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Identification of a novel IRGM promoter single nucleotide polymorphism associated with tuberculosis
Clinica Chimica Acta 411 (2010) 1645–1649
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Identification of a novel IRGM promoter single nucleotide polymorphism associated with tuberculosis Nanying Che a, Song Li a,b, Tiejie Gao c, Zhiguo Zhang c, Yuefei Han c, Xuxia Zhang a, Yong Sun a, Yi Liu a, Zhaogang Sun a, Jianyuan Zhang a, Weicong Ren a, Miao Tian a, Yan Li a, Wensheng Li a, Jun Cheng a, Chuanyou Li a,⁎ a b c
Department of Bacteriology and Immunology, Beijing Tuberculosis & Thoracic Tumor Research Institute, Tongzhou District, Beijing, 101149, PR China College of Life Sciences, Jilin Agricultural University, Changchun, 130000, PR China Beijing Changping Center for Tuberculosis Control and Prevention, Changping District, Beijing, 102200, PR China
a r t i c l e
i n f o
Article history: Received 30 April 2010 Received in revised form 7 June 2010 Accepted 7 June 2010 Available online 11 June 2010 Keywords: IRGM gene Susceptibility Tuberculosis DNA extraction Clotted blood
a b s t r a c t Background: Human immunity-related GTPase M (IRGM) is found to play an important role in defense against intracellular pathogen Mycobacterium tuberculosis in vitro by regulating autophagy. To verify whether single nucleotide polymorphisms (SNPs) in the promoter region of IRGM gene are associated with tuberculosis (TB) 1.7 kb IRGM promoter region was sequenced and SNP analysis was conducted in TB patients and healthy controls. Methods: A simple and rapid procedure for extracting DNA from clotted-blood was developed in this study. A 1.7 kb IRGM promoter region was amplified and sequenced for nucleotide polymorphism search. Then, 3 SNPs were selected and analyzed in 216 TB patients and 275 healthy subjects by ligase detection reaction technique. Results: DNA extracted by our method was of high quality and suitable for PCR, sequencing, and genotyping. We identified 29 polymorphisms in the 1.7 kb IRGM promoter region, including 11 novel polymorphisms not yet reported. Large population analysis showed that frequencies of −1208A allele (P= 0.031), −1208AA genotype (P= 0.042), and −1208A/−1161C/−947C (P= 0.035) and −1208G/−1161C/−947C (P= 0.030) haplotypes in cases were significantly different from those in controls. Conclusions: In 1.7 kb IRGM promoter region, only −1208A/G polymorphism is associated with susceptibility to TB. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Autophagy is an important immune mechanism against intracellular pathogens in macrophages by depositing them into an autophagosome for subsequent autolysosomal acidification and peptidase-mediated degradation [1]. Recently, immunity-regulated GTPases (IRGs) are reported to play a critical role in defense against intracellular pathogens by regulating autophagy formation [2,3]. The initial connection between IRG and autophagy is confirmed by studies on macrophage infection with Mycobacterium tuberculosis [4]. There are 3 IRG genes, IRGC, IRGQ, and IRGM, in human genome with only IRGM proved to be functional [3,5]. Inhibition of IRGM expression by siRNA caused impaired autophagy and also an extended survival of Mycobacterium bovis BCG in macrophages [3]. Recently, genetic variants of IRGM are found to be related to risks of developing diseases. A 20 kb upstream deletion polymorphism in IRGM has been identified to be associated with altered IRGM expression and Crohns disease [6]. By sequencing 1053 bp 5′UTR,
⁎ Corresponding author. Tel.: + 86 10 69545354; fax: + 86 10 69546819. E-mail address: [email protected] (C. Li). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.06.009
546 bp coding region and 250 bp 3′ UTR of IRGM several novel genetic variants have been identified, including TB related −261T (position relative to translation start site ATG) allele [7]. However, in promoter region, no polymorphism is yet reported to be related to TB. The 20 kb deletion polymorphism, reported to be related to Crohn's disease, was not directly associated with susceptibility to TB. It was only one of eight polymorphisms used for haplotype association analysis [7]. To determine whether there are any TB susceptible genetic polymorphisms in IRGM promoter region, we sequenced 60 samples (30 TB patients, 30 healthy controls) to search genetic variants, and genotyped them in large population (216 patients, 275 healthy controls). Analysis of polymorphisms or mutations in studies of disease susceptibility requires large numbers of genomic DNA samples. As an important resource for DNA recovery, blood clots are widely available after serum tests in the clinical laboratory. Utilizing these samples avoids further involvement of participants after regular medical examination and notably improves operability. However, DNA extraction from the clots is a significant challenge. Slicing clots with a sharp instrument is dangerous and cumbersome [8,9]; homogenization requires careful cleaning of the probes to avoid crosscontamination [9,10]; and direct proteinase digestion takes hours or
