Association of the FTO gene polymorphism with the features of the clinical course of tuberculosis infection

Abstract

Objectives

Severe tuberculosis, characterized by high mortality and disability rates, presents a significant challenge in the prevention and treatment of the disease. The pathogenesis of this severe form of tuberculosis remains largely elusive. While numerous studies have established a link between host gene polymorphisms and susceptibility to tuberculosis, there is a paucity of researchon genetic susceptibility specifically related to severe TB. This study delves deeper into the relationship between host gene polymorphisms and severe tuberculosis, and provides functional verification of dual-luciferase reporter genes.

Methods

The study encompassed Han Chinese tuberculosis patients, aged between 15 and 45 years (both inclusive), who were treated at the West China Hospital, a subsidiary of Sichuan University, from January 2013 to December 2020. A total of 558 severe and 579 mild tuberculosis cases were selected for a case-control analysis. Two Tag SNPs sites within the FTO gene were identified to explore the correlation between gene polymorphism and tuberculosis severity. The identified SNPs loci associated with TB severity in the association analysis. Predicting the function of loci associated with tuberculosis severity and completing dual-luciferase reporter gene functional validation.

Results

The correlation analysis revealed that the minor allele G of FTO rs16952730 is associated with a decreased risk of severe tuberculosis (OR^a 0.828, 95% CI 0.691–0.993, P^a=0.041). In contrast, the GG genotype was found to be associated with a lower risk of severe tuberculosis compared to the AA genotype (OR^a 0.636, 95% CI 0.421–0.961, P^a=0.032). The genetic model analysis also indicated that the additive model was associated with a reduced risk of severe tuberculosis (OR^a 0.834, 95% CI 0.698–0.996, P^a=0.045). During the functional validation of the dual luciferase reporter gene, a comparative analysis was conducted between the rs16952730-PGL3-promoter-AA and rs16952730-PGL3-promoter-GG recombinant plasmids. This comparison unveiled a statistically significant variance in the relative luciferase expression activity, evidenced by a P value of 0.00295.

Conclusions

The FTO gene rs16952730 may serve as a potential functional locus influencing gene expression, potentially correlating with the severity of tuberculosis.

Clinical trial

Not applicable.

Introduction

Severe tuberculosis (TB) presents a major clinical challenge, characterized by pronounced symptoms, prolonged treatment, significant therapeutic difficulties, and high rates of mortality and disability [1]. Despite its severe impact on patients’ health and socioeconomic well-being, a universally accepted definition and clear diagnostic criteria for severe TB are still lacking, hampering both clinical management and fundamental research. A critical gap in understanding the pathogenesis of severe TB persists.

Host genetic factors are known to influence TB susceptibility, as demonstrated by numerous studies linking gene polymorphisms to the risk of developing the disease [2–4]. However, whether specific genetic variants determine the progression to severe forms of TB remains poorly investigated, especially in the Chinese population. This study therefore aims to identify genetic susceptibility factors associated with TB severity using a candidate gene approach.

The Fat Mass and Obesity-Associated (FTO) gene has emerged as a compelling candidate. While initially characterized for its role in energy metabolism and obesity [5], evidence now implicates FTO polymorphisms in a broader spectrum of diseases, including infectious diseases. For instance, associations between FTO variants and TB risk have been reported in Iranian and Chinese populations [6, 7]. Given that obesity can impair immune function, one plausible link is that FTO-mediated metabolic alterations might influence host defense mechanisms against Mycobacterium tuberculosis. This leads to the hypothesis that FTO polymorphisms could affect not only susceptibility to TB but also its clinical severity.

To systematically investigate this, our study had two main objectives: First, to assess the association between FTO gene polymorphisms and severe TB in the Chinese Han population. Second, to functionally validate the potential regulatory impact of the identified risk polymorphisms using dual-luciferase reporter assays, thereby bridging genetic association with a potential molecular mechanism.

