/GG: p = 0.044; log - additive model: p = 0.022, respectively) and rs11072552 (adjusted, C/C vs. A/A: p = 0.048; C/C vs. CA/AA: p = 0.010, respectively)were signicantly related to LDH risk reduction. After age stratication, rs76107647 (cid:0) adjusted, A/A vs. G/G: p = 0.036; A/G vs. G/G: p = 0.008, ; AA/AG vs. G/G: p = 0.002; log–additive model: p = 0.001, respectively) can reduce the risk of LDH for young people ≤ 49 years old, while rs11072552 (adjusted, CC vs. CA/AA: p = 0.009) can signicantly reduce the risk of LDH in patients over 49 years old. With gender stratication, rs76107647 (adjusted, A/A vs. G/G: p = 0.017, ; A/A vs. AG/GG: p = 0.019; log - additive model: p = 0.018, respectively) was signicantly associated with reduced LDH risk in female. Conclusion: Our research suggests that PTPN9 polymorphisms may have protective effects on the risk of LDH in Chinese Han population.


Introduction
Lumbar disc herniation (LDH) is a spinal disease caused by degeneration of the intervertebral disc space, nuclear displacement, or circular brosis [1]. It is considered a worldwide health problem even though it is a benign disease. Because it can cause physical weakness, negatively affect work activities and the body, reduce the comfort of life, and cause distress in people's hearts [2]. About 90% of sciatica is caused by symptomatic LDH, and it is also the most common manifestation of symptomatic LDH [3]. Although mechanical deformation may lead to radiation-induced pain, it is currently believed that autoimmune response and related in ammatory factors are the major factors leading to this symptom [4]. Epidemiological studies have shown that smoking, age, gender, BMI, occupation type and physical activity are the main risk factors for lumbar disc degeneration and hernia [5]. Recent studies have shown that heritability plays a vital role in the process of LDH, and its heritability is 52%-68% [6]. Because LDH is often seen in adults and of course occurs in adolescents [7], understanding the relationship between LDH and genetics can give patients new preventive and therapeutic measures. The study of LDHrelated genes has laid a foundation for the interpretation of its genetics, and at the same time opened up a new world for LDH ethics research [8].
PTP-MEG2 (also known as PTPN9) is a type of non-selective PTP. It was rst cloned from the cDNA library of human umbilical cord endothelial cells (HUVECs) and megakaryocytes, which is different from other PTP mammals, because there is an assumed lipid eld at the end of NH2. [9].It is a cytoplasmic protein whose function is mainly to regulate cell growth, cell cycle and malignant transformation [10]. Studies have shown that it plays an important role in multiple cancer processes. In breast cancer cells, PTP-Meg2 directly dephosphorylates EGFR and inhibits EGFR induced signal transduction [11]. Overexpressed in esophageal squamous cell carcinoma (ESCC) specimens, which indicates that PTPN9 may be an oncogene during ESCC [12]. Moreover, the expression of PTPN9 is down-regulated in human hepatocellular carcinoma (HCC) tumor tissues, and the decrease of PTPN9 expression is related to the deterioration of the overall survival of HCC patients. Its consumption inhibits apoptosis and promotes the proliferation of HCC cells [13]. In colorectal cancer, PTPN9 inhibits cell growth and survival by inhibiting the expression of Stat3, which suggests an important basic mechanism for regulating cell growth and provides a new target received IVD operation [4]. All the subjects are Han Chinese, which can be traced back to at least three generations. Prior to the study, we obtained informed consent from all participants. This study is in line with the declaration of Helsinki and approved by the ethics committee of Tibet University for nationalities.

Genotyping assay
We used the venipuncture method to draw the fasting peripheral blood of the patient into the anticoagulation blood vessel and store it at −80℃. We used GoldMag-Mini Whole Blood Genomic DNA Puri cation Kit (GoldMag. Co. Ltd.) to extract DNA, following the manufacturer's agreement. Then the DNA concentration was determined by spectrophotometry (NanoDrop 2000). The rs76107647, rs10851882, rs753992 and rs11072552 found in PTPN9 are based on our 1000 genome project data http://www.internationalgome.org/), and their micro allele frequency (MAF) exceeded 5%. To design four SNP primers for ampli cation and extension, we used Agena Bioscience Assay Design Suite V2.0 software (https://agenacx.com/online-tools/). Multiple SNP MassEXTEND analysis was designed using Agena-MassARRAY analysis design 4.0 software, and according to the manufacturer's explanation, SNP genotyping was detected by Agena massarray RS1000. Data management and analysis were performed using Agena Typer 4.0 software. Then, we conducted haplotype analysis using Haploview 4.2 software.

Statistical analysis
We analyzed the data using SPSS 19.0 software (SPSS Inc., Chicago, IL). The Hardy-Weinberg equilibrium (HWE) test was performed on the genotype frequency of the control group by chi-square test, and the allele frequency and genotype frequency of each SNP in the two groups were tested by Pearson chisquare test or t test, so as to determine the association between genotype and LDH risk. Logistic regression analysis was used to calculate the 95% con dence intervals(CIs) and odds ratios (ORs) for four different genetic models (co-dominance, dominance, recessive, log-additive) established by PLINK software (http://zzz.bwh.harvard.edu/plink/ ld.shtml) and adjusted for age and gender to estimate the risk of genotype-related lumbar disc herniation. The statistical signi cance is considered to be p < 0.05 (both sides).

