SP110 Polymorphisms Are Genetic Markers for Vulnerability to Latent and Active Tuberculosis Infection in Taiwan

One-fourth of the human population is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) and carries the infection in its latent form. This latent infection presents a lifelong risk of developing active tuberculosis (TB) disease, and persons with latent TB infection (LTBI) are significant contributors to the pool of active TB cases. Genetic polymorphisms among hosts have been shown to contribute to the outcome of Mtb infection. The SP110 gene, which encodes an interferon-induced nuclear protein, has been shown to control host innate immunity to Mtb infection. In this study, we provide experimental data demonstrating the ability of the gene to control genetic susceptibility to latent and active TB infection. Genetic variants of the SP110 gene were investigated in the Taiwanese population (including 301 pulmonary TB patients, 68 LTBI individuals, and 278 healthy household contacts of the TB patients), and their association with susceptibility to latent and active TB infection was examined by performing an association analysis in a case-control study. We identified several SNPs (rs7580900, rs7580912, rs9061, rs11556887, and rs2241525) in the SP110 gene that are associated with susceptibility to LTBI and/or TB disease. Our studies further showed that the same SNPs may have opposite effects on the control of susceptibility to LTBI versus TB. In addition, our analyses demonstrated that the SP110 rs9061 SNP was associated with tumor necrosis factor-α (TNFα) levels in plasma in LTBI subjects. The results suggest that the polymorphisms within SP110 have a role in controlling genetic susceptibility to latent and active TB infection in humans. To the best of our knowledge, this is the first report showing that the SP110 variants are associated with susceptibility to LTBI. Our study also demonstrated that the identified SP110 SNPs displayed the potential to predict the risk of LTBI and subsequent TB progression in Taiwan.


Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains one of the top ten causes of death in the world [1]. Approximately one-fourth of the world population has been infected with Mtb [2], but only 10% of these infected persons develop progressive disease during their lifetimes [3]. The majority of infected individuals remain healthy and noninfectious but carry Mtb in a latent form. Latent TB infection (LTBI) is a state during which a persistent host immune response to stimulation by Mtb antigens is sustained without evidence of clinically manifested active TB [4,5]. As many as 10% of people with LTBI will go on to develop progressive disease in the near or remote future (a process named "TB reactivation"), and the risk is significantly higher in the presence of predisposing factors, such as coinfection with human immunodeficiency virus (HIV).
Persons with LTBI can be diagnosed by skin (tuberculin skin test (TST)) and/or blood (interferon-gamma (IFNγ) release assay (IGRA)) tests [6]. A positive result from these assays indicates an immune response to stimulation by Mtb antigens in the LTBI population, despite the fact that these individuals have a negative bacteriological test. However, neither TST nor IGRA can distinguish between LTBI and active TB disease or predict who will progress to active TB [7]. Given that the LTBI individuals represent a potential reservoir for future active TB cases, preventive therapy for LTBI is as important a goal as timely anti-TB treatment to reduce the burden of TB [8]. Therefore, identifying and treating cases with LTBI will contribute to TB elimination. Fundamental research for the development of diagnostic assays with improved performance and predictive assessment for TB reactivation will have practical applications and offer a substantial benefit for LTBI management.
In both humans and experimental animal models, genetic polymorphisms among hosts have been shown to contribute to the outcome of Mtb infection [9][10][11][12][13]. In mice, the Ipr1 (intracellular pathogen resistance 1) gene is located within the sst1 (supersusceptibility to tuberculosis 1) locus on chromosome 1 (49-54 cM) [14] and has been identified as a genetic determinant conferring host innate immunity to Mtb infection [15]. The previous studies indicate that the Ipr1 gene may function to integrate mechanisms on controlling cell death, innate immunity, and pathogenesis during intracellular pathogen infections [15,16]. The gene orthologous to the mouse Ipr1 in humans is SP110, located on chromosome 2q37.1. Expression of both Ipr1 and SP110 genes is intensively regulated by IFNs, suggesting that the function of both genes is related to the IFN-mediated immune response [17].
Genetic defects in the SP110 gene have been found to be responsible for hepatic veno-occlusive disease and immunodeficiency [18,19], indicating that the gene plays important roles in immunity [20]. The SP110 gene encodes the SP110 nuclear body protein, which has at least three isoforms, including the dominantly expressed SP110a, b, and c isoforms that are believed to be the result of alternative mRNA splicing. Our recent study demonstrated that SP110b, which is most similar to mouse Ipr1 and is expressed more abundantly than SP110a and SP110c, modulates nuclear factor-κB (NF-κB) activity resulting in the downregulation of tumor necrosis factor-α (TNFα) production and concomitant upregulation of NF-κB-induced antiapoptotic gene expression, thereby suppressing IFNγ-mediated monocyte/ macrophage cell death [21]. This indicates that the protein is crucial in the control of the activation of macrophages, the reservoir for Mtb persistence.
Although a number of genetic variants of the SP110 gene have been reported to be associated with susceptibility to human TB, the results of studies regarding the relationship between SP110 polymorphisms and TB susceptibility are inconsistent [22][23][24][25][26][27][28][29]. A family-based study in West Africa identified 3 SP110 polymorphisms that are associated with TB susceptibility [22]; however, no significant associations between SP110 and disease susceptibility were identified by other, larger case-control studies conducted on various populations [24][25][26]. After screening Taiwanese populations for polymorphisms in SP110, we identified some single-nucleotide polymorphisms (SNPs) in SP110 that are significantly associated with susceptibility to LTBI as well as TB disease. These results suggest that the SP110 variants may provide novel predictive markers for TB infection status and disease outcome.