Materials and methods

Ethics statement and study population

This study was approved by the Ethics Committee of West China Hospital, Sichuan University [Approval No.: 932(2019)]. All participants provided written informed consent prior to enrollment. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

A total of 1137 clinically or bacteriologically confirmed tuberculosis patients were included, all of whom were unrelated individuals of Chinese Han ethnicity aged 15–45 years. These patients were inpatients in the Tuberculosis Ward of West China Hospital, Sichuan University, between 2013 and 2020. The cohort consisted of 558 patients with severe tuberculosis and 579 with mild tuberculosis. The severe group included 308 males and 250 females, while the mild group included 298 males and 281 females.

Inclusion and exclusion criteria

Inclusion Criteria: Patients were included if they had a clinical diagnosis or bacteriological confirmation of tuberculosis. Clinical diagnosis was based on symptoms, physical signs, laboratory tests, and imaging findings, while excluding tumors, non-tuberculous mycobacteria, and fungal infections. Bacteriological confirmation was defined as a positive result from TB-DNA testing, GeneXpert, mycobacterial culture, or histopathology [8, 9].

Exclusion Criteria: Patients with diabetes, hepatitis B virus infection, AIDS, tumors, autoimmune diseases, or congenital immunodeficiency (as assessed by clinical presentation and family history) were excluded from the study.

Definition of severe tuberculosis

Severe tuberculosis was defined based on previously published criteria [2, 3, 5–8] as meeting at least one of the following conditions: bilateral pulmonary lesions involving more than two-thirds of the lung fields on imaging; tuberculous meningitis; tuberculosis involving two or more organ systems; pulmonary cavity diameter greater than 4 cm on radiography; or hematogenous disseminated pulmonary tuberculosis.

Study design

This candidate gene association study investigated the relationship between FTO gene polymorphisms and tuberculosis severity. Genomic DNA was extracted from blood samples, and selected SNP loci were genotyped using high-throughput technology. Differences in genotype frequency distributions between the mild and severe tuberculosis groups were evaluated under four genetic models: allelic, additive, dominant, and recessive.

Tag SNP selection

Tag SNPs were selected using genotype data from the Han Chinese in Beijing (CHB) population of the 1000 Genomes Project (http://www.1000genomes.org), accessed via the Genome Variation Server platform (https://gvs.gs.washington.edu/GVS150/).

The selection region covered 3000 bp upstream to 300 bp downstream of the 5′ end of the FTO gene. Tag SNPs were required to have a minor allele frequency (MAF) ≥ 5% and an r² value between 0.8 and 1.0. Two SNPs in the FTO gene, rs16952577 and rs16952730, were included.

DNA extraction and genotyping

Genomic DNA was extracted using the Tiangen Bio-DNA Extraction Kit (Beijing, China). DNA concentration and purity were assessed, and only samples with a concentration>15ng/µL and an A260/A280 ratio between 1.8 and 2.0 were used for genotyping. SNP genotyping was performed using the SNPscan™ multiplex SNP typing technique based on ligase detection reaction.

Dual-luciferase reporter assay

A recombinant plasmid carrying the rs16952730 AA genotype was constructed using the pGL3-Promoter vector. Site-directed mutagenesis by PCR was used to generate the corresponding GG genotype plasmid. Both plasmids were transfected into 293T cells. A PE multifunctional microplate reader was used to measure dual-luciferase expression levels across genotypes. The pGL3-Promoter and pGL3-Basic vectors were used as controls.

Statistical analysis

Allele and genotype frequency distributions were compared between groups using the chi-square test. Associations between SNPs and tuberculosis severity were evaluated under four genetic models using binary logistic regression, with results expressed as odds ratios (ORs) and corresponding P-values; adjustments were made for age and gender as confounding factors. The chi-square test was also used to assess Hardy–Weinberg equilibrium (HWE). For the dual-luciferase assay, statistical differences in luciferase expression among plasmids were analyzed using GraphPad Prism software.

Results

Study population

Demographic and clinical parameters of mild and severe tuberculosis are summarized in Table 1. The Hardy-Weinberg equilibrium test was conducted independently within the mild TB group and the severe TB group, yielding p values exceeding 0.05. This suggests that the chosen populations were in accordance with HWE equilibrium.

