Collagen type XVIII alpha 1 chain (COL18A1) variants affect the risk of anti‐tuberculosis drug‐induced hepatotoxicity: A prospective study

Abstract Background The role of collagen type XVIII alpha 1 chain (COL18A1) in anti‐tuberculosis drug‐induced hepatotoxicity (ATDH) has not been reported. This study aimed to explore the association between of COL18A1 variants and ATDH susceptibility. Methods A total of 746 patients were enrolled in our study from December 2016 to April 2018, and all subjects in the study signed an informed consent form. The custom‐by‐design 2x48‐Plex SNPscanTM kit was used to genotype all selected 11 SNPs. Categorical variables were compared by chi‐square (χ2) or Fisher's exact test, while continuous variables were compared by Mann‐Whitney's U test. Plink was utilized to analyze allelic and genotypic frequencies, and genetic models. Multivariate logistic regression analyses were used to adjust potential factors. The odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were also calculated. Results Among patients with successfully genotyping, there were 114 cases and 612 controls. The mutant A allele of rs12483377 conferred the decreased risk of ATDH (OR = 0.13, 95%CI: 0.02–0.98, P = 0.020), and this significance still existed after adjusting age and gender (P = 0.024). The mutant homozygote AA genotype of rs12483377 was associated with decreased total protein levels (P = 0.018). Conclusion Our study first revealed that the A allele of COL18A1 rs12483377 was associated with the decreased risk of ATDH in the Western Chinese Han population, providing new perspective for the molecular prediction, precise diagnosis, and individual treatment of ATDH.


| INTRODUC TI ON
Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is the most serious adverse drug reaction during the course of tuberculosis (TB) therapy. 1 ATDH is defined as the inflammation of hepatocytes caused by idiosyncratic reaction to the anti-TB drugs. 2 The following 4 mechanisms are considered as the pathogenesis of ATDH: I) the enzymes and pathways about drug metabolizing, such as glutathione S-transferase (GST) and N-acetyl transferase 2 (NAT2); II) the accumulation of bile acids, lipids, and heme metabolites; III) the toxicity mediated by immune system; IV) the increasing oxidant stress. [3][4][5][6] It is noted that ATDH can be curable in the early stage, 7 although this disease has high mortality (22.7%) and morbidity (28%) [8][9][10] and adverse effects on the anti-TB treatment efficiency. 11 However, the ambiguous diagnostic criteria and atypical symptoms interfere with early prediction and diagnosis of ATDH.
Even worse, the delayed diagnosis of ATDH aggravates the severity of the disease and increases the disease burden. 12,13 Clearly, it is urgent to explore new biomarkers for diagnosis of ATDH. With the development of molecular detection methods, genetic factors are gradually well-recognized and considered as the crucial elements in the pathogenesis, prediction, diagnosis and treatment of many diseases. [14][15][16] Nowadays, a growing body of evidence proves that single nucleotide polymorphisms (SNPs), such as pregnane X receptor (PXR) rs7643645 17,18 and phase I cytochrome P450 enzyme (CYP2E1), 8 play important roles in the prediction, diagnosis, and treatment of ATDH. However, these SNPs are not applied in clinic due to limited predictive capacity or reliability. Thus, there is a promising future for exploring the association between more meaningful SNPs and ATDH to achieve precise prediction and treatment of ATDH.
Collagen type XVIII alpha 1 chain (COL18A1) is located on chromosome 21q22.3, encoding the alpha XVIII collagen. The product of alpha XVIII collagen, endostatin (EST), is mainly present in the liver sinusoidal and basement. 19 The close relationship between EST and liver diseases has been reported in many studies. [20][21][22] Many researchers find that EST can initiate the nicotinamide adenine dinucleotide phosphate oxidase (NOX) redox signaling cascade. 20,23,24 While the activation of NOX can generate reactive oxygen species (ROS) to increase oxidant stress which is one of the pathogenesis of ATDH as described before, and thus leads to the exacerbation of liver injury. 4,23,25 Moreover, Wnt/β-catenin signaling directs multiple liver cell processes and it is the essential signal for protecting hepatocyte from oxidative stress-induced cell deaths. 26 Moreover, it is has been published that EST can inhibit Wnt/β-catenin signaling through promoting the degradation of β-catenin. 27 Hence, we speculated that COL18A1 plays a role in ATDH by involving in the Wnt/β-catenin signaling, oxidative stress, and other various ways. [28][29][30] It is a pity that few studies have paid attention to explore the correlation between COL18A1 and ATDH. Regarding the high burden of ATDH in Western China, 31 we conducted this prospective study to evaluate the association between COL18A1 polymorphisms and the risk of ATDH in the Western Chinese Han population for the first time. We aimed to explore novel targets for the pathogenesis and personal treatment of ATDH patients.

| Study population
In this prospective study, 746 subjects were recruited from the West  During the anti-TB therapy, patients would be tested liver function regularly to monitor their liver function and the baseline levels of laboratory indicators before anti-TB treatment were tested. ATDH was defined as follows 33 : (a) an increase in alanine aminotransferase (ALT) levels more than 2 times upper limit of normal (ULN); (b) an increase in ALT 2 times upper ULN combined a rise in aspartate aminotransferase (AST) or total bilirubin (TB) levels.