Materials and Methods
2.1. Human Subject Study. The study was conducted in accordance with the terms of the informed consent that was provided to, and received from, participants prior to inclusion in the study. This study was approved by the National Taiwan University Hospital Institutional Review Board (IRB No. 200612009M and IRB No. 201512169RINA). Human blood was ethically collected from patients with cultureconfirmed pulmonary TB and their household contacts as described previously [30]. Briefly, the participating contacts received chest radiography, and mycobacteriology studies were conducted for 3 sputum samples (including acid-fast smear and mycobacterial culture) to exclude the possibility of active TB disease. Because routine BCG vaccination for newborns in Taiwan could affect the accuracy of tuberculin skin test, all enrolled contacts were then tested for LTBI using a T-SPOT.TB assay (Oxford Immunotec Ltd., Abingdon, UK) or QuantiFERON-TB Gold In-Tube assay (QFT) (Qiagen, Hilden, Germany), and the assays were interpreted according to the manufacturers' criteria. Both patients and contacts were excluded if they were tested positive for HIV infection. In total, 301 pulmonary TB patients, 68 individuals with LTBI, and 278 healthy household contacts were included in the study, and genomic DNA was extracted from their peripheral blood (1-2 mL) using a kit from Qiagen according to the manufacturer's protocol. SP110 polymorphisms were identified from the National Center for Biotechnology Information dbSNP database (https://www.ncbi.nlm.nih. gov/snp). The SNPs were genotyped using the MassARRAY System (Sequenom, San Diego, CA, US), and the primer extension products were analyzed by MALDI-TOF mass spectrometry as previously described [31,32]. Details of the primers that were used are listed in Table S1 in the Supplementary Materials. Plasma samples were prepared from blood samples by centrifugation and then stored at −80°C until analysis. The TNFα levels in plasma samples were determined by a MAG-PIX® platform (Luminex Corp., Austin, TX. US) with a MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel I kit (Merck Millipore, Billerica, MA, US) according to the manufacturer's instructions.

Statistical
Analyses. The associations of gene polymorphisms with LTBI and TB disease were analyzed by SAS 9.4 software (SAS Institute, Cary, NC, US) [33]. Linkage disequilibrium and haplotype analyses were performed using Haploview (https://www.broadinstitute.org/haploview) [34]. Chisquare tests were used to compare frequencies. Odds ratios (ORs) were calculated with 95% confidence intervals (CIs). Bonferroni correction or false discovery rate (FDR) correction was applied for multiple comparison adjustments as indicated. The difference of the TNFα levels in plasma between two genotype groups of samples was calculated using a two-tailed unpaired t-test with Prism software (GraphPad, San Diego, CA, US). p values less than 0.05 were considered to indicate statistical significance, and the number of asterisks represents the degree of significance with regard to the p values.

