Association of VAMP8 rs1010 Polymorphism with Host Susceptibility to Pulmonary Tuberculosis in a Chinese Han Population

Abstract

Aims:

Autophagic eradication of pathogenic microbes, including Mycobacterium tuberculosis (Mtb), is an effective host immune process that protects hosts from developing diseases associated with intracellular pathogens. This study was designed to investigate the association between the single nucleotide polymorphisms (SNPs) of the autophagy-related genes VAMP8 and VTI1B, and the susceptibility to pulmonary tuberculosis (PTB) in a Chinese Han population.

Materials and Methods:

Two SNPs, rs1010 from the VAMP8 gene and rs15493 from the VTI1B gene, were examined in 202 PTB patients and 216 healthy controls using high-resolution melt-polymerase chain reaction.

Results:

The rs1010 SNP genotypes AG (p = 0.028) and GG (p = 0.016) were associated with increased susceptibility to PTB. However, the VTI1B rs15493 SNP had no impact on the susceptibility to PTB (p > 0.05).

Conclusions:

Our study demonstrated that the rs1010 SNP of VAMP8 gene was significantly associated with the susceptibility to PTB. This result suggests that rs1010 genotyping could be used as prognostic biomarker to predict the risk of Mtb infection and/or PTB disease development after Mtb infection in the Chinese Han population.

Introduction

Tuberculosis (TB) is the top infectious cause and one of the top 10 causes of deaths worldwide. As an airborne contagious pathogen, it can easily be spread via droplets from coughs or sneezes of TB patients in countries with a high population density, such as China. However, only 80-90% of subjects are susceptible to the Mycobacterium tuberculosis (Mtb) infection after exposure to Mtb. Even after an infection, the majority of infected adults never develop any overt clinical symptom, and only 5-10% of infected adults develop clinical TB later in life (Abel et al., 2014). This might be explained by host's genetic factors (Comstock, 1978; Thye et al., 2010; Boisson-Dupuis et al., 2015); therefore, it would be beneficial to study the genetic polymorphisms that might contribute to the variation in subjects' susceptibility to Mtb infection or clinical TB development.

Evidence in vitro has shown that the induction of autophagy through physiological or pharmacological methods could enhance the intracellular eradication of Mtb (Jagannath et al., 2009; Gupta et al., 2014; Lawlor et al., 2016). Autophagy is a crucial homeostasis process for both intracellular nutrient regeneration and eradication of pathogenic microorganisms, and it also plays a regulatory role by controlling excessive inflammation and promoting the antigen-presenting process (Deretic et al., 2006). The autophagy process not only involves the proteins coded by the autophagy-related genes (the ATG family), but is also regulated by the immunity-related GTPase M (IRGM) and other membrane fusion-related proteins, such as target-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) and vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs). Previous candidate-gene association studies found that at least five genetic variants of the IRGM gene were associated with a decreased susceptibility to clinical TB development in different populations (Intemann et al., 2009; Che et al., 2010; Bahari et al., 2012), indicating that genetic polymorphisms in autophagy regulatory genes may influence host susceptibility to Mtb infection or clinical TB development.

As two of the important autophagy regulators, vesicle-associated membrane protein 8 (VAMP8) and vesicle transport through interaction with t-SNAREs 1b (VTI1B) are both essential proteins that interact with t-SNAREs and are involved in the autophagy process through the direct control of membrane fusion between autophagosome and lysosome (Pushparaj et al., 2009; Nair et al., 2011; Nozawa et al., 2017). Confocal microscopy showed that VAMP8 and VTI1B function as a t-SNARE and a v-SNARE, respectively, to work in combination during the group A Streptococcus (GAS) invasion (Furuta et al., 2010). Furthermore, knockout of VAMP8 or VTI1B with siRNAs significantly lowered cellular bactericidal efficiency functionally (although showing no influence on GAS invasion efficiency) and thus impaired antimicrobial autophagy, suggesting that both VAMP8 and VTI1B mediate autophagic degradation by influencing fusion between antimicrobial autophagosomes and lysosomes (Furuta et al., 2010). However, no studies have been conducted, to our knowledge, to investigate the association between the genetic variants of these two genes and the susceptibility to Mtb infection nor to clinical TB development.