| Genotyping
Peripheral whole blood specimens were collected from each enrolled patient. All these samples were used to extract genomic deoxyribonucleic acid (DNA) via QLAamp ® DNA Blood Mini Kit (Qiagen, Germany). Then, the custom-by-design 2x48-Plex SNPscanTM kit (Genesky Biotechnologies Inc, Shanghai, China) was utilized for genotyping all SNPs. All processes were carried out in accordance with the instructions.

| Statistical analysis
Categorical variables such as gender and drinking statuses were compared by chi-square (χ 2 ) or Fisher's exact test, whereas continuous variables such as age and serum ALT levels were com-

| Study characteristics
A total of 746 TB patients were included in our study, while 726 patients were successfully genotyped with all selected SNPs ( Figure 1).

| Single selected SNP association with ATDH
All of the 11 SNPs genotypes from the controls did not deviate from the HWE. The mutant A allele frequency of rs12483377 was 0.46% and 3.29% in the cases and the controls, respectively. The mutant A allele conferred the decreased risk of ATDH (OR = 0.13, 95%CI: 0.02-0.98, P = 0.020). Logistic regression showed that this significance still existed after adjusting age and gender (P = 0.024), and the statistical power is 0.77 (Table 1). No case harbored AA genotype of rs12483377, while there were 2 patients with AA genotype in the controls. For the AG genotype of rs12483377, there was 1 and 35 subjects in the ATDH group and non-ATDH group, respectively. However, as shown in Table 2, comparable risk of ATDH was identified in these 3 genetic models of rs12483377. As for the genetic models of the other 10 selected SNPs, none of them reach the threshold value of statistical significance.

| The correlation within SNPs and laboratory indicators
For rs12483377, mutant homozygote AA genotype was associated with lower total protein (P = 0.018). However, no significant findings on the relationship between rs12483377 and other clinical characteristics were observed (Figure 3). The total protein levels under different genotypes were displayed in Table 4.

| D ISCUSS I ON
In this present study, we investigated the relationship between COL18A1 polymorphisms and the risk of ATDH in the Western Chinese Han population. We revealed that the mutant A allele of CLO18A1 rs12483377 was associated with decreased risk of ATDH.
Furthermore, the statistical significance of rs12483377 on total protein had been identified.
COL18A1, highly expressing in the liver (http://biogps. org/#goto=gener eport &id=80781), is closely related to liver diseases. 25,42,43 Musso et al 44 have reported the relationship between COL18A1 and liver fibrosis. Type XVIII collagen, encoded by COL18A1, can increase before and during the fibrotic stages of liver fibrosis. 45,46 The increased type XVIII collagen upregulates its own product, EST. EST is able to resist liver fibrosis by inhibiting the expression of TGF-β1 mRNA through RhoA/ROCK signal pathways in hepatic stellate cells (HSCs). 47,48 In addition, liver fibrosis refers to the progression of extracellular matrix excessive deposition, which is often promoted by the activation of HSCs. HSCs can transdifferentiate into cells which can secrete extracellular matrix. [49][50][51] Therefore, COL18A1 is associated with liver fibrosis via regulating the expression of EST COL18A1 is also related to hepatic carcinoma by EST. 52 EST can inhibit endogenous angiogenesis by suppressing the production of angiogenic factors, while angiogenesis is a common physiological and pathological process in liver cancer. 53,54 Besides, the relationship between COL18A1 polymorphisms and liver cancer is also reported. Wu et al 36  In our study, we firstly reported the relationship between COL18A1 variants and ATDH susceptibility. We found that rs12483377 is associated with decreased risk of ATDH. It has been verified that the mutant A allele of rs2483377 could decrease the ability of EST to bind other molecules and the function to inhibit angiogenesis. 58 Considering the role of EST in the occurrence of ATDH as we described before, we deduced that rs12483377 may influence ATDH by regulating the expression of EST which participates in oxidative stress. 20 Besides, in our study, there were significant differences in fever, weight loss, total bilirubin levels, serum ALT levels, serum AST levels, uric acid levels, ALP levels, and GGT levels between the case and control group. Fever and weight loss are the common symptoms of tuberculosis poisoning. 63 After Mycobacterium tuberculosis infected the body, it will produce toxins and metabolites, which can not only cause allergic reactions such as fever, fatigue, and so on, but also will stimulate the central nervous system, resulting in dysfunction of the autonomic nervous system which lead to night sweats. 63,64 Bilirubin, ALT, AST, ALP, and GGT are all associated with the liver metabolism. 65,66 As is stated

| Strengths and limitations
We firstly investigated the relationship between COL18A1 polymorphisms and ATDH susceptibility in Western Chinese Han population.
Based on available evidence, we also speculated the potential mechanisms that how COL18A1 polymorphisms affect ATDH susceptibility, contributing to the deep understanding of ATDH etiology to some extent. Besides, our finding is beneficial to explore more novel biomarkers of ATDH and decrease the burden brought by ATDH to some degree. Nevertheless, there were still some limitations in our study.
The design of single center study restricts us to verify our findings in different ethnicities. Functional experiment about rs12483377 should have been further performed to validate our speculation.
More high-quality studies with lager cohorts are warranted.

| CON CLUS ION
Collectively, our study revealed that CLO18A1 rs12483377 is related to the risk and specific characteristic of ATDH in the Western Chinese Han population, mining and further emphasizing the role of CLO18A1 variants in ATDH.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data to this article can be found at the end of this manuscript.