3.4.
Association between the SP110 rs9061 SNP and the TNFα Production in LTBI Subjects. TNFα plays a crucial role in controlling Mtb infection and TB reactivation; however, overproduction of TNFα may cause pathology [35,36]. Our previous studies demonstrated that the SP110b protein, which is encoded by the SP110 gene and whose expression is upregulated by IFNs, downregulates TNFα production in monocyte/macrophage cells activated by IFNγ, thereby alleviating cell death [21]. This finding indicates that the protein functions as a regulator of proinflammatory cytokines of host immunity contributing to a reduction in tissue damage caused by excessive inflammation [21]. To further investigate the potential association between the SP110 SNPs and disease status, we next analyzed clinical parameters in the studied subjects. We measured TNFα levels in plasma from LTBI individuals who carry the different genotypes of the SP110 SNPs and demonstrated that the "GA" genotype of rs9061 in LTBI individuals was associated with lower TNFα levels in plasma compared to "GG" LTBI subjects (Figure 2). The TNFα levels of healthy controls with both genotypes were undetectable (not shown). These data are in agreement with the protective role of the "GA" genotype of rs9061 in LTBI (Table 3) and further support our recent finding showing that the SP110b protein prevents cell death and tissue damage by downregulating TNFα production.
SNP rs9061 (G→A) introduces an amino acid change from glutamic acid to lysine at codon position 207 of the SP110 protein. Transforming an acidic amino acid to a basic amino acid may alter the protein structure or posttranslational modification of the SP110 protein leading to better downregulation of TNFα production. It has been suggested that the A allele may cause changes in α-helices and β-sheets in the secondary structure of the SP110 protein compared with the G allele [38]. In addition, we analyzed the SP110 protein using the ELM database (http://elm.eu.org/) and found that this amino acid change generates a potential binding motif for the C-terminal ubiquitin-like domain (CTD) of ubiquitin specific protease 7 (USP7), one of the most abundant deubiquitinases [39]. USP7 plays important roles in various biological activities, including cell survival, proliferation, apoptosis, and tumorigenesis [40,41]. As shown in Figure 2, the data demonstrated an association between the "GA" genotype of rs9061 with lower TNFα levels in plasma from LTBI individuals compared to "GG" LTBI subjects. One possible explanation for this result is that SP110 with the "GA" genotype at rs9061 may interact with USP7 and thus is more stable than the SP110 with the "GG" genotype, resulting in a more efficient downregulation of TNFα production. However, further functional studies are needed to elucidate the exact effects of SNP rs9061 on the SP110 gene and disease risk.
Individuals with LTBI, based on a positive result in the TST or the IGRA, usually show no disease symptoms and acquire an effective adaptive immunity. However, a proportion of people with LTBI might reactivate and develop clinical disease. The positive results from these assays indicate an immune response to stimulation by Mtb antigens in the LTBI population; however, neither TST nor IGRA can distinguish between LTBI and active TB. In addition, these assays also cannot predict which LTBI cases will progress to active TB [7]. To control and eliminate TB, worldwide TB eradication endeavors have been focused on identifying and treating cases with LTBI. Therefore, our studies identified several SNPs in SP110 that are significantly associated with controlling susceptibility to LTBI and TB disease and thus may provide novel predictive markers for latent and active TB infection. This study may also yield an improved strategy that can identify persons at increased risk of the disease. There were some limitations in our study. First, this study dealt with relatively small sample sizes (301, 68, and 278 for TB patients, LTBI cases, and healthy household contacts, respectively). Further large-scale studies are, therefore, needed to validate the predictive value of the SP110 SNPs identified in this study. Second, the LTBI number of our study group is small, and besides, the results on plasma TNFα levels were analyzed from groups with even smaller sample sizes; therefore, the difference in TNFα levels in plasma of LTBI cases with two genotypes may be underestimated and should be verified in a different population. In addition, the analysis of TNFα levels in plasma of active TB patients with the different genotypes is in progress. The result may help support our finding in LTBI individuals and clarify the potential association between the SP110 SNPs and disease status.

Conclusions
The results suggest that the SP110 variants have a role in controlling genetic susceptibility to latent and active TB infection in humans. To the best of our knowledge,  Figure 1: Haplotype block maps for SP110 with 3 SNPs (rs9061, rs7580900, and rs7580912). The haplotype blocks were analyzed in LTBI cases vs. healthy controls (a) and TB cases vs. LTBI individuals (b), respectively. this is the first report demonstrating associations of polymorphisms in SP110 with LTBI susceptibility. Additionally, we provide evidence that the SP110 rs9061 SNP is associated with plasma TNFα levels in LTBI individuals. These data suggest that the identified SP110 SNPs may serve as a biomarker for latent and active TB infection in Taiwan.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflict of interest.  Figure 2: Association between the SP110 rs9061 SNP and the TNF levels in plasma of LTBI subjects. The TNF production was determined in plasma samples from LTBI cases carrying the different genotypes of the rs9061 SNP by a MAGPIX instrument with a MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel I kit. Sample numbers are 14 for "GG" and 8 for "GA" genotypes, respectively. Statistical significance was calculated using a two-tailed unpaired t-test. * p < 0 05.