Taking all these into account, we hypothesized that the genetic polymorphisms on VAMP8 and VTI1B are related to the susceptibility to Mtb infection and TB development. We selected two single nucleotide polymorphisms (SNPs), including the rs1010 polymorphism, which lies in a 3′UTR of VAMP8, and the rs15493 on VTI1B. In this case-control study, we investigated the association between two SNPs on these genes and the susceptibility to Mtb infection or TB development using high-resolution melt-polymerase chain reaction (HRM-PCR) in a Chinese Han population.

Materials and Methods

Study population

A total of 202 case group samples were obtained from pulmonary tuberculosis (PTB) patients (143 males and 59 females) who had been referred to the Tuberculosis Research Institute in Shenyang Tenth People's Hospital from July to December 2016. Diagnoses were made according to the detection of acid-fast bacilli on a sputum smear and/or positivity for culturing Mtb organisms on 3% Ogawa medium, together with positive results of chest X-rays. Participants who were related, infected with HIV, or taking immunosuppressant drugs were excluded. Healthy controls (n = 216; 146 males, 70 females) were recruited from people admitted to the First Hospital of China Medical University for physical examinations from November 2015 to March 2016, who had no history of TB and no radiographic results of pulmonary TB. Written informed consent was obtained from patients or their family members. The study procedures and protocols were designed according to the guidelines of the Declaration of Helsinki, and approval was granted from the Institutional Ethics Committee of China Medical University.

Genotyping

Whole-blood samples were collected for whole-genome DNA extraction using a QIAamp DNA Micro Kit (Qiagen, Germany). The DNA pellet was then dissolved to 30 ng/μL and stored at −80°C (ThermoFisher Scientific). We then subjected the DNA samples to SNP genotyping by HRM analysis using the LightCycler480 High-Resolution Melting Master reagent kit on the LightCycler480 device (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's manual. Gene-specific primer pairs were designed using Primer Premier 6 and were used for both HRM genotyping and Sanger sequencing verification (Table 1). With the original setup, the samples were only classified as either homozygotes or heterozygotes. To further distinguish mutant homozygotes from wild-type homozygotes, all samples were spiked with a known wild-type homozygote sample in a volume ratio of 1:9 (known to testing). Then, the PCR mix was prepared according to Table 2, while the PCR program was set up according to Table 3.

Table 1.

Polymerase Chain Reaction Primers and Amplicons for High-Resolution Melt Genotyping and Sanger Sequencing

Gene	SNP	Direction	Sequence	Annealing temperature (°C)	Amplicon size (bp)
VAMP8	rs1010	Forward	5′-TCAATGGCTGCGAAAGAAT-3′	59.37	127
VAMP8	rs1010	Reverse	5′-TGACTACTCCCCACTTTGCTT-3′	58.85	127
VTIB	rs15493	Forward	5′-GCCAAGAGGGTCTCCTTTCC-3′	60.03	188
VTIB	rs15493	Reverse	5′-CTAGGTCTCTCCAGCCCACT-3′	60.03	188

SNP, single nucleotide polymorphism; VAMP8, vesicle-associated membrane protein 8.

Table 2.

Polymerase Chain Reaction Mix Preparation

LightCycle^® 480 High Resolution Melting Master (Roche, Germany)	5 μL
MgCl₂	1 μL
Double Distilled Water (Roche)	2.5 μL
Primer (0.2 μmol/L)
Forward	0.5 μL
Reverse	0.5 μL
Homozygote wild-type sample (spiking control)	0.05 μL
Testing sample	0.45 μL
Total volume	10 μL

Table 3.

