Conditional haploinsufficiency of NCF1 (encoding p47phox), a signaling gene with a heterozygous phenotype potentially subject to natural selection
Introduction
Haploinsufficiency refers to the phenotype associated with inactivation of a single allele in a diploid organism [1], and has been termed a condition in which “half a loaf is not enough” [2]. The products of most genes are made in excess (presumably, as J.B.S. Haldane proposed, as a result of natural selection for more efficient alleles) but a few are made at the rate limiting level required for haploinsuffiency, where this may result in dominant Mendelian disease. Interest in the condition has been growing for several reasons. As a probe of gene expression, it helps identify the group of regulatory and signaling genes whose products operate at rate-limiting concentration. Significantly, transcription factors are over represented among the gene products responsible for monofactorial dominant inherited disease [2]. Deficiencies of regulatory proteins such as antithrombin III (ATIII) belong to the same category [3], where the disease of heterozygotes has little impact on genetic fitness, as in ATIII deficiency, higher gene frequencies are expected and protein supplementation becomes an attractive therapeutic strategy.
A general point about the likely impact of the condition on disease frequency is illustrated in Fig. 1, where in two forms of immunodeficiency each mediated by a variety of autosomal and X-linked genes, it is the X-linked disease that occurs at the highest frequency. This, it has been suggested, may reflect a degree of haploinsufficiency that is too low to cause overt disease, but can still reduce the fitness of heterozygotes and thus lower the frequency of the homozygous deficiency responsible for overt disease [4]. This is in contrast to X-linked disease, where the contribution to fitness of the heterozygous mutant allele hardly affects the gene frequency. In accordance with this view, although the autosomal forms of chronic granulomatous disease are recessive, a defect in the respiratory burst of NCF1 +/− granulocytes has been recorded [5].
Here we report a small study of haploinsufficiency of the NCF1 gene of the mouse using our previously described knockout [6]. Our long-term aim was to determine the feasibility of using a mouse model to test the fitness of heterozygotes in resistance to infection or in other functions. The gene encodes p47phox, a protein that along with the others referred to in Fig. 1(b) mediates the respiratory burst in granulocytes and macrophages that is essential for bactericidal activity. However, the view that the burst is directly responsible for killing microbes has been challenged by evidence that it is in fact cellular proteases that do so, thus making release of these enzymes the principle function of the burst [7], [8]. Particularly convincing is the finding that mice deficient in granulocyte proteases compared with mice deficient in p47phox show the same heightened susceptibility to bacterial and fungal infection, and the same lack of killing activity in vitro. Recently another function of the NCF1 gene has emerged, as a regulator of inflammation [9], [10]. Haploinsufficiency may help sort out these two functions, and we hope that the use of cytometry to localize the respiratory burst in distinct cells, as described here, will also help in this task.
Section snippets
Materials and methods
The p47phox knockout mice used here were of strain 129.S2/P2 and have been fully described [6]. The human phox gene obligatory heterozygotes were identified as parents of CGD patients. As expected, they were asymptomatic and had normal blood counts.
The respiratory burst was measured by the dihydrorhodamine method [11]. In contrast to others such as the lucigenin assay that is better adapted to large scale use [12], this method allows granulocytes and macrophages to be assayed simultaneously in
Results
Although peritoneal exudate is a standard source of myeloid cells from mice, the distinguishing of unstained cells of the two types taken from a single individual has not previously been described. Our procedure is illustrated in Fig. 1. The forward/side scatter plot reveals six regions, with R1 composed mainly of granulocytes (91%Gr1+) and R2 of macrophages (96%F4/80+). Each of these populations made up 9% of the total. R3 and R4 are composed of unidentified F4/80 dull cells (staining not
Discussion
Functional haploinsufficiency of the p47phox gene in mice is here clearly evident in the respiratory burst from PMA-treated cells. The +/− versus +/+ difference is highly significant for granulocytes, but only marginally so for macrophages. Thus the effect observed here qualifies as conditional. We conclude that the way is now open for more systematic study of the effect of this haploinsufficiency on resistance to infection and arthritic disease (referred to above). The findings of this small
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