No abstract

The 1513C allele increases susceptibility to extrapulmonary TB, and this defect is associated with the reduction in the capacity of macrophages to kill M. tuberculosis.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.

Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X7 pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-primed, mycobacteria-infected macrophages from wild-type individuals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from individuals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X7-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.

Summary Mycobacterial disease remains a serious global health problem. Tuberculosis causes more than 2 million deaths a year, and leprosy is still a cause of severe disability in many parts of the world. As a result of the study of individuals with marked susceptibility to usually nonpathogenic mycobacteria, as well as case-control studies with candidate genes and genome-wide screens of affected populations, there is substantial evidence for the role of genetic factors in the susceptibility to mycobacterial disease. These studies have defined immunological processes essential for the control of mycobacteria infections in humans.