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even days to complete [10–12]. Moreover, traditional DNA extraction procedures following pretreatment of clots usually contain organic solvent extraction and alcohol precipitation that are toxic and timeconsuming [8–10]. To solve these problems, we developed a rapid, simple, and efficient clot dispersing and DNA extraction procedure in this study. 2. Materials and methods 2.1. Patients and controls Study participants were all recruited in Beijing, China. A total of 216 TB patients (138 male/78 female, 46.1 ± 20.8 years) were enrolled at Beijing Tuberculosis & Thoracic Tumor Institute. 97 patients were smear-positive and culture-positive, 4 were smearnegative and culture-positive, 2 were smear-positive and culturenegative, and 113 were smear-negative and culture-negative. TB status, especially for smear- and culture-negative patients, was confirmed by radiographic abnormalities and clinical observations for no response to a course of broad-spectrum antibiotics but sensitive to antituberculosis chemotherapy. There were 165 patients with pulmonary tuberculosis, 19 with tuberculous pleurisy, 32 with both pulmonary tuberculosis and tuberculous pleurisy. Serological HIV test was also performed to confirm that only HIV-negative patients were recruited. Also, 275 healthy controls (156 male/119 female, 38.3 ± 11.5 years) were recruited from local units during regular physical examinations at Beijing Tuberculosis & Thoracic Tumor Institute and Beijing Changping Center for Tuberculosis Control and Prevention. Tuberculin skin test and chest X-ray radiography were performed to confirm selected healthy subjects are not related to TB. Blood sample
collection and genotyping were performed under protocols approved by the Ethical and Institutional Review Boards for Human investigation at the Beijing Tuberculosis & Thoracic Tumor Institute and Beijing Changping Center for Tuberculosis Control and Prevention. 2.2. DNA extraction from clotted blood Clotted-blood samples were collected after serological tests from TB patients and healthy subjects and stored at − 70 °C before extraction. The blood samples were thawed at 37 °C in a water bath for 3 min and clotted blood was placed in a 5-mL disposable syringe (BD, USA) containing two layers of sterile gauze. The blood was gently squeezed into a second 5-mL syringe containing four layers of sterile gauze, then squeezed into a clean tube. These steps, finished within 1 min for each sample, completely scattered the clotted blood to an aqueous status like whole-blood. Then DNA was extracted through common whole-blood genomic DNA extraction procedures using the Whole Blood DNA Extraction Kit (Bioteke, Beijing, China). The quality of DNA was determined by gel electrophoresis. 2.3. DNA sequencing for SNP search in IRGM promoter In this study, 1.7 Kb IRGM promoter regions were amplified by using DNA extracts from 30 TB patients, 10 PPD-positive and 10 PPD-negative controls. Region 1 (−1354 to −623) and Region 2 (−718 to +303) were amplified by forward primer 1 (5′-GTAAGTAAACATGGCTGGGCAT-3′)/reverse primer 1 (5′-CACCAGACTCAGGAGCCCAGC-3′) and forward primer 2 (5′-TCCACTGCCCAAGGGCTGAAG-3′)/reverse primer 2 (5′-GATGTTTGGATGTGTTCAGAGTT-3′), respectively. Amplified
Table 1 IRGM promoter variants identified by sequencing. Position
db SNP rs number
− 1208 − 1161a − 1133a − 1049a − 1030b − 1017a − 972a − 947c − 886a − 708a − 670b − 539a − 50a
Allele (frequency)
Genotype (frequency)
rs4958842 rs4958843 rs4958423 rs4958424 rs79665755 rs2004711 rs751627 rs4958846 rs34005003 rs35707106 rs75536889 N N
A (0.37)/G (0.63) C (0.45)/T (0.55) A (0.45)/G (0.55) C (0.55)/T (0.45) C (0.81)/T (0.19) C (0.55)/T (0.45) A (0.42)/G (0.58) C (0.65)/T (0.35) C (0.55)/T (0.45) A (0.45)/G (0.55) C (0.19)/T (0.81) A (0.55)/G (0.45) 2 ‘TGGG’ (0.55)/ 3 ‘TGGG’ (0.45)
− 11a + 11c + 24a + 47d + 57a + 79a + 83a + 88a + 120a + 146a + 160a + 169
N rs12654043 N N N N N N rs35898555 rs34156253 rs58398445 N
+ 172a + 177d + 179a + 208a
rs33993564 N rs61270113 rs11748151
A (0.55)/G (0.45) A (0.35)/G (0.65) C (0.45)/T (0.55) −(0.95)/T (0.05) A (0.45)/G (0.55) C (0.55)/T (0.45) −(0.55)/T (0.45) C (0.55)/T (0.45) C (0.45)/T (0.55) A (0.55)/G (0.45) G (0.55)/T (0.45) C (0.02)/ G (0.97)/ T (0.01) −(0.45)/G (0.55) A (0.95)/G (0.05) A (0.45)/G (0.55) C (0.55)/T (0.45)
AA (0.17)/AG (0.40)/GG (0.43) CC (0.21)/CT (0.47)/TT (0.32) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21) CC (0.67)/CT (0.28)/TT (0.05) CC (0.32)/CT (0.47)/TT (0.21) AA (0.21)/AG (0.47)/GG (0.32) CC (0.48)/CT (0.33)/TT (0.19) CC (0.32)/CT (0.47)/TT (0.21) AA (0.21)/AG (0.47)/GG (0.32) CC (0.05)/CT (0.28)/TT (0.67) AA (0.32)/AG (0.47)/GG (0.21) 2 ‘TGGG’&2 ‘TGGG’ (0.32)/ 2 ‘TGGG’&3 ‘TGGG’ (0.47)/ 3 ‘TGGG’&3 ‘TGGG’ (0.21) AA (0.32)/AG (0.47)/GG (0.21) AA (0.19)/AG (0.33)/GG (0.48) CC (0.21)/CT (0.47)/TT (0.32) −(0.90)/−T (0.10)/TT (0) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21) −(0.32)/−T (0.47)/TT (0.21) CC (0.32)/CT (0.47)/TT (0.21) CC (0.21)/CT (0.47)/TT (0.32) AA (0.32)/AG (0.47)/GG (0.21) GG (0.32)/GT (0.47)/TT (0.21) CC (0)/CG (0.03)/ CT (0)/GG (0.95) / GT (0.02)/TT (0) −(0.21)/−G (0.47)/GG (0.32) AA (0.90)/AG (0.10)/GG (0) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21)
Position means distance relative to transcription start site. Variants with the same symbol (a, b, c or d) on the top of the position numbers are of perfect linkage disequilibria (LD). ‘N’ means new polymorphism identified in this study.
