Identification of a major locus, TNF1, that controls BCG-triggered tumor necrosis factor production by leukocytes in an area hyperendemic for tuberculosis

Abstract

Background: Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy.

Methods: We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-γ) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihood-binomial method for quantitative trait loci to multivariate analysis.

Results: Stimulation of whole blood by either BCG or BCG plus IFN-γ resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r = 0.81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P < 10(-5)) on chromosome region 11p15, termed TNF locus 1 (TNF1), that controlled TNF production after stimulation by both BCG alone and BCG plus IFN-γ.

Conclusions: The TNF1 locus was mapped in the vicinity of the TST1 locus, previously identified in the same family sample, that controls tuberculin skin test (TST) negativity per se, that is, T-cell-independent resistance to Mycobacterium tuberculosis infection. This suggested that there is a connection between TST negativity per se and TNF production.

Keywords: TNF; multivariate linkage analysis; pleiotropic locus; tuberculosis.

Figure 1.

Distribution of tumor necrosis factor (TNF) values following stimulation by BCG vaccine (TNF-BCG) only and BCG plus interferon-γ (TNF-BCG + γ). A, Distribution of TNF-BCG values (light gray bars) and TNF-BCG + γ values (dark gray bars) in 392 children. The concentration of TNF measured in the supernatant of whole blood assays following stimulation for 18 hours is given in picograms per milliliter (pg/mL) bins on the x-axis. The count of subjects is plotted on the y-axis. B, For 392 children, the concentration of TNF in pg/mL in whole blood assay supernatants following stimulation with TNF-BCG + γ (y-axis) is plotted against TNF-BCG values (x-axis). Dot color is a function of the local dot density (light gray < gray < black). Abbreviations: TNF, tumor necrosis factor; TNF-BCG, tumor necrosis factor induced by BCG vaccine; TNF-BCG + γ, tumor necrosis factor induced by BCG vaccine plus interferon-γ.

Figure 2.

Univariate genome-wide model-free linkage analysis of tumor necrosis factor (TNF) induced by BCG vaccine or BCG plus interferon- γ (IFN-γ). Employing a univariate phenotype approach, multipoint linkage analyses were conducted for TNF secreted into supernatants following stimulation with BCG (A) or BCG plus IFN-γ (B). Evidence for linkage is displayed as the negative log₁₀ of the P value (black lines; left y-axis) plotted against the 22 autosomes on the x-axis. Information content (gray line; right y-axis) is plotted along the 22 chromosomes. Abbreviations: TNF-BCG, tumor necrosis factor induced by BCG vaccine; TNF-BCG + γ, tumor necrosis factor induced by BCG vaccine plus interferon-γ.

Figure 3.

Schematic view of the multivariate linkage analysis strategy. First, we transformed the tumor necrosis factor (TNF) produced in response to BCG vaccine and TNF produced in response to BCG plus interferon-γ into a set of 2 uncorrelated new variables, PC1 and PC2, by means of principal component analysis. Then, univariate quantitative model-free linkage analyses of PC1 and PC2 were conducted using the new maximum-likelihood-binomial method for quantitative traits (nMLB-QTL) approach. A multivariate test statistic T was defined as T = LRT_PC1 + LRT_PC2, where LRT_PC1 and LRT_PC2 denote the univariate nMLB-QTL likelihood ratio tests of PC1 and PC2, respectively. Finally we derived the asymptotic distribution of T as being a mixture of 3 χ² distributions with 0, 1, and 2 degrees of freedom. Abbreviations: LRT, likelihood ratio test; nMLB-QTL, new maximum-likelihood-binomial method for quantitative traits; PC, principal component; PCA, principal components analysis; TNF-BCG, tumor necrosis factor induced by BCG vaccine; TNF-BCG + γ, tumor necrosis factor induced by BCG vaccine plus interferon-γ.

Figure 4.

Bivariate genome-wide model-free linkage analysis of tumor necrosis factor (TNF) production induced by BCG vaccine or BCG plus interferon-γ. A, Multipoint evidence of linkage expressed as negative log₁₀ of the P value (black line; left y-axis) and the information content (gray line; right y-axis) are plotted along the 22 autosomes. The 2 dotted horizontal lines indicate the formal cutoff points for genome-wide suggestive or significant linkage. B, Expanded view of the region with the highest negative log₁₀ of P (y-axis) along chromosome 11 from 0 to 40 megabases (Mb; x-axis). The multipoint evidence of linkage (black line) and information content at marker positions (solid gray line) are given. Left and right y-axes indicate negative log₁₀ of the P value and the extent of information content, respectively. The 2 dotted horizontal lines indicate the formal cutoff points for genome-wide suggestive or significant linkage.