Table 1.

Characteristics of patients with mild and severe tuberculosis

Features	Severe tuberculosis, N(%)	Mild tuberculosis, N(%)	P
Number, N	558	579
Mean Age, Years	26.79 ± 8.166	29.01 ± 8.199	< 0.001
Gender, N(%)
Male	308(55.2)	298(51.5)	0.208
Female	250(44.8)	281(48.5)
Living environment, N(%)			0.901
Rural	421(75.4)	435(75.1)
Town	137(24.6)	144(24.9)

The severe tuberculosis group comprised 558 patients, while the mild tuberculosis group consisted of 579 patients. No statistically significant disparities were observed in terms of gender distribution or living conditions between the two groups. The mean age of the patients in the severe tuberculosis group was 26.79 ± 8.166, whereas that of the mild tuberculosis group was 29.01 ± 8.199. A notable statistical disparity was identified in the age distribution between these two patient cohorts, as detailed in Table 1.

Association analysis of genetic susceptibility to severe tuberculosis

Frequency distribution and genetic model analysis of FTO gene polymorphism in patients are shown in Table 2. The study examined the correlation between tuberculosis severity and two TagSNPs loci, rs16952577 and rs16952730, of the FTO gene. A logistic regression analysis demonstrated that the minor allele G of rs16952730 (A > G) diminished the risk of severe tuberculosis (OR 0.822, 95%CI 0.687–0.983, P = 0.032). When compared to the AA genotype, the GG genotype significantly lowered the risk of severe tuberculosis (OR 0.630, 95%CI 0.418–0.947, P = 0.026). After accounting for age and gender confounding factors in both groups, the statistical significance of the polymorphism of rs16952730 remained, with the minor allele G further reducing the risk of severe tuberculosis (OR^a 0.828, 95%CI 0.691–0.993, P^a=0.041). In comparison to the AA genotype, the GG genotype also decreased the risk of severe tuberculosis (OR^a 0.636, 95%CI 0.421–0.961, P^a=0.032). In a genetic model analysis, the additive model was associated with a reduced risk of severe tuberculosis (OR^a 0.834, 95%CI 0.698–0.996, P^a=0.045). However, no correlation was found between rs16952577 and severe tuberculosis (Table 2).

Table 2.

Frequency distribution and genetic model analysis of FTO gene polymorphism in severe and mild tuberculosis groups

SNPs	Genetic models	Allele/ Genotype	Severe TB N(%)	Mild TB N(%)	OR(95%CI)	P	ORa(95%CI)	P a
rs16952577 (G> T)		G	575 (51.5)	600 (51.8)	1	Reference	1	Reference
		T	541 (48.5)	558 (48.2)	1.012 (0.858–1.293)	0.890	1.017 (0.861-1.200)	0.846
		GG	158 (28.3)	161 (27.8)	1	Reference	1	Reference
		GT	259 (46.4)	278 (48.0)	0.949 (0.720–1.253)	0.713	0.950 (0.718–1.256)	0.717
		TT	141 (25.3)	140 (24.2)	1.026 (0.745–1.414)	0.874	1.036 (0.749–1.432)	0.830
	Additive	2TT+GTvsGG			1.011 (0.861–1.187)	0.893	1.016 (0.864–1.194)	0.850
	Dominant	GT+TTvsGG			0.975 (0.753–1.263)	0.849	0.978 (0.754–1.270)	0.870
	Recessive	TTvs GG+GT			0.979 (0.896–1.069)	0.635	0.978 (0.894–1.069)	0.625
rs16952730 (A> G)		A	799 (71.6)	781 (67.4)	1	Reference	1	Reference
		G	317 (28.4)	377 (32.6)	0.822 (0.687–0.983)	0.032	0.828 (0.691–0.993)	0.041
		AA	287 (51.4)	271 (46.8)	1	Reference	1	Reference
		GA	225 (40.3)	239 (41.3)	0.889 (0.695–1.137)	0.349	0.899 (0.701–1.153)	0.402
		GG	46 (8.2)	69 (11.9)	0.630 (0.418–0.947)	0.026	0.636 (0.421–0.961)	0.032
	Additive	2GG+AGvsAA			0.827 (0.694–0.987)	0.035	0.834 (0.698–0.996)	0.045
	Dominant	AG+GGvsAA			0.831 (0.658–1.049)	0.119	0.972 (0.895–1.056)	0.498
	Recessive	GGvs AA+AG			0.968 (0.892–1.051)	0.438	0.840 (0.664–1.063)	0.147

