Significance of LncRNA CASC8 genetic polymorphisms on the tuberculosis susceptibility in Chinese population

Abstract Background Tuberculosis remains an important disease threatening the security of public health, and no effective targets have been found for the immunological diagnosis or therapy of tuberculosis. The aim of this study was to explore the associations between lncRNA CASC8 genetic polymorphism and tuberculosis risk. Method A total of 900 tuberculosis patients and 1534 healthy individuals in the Western Chinese Han population were recruited for our study. Candidate SNPs of CASC8 were initially filtered by importing the 1000 genomes database into Haploview, and subsequently genotyped using modified multiplex ligation detection reactions. Results The lncRNA CASC8 genetic variant rs7836840 was associated with an increased tuberculosis risk with a P‐value of .034, but .134 after Bonferroni correction. Using subtype analysis, the C allele in rs7836840 showed a significant association with tuberculosis susceptibility (OR = 1.196, 95% CI = 1.05‐1.362, P = .02739 after Bonferroni correction). Patients carrying genotype AG and GG of rs7825118 and rs9297758 exhibited lower Hb concentrations (P = .006) and neutrophil counts (P = .015), respectively, while genotype AG and AA in rs6981424 demonstrated higher levels of ALT (P = .005) and AST (P = .033) in a dominant model, which were consistent with a tendency toward increased TB risk. Conclusions This study was the first to explore the association between lncRNA CASC8 polymorphisms and TB infection risk and clinical manifestations. Our results provide evidence that CASC8 may act as a biomarker for the progression of clinical tuberculosis.

people die from tuberculosis each year. 1 Although an estimated 1.7 billion people are infected with MTB worldwide, only 5%-10% of latent tuberculosis infection (LTBI) individuals will go on to develop active TB. 2 This indicates that host genetic factors, mainly associated with "candidate" genes, including the genes encoding human leukocyte antigen (HLA), killer immunoglobulin-like receptor (KIR), toll-like receptors (TLRs), cytokine/chemokines and their receptors, vitamin D receptor (VDR) and SLC11A1 play important roles in determining individual susceptibility to infectious TB. 3 However, the roles of genetic polymorphisms in the regulation of TB have rarely been described, and therefore, there is an urgent need to explore more genetic loci which affect the clinical phenotypes and might assist in the diagnosis of clinical TB.
Long non-coding RNAs (lncRNAs) refer to types of ncRNA that are longer than 200 nucleotides and have no protein-coding potential. Studies are increasingly focused on lncRNAs and their underlying roles in the occurrence and development of human diseases. It is well known that lncRNAs are involved in the regulation of mRNA translations, RNA maturation and transport, protein synthesis, 4 cell differentiation, and tissue development. According to recent reports, lncRNAs have been shown to be involved in the dysregulation of cancer, 5 cardiovascular diseases 6 and especially infectious diseases. 7 For instance, aberrantly expressed lncRNAs HEAL in T cells are involved in HIV infection. 8 Additionally, many lncRNAs, like MEG3 9 and PCED1B-AS1 10 in macrophages, lncRNAs CD244 11 in CD8 + T cells and some lncRNAs in CD4 + T cells were differentially expressed in patients with TB. Furthermore, our research team previously reported the presence of differentially expressed lncRNA in peripheral blood mononuclear cells (PBMCs) in response to multidrug-resistant TB (MDR-TB) infection. 12 These data reveal that lncRNAs play important roles in TB infection, and therefore, we selected to investigate lncRNA polymorphisms and their association with human susceptibility to clinical TB.
In human TB, CD8+ T cells have been proven to contribute to host defenses by releasing Th1 cytokines or directly killing Mtbinfected macrophages. Fu et al 13 conducted microarray analysis to investigate differentially expressed lncRNA and mRNA genes in CD8+ T cells isolated from active TB group and healthy control group and recorded these profiles in the GEO database (GSE97530). For a more comprehensive lncRNA profile, we reannotated microarray probes with GENCODE Release 32 (GRCh38. p13) and re-analyzed this microarray. Differentially expressed ln-cRNAs profile (Figure 1) showed that the lncRNA transcript CASC8 encoded by gene CASC8 (Ensembl ID ENSG00000246228) was upregulated in CD8+ T cells from active TB (ATB) when compared to healthy controls. It is possible that the lncRNA CASC8 gene may be involved in the establishment and progression of TB infection.
CASC8 (also known as LINC00860), located at the 8q24.21, is a gene that is associated with an increased risk of cleft lip/palate 14,15 and several malignancies including colorectal cancer. 16 Recent evidence has indicated that the development of colorectal cancer (CRC) requires a series of inflammatory and immunological factors to enable and shape a tumorigenic milieu. 17 It is also reported that the expression of CASC8 is correlated with the SNPs inside the CASC8 gene body. 18  F I G U R E 1 Differentially expressed LncRNA CASC8 between ATB and control group. High relative expression was indicated by red color, and low relative expression was indicated by blue color 2 | ME THODS AND MATERIAL S