Polymerase Chain Reaction Program

Procedure	Temperature (°C)	Time	Cycles
Preincubation	95	5 min	1
Amplification
Denaturation	95	10 s	50
Annealing	65-53^a	30 s
Extension	72	10 s
HRM
Denaturation	95	30 s	1
Pairing	40	30 s	1
Preheating	65	1 s	1
Melting	65-95^b		1
Cooling	40	10 s	1

Touchdown annealing: annealing temperature decreased 0.5°C per cycle for the first 24 cycles.

Capturing of the HRM curve: the fluorescence signal was detected at 25 times per °C.

HRM, high-resolution melt.

PCR products were heated from 65°C to 95°C in the high-resolution melting process, and LightCycler480 Gene Scanning software captured fluorescence data at a rate of 25 acquisitions per 1°C and drew an HRM curve for genotype analysis. The melting temperatures obtained from HRM analysis for VAMP8 for all rs1010 genotypes are shown in the Supplementary Figure S1. Independent Sanger sequencing assays were performed for 30 randomly selected samples on an ABI7000 sequence detection system (Applied Biosystems) to verify the results obtained by HRM-PCR for both VAMP8 and VTI1B, and the sequencing results verified all genotypes.

Statistical analysis

All analyses were performed using SPSS 19.0. To measure possible associations between SNPs and disease risk, unconditional logistic regression analyses were calculated and reported as estimated odds ratio (OR) and 95% confidence interval (CI). To examine the associations, genotypic and allelic differences between the two groups were compared using χ² tests, which were also used to access the Hardy-Weinberg equilibrium in both groups (cases and controls). Association tests for VAMP8 rs1010 were performed for several inheritance models, including AG versus GG, AA versus GG, GG versus AG+AA (the recessive model), AA versus AG+GG (the dominant model), and AG versus GG+AA (the additive model), as shown in Table 5. In all models, a p (two-tailed) value <0.05 was considered statistically significant.

Structural prediction

To study the potential effect of rs1010, MFOLD web server (Zuker, 2003) was used to predict the molecular structure of VAMP8 mRMA. The reference sequences entered into MFOLD were downloaded from the National Center for Biotechnology Information website (NCBI reference sequence: NM_003761.4). The original sequence was named “wild,” and the sequence with G polymorphism was named “variant.” Both structure graphs were generated by the MFOLD web server using default settings. Although the structural differences between the G and A alleles were also present in other predicted structures from the MFOLD program, only the top predicted structures are provided in Supplementary Figures S2 and S3.

Results

Demography of study subjects

As shown in Table 4, there is no statistical differences in gender or mean age between the two groups, according to the results of a χ² test (p = 0.479) and the Student's t-test (p = 0.663), respectively.

Table 4.

Demography of Study Subjects

	Case	Control	p
Gender
Female	59	70	0.479
Male	143	146	0.479
Age (years)	47.25 ± 19.36	46.56 ± 12.70	0.663

Association between selected SNPs and TB susceptibility

For the 30 randomly selected samples for verification, the independent Sanger sequencing assays obtained the same genotyping results as those obtained by HRM-PCR. The genotypic and allelic distribution in both groups, as well as the results of risk analyses are shown in Table 5. The frequency of VAMP8 rs1010 SNP AG genotype in healthy individuals was significantly higher than in the PTB group (OR = 0.646; 95% CI = 0.437-0.956; p = 0.028), suggesting that the AG genotype may be linked with increased protection against Mtb infection, or that this polymorphism may also be linked with decreased susceptibility to PTB development in infected patients. Conversely, the frequency of the GG genotype at this locus was significantly higher in the PTB group (OR = 1.893; 95% CI = 1.119-3.202; p = 0.016). The AA genotype (p = 0.708) and A/G alleles (p = 0.104) did not appear to be related to risk/protection for PTB.

Table 5.