Abbreviation: TB tuberculosis; SNPs single nucleotide polymorphisms; OR odds ratio; CI confidence interval; a, adjusted for age and gender

Dual luciferase reporter gene assay results

Figure 1 illustrates the dual-luciferase expression activity.The potential biological function of the FTO rs16952730 polymorphismwas assessed using two online software tools, RegulomeDB (http://www.regulomedb.org/) and HaploReg v4 (http://compbio.mit.edu/HaploReg). These analyses suggested that this gene polymorphism could potentially influence biological functions. Consequently, a dual-luciferase reporter gene function verification assay was performed to further investigate this hypothesis. We constructed a recombinant plasmid carrying the rs16952730 genotype AA based on the pGL3-promoter. Then, we used site-directed mutagenesis PCR to generate a recombinant plasmid with the rs16952730 genotype GG. The dual luciferase assay results showed that the rs16952730-PGL3-promoter-AA and rs16952730-PGL3-promoter-GG recombinant plasmids had significantly higher luciferase expression activity than the PGL3-basic control plasmid (P = 0.034 and P = 0.001, respectively). The luciferase expression activities of the genotype recombinant plasmids were significantly lower than those of the PGL3-promoter empty vector without insert (P < 0.0001 and P = 0.0005, respectively). There was a significant difference in the relative luciferase expression activities between the rs16952730-PGL3-promoter-AA and rs16952730-PGL3-promoter-GG recombinant plasmids (P = 0.00295), as detailed in Fig. 1.

Fig. 1.

FTO rs16952730 dual luciferase expression activity. Abbreviation: Promoter: PGL3-promoter; A: rs16952730 genotype AA and PGL3-promoter recombinant plasmid; G:rs16952730 genotype GG and PGL3-promoter recombinant plasmid; Basic: PGL3-basic

Discussion

The regulatory role of host genetic factors in tuberculosis susceptibility has been well established by multiple studies [10–14]. However, investigations into the genetic susceptibility mechanisms underlying the progression to severe tuberculosis remain limited. Elucidating the genetic factors influencing tuberculosis severity is of great significance for understanding disease progression and developing targeted prevention and control strategies. The FTO gene, classically known as an obesity-associated gene, plays a central role in energy metabolism and adiposity regulation [5]. Recent studies have implicated FTO polymorphisms in susceptibility to Mycobacterium tuberculosis infection [15–17], with associations between FTO variants and tuberculosis risk reported in both Iranian and Chinese populations [6, 7]. Given that obesity can impair host defense against Mycobacterium tuberculosis by suppressing both innate and adaptive immune functions, it is plausible that FTO-mediated metabolic regulation may influence not only tuberculosis susceptibility but also disease severity [18–20]. Therefore, this study focused on the Chinese Han population to investigate the association between FTO gene polymorphisms and the progression to severe tuberculosis, and to validate the biological effects through functional experiments, thereby providing new insights into the genetic mechanisms underlying tuberculosis severity.