| Study subjects
Our study enrolled 900 tuberculosis patients and 1534 healthy controls. All subjects were recruited from the West China Hospital of Sichuan University between January 2014 and February 2016. In the case study group, we recruited patients that had a primary diagnosis of TB that was confirmed by two independent experienced respiratory physicians. The criteria for all enrolled patients included: typical symptoms, recent radiographic evidence of TB cavitation, and positive laboratory data (smear/culture/TB-DNA).
Patients with HIV infection, diseases associated with immunodeficiency, hepatitis virus infection, and other lung diseases were excluded from this study. The control subjects were selected from a pool of patients with no history of TB that had received a recent physical examination with normal laboratory and imaging data.

| Single nucleotide polymorphisms (SNPs) selection and genotyping
Genetic variants of CASC8 were obtained from the dbSNP database (http://www.ncbi.nlm.nih.gov/proje cts/SNP/). Candidate SNPs were filtered as dbSNP of CASC8 from the 1000 genomes database into Haploview, and tagSNPs were selected if they were represented with a minor allele frequency (MAF) ≥ 0.05 in the Han Chinese population of Beijing (CHB). Then, the tag-SNPs located in potentially functional regions like promoters, exons, untranslated regions (UTRs), and introns were preferentially selected. We selected 4 SNPs of the CASC8 gene including rs7825118, rs6981424, rs9297758, and rs7836840 for subsequent genotyping.
Our study used the QIAamp ® DNA Blood Mini Kit (Qiagen) to isolate genomic DNA from peripheral blood samples and candidate SNPs were genotyped using a modified multiplex ligation assay (Genesky Biotechnologies Inc). A 0.5 μL ligation product was fractionated and loaded in ABI 3730XL, and GeneMapper v4.1 software was employed to analyze the raw data. Fluorescent labels of allele-specific oligonucleotide probe pairs distinguished specific alleles of each SNP, and the differentiated extension length of the different SNPs at the 3′ end site. Genotyping was performed as a blinded experiment so that the experimenter was unaware of the status of the case controls. Moreover, we introduced ddH 2 O as a negative control in the experiment to monitor genotyping quality. Furthermore, we randomly selected approximately 10% for genotyping, with a 100% concordance rate.

| Statistical analysis
In our experiment, the chi-square test and the Mann-Whitney U test were used to analyze categorical variables and continuous variables, respectively. The goodness-of-fit chi-squared test was applied to assess the Hardy-Weinberg equilibrium (HWE) for all SNPs in all samples. The statistical method was performed using SPSS version 19.0 (IBM Corp.). The associations between candidate SNPs and TB infections was determined based on the distribution of allele and genotype frequencies as well as genetic models (dominant and recessive models). Furthermore, an unconditional logistic regression analysis by PLINK v1.07 was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) while correcting for age and gender. Calibration of the tests was carried out using the Bonferroni method. Fernando found that SNPs are preferentially associated with TB sub-phenotypes.
Correlation analysis between candidate SNPs and TB was then Furthermore, all TB subjects' samples were used for evaluating the correlation between genotypes and clinical phenotypes. The closely related clinical laboratory data (ALB and GLB related to impaired liver function in tuberculosis; WBC, Hb, ESR, and CRP involved in inflammatory responses in tuberculosis), apart from some missing data, were collected for this analysis. The chi-square test was used, all statistical tests were 2-sided, and P < .05 was considered statistically significant.

