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Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration.

Curtis J, Luo Y, Zenner HL, Cuchet-Lourenço D, Wu C, Lo K, Maes M, Alisaac A, Stebbings E, Liu JZ, Kopanitsa L, Ignatyeva O, Balabanova Y, Nikolayevskyy V, Baessmann I, Thye T, Meyer CG, Nürnberg P, Horstmann RD, Drobniewski F, Plagnol V, Barrett JC, Nejentsev S - Nat. Genet. (2015)

Bottom Line: In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514).The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes.Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.

ABSTRACT
Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

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ASAP1 expression in peripheral blood leukocytes and monocyte-derived macrophages and dendritic cells (DCs)(a) Levels of the ASAP1 mRNA expression in primary leukocytes of healthy volunteers each measured in triplicate by quantitative reverse transcriptase PCR and shown relative to the level of expression in monocytes. CD14+ monocytes were isolated from peripheral blood of healthy volunteers and cultured either with M-CSF for 5 days to derive macrophages or with GM-CSF and IL-4 for 6 days to derive DCs. DCs were infected with live M. bovis BCG at multiplicity of infection 3:1 for 24 hours. Horizontal bars show mean levels. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.(b) Box plot showing the level of reduction of ASAP1 mRNA expression in DCs after M. tuberculosis infection for 65 subjects with different rs10956514 genotypes, 23 (AA), 30 (AG) and 12 (GG). The bold line in the boxplot is the median; the whiskers are 1.5 IQR away from the first and third quartiles. Data are from ref 23 after adjustment for 5 and 8 principle components in non-infected and infected DCs, respectively (see Methods).
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Figure 2: ASAP1 expression in peripheral blood leukocytes and monocyte-derived macrophages and dendritic cells (DCs)(a) Levels of the ASAP1 mRNA expression in primary leukocytes of healthy volunteers each measured in triplicate by quantitative reverse transcriptase PCR and shown relative to the level of expression in monocytes. CD14+ monocytes were isolated from peripheral blood of healthy volunteers and cultured either with M-CSF for 5 days to derive macrophages or with GM-CSF and IL-4 for 6 days to derive DCs. DCs were infected with live M. bovis BCG at multiplicity of infection 3:1 for 24 hours. Horizontal bars show mean levels. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.(b) Box plot showing the level of reduction of ASAP1 mRNA expression in DCs after M. tuberculosis infection for 65 subjects with different rs10956514 genotypes, 23 (AA), 30 (AG) and 12 (GG). The bold line in the boxplot is the median; the whiskers are 1.5 IQR away from the first and third quartiles. Data are from ref 23 after adjustment for 5 and 8 principle components in non-infected and infected DCs, respectively (see Methods).

Mentions: The role of ASAP1 in macrophages, DCs and other immune cells has not been studied previously. To investigate it, we initially characterized the ASAP1 mRNA expression by quantitative reverse transcriptase-PCR (qRT-PCR) in primary leukocytes of healthy donors and found that monocytes, B lymphocytes, CD4+ and CD8+ T lymphocytes and neutrophils express relatively low levels of ASAP1. However, when monocytes were differentiated into macrophages or DCs, the expression of ASAP1 increased (Fig. 2a). We then infected DCs with Mycobacterium bovis BCG and found that it led to the reduction of ASAP1 expression (Fig. 2a). We also analyzed ASAP1 expression in DCs from 65 subjects that were previously studied using microarrays23. Similar to our results, in this dataset the ASAP1 level in the M. tuberculosis-infected DCs was significantly lower than in non-infected DCs (log2 fold change = −0.49, Pcorrected = 2.9 × 10−18, ref 23). To study if any of the TB-associated SNPs in the ASAP1 gene affects its expression, we first analyzed rs10956514 and rs4733781 that were genotyped in this dataset. The SNP rs10956514 showed evidence of association with ASAP1 expression in non-infected samples (P = 4.6 × 10−3) and with the relative level of reduction in ASAP1 expression after M. tuberculosis infection (P = 4.6 × 10−3). Homozygotes of allele A, which was associated with higher TB risk in our GWAS, showed stronger reduction of ASAP1 expression after infection than homozygotes of allele G, which was associated with lower TB risk (Fig. 2b). We imputed genotypes of other TB-associated ASAP1 SNPs and found that they had weaker association with the level of reduction of ASAP1 expression after M. tuberculosis infection than rs10956514 (Supplementary Table 5). The lack of association between ASAP1 expression and rs4733781 reflects different linkage disequilibrium patterns in this region (e.g. r2 between rs4733781 and rs10956514 was 0.8 in the Russian dataset, but only 0.62 in 65 non-Russian Caucasians23). These results indicate that larger studies in different populations will be needed to pinpoint the causative variant affecting ASAP1 expression, and thence the risk of pulmonary TB. Nevertheless, our results suggest that either rs10956514 or a polymorphism in strong linkage disequilibrium with it may be responsible for the control of ASAP1 expression in DCs. Since rs10956514 is also one of the candidates associated with the risk of active pulmonary TB (Table 1), genetically regulated levels of ASAP1 expression in DCs are likely to be important in TB pathogenesis.


Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration.