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3. Results 3.1. DNA extracted from blood clots We determined DNA quality by PCR and sequencing .In gel electrophoresis, extracted genomic DNA samples showed sharp high molecular weight bands without any degradation, indicating high quality DNA (Fig. 1A). Amplification of IRGM promoter regions by PCR was efficient and the products were suitable for sequencing (Fig. 1B, C). 3.2. Novel polymorphisms indentified in IRGM promoter by sequencing In this study, 29 polymorphisms were detected in 1.7 kb IRGM promoter region by sequencing of 60 DNA samples, including 11 novel variants − 539A/G, − 50 2 copies of GTTT/3 copies of GTTT, − 11A/ G, + 24C/T, + 47 T/deletion, + 57A/G, + 79C/T, + 83T/deletion, + 88C/T, + 169C/G/T, and + 177A/G (Table 1). Interestingly, distribution frequencies of allele and genotype showed that −1161C/ T was of perfect LD with −1133A/G, −1049C/T, −1017C/T, −972A/G, −886C/T, −708A/G, −539A/G, −50 2 copies of GTTT/3 copies of GTTT, −11A/G, +24C/T, +57A/G, +79C/T, +83T/deletion, +88C/T, + 120C/T, + 146A/G, + 160G/T, + 172G/deletion, + 179A/G, and +208C/T; −1030C/T with −670C/T; −947C/T with +11A/G; +47T/ deletion with +177A/G (Table 1). We also observed that the minor allele frequency of −1030C/T, −670C/T, +47T/deletion, +177A/G, and +169C/G/T was lower than 20%. So we selected three representative SNPs −1208A/G, −1161C/T, and −947C/T for large population genotyping. 3.3. Allele, genotype and haplotype analysis associated with TB
Fig. 1. Genomic DNA extraction from clotted blood and IRGM polymorphism search by PCR amplification and sequencing. (A) Gel electrophoresis of genomic DNA (lanes 1–4) extracted from clotted blood. Lane M, DNA marker. (B) Gel electrophoresis of PCR products amplifying − 1354 to − 623 region (lanes 1–4) and − 718 to + 303 region (lanes 5–8) of IRGM promoter with 100 ng genomic DNA. Lane M, DNA marker. (C) Detection of the − 1208A/G SNP site in the IRGM promoter by DNA sequencing.
products were sequenced (Generay, Shanghai, China) and all the detectable SNP variants were searched out. 2.4. LDR based sequencing for SNP genotyping Based on the SNP analysis by sequencing, three SNPs (rs4958842, rs4958843, and rs4958846) were selected for genotyping. Further variants that were identified by sequencing were not subjected to genotyping, as they were either in perfect LD with other SNP sites, or too low (minor allele frequency lower than 20%) in their frequencies to allow for reasonable statistical calculations (Table 1). SNP genotyping was done by LDR based sequencing (Generay, Shanghai, China) [13]. 2.5. Statistical analysis The differences in allelic and genotypic frequencies between TB patients and controls were evaluated by χ2 test with odds ratio (OR) (SPSS statistics 17.0, SPSS inc., Chicago, USA). The Hardy–Weinberg equilibria (HWE), Linkage disequilibria (LD), and haplotype association were analyzed by SHEsis software (http://analysis.bio-x.cn) [14,15].
