Age-and gender-dependent association of SLC11A1 polymorphisms with tuberculosis susceptibility

Background: Tuberculosis (TB) is an important health issue in our world. It is reported that various factors may effect on its pathogenesis. In this current study, we aimed to investigate the association between SLC11A1 polymorphism and the risk of TB among 510 TB patients and 508 healthy controls. Methods: Agena MassARRAY platform was conducted for genotyping. Odds ratios (ORs) and 95% con�dence intervals (CIs) were analyzed through unconditional logistic regression adjustment confound factors, such as age and gender. Results: The results suggested that the allele and genotype frequencies of polymorphisms in SLC11A1 were not observed associated with TB risk. Subsequently, strati�ed analysis by age and gender con�rmed that rs7608307 “A/A” and “C/T-T/T” genotypes were related with increased TB risk in age ≤ 41 group (p = 0.021) and males (p = 0.013), respectively. Besides, rs13062 “A/A” genotype was reduced TB risk in age > 41 group (p = 0.043). In addition, we observed that the “C/C” genotype of rs4674301 was noteworthy correlated with increased TB risk in females (p = 0.043). Conclusion: Our results demonstrated the relationship between SLC11A1 polymorphism and TB risk and con�rmed for the �rst time that the correlation was restricted to age and gender in northwest Chinese population.


Introduction
Tuberculosis (TB) is a common public health issue with a high morbidity and infection rate in global (1,2).According to the WHO reported in 2017, approximately 10.4 million persons were newly identi ed infection with TB and 1.3 million deaths occurred.Epidemiological investigation con rmed that the disease was main caused by Mycobacteria tuberculosis (MTB).However, approximately 10% of persons infected MTB develop clinical diseases during their lifetimes (3).Previous studies have provided evidences con rmed that several environment factors including age, gender, poverty and diabetes may affect TB development (4,5).Recently researches demonstrated that host genetic factors also play a crucial role in the occurrence and development in TB.So far, multiples TB sensibility genes were con rmed through genome-wide association studies (GWAS) (6, 7).Nevertheless, the detailed molecular mechanisms of the genetic predisposition to TB still remain unknown.
The human solute carrier family 11 member 1 (SLC11A1) was also known as natural resistanceassociated macrophage macrophage protein (NRAMP1) (8).Studies reported that the gene is located on 2q35, and encode a transport protein with multiple functions including the regulation of erythrophagocytosis and infections (9).Numerous studies have shown that the polymorphisms of SLC11A1 are interacted with various in ammatory diseases, such as tuberculosis (10).The rst casecontrol study was conducted among the Gambia, and demonstrated that SLC11A1 polymorphisms were associated with TB susceptibility (11).Subsequently, plenty researches found the association between SLC11A1 polymorphisms and TB susceptibility in Americans, Japanese and Turkish and so on (12,13).
Although, there were some literatures reported the interaction between SLC11A1 polymorphisms and the risk of TB in Chinese populations including Chinese Kazakh Population and Taiwanese (14,15), no study investigated the correlation between SLC11A1 polymorphisms and TB sensibility in northwest Chinese population.
Here, we performed this study to investigate the relationship between SLC11A1 (rs11695562, rs7608307, rs4674301, rs2695343, rs13062 and rs1555529) polymorphism and TB risk in northwest Chinese population.These results provide important insights into SLC11A1 function in the occurrence development of TB.

Subjects
All of 1018 subjects including 510 TB patients and 508 unrelated controls were enrolled from the Xi'an Chest Hospital, Shaanxi Province in the current study.All of the subjects were unrelated ethnic Chinese.TB patients were diagnosed through clinical characteristics, positive sputum smear and chest x-ray examination.Patients with familial hereditary diseases, chronic in ammatory and other autoimmune diseases were excluded.Healthy controls, without any clinical characteristics of TB and autoimmune history, were recruited from the same hospital.The guideline of this research was authorized by the clinical investigative ethical committee of the same hospital, and then all participants signed the written informed documents.Subsequently, blood samples were extracted from each subject for molecular analysis.

SNP selection and genotyping
GoldMag DNA puri cation kit (GoldMag, China) was used to isolate the genomic DNA from samples, according to manufacturer's instructions.Soon afterwards, DNA purity and concentration were detected using NanoDrop 2000 (Thermo Fisher, USA).In accordance with minor allele frequency (MAF) > 0.05, ve candidate single nucleotide polymorphisms (SNPs) involving in SCL11A1 gene were selected from the 1000 Genomes Project data (http://www.internationalgenome.org/).The ampli cation and extension primer of each SNP was design through the Agena Bioscience Assay Design Suite V2.0 software.Moreover, the SNPs genotype and data management were performed by the MassARRAY iPLEX platform and Agena Bioscience TYPER 4.0 software, respectively.

Statistical analysis
The demographic characteristics of subjects involving age and sex were performed through the SPSS 20.0 software.Hardy-Weinberg equilibrium (HWE) p values of all controls were obtained from the χ 2 test.The values of odds ratios (ORs) and 95% con dence intervals (CIs) were used to estimate the correlation between individual alleles and genotype and TB risk.In addition, the PLINK software (version 1.07) was conducted to evaluate the relationship between SCL11A1 polymorphism and TB sensibility under four genetic models.Furthermore, the strati ed analyses by age and gender were also performed.In this study, p < 0.05 in all statistical tests was thought to be statistically signi cant.