Basic information of cases and controls
In this study, 504 patients with lumbar disc herniation and 503 healthy controls were included. Table 1 shows the characteristics of cases of lumbar disc herniation and healthy controls. There was no signi cant difference in age and gender between the LDH group and the healthy group (P > 0.05). The average age of patients was 49.30 ± 14.92 years old, and that of the control group was 49.25 ± 13.58 years old. The primer sequences of the four sites we designed are shown in Table 2.The basic information and allele frequencies of the four SNPs (rs76107647, rs10851882, rs75393192, rs11072552) are shown in Table 3. Both SNPs in the control group were in the Hardy-Weinberg equilibrium (HWE) (p > 0.05). We used chi-square test to analyze the allele distribution of the lumbar disc herniation case group and the healthy control group, and learned that the SNP rs76107647 allele A was associated with a decreased risk of lumbar disc herniation (p = 0.023, OR = 0.75, 95%CI = 0.58-0.96).  Genetic model analysis between PTPN9 gene and LDH risk The genetic relationships between the four selected single nucleotide polymorphisms and the risk of lumbar disc herniation are shown in Table 4

Strati cation analysis by age
We also performed a strati ed analysis to assess the effect of SNPs genotypes in four genetic models on the risk of lumbar disc herniation. Since the average age of the case group and the control group in our study population was 49 years, we strati ed by the age of 49 years. Table 5 shows the relationship between genotype and risk of lumbar disc herniation after age strati cation. In age > 49 years, the rs11072552 gene was associated with risk of low LDH (adjusted, CC vs. CA/AA: p = 0.009, OR = 0.49, 95%CI = 0.29-0.84). In age ≤ 49 years, we found that rs76107647 of PTPN9 gene was signi cantly related to the reduction of LDH risk(adjusted, A/A vs. G/G: p = 0.036, OR = 0.19, 95%CI = 0.04-0.90; A/G vs. G/G: p = 0.008, OR = 0.58, 95%CI = 0.38-0.87; AA/AG vs. G/G: p = 0.002, OR = 0.54, 95%CI = 0.36-0.80; log -additive model: p = 0.001, OR = 0.54, 95%CI = 0.38-0.78, respectively).   6 lists the relationship between SNP genotype and risk of lumbar disc herniation in gender strati cation. In females, the rs76107647 was related to a decrease the risk of LDH (adjusted, A/A vs. G/G: p = 0.017, OR = 0.08, 95%CI = 0.01-0.64; A/A vs. AG/GG: p = 0.019, OR = 0.09, 95%CI = 0.01-0.67; log -additive model: p = 0.018, OR = 0.64, 95%CI = 0.44-0.93, respectively). In males, however, we did not nd a signi cant association lumbar disc herniation in any genetic model. Our study shows that rs76107647 and rs11072552 on PTPN9 are signi cantly associated with reduced susceptibility to lumbar disc disease. As far as we know, there are not any reports on the risks of these two SNPS. In addition, we tested the age and gender strati cation of the PTPN9 and analyzed the correlation between their SNPs and LDH risks. In age strati cation, rs11072552 has a protective effect on LDH risk of people over 49 years old. Rs76107647 can reduce LDH risk of people under 49 years old. In the gender strati cation group, rs76107647 reduced LDH risk of female subjects. Therefore, we speculate that the PTPN9 may play a role in reducing risk factors for LDH progression.
Protein tyrosine phosphatase non receptor 9 (PTPN9), also known as PTPMeg2 or Meg2, is a cytoplasmic PTPs, which is widely expressed in brain tissues, white blood cells, endocrine cells and other tissues. It has been shown that multiple signaling pathways and physiological processes can be regulated by PTPN9 [14]. In our study, we found that new genes PTPN9 affect the risk of LDH. It has been identi ed as protective factors of LDH. Therefore, when a disc ruptures, NGF protein levels rise in the ruptured disc, possibly leading to pathologic nerve growth to the disc. So, we believe that NGF may be the key factor leading to the discogenic pain. And PTPN9 can down-regulate NGF/TrkA signal transduction [18].Moreover, VEGF is expressed in the degenerative disc tissue in the patients with lumbar disc herniation, which acts on the formation and in ltration of new blood vessels and accelerates the degeneration of the disc tissue [19], while PTP-Meg2 negatively regulates the downstream signal transduction of vascular endothelial growth factor (VEGF) -induced receptors in endothelial cells [9]. Moreover, immune cells and nonimmune cell, such as NP cells, broblasts and endothelial cells, can store and secrete a variety of cytokines, such as IL-1, IL-6, TNF-α and granulocyte macrophage colony stimulating factor, which may lead to the nal occurrence of sciatica [20]. Among them, IL-6 also induces the activation of JAK/STAT3 signaling pathway and plays a role in in ammation [21]. In NP tissues, IL-21 mainly induces the expression of TNF-α through JAK-STAT3 signaling pathway, which aggravates the degeneration of the intervertebral disc [22]. In addition, activation of RAGE/STAT3 pathway plays an important role in LDH persistent pain [23]. PTPN9 can dephosphorylate and inactivate vascular endothelial growth factor receptor 2 and STAT-3 [14], and negatively regulate STAT3 signaling pathway [24]. Therefore, PTPN9 is a new target for the treatment of LDH.
It should be pointed out that this study has some limitations. First of all, all participants in this study are from the same hospital, and there may be selection bias in the study population. There are many factors that affect LDH susceptibility, such as lifestyle factors and environmental factors. Due to lack of information or objective reasons, we are unable to assess the impact of all factors on LDH risk. Therefore, it necessary to further studies this study.

Conclusion
In conclusion, the polymorphisms of PTPN9 in the Chinese Han population are related to the susceptibility to lumbar disc herniation, which can be seen from case-control studies. This study provides new directions for the treatment of LDH. Therefore, further comprehensive functional studies and prospective studies based on a wide population are needed to provide accurate evidence of the effect of genotype on lumbar disc herniation.

Consent to publish
All patients agreed to publish the manuscript.