The genotypic distributions in TB patients and controls were checked for HWE test. No deviation was observed for all the polymorphisms (P N 0.05). Distribution frequencies of allele and genotype in 216 TB patients and 275 controls showed that − 1208A allele (P value 0.031, OR 0.752, 95% CI 0.580–0.975) and − 1208AA genotype (P value 0.042, OR 0.579, 95% CI 0.341–0.984) was negatively associated with TB. In the other two variants, no allelic or genotypic association with disease or resistance was observed (Table 2). As haplotype analysis is suggested to be more powerful than individual SNP analysis [16]. LD and haplotype were reconstructed with SHEsis software. Analysis of three SNPs showed significant LD in both groups of TB patients and controls (Fig 2). Haplotype analysis predicted six distinct haplotypes ACC, GCC, GTC, GTT, ACT, and GCT (Table 2). Further χ2 test showed that ACC haplotype was associated with resistance to TB (P value 0.035, OR 0.756, 95% CI 0.583–0.980), while GCC haplotype was associated with risk to TB (P value 0.030, OR 1.808, 95% CI 1.052–3.108). GTC and GTT haplotypes showed no significant association (P N 0.05). As frequencies of ACT and GCT haplotypes in both TB and control groups were lower than 2%, they were not evaluated. 4. Discussion Autophagy plays critical roles in controlling replication and survival of intracellular pathogens. As the only human IRG gene functionally identified, IRGM has been proved to be a key factor in initiation and development of autophagosome [17–19]. Till now, only one SNP located in 5′ UTR was reported to be associated with TB susceptibility [7]. In this study, we sequenced 1.7 kb IRGM promoter region and identified 29 polymorphisms including 11 novel sites. Genotyping in large population indicated that −1208A allele and −1208AA genotype of IRGM were associated with decreased susceptibility to TB. Further analysis also suggested that −1208A/−1161C/−947C haplotype was negatively associated with TB, while −1208G/−1161 T/−947T haplotype was
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Table 2 IRGM promoter allele, genotype, and haplotype association with TB. Allele rs4958842 rs4958843 rs4958846
A G C T C T Genotype
rs4958842
rs4958843
rs4958846
a b
AA AG GG CC CT TT CC CT TT
TB (%) 153 279 186 246 275 157
Haplotypea
TB (%)
ACC GCC GTC GTT A C Tb G C Tb
153.00 32.99 89.01 156.99 0.00 0.01
P
OR (95% CI)
4.646
0.031
1.191
0.275
0.678
0.410
0.752 (0.58–0.975) 1 0.868 (0.674–1.119) 1 0.895 (0.687–1.165) 1
χ2
P
OR (95% CI)
4.123 1.514
0.042 0.219
0.912 0.002
0.340 0.961
0.135 0.109
0.713 0.742
0.579 (0.341–0.984) 0.782 (0.529–0.157) 1 0.777 (0.462–1.305) 0.990 (0.658–1.489) 1 0.902 (0.520–1.564) 1.079 (0.633–1.902) 1
Control (%)
χ2
P
OR (95% CI)
230.79 24.00 109.21 184.79 1.21 0.00
4.471 4.706 0.074 0.758 – –
0.035 0.030 0.785 0.384 – –
0.756 (0.583–0.980) 1.808 (1.052–3.108) 1.045 (0.763–1.429) 1.124 (0.863–1.465) – –
(35) (65) (43) (57) (64) (36)
TB (%) 30 93 93 38 110 68 89 97 30
χ2
Control (%) 232 318 256 294 364 186
(42) (58) (46) (54) (66) (34)
Control (%) (14) (43) (43) (18) (51) (31) (41) (45) (14)
(35.4) (7.6) (20.6) (36.3) (0.0) (0.0)
54 124 97 59 134 82 125 112 38
(20) (45) (35) (21) (49) (30) (45) (41) (14)
(42.0) (4.4) (19.9) (33.6) (0.2) (0.0)
Haplotype was constructed with three polymorphisms rs4958842, rs4958843 and rs4958846. Frequencies lower than 2% both in TB and control groups were not evaluated by χ2 test.
positively associated with TB. These results provided further evidences that IRGM gene was related to susceptibility to TB in population. Although IRG genes are essential for autophagy both in mice and human, they function in quite different ways. There are 23 IRG family genes in the mouse [5], but only 3 paralogs in humans [3]. In mice, expression of 19 IRG genes is regulated by interferon by interaction with interferon-stimulated response element (ISRE) or γ-activated sequence (GAS) element [5,20,21]. These IRGs constitute a powerful resistance system against intracellular pathogens [22,23]. In human cells, IRGM is required for full autophagy activation stimulated with interferon-γ (IFN-γ), starvation or rapamycin [3]. However, human IRGM is constitutively expressed in human cells and is not regulated by IFN-γ [5]. These suggest that IRGM in human and IRGs in mice may promote authophagy in different mechnisms. The IRGM gene is predicted to be moved to the present position by translocation or retroposition from an ancestral position elsewhere in the genome [6]. Haplotype analysis of IRGM promoter polymorphism indicated that there were 4 groups of variants in perfect LD. One group consists of 21 variants, distributing in a 1.3 kb region (− 1161 to +179 bp relative to transcription start site) (Table 1). Together with other SNPs in IRGM reported to be in complete linkage [6], all these evidences supported the opinion that large region recombination occurred in IRGM. As IRG family members alters greatly in human, mice, dogs or fish genome
[5], evolutionary change might have been occurred in a quite ancient time. How these evolutions influence the expression and function of IRG genes remains to be answered. In this study, we also reported a simple and efficient method for DNA extraction from clotted blood. Compared to traditional methods, our procedure has several clear advantages. First, the protocol is easy to operate, saving a lot of time. The whole extraction procedure was finished within 1 h, while other methods take several hours or up to one day [8–12]. Second, purification of DNA by nucleic acid binding column avoids using toxic organic reagents such as phenol or chloroform. Third, disposable syringes and gauze are inexpensive, easy to obtain, and efficiently avoid cross-contamination. So this method is especially practical for clinical laboratories to utilize clotted blood, which is abundant but usually discarded after serum tests, as an important source for DNA.
Acknowledgments This work was supported by the grants from the National Key Science and Technology Specific Projects on Infectious Diseases (Grant Numbers: 2008ZX10003-007 and 2008ZX10003-005) and grants from Beijing Health System Training Program for HighQualified Talents (Grant Number: 2009-3-50).