| Correlation analysis of single SNPs
In the case-control population, 4 SNPs (Table S1)  The genotype distribution and allele frequency data of 4 selected SNPs, among all TB cases and controls, associated with CASC8 gene are presented in Table 2. Rs7836840 with the mutant allele C (A>C) showed significant differences in both genotype distribution (P = .048) and allele frequency (P = .034, OR = 1.135, 95% CI = 1.01-1.28) between TB patients and the control group after adjusting for age and gender. However, after adjustments were made by Bonferroni correction, the underlying association disappeared (P = .191 and 0.134). Furthermore, genetic model analyses (dominant model and recessive model) were carried out to further explore the differences in genotype distributions. Table 3 (Table 6).

| Correlation between genotypes and clinical phenotypes
We further explored whether the four candidate SNPs influence the clinical features of TB patients.  and rs9297758 were not shown to be significantly associated with TB susceptibility in the above SNP analysis, they were closely associated with Hb and neutrophil (Neu) levels (P = .006 and 0.015, respectively). Lower levels of Hb and Neu were displayed among patients possessing the AG+AA genotypes. Additionally, significant associations were discovered between rs6981424 and indices of liver function in patients, and more patients with the AA+AG genotype exhibited higher ALT and AST activities (P = .005 and .033 respectably) (Figure 3).

| D ISCUSS I ON
With the high prevalence and rapid dissemination of MDR-TB in recent years, the regional disease burden of TB has become high, threatening health of the public in China. 19 Our study is the first to identify the association between the 4 SNPs of LncRNA CASC8 and TB susceptibility. We demonstrated that tagSNP rs7836840 may be involved in the risk of the development of TB, particularly in the PTB subgroups. Further research on candidate SNP rs6981424 may determine the influence of Hb levels in TB patients. To the best of our knowledge, this is the first study to comprehensively analyze the strong associations between CASC8 and TB risk, and CASC8 may become a promising biomarker in the diagnosis of TB.
Long non-coding RNAs are newly discovered transcripts that are more than 200 nt in length but without the capacity of protein encoding, 20 and they are involved in regulating gene expression through a variety of molecular mechanisms. LncRNAs are highly tissue-specific when compared to mRNA 21,22 and have the potential to be new targets for monitoring disease progression. However, there are currently insufficient data to demonstrate the impact of lncRNA genetic polymorphisms on TB susceptibility and clinical presentation. With this in mind, we conducted a case-control study in the western Han Chinese population for correlation analysis.
CASC8 is located in the 8q24 region with few functional annotation genes, meaning that it is possible to carry multiple non-coding transcripts to perform a variety of functions. 23 28 In our disease association study, the rs7836840 genetic variant in the intron region was determined to be strongly associated with TB risk. This may affect the functional expression of genes through mechanisms such as transcription levels within intron region mutations, intron splicing enhancers, or silencers. 29 Our findings provide useful information for the future investigation of the potential biological functions of SNP rs7836840 in the development of TB.
Gene levels and transcription levels are closely related to the development of disease. Our study is based on the correlation analysis between the expression of SNP by lncRNA and TB risk. In the next phase of our work, we will analyze the structural variation of genes and changes in downstream pathway regulation from the genetic level to explore the occurrence and development of TB.
Our study describes the first data describing the associations between CASC8 polymorphisms and TB in the western Han Chinese population. However, our study has some limitations: First, the gene-environment or gene-gene interaction was not further analyzed in SNPs studies without significant association with tuberculosis.
Second, the sample size of our study is limited. Third, the heterogeneity of the population was limited as the samples collected were mainly in western China, so some polymorphisms with significant effects may have been undetected. Lastly, several samples in this study lacked sufficient clinical characterization due to poor patient compliance; ideally, our results should be confirmed in larger population-based studies with more clinical detail.
In conclusion, this study investigated whether the SNP rs7836840 C allele of lncRNA CASC8 may act as potential risk factor associated with TB susceptibility. The AG+GG genotype in other 3 SNPs was correlated with serum Hb and Neu levels and hepatic enzyme activity in a dominant model. Our findings suggest that genetic polymorphisms of LncRNA CASC8 may play an important role in TB risk and inflammatory response and are expected to serve as a new target for diagnosing tuberculosis.