Genotypic and Allelic Frequencies of Two Single Nucleotide Polymorphisms in Pulmonary Tuberculosis Cases and Controls

	Case (%)	Control (%)	OR	95% CI	χ²	p
VAMP8 rs1010
HWE	χ² = 11.816	χ² = 0.118
HWE	p = 0.001	p = 0.731
Genotype
GG	43 (21.3%)	27 (12.5%)	Reference		Reference
AG	74 (36.6%)	102 (47.2%)	0.456	0.258-0.803	7.544	0.006 ^**
AA	85 (42.1%)	87 (40.3%)	0.613	0.348-1.081	2.880	0.090
GG+AA	128 (63.4%)	114 (52.8%)	Reference		Reference
AG	74 (36.6%)	102 (47.2%)	0.646	0.437-0.956	4.801	0.028 ^*
AG+AA	159 (78.7%)	189 (87.5%)	Reference		Reference
GG	43 (21.3%)	27 (12.5%)	1.893	1.119-3.202	5.781	0.016 ^*
AG+GG	117 (57.9%)	129 (59.7%)	Reference		Reference
AA	85 (42.1%)	87 (40.3%)	1.0772	0.730-1.591	0.140	0.708
Allele
A	244 (60.4%)	276 (63.9%)	0.862	0.652-1.140	2.644	0.104
G	160 (39.6%)	156 (36.1%)	0.862	0.652-1.140	2.644	0.104
Vti1b rs15493
HWE	χ² = 30.877	χ² = 17.241
HWE	p = 2.749E-08	p = 3.293E-05
Genotype
GG	81 (40.1%)	102 (47.2%)	Reference		Reference
AG	119 (58.9%)	110 (50.9%)	1.362	0.922-2.013	2.416	0.120
AA	2 (1.0%)	4 (1.9%)	0.630	0.112-3.524	0.289	0.591
Allele
A	123 (30.4%)	118 (27.3%)	1.165	0.863-1.571		0.318
G	281 (69.6%)	314 (72.7%)	1.165	0.863-1.571		0.318

Bold values indicate that the differences were significant (p < 0.05).

p < 0.05, ^**p < 0.01.

CI, confidence interval; OR, odds ratio.

Regarding VTI1B rs15493 SNP, no statistical differences were found between the two groups at the genotypic or the allelic level.

Structural prediction

As predicted by the MFOLD program (Zuker, 2003), the region containing the VAMP8 rs1010 locus contains a hairpin loop structure. At the very bottom of the pictures, the structure reveals a change from two-small-hairpin loop structure (the rs1010 wild (A) genotype; see Supplementary Fig. S2) to one-bigger-hairpin loop structure [the rs1010 variant (G) genotype; see Supplementary Fig. S3].

Discussion

In this study, we genotyped two polymorphisms for 202 PTB patients and 216 healthy controls in a Chinese Han population. Results indicated that the VAMP8 rs1010 SNP was significantly associated with PTB. To our understanding, this is the first time that a variant on a SNARE gene showed an association with resistance/susceptibility to PTB. Our findings could provide new clues for deciphering the role of autophagy in TB. Furuta et al. (2010) demonstrated that VAMP8 and VTI1B can work together to fuse lysosomes with autophagosomes and play a major role in the autophagic eradication of GAS, suggesting that they may also mediate antimicrobial autophagy of other similar pathogens such as Mtb. The infection mechanism of Mtb is similar to GAS as they both exploit endocytosis to invade human cells, then avoid lysosomal degradation by escaping from endosomes to the cytoplasm, where they become engulfed by autophagosomes, which later fuse with lysosomes for degradation (Sia et al., 2015). Based on these conclusions, we can speculate that VAMP8 and VTI1B may also affect the autophagic eradication of Mtb in macrophages. Therefore, to elucidate the exact mechanism by which rs1010 affects susceptibility to TB, functional studies are needed to determine whether rs1010 can cause changes in mRNA or protein expression of VAMP8, and whether these changes ultimately affect the efficiency of bacilli clearance.

As mentioned above, the variant rs1010 lies in a 3′UTR portion on the VAMP8 mRNA transcript, a portion also containing suggested binding sites targeted by multiple miRNAs (miR-15, miR-96, miR-370, and miR-3945) (Lewis et al., 2005; Liu et al., 2012; Stegeman et al., 2015).