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Fig. 2. Pairwise linkage disquilibria between rs4958842, rs4958843, and rs495884 in groups of TB patients and controls.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Identification of a novel IRGM promoter single nucleotide polymorphism associated with tuberculosis Nanying Che a, Song Li a,b, Tiejie Gao c, Zhiguo Zhang c, Yuefei Han c, Xuxia Zhang a, Yong Sun a, Yi Liu a, Zhaogang Sun a, Jianyuan Zhang a, Weicong Ren a, Miao Tian a, Yan Li a, Wensheng Li a, Jun Cheng a, Chuanyou Li a,⁎ a b c
Department of Bacteriology and Immunology, Beijing Tuberculosis & Thoracic Tumor Research Institute, Tongzhou District, Beijing, 101149, PR China College of Life Sciences, Jilin Agricultural University, Changchun, 130000, PR China Beijing Changping Center for Tuberculosis Control and Prevention, Changping District, Beijing, 102200, PR China
a r t i c l e
i n f o
Article history: Received 30 April 2010 Received in revised form 7 June 2010 Accepted 7 June 2010 Available online 11 June 2010 Keywords: IRGM gene Susceptibility Tuberculosis DNA extraction Clotted blood
a b s t r a c t Background: Human immunity-related GTPase M (IRGM) is found to play an important role in defense against intracellular pathogen Mycobacterium tuberculosis in vitro by regulating autophagy. To verify whether single nucleotide polymorphisms (SNPs) in the promoter region of IRGM gene are associated with tuberculosis (TB) 1.7 kb IRGM promoter region was sequenced and SNP analysis was conducted in TB patients and healthy controls. Methods: A simple and rapid procedure for extracting DNA from clotted-blood was developed in this study. A 1.7 kb IRGM promoter region was amplified and sequenced for nucleotide polymorphism search. Then, 3 SNPs were selected and analyzed in 216 TB patients and 275 healthy subjects by ligase detection reaction technique. Results: DNA extracted by our method was of high quality and suitable for PCR, sequencing, and genotyping. We identified 29 polymorphisms in the 1.7 kb IRGM promoter region, including 11 novel polymorphisms not yet reported. Large population analysis showed that frequencies of −1208A allele (P= 0.031), −1208AA genotype (P= 0.042), and −1208A/−1161C/−947C (P= 0.035) and −1208G/−1161C/−947C (P= 0.030) haplotypes in cases were significantly different from those in controls. Conclusions: In 1.7 kb IRGM promoter region, only −1208A/G polymorphism is associated with susceptibility to TB. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Autophagy is an important immune mechanism against intracellular pathogens in macrophages by depositing them into an autophagosome for subsequent autolysosomal acidification and peptidase-mediated degradation [1]. Recently, immunity-regulated GTPases (IRGs) are reported to play a critical role in defense against intracellular pathogens by regulating autophagy formation [2,3]. The initial connection between IRG and autophagy is confirmed by studies on macrophage infection with Mycobacterium tuberculosis [4]. There are 3 IRG genes, IRGC, IRGQ, and IRGM, in human genome with only IRGM proved to be functional [3,5]. Inhibition of IRGM expression by siRNA caused impaired autophagy and also an extended survival of Mycobacterium bovis BCG in macrophages [3]. Recently, genetic variants of IRGM are found to be related to risks of developing diseases. A 20 kb upstream deletion polymorphism in IRGM has been identified to be associated with altered IRGM expression and Crohns disease [6]. By sequencing 1053 bp 5′UTR,
⁎ Corresponding author. Tel.: + 86 10 69545354; fax: + 86 10 69546819. E-mail address: [email protected] (C. Li). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.06.009
546 bp coding region and 250 bp 3′ UTR of IRGM several novel genetic variants have been identified, including TB related −261T (position relative to translation start site ATG) allele [7]. However, in promoter region, no polymorphism is yet reported to be related to TB. The 20 kb deletion polymorphism, reported to be related to Crohn's disease, was not directly associated with susceptibility to TB. It was only one of eight polymorphisms used for haplotype association analysis [7]. To determine whether there are any TB susceptible genetic polymorphisms in IRGM promoter region, we sequenced 60 samples (30 TB patients, 30 healthy controls) to search genetic variants, and genotyped them in large population (216 patients, 275 healthy controls). Analysis of polymorphisms or mutations in studies of disease susceptibility requires large numbers of genomic DNA samples. As an important resource for DNA recovery, blood clots are widely available after serum tests in the clinical laboratory. Utilizing these samples avoids further involvement of participants after regular medical examination and notably improves operability. However, DNA extraction from the clots is a significant challenge. Slicing clots with a sharp instrument is dangerous and cumbersome [8,9]; homogenization requires careful cleaning of the probes to avoid crosscontamination [9,10]; and direct proteinase digestion takes hours or