Curtis J, Luo Y, Zenner HL, Cuchet-Lourenço D, Wu C, Lo K, Maes M, Alisaac A, Stebbings E, Liu JZ, Kopanitsa L, Ignatyeva O, Balabanova Y, Nikolayevskyy V, Baessmann I, Thye T, Meyer CG, Nürnberg P, Horstmann RD, Drobniewski F, Plagnol V, Barrett JC, Nejentsev S - Nat. Genet. (2015)

ASAP1 expression in peripheral blood leukocytes and monocyte-derived macrophages and dendritic cells (DCs)(a) Levels of the ASAP1 mRNA expression in primary leukocytes of healthy volunteers each measured in triplicate by quantitative reverse transcriptase PCR and shown relative to the level of expression in monocytes. CD14+ monocytes were isolated from peripheral blood of healthy volunteers and cultured either with M-CSF for 5 days to derive macrophages or with GM-CSF and IL-4 for 6 days to derive DCs. DCs were infected with live M. bovis BCG at multiplicity of infection 3:1 for 24 hours. Horizontal bars show mean levels. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.(b) Box plot showing the level of reduction of ASAP1 mRNA expression in DCs after M. tuberculosis infection for 65 subjects with different rs10956514 genotypes, 23 (AA), 30 (AG) and 12 (GG). The bold line in the boxplot is the median; the whiskers are 1.5 IQR away from the first and third quartiles. Data are from ref 23 after adjustment for 5 and 8 principle components in non-infected and infected DCs, respectively (see Methods).
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Related In: Results  -  Collection

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Figure 2: ASAP1 expression in peripheral blood leukocytes and monocyte-derived macrophages and dendritic cells (DCs)(a) Levels of the ASAP1 mRNA expression in primary leukocytes of healthy volunteers each measured in triplicate by quantitative reverse transcriptase PCR and shown relative to the level of expression in monocytes. CD14+ monocytes were isolated from peripheral blood of healthy volunteers and cultured either with M-CSF for 5 days to derive macrophages or with GM-CSF and IL-4 for 6 days to derive DCs. DCs were infected with live M. bovis BCG at multiplicity of infection 3:1 for 24 hours. Horizontal bars show mean levels. P-values were calculated using two-tailed Student T test. Unadjusted P-values are shown.(b) Box plot showing the level of reduction of ASAP1 mRNA expression in DCs after M. tuberculosis infection for 65 subjects with different rs10956514 genotypes, 23 (AA), 30 (AG) and 12 (GG). The bold line in the boxplot is the median; the whiskers are 1.5 IQR away from the first and third quartiles. Data are from ref 23 after adjustment for 5 and 8 principle components in non-infected and infected DCs, respectively (see Methods).
Mentions: The role of ASAP1 in macrophages, DCs and other immune cells has not been studied previously. To investigate it, we initially characterized the ASAP1 mRNA expression by quantitative reverse transcriptase-PCR (qRT-PCR) in primary leukocytes of healthy donors and found that monocytes, B lymphocytes, CD4+ and CD8+ T lymphocytes and neutrophils express relatively low levels of ASAP1. However, when monocytes were differentiated into macrophages or DCs, the expression of ASAP1 increased (Fig. 2a). We then infected DCs with Mycobacterium bovis BCG and found that it led to the reduction of ASAP1 expression (Fig. 2a). We also analyzed ASAP1 expression in DCs from 65 subjects that were previously studied using microarrays23. Similar to our results, in this dataset the ASAP1 level in the M. tuberculosis-infected DCs was significantly lower than in non-infected DCs (log2 fold change = −0.49, Pcorrected = 2.9 × 10−18, ref 23). To study if any of the TB-associated SNPs in the ASAP1 gene affects its expression, we first analyzed rs10956514 and rs4733781 that were genotyped in this dataset. The SNP rs10956514 showed evidence of association with ASAP1 expression in non-infected samples (P = 4.6 × 10−3) and with the relative level of reduction in ASAP1 expression after M. tuberculosis infection (P = 4.6 × 10−3). Homozygotes of allele A, which was associated with higher TB risk in our GWAS, showed stronger reduction of ASAP1 expression after infection than homozygotes of allele G, which was associated with lower TB risk (Fig. 2b). We imputed genotypes of other TB-associated ASAP1 SNPs and found that they had weaker association with the level of reduction of ASAP1 expression after M. tuberculosis infection than rs10956514 (Supplementary Table 5). The lack of association between ASAP1 expression and rs4733781 reflects different linkage disequilibrium patterns in this region (e.g. r2 between rs4733781 and rs10956514 was 0.8 in the Russian dataset, but only 0.62 in 65 non-Russian Caucasians23). These results indicate that larger studies in different populations will be needed to pinpoint the causative variant affecting ASAP1 expression, and thence the risk of pulmonary TB. Nevertheless, our results suggest that either rs10956514 or a polymorphism in strong linkage disequilibrium with it may be responsible for the control of ASAP1 expression in DCs. Since rs10956514 is also one of the candidates associated with the risk of active pulmonary TB (Table 1), genetically regulated levels of ASAP1 expression in DCs are likely to be important in TB pathogenesis.

Bottom Line: In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514).The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes.Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge, UK.

ABSTRACT
Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

Show MeSH
Related in: MedlinePlus