Kondkar et al. transfected the VAMP8-expressing colorectal cancer cell line HCT116Dicer-KO 2 with miR-96 and observed a significant reduction in VAMP8 mRNA expression and a dose-dependent decrease in VAMP8 protein (Kondkar et al., 2010), suggesting that miR-96 binding could affect VAMP expression. In our study, as predicted by the MFOLD program (Zuker, 2003), this region contains a two-hairpin loop structure, which becomes a single-hairpin-loop structure when the allele A at rs1010 is replaced by allele G. With the change in hairpin structure, the targeted binding of miR96 might be disabled, which might further affect the regulation of VAMP8 expression and thus the protein function. This may suggest that the rs1010 SNP possibly affects the affinity of VAMP8 with miR-96 and further influences the mRNA expression and protein function of VAMP8.

For other miRNAs, no functional study was done to investigate the influence on mRNA expression or protein function of VAMP8, but in silico analyses predicted that both miR-370G>A and miR-3945C>T polymorphisms might cause a decrease in VAMP8 mRNA expression (Liu et al., 2012; Stegeman et al., 2015). Therefore, the rs1010 polymorphism that occurs in the binding region related to miR-370 and miR-3945 might also influence VAMP8 mRNA expression. However, due to a lack of evidence, it is uncertain how, and to what degree, the autophagy pathway could be influenced by rs1010 polymorphism in the immune responses to Mtb infection.

In our study, it was observed that neither the genotype nor allele frequencies were statistically correlated with susceptibility to PTB at the rs15493 SNP of VTI1B. At present, the research into gene polymorphism in VTI1B is relatively limited and has not been reported to be associated with any human diseases. It has been proposed that VTI1B works in combination with its partner, VAMP8. During autophagy membrane fusion, VTI1B forms a parallel four-helix bundle structure with the VAMP8 molecule. These two proteins then work together and are assembled into a highly stable SNARE complex (Nair et al., 2011). However, other molecules may replace the role of VTI1B in membrane fusion. There is evidence that VAMP8 can also interact with ATG14 and STX17 to induce membrane fusion, thereby reducing the importance of VTI1B in this process (Bernard and Klionsky, 2015; Liu et al., 2015). This may partly explain the lack of association of VTI1B polymorphism with TB.

In addition, the following limitations might affect the accuracy of our study. Firstly, although the sample size in this study fulfilled the basic standards for controlling systematic errors, it was still limited and thus had insufficient statistical power. Secondly, healthy controls were not evaluated for latent Mtb infection. As a result, it was not possible to distinguish between infected and virtually uninfected individuals. A possible clinical significance of rs1010 could also be explained by meta-analysis. Moreover, we only selected two variations that we think are important from limited regions on VAMP8 and VTI1B. Thus, it is necessary to expand the SNP coverage so that the relationship between these two genes and PTB can be fully reflected. Further studies of both innate and adaptive immune pathways during Mtb infection and PTB development, including autophagic pathways and relevant genes, will be critical for developing novel host-directed immune therapies and novel effective vaccines against TB.

Conclusions

Our study indicates that the functional rs1010 SNP of VAMP8 is significantly associated with the susceptibility to TB in a Chinese Han population. This suggests that rs1010 may be a possible novel prognostic or predictive biomarker for TB to be validated in future studies and autophagy-related genes, such as VAMP8 and VTI1B, which are good candidate genes for population genetic analysis of autophagy in the context of PTB susceptibility. Case-control studies based on diversified populations are needed to confirm our results.

Footnotes

Acknowledgments

We thank our colleagues at the Laboratory Medicine and Respiratory Departments of the First Hospital of China Medical University for their help in collecting clinical information and samples from patients. Moreover, we greatly appreciate our patients' input and their willingness to cooperate. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Disclosure Statement

No competing financial interests exist.

Supplementary Material

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Supplementary Material

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