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even days to complete [10–12]. Moreover, traditional DNA extraction procedures following pretreatment of clots usually contain organic solvent extraction and alcohol precipitation that are toxic and timeconsuming [8–10]. To solve these problems, we developed a rapid, simple, and efficient clot dispersing and DNA extraction procedure in this study. 2. Materials and methods 2.1. Patients and controls Study participants were all recruited in Beijing, China. A total of 216 TB patients (138 male/78 female, 46.1 ± 20.8 years) were enrolled at Beijing Tuberculosis & Thoracic Tumor Institute. 97 patients were smear-positive and culture-positive, 4 were smearnegative and culture-positive, 2 were smear-positive and culturenegative, and 113 were smear-negative and culture-negative. TB status, especially for smear- and culture-negative patients, was confirmed by radiographic abnormalities and clinical observations for no response to a course of broad-spectrum antibiotics but sensitive to antituberculosis chemotherapy. There were 165 patients with pulmonary tuberculosis, 19 with tuberculous pleurisy, 32 with both pulmonary tuberculosis and tuberculous pleurisy. Serological HIV test was also performed to confirm that only HIV-negative patients were recruited. Also, 275 healthy controls (156 male/119 female, 38.3 ± 11.5 years) were recruited from local units during regular physical examinations at Beijing Tuberculosis & Thoracic Tumor Institute and Beijing Changping Center for Tuberculosis Control and Prevention. Tuberculin skin test and chest X-ray radiography were performed to confirm selected healthy subjects are not related to TB. Blood sample
collection and genotyping were performed under protocols approved by the Ethical and Institutional Review Boards for Human investigation at the Beijing Tuberculosis & Thoracic Tumor Institute and Beijing Changping Center for Tuberculosis Control and Prevention. 2.2. DNA extraction from clotted blood Clotted-blood samples were collected after serological tests from TB patients and healthy subjects and stored at − 70 °C before extraction. The blood samples were thawed at 37 °C in a water bath for 3 min and clotted blood was placed in a 5-mL disposable syringe (BD, USA) containing two layers of sterile gauze. The blood was gently squeezed into a second 5-mL syringe containing four layers of sterile gauze, then squeezed into a clean tube. These steps, finished within 1 min for each sample, completely scattered the clotted blood to an aqueous status like whole-blood. Then DNA was extracted through common whole-blood genomic DNA extraction procedures using the Whole Blood DNA Extraction Kit (Bioteke, Beijing, China). The quality of DNA was determined by gel electrophoresis. 2.3. DNA sequencing for SNP search in IRGM promoter In this study, 1.7 Kb IRGM promoter regions were amplified by using DNA extracts from 30 TB patients, 10 PPD-positive and 10 PPD-negative controls. Region 1 (−1354 to −623) and Region 2 (−718 to +303) were amplified by forward primer 1 (5′-GTAAGTAAACATGGCTGGGCAT-3′)/reverse primer 1 (5′-CACCAGACTCAGGAGCCCAGC-3′) and forward primer 2 (5′-TCCACTGCCCAAGGGCTGAAG-3′)/reverse primer 2 (5′-GATGTTTGGATGTGTTCAGAGTT-3′), respectively. Amplified
Table 1 IRGM promoter variants identified by sequencing. Position
db SNP rs number
− 1208 − 1161a − 1133a − 1049a − 1030b − 1017a − 972a − 947c − 886a − 708a − 670b − 539a − 50a
Allele (frequency)
Genotype (frequency)
rs4958842 rs4958843 rs4958423 rs4958424 rs79665755 rs2004711 rs751627 rs4958846 rs34005003 rs35707106 rs75536889 N N
A (0.37)/G (0.63) C (0.45)/T (0.55) A (0.45)/G (0.55) C (0.55)/T (0.45) C (0.81)/T (0.19) C (0.55)/T (0.45) A (0.42)/G (0.58) C (0.65)/T (0.35) C (0.55)/T (0.45) A (0.45)/G (0.55) C (0.19)/T (0.81) A (0.55)/G (0.45) 2 ‘TGGG’ (0.55)/ 3 ‘TGGG’ (0.45)
− 11a + 11c + 24a + 47d + 57a + 79a + 83a + 88a + 120a + 146a + 160a + 169
N rs12654043 N N N N N N rs35898555 rs34156253 rs58398445 N
+ 172a + 177d + 179a + 208a
rs33993564 N rs61270113 rs11748151
A (0.55)/G (0.45) A (0.35)/G (0.65) C (0.45)/T (0.55) −(0.95)/T (0.05) A (0.45)/G (0.55) C (0.55)/T (0.45) −(0.55)/T (0.45) C (0.55)/T (0.45) C (0.45)/T (0.55) A (0.55)/G (0.45) G (0.55)/T (0.45) C (0.02)/ G (0.97)/ T (0.01) −(0.45)/G (0.55) A (0.95)/G (0.05) A (0.45)/G (0.55) C (0.55)/T (0.45)
AA (0.17)/AG (0.40)/GG (0.43) CC (0.21)/CT (0.47)/TT (0.32) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21) CC (0.67)/CT (0.28)/TT (0.05) CC (0.32)/CT (0.47)/TT (0.21) AA (0.21)/AG (0.47)/GG (0.32) CC (0.48)/CT (0.33)/TT (0.19) CC (0.32)/CT (0.47)/TT (0.21) AA (0.21)/AG (0.47)/GG (0.32) CC (0.05)/CT (0.28)/TT (0.67) AA (0.32)/AG (0.47)/GG (0.21) 2 ‘TGGG’&2 ‘TGGG’ (0.32)/ 2 ‘TGGG’&3 ‘TGGG’ (0.47)/ 3 ‘TGGG’&3 ‘TGGG’ (0.21) AA (0.32)/AG (0.47)/GG (0.21) AA (0.19)/AG (0.33)/GG (0.48) CC (0.21)/CT (0.47)/TT (0.32) −(0.90)/−T (0.10)/TT (0) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21) −(0.32)/−T (0.47)/TT (0.21) CC (0.32)/CT (0.47)/TT (0.21) CC (0.21)/CT (0.47)/TT (0.32) AA (0.32)/AG (0.47)/GG (0.21) GG (0.32)/GT (0.47)/TT (0.21) CC (0)/CG (0.03)/ CT (0)/GG (0.95) / GT (0.02)/TT (0) −(0.21)/−G (0.47)/GG (0.32) AA (0.90)/AG (0.10)/GG (0) AA (0.21)/AG (0.47)/GG (0.32) CC (0.32)/CT (0.47)/TT (0.21)
Position means distance relative to transcription start site. Variants with the same symbol (a, b, c or d) on the top of the position numbers are of perfect linkage disequilibria (LD). ‘N’ means new polymorphism identified in this study.