This case-control study revealed that the minor allele G of the FTO rs16952730 polymorphism was significantly associated with a reduced risk of severe tuberculosis in the Chinese Han population. This protective effect remained statistically significant after adjusting for age and sex (OR^a 0.828, 95% CI 0.691–0.993, P^a=0.041). Compared with the AA genotype, individuals carrying the GG genotype had a significantly lower risk of developing severe tuberculosis (OR^a 0.636, 95% CI 0.421–0.961, P^a=0.032), and the additive genetic model further validated this protective association (OR^a 0.834, 95% CI 0.698–0.996, P^a=0.045). In contrast, no association was found between another tag SNP of the FTO gene, rs16952577, and severe tuberculosis, suggesting a locus-specific effect of FTO in regulating tuberculosis severity. Building upon previous studies linking FTO polymorphisms to tuberculosis susceptibility, our findings provide the first evidence that the rs16952730 variant is associated with the clinical severity of tuberculosis, offering a novel candidate target for genetic susceptibility screening in severe tuberculosis.

To validate the biological function of the rs16952730 polymorphism, dual-luciferase reporter assays were performed. The results revealed a significant difference in relative luciferase expression activity between recombinant plasmids carrying the AA and GG genotypes (P = 0.00295), confirming that this locus is a functional polymorphism and that genetic variation at this site can directly affect FTO gene expression. Based on the known role of FTO in metabolic regulation, it is hypothesized that this polymorphism may influence FTO expression, thereby altering energy and lipid metabolism, which in turn affects the activation, proliferation, and cytokine secretion of host immune cells. Ultimately, this may modulate the host’s ability to clear Mycobacterium tuberculosis and impact the clinical progression and severity of the disease. These functional validation results provide direct molecular evidence supporting the genetic association findings, establishing a functional link between FTO polymorphism and tuberculosis severity, and laying an experimental foundation for further investigation of the underlying regulatory pathways.

This study has several limitations. First, due to the lack of systematically recorded height and weight data in the historical dataset, body mass index (BMI) could not be calculated or included in the regression analysis as a confounding factor. As a classic phenotypic marker of the FTO gene, BMI is a key indicator linking the gene, metabolism, and immune function [21–23]. The absence of BMI data precludes determination of whether the FTO polymorphism directly regulates tuberculosis severity or indirectly influences disease outcomes through BMI-mediated effects, nor can we exclude potential confounding by BMI. Second, the study population was restricted to Han Chinese tuberculosis patients aged 15–45 years from Southwest China, limiting the generalizability of the findings. Additionally, only age and sex were adjusted for in the analysis; other potential confounding factors that may influence tuberculosis severity—such as environmental exposures, Mycobacterium tuberculosis strain virulence, and timing of clinical treatment—were not included.

In conclusion, this study demonstrates that the G allele of the FTO rs16952730 polymorphism may be a protective genetic factor against severe tuberculosis in the Chinese Han population. This locus is functional and may participate in regulating tuberculosis severity through modulation of gene expression. These findings provide new insights into the genetic mechanisms underlying severe tuberculosis and offer a potential molecular target for risk stratification and precision intervention. Future studies should systematically collect BMI data to clarify its role in the gene-disease association, expand sample sizes to include diverse geographic regions, ethnicities, and age groups, and adjust for additional confounding factors. Further investigation into the specific molecular pathways through which FTO regulates tuberculosis severity is warranted to validate these findings and facilitate their clinical translation.

Conclusions

This study suggests that the FTO rs16952730 G allele may be associated with reduced tuberculosis severity in the Chinese Han population.

Abbreviations

TB

Tuberculosis

SNPs

Single nucleotide polymorphisms

OR

Odds ratio

CI

Confidence interval

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

CRP

C-reactive protein

ESR

Erythrocyte sedimentation rate

PCT

Procalcitonin

ICU

Intensive care unit

Author contributions

JQH made a substantial contribution to the study design. MJW contributed to the data analysis and manuscript writing. FJ contributed to the data analysis. JZ was involved in data collection, data analysis, and also participated in the manuscript writing. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81870015).

Data availability

The datasets generated and/or analysed during the current study are available in the Figshare database repository (https://figshare.com/s/91098dabecc7a1d9d786).

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of West China Hospital of Sichuan University [Approval No.: 932 (2019)]. All subjects comprehended the objective and methodology of the study, and subsequently signed informed consent forms.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.