Demographics
As summarized in table 1, the demographics characteristics of 510 TB cases and 508 unrelated controls were described.The mean age of TB cases was 41.90 ± 14.83 years old including 318 (62.35%) males and 192 (37.65%) females.For healthy controls, the mean age was 41.14 ± 18.42 year old containing 316 (62.20%) males and 192 (37.80%) females.Statistical analysis suggested that the age and gender among all participants were matched.No difference was observed regarding age and gender groups.The p values were 0.469 and 0.961, respectively.

Association between SLC11A1 polymorphism and TB risk
The detailed characteristics of ve candidate SNPs in SLC11A1 gene were displayed in table 2. In our study subjects, the MAF of SNPs was more than 0.05.At the same time, all SNPs were accordance with HWE (p > 0.05) among healthy controls.The allele and genotype frequencies distribution of all SNPs between participants were analyzed by χ 2 test, and the results were shown in table 3.However, all candidate SNPs of SLC11A1 polymorphism did not present any difference in allele and genotype frequencies among TB patients and healthy controls (all p > 0.05).
Strati ed analysis to assess the association between SLC11A1 polymorphism and TB risk Subsequently, we performed the strati cation analysis by age and gender.After analyzing the strati cation by age, our results suggested that SLC11A1 rs7608307 "C/T" genotype gene was signi cantly interacted with improved TB risk in the younger group (age ≤ 41) under the co-dominant genetic models (OR = 1.66, 95% CI = 1.04 -2.65, p = 0.035).In contrast, rs13062 "A/A" genotype was associated with reduced risk of TB in the old group under the co-dominant genetic model (OR = 0.44, 95% CI = 0.20 -0.98, p = 0.043) and the recessives genetic model (OR = 0.40, 95% CI = 0.18 -0.87, p = 0.021) (Table 4).However, there was no signi cant difference between these remaining SNPs polymorphism and TB susceptibility (p > 0.05), all data was not shown.

Discussion
In the current case-control researches, there was a population-based inheritance correlation study conducted among unrelated Chinese Han population in northwest in China.We investigated the SLC11A1 polymorphism and TB susceptibility.Our results revealed that SLC11A1 polymorphism does not present any difference in allele and genotype frequencies among TB patients and healthy controls (p > 0.05).
Subsequently, strati ed analysis by age and gender con rmed that SLC11A1 rs7608307, rs13062 and rs4674301 polymorphism was correlated with TB susceptibility (p < 0.05).
SLC11A1 was identi ed as a proton cation antiporter which localizes to lysosomes or late endosome.Several studies suggested that it might change the microenvironment of the phagosome to in uence microbial killing (11).Epidemiology investigation have provided evidence that SLC11A1 was a candidate gene for genetic susceptibility to disease caused by intracellular pathogens (16,17).Recently, numerous studies demonstrated that SLC11A1 polymorphism was associated with TB risk (13,18).A study investigated 855 individuals (435 families) and reported that SLC11A1 polymorphism rs3731865 was associated with TB in African-Americans (19).In addition, four SNPs distributed across the SLC11A1 gene, such as rs17235416 in the 3′ untranslated region, a GTn repeat in the 5′ promoter region and two SNPs in intron 4 (rs3731865) and exon 15 (rs17235409) were signi cantly considered to be related with TB risk in West Africans (20,21).The results of Two meta-analysis researches, which respectively identi ed 82 case-control studies in 35 articles and 131 case-control studies, showed that SLC11A1 polymorphism was associated with TB risk (18,22).Nevertheless, this is the rst time to assess the relationship between SLC11A1 polymorphism and TB risk in northwest China.The results con rmed that SLC11A1 rs11695562, rs7608307, rs4674301, rs2695343, rs13062 and rs1555529 polymorphisms were not associated with TB risk in northwest China.
Several studies reported that demographic factors including age and gender as confounding factors affected the candidate gene with TB susceptibility (12,23).A case-control study in which individuals were unrelated ethnic Chinese in Hong Kong, suggesting that SLC11A1 polymorphism was correlated with TB risk in age ≤ 65 years group and the females.However, no differences were noted in either the older age group or the males (24).In this study, we observed that the "C/T" and "C/T -T/T" genotypes of rs7608307 and rs13062 "C/A" genotype were associated with increased risk of TB in the younger group (age ≤ 41) and males, respectively.However, the "A/A" genotype of rs13062 was correlated with reduced TB risk in the old group (age > 41).

Conclusion
Although, there are several problems need to be considered in our study, such as the molecular mechanism which considered to be perform in future research, this is the rst time to con rm the relationship between SLC11A1 polymorphism and TB risk in northwest China.The study provides an important direction to understand the occurrence and development mechanism of TB in Chinese population.

Table 1 .
Characteristics of samples

Table 2 .
Basic characteristics and allele frequencies among SLC11A1 SNPs

Table 3 .
The association between SNPs within the SLC11A1 gene and the risk of tuberculosis CI, con dence interval; OR, odds ratio; SNP: single nucleotide polymorphism.*p a < 0.05 indicates statistical signi cance.

Table 4 .
The association between three SNPs within the SLC11A1 gene and the risk of tuberculosis strati ed by gender CI, con dence interval; OR, odds ratio; SNP: single nucleotide polymorphism.*p < 0.05 indicates statistical signi cance.

Table 5 .
The association between two SNPs within the SLC11A1 gene and the risk of tuberculosis strati ed by age