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3. Results 3.1. DNA extracted from blood clots We determined DNA quality by PCR and sequencing .In gel electrophoresis, extracted genomic DNA samples showed sharp high molecular weight bands without any degradation, indicating high quality DNA (Fig. 1A). Amplification of IRGM promoter regions by PCR was efficient and the products were suitable for sequencing (Fig. 1B, C). 3.2. Novel polymorphisms indentified in IRGM promoter by sequencing In this study, 29 polymorphisms were detected in 1.7 kb IRGM promoter region by sequencing of 60 DNA samples, including 11 novel variants − 539A/G, − 50 2 copies of GTTT/3 copies of GTTT, − 11A/ G, + 24C/T, + 47 T/deletion, + 57A/G, + 79C/T, + 83T/deletion, + 88C/T, + 169C/G/T, and + 177A/G (Table 1). Interestingly, distribution frequencies of allele and genotype showed that −1161C/ T was of perfect LD with −1133A/G, −1049C/T, −1017C/T, −972A/G, −886C/T, −708A/G, −539A/G, −50 2 copies of GTTT/3 copies of GTTT, −11A/G, +24C/T, +57A/G, +79C/T, +83T/deletion, +88C/T, + 120C/T, + 146A/G, + 160G/T, + 172G/deletion, + 179A/G, and +208C/T; −1030C/T with −670C/T; −947C/T with +11A/G; +47T/ deletion with +177A/G (Table 1). We also observed that the minor allele frequency of −1030C/T, −670C/T, +47T/deletion, +177A/G, and +169C/G/T was lower than 20%. So we selected three representative SNPs −1208A/G, −1161C/T, and −947C/T for large population genotyping. 3.3. Allele, genotype and haplotype analysis associated with TB
Fig. 1. Genomic DNA extraction from clotted blood and IRGM polymorphism search by PCR amplification and sequencing. (A) Gel electrophoresis of genomic DNA (lanes 1–4) extracted from clotted blood. Lane M, DNA marker. (B) Gel electrophoresis of PCR products amplifying − 1354 to − 623 region (lanes 1–4) and − 718 to + 303 region (lanes 5–8) of IRGM promoter with 100 ng genomic DNA. Lane M, DNA marker. (C) Detection of the − 1208A/G SNP site in the IRGM promoter by DNA sequencing.
products were sequenced (Generay, Shanghai, China) and all the detectable SNP variants were searched out. 2.4. LDR based sequencing for SNP genotyping Based on the SNP analysis by sequencing, three SNPs (rs4958842, rs4958843, and rs4958846) were selected for genotyping. Further variants that were identified by sequencing were not subjected to genotyping, as they were either in perfect LD with other SNP sites, or too low (minor allele frequency lower than 20%) in their frequencies to allow for reasonable statistical calculations (Table 1). SNP genotyping was done by LDR based sequencing (Generay, Shanghai, China) [13]. 2.5. Statistical analysis The differences in allelic and genotypic frequencies between TB patients and controls were evaluated by χ2 test with odds ratio (OR) (SPSS statistics 17.0, SPSS inc., Chicago, USA). The Hardy–Weinberg equilibria (HWE), Linkage disequilibria (LD), and haplotype association were analyzed by SHEsis software (http://analysis.bio-x.cn) [14,15].
The genotypic distributions in TB patients and controls were checked for HWE test. No deviation was observed for all the polymorphisms (P N 0.05). Distribution frequencies of allele and genotype in 216 TB patients and 275 controls showed that − 1208A allele (P value 0.031, OR 0.752, 95% CI 0.580–0.975) and − 1208AA genotype (P value 0.042, OR 0.579, 95% CI 0.341–0.984) was negatively associated with TB. In the other two variants, no allelic or genotypic association with disease or resistance was observed (Table 2). As haplotype analysis is suggested to be more powerful than individual SNP analysis [16]. LD and haplotype were reconstructed with SHEsis software. Analysis of three SNPs showed significant LD in both groups of TB patients and controls (Fig 2). Haplotype analysis predicted six distinct haplotypes ACC, GCC, GTC, GTT, ACT, and GCT (Table 2). Further χ2 test showed that ACC haplotype was associated with resistance to TB (P value 0.035, OR 0.756, 95% CI 0.583–0.980), while GCC haplotype was associated with risk to TB (P value 0.030, OR 1.808, 95% CI 1.052–3.108). GTC and GTT haplotypes showed no significant association (P N 0.05). As frequencies of ACT and GCT haplotypes in both TB and control groups were lower than 2%, they were not evaluated. 4. Discussion Autophagy plays critical roles in controlling replication and survival of intracellular pathogens. As the only human IRG gene functionally identified, IRGM has been proved to be a key factor in initiation and development of autophagosome [17–19]. Till now, only one SNP located in 5′ UTR was reported to be associated with TB susceptibility [7]. In this study, we sequenced 1.7 kb IRGM promoter region and identified 29 polymorphisms including 11 novel sites. Genotyping in large population indicated that −1208A allele and −1208AA genotype of IRGM were associated with decreased susceptibility to TB. Further analysis also suggested that −1208A/−1161C/−947C haplotype was negatively associated with TB, while −1208G/−1161 T/−947T haplotype was
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Table 2 IRGM promoter allele, genotype, and haplotype association with TB. Allele rs4958842 rs4958843 rs4958846
A G C T C T Genotype
rs4958842
rs4958843
rs4958846
a b
AA AG GG CC CT TT CC CT TT
TB (%) 153 279 186 246 275 157
Haplotypea
TB (%)
ACC GCC GTC GTT A C Tb G C Tb
153.00 32.99 89.01 156.99 0.00 0.01
P
OR (95% CI)
4.646
0.031
1.191
0.275
0.678
0.410
0.752 (0.58–0.975) 1 0.868 (0.674–1.119) 1 0.895 (0.687–1.165) 1
χ2
P
OR (95% CI)
4.123 1.514
0.042 0.219
0.912 0.002
0.340 0.961
0.135 0.109
0.713 0.742
0.579 (0.341–0.984) 0.782 (0.529–0.157) 1 0.777 (0.462–1.305) 0.990 (0.658–1.489) 1 0.902 (0.520–1.564) 1.079 (0.633–1.902) 1
Control (%)
χ2
P
OR (95% CI)
230.79 24.00 109.21 184.79 1.21 0.00
4.471 4.706 0.074 0.758 – –
0.035 0.030 0.785 0.384 – –
0.756 (0.583–0.980) 1.808 (1.052–3.108) 1.045 (0.763–1.429) 1.124 (0.863–1.465) – –
(35) (65) (43) (57) (64) (36)
TB (%) 30 93 93 38 110 68 89 97 30
χ2
Control (%) 232 318 256 294 364 186
(42) (58) (46) (54) (66) (34)
Control (%) (14) (43) (43) (18) (51) (31) (41) (45) (14)
(35.4) (7.6) (20.6) (36.3) (0.0) (0.0)
54 124 97 59 134 82 125 112 38
(20) (45) (35) (21) (49) (30) (45) (41) (14)
(42.0) (4.4) (19.9) (33.6) (0.2) (0.0)
Haplotype was constructed with three polymorphisms rs4958842, rs4958843 and rs4958846. Frequencies lower than 2% both in TB and control groups were not evaluated by χ2 test.
positively associated with TB. These results provided further evidences that IRGM gene was related to susceptibility to TB in population. Although IRG genes are essential for autophagy both in mice and human, they function in quite different ways. There are 23 IRG family genes in the mouse [5], but only 3 paralogs in humans [3]. In mice, expression of 19 IRG genes is regulated by interferon by interaction with interferon-stimulated response element (ISRE) or γ-activated sequence (GAS) element [5,20,21]. These IRGs constitute a powerful resistance system against intracellular pathogens [22,23]. In human cells, IRGM is required for full autophagy activation stimulated with interferon-γ (IFN-γ), starvation or rapamycin [3]. However, human IRGM is constitutively expressed in human cells and is not regulated by IFN-γ [5]. These suggest that IRGM in human and IRGs in mice may promote authophagy in different mechnisms. The IRGM gene is predicted to be moved to the present position by translocation or retroposition from an ancestral position elsewhere in the genome [6]. Haplotype analysis of IRGM promoter polymorphism indicated that there were 4 groups of variants in perfect LD. One group consists of 21 variants, distributing in a 1.3 kb region (− 1161 to +179 bp relative to transcription start site) (Table 1). Together with other SNPs in IRGM reported to be in complete linkage [6], all these evidences supported the opinion that large region recombination occurred in IRGM. As IRG family members alters greatly in human, mice, dogs or fish genome
[5], evolutionary change might have been occurred in a quite ancient time. How these evolutions influence the expression and function of IRG genes remains to be answered. In this study, we also reported a simple and efficient method for DNA extraction from clotted blood. Compared to traditional methods, our procedure has several clear advantages. First, the protocol is easy to operate, saving a lot of time. The whole extraction procedure was finished within 1 h, while other methods take several hours or up to one day [8–12]. Second, purification of DNA by nucleic acid binding column avoids using toxic organic reagents such as phenol or chloroform. Third, disposable syringes and gauze are inexpensive, easy to obtain, and efficiently avoid cross-contamination. So this method is especially practical for clinical laboratories to utilize clotted blood, which is abundant but usually discarded after serum tests, as an important source for DNA.
Acknowledgments This work was supported by the grants from the National Key Science and Technology Specific Projects on Infectious Diseases (Grant Numbers: 2008ZX10003-007 and 2008ZX10003-005) and grants from Beijing Health System Training Program for HighQualified Talents (Grant Number: 2009-3-50).
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Fig. 2. Pairwise linkage disquilibria between rs4958842, rs4958843, and rs495884 in groups of TB patients and controls.
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