Blood, 15 April 2003, Vol. 101, No. 8, pp. 3337-3337
CORRESPONDENCE
To the editor:
Association between p47phox pseudogenes and
inflammatory bowel disease
We read with great interest the recent article by Heyworth et
al,1 which describes the ratio between the
p47phox pseudogenes (
NCF1) and the
p47phox gene (NCF1). Gene duplication has
prevented elucidation of the genomic sequence at 7q11.23, although the
NCF1/NCF1 ratio had been assumed2-4 to be
2:1. Specifically, the location and quantity of NCF1
pseudogenes is unknown. Heyworth et al1 demonstrated the
ratio to be 1:1 and 1:2 in 13% and 4% of healthy individuals, respectively, the rest being 2:1. Using a family study, they elegantly showed that variability in the ratio probably occurred following DNA
exchange by recombination or, conceivably, gene conversion between
NCF1 and NCF1, to produce a gene hybrid (type
II NCF1). Similar to NCF1, type II
NCF1 contains a GT repeat (GTGT) at the start of exon 2, and therefore its transcription product encodes a full-length protein
that is homologous to NCF1. NCF1, however, contains a dinucleotide deletion at this allele (
GT) that results in
a premature stop codon. The functional significance of type II
NCF1 remains unknown.
We adapted a Genescan method5 to try to identify
NCF1 heterozygotes using the GT/GTGT ratio in 138 patients with inflammatory bowel disease (IBD) and 37 healthy
individuals. Several findings suggested that NCF1
haploinsufficiency could be a susceptibility factor for IBD. First,
chronic bowel inflammation is a feature of chronic granulomatous
disease (CGD) caused by p47phox deficiency. Second,
as with CGD, defects in innate immunity are found in
Crohn disease (CD).6,7 Third, significant linkage has been demonstrated in IBD8 to microsatellite
markers spanning a 22-centimorgan (cM) region of chromosome 7, which encompasses the NCF1 locus. Finally,
p47phox heterozygotes have reduced neutrophil oxygen
consumption in response to phorbol myristate acetate but have
normal neutrophil oxygen consumption in response to opsonized
zymosan,9 and we noted the same phenomenon in a
minority of patients with CD (M.H., unpublished observation, March 2002).
We were surprised to find an excess of IBD patients with a GT/GTGT
ratio of approximately 1:1, which was greater in patients with CD (CD,
22.4%; control, 8.1%; Fisher exact test, P < .05, odds
ratio 3.3) than in those with ulcerative colitis (UC) (UC, 14.1%;
P = .28) (Figure
1).
This suggested that type II NCF1 might be a
susceptibility factor for CD. Therefore, we assessed the effect of the
GT/GTGT ratio on cellular migration into an acute inflammatory
cantharidin blister.10 In patients with a ratio lower than
1.2 (n = 10; 5 CD, 3 healthy subjects) there was a significant
increase in blister cell number compared to those with a ratio higher
than 1.2 (n = 62; 28 CD, 26 healthy subjects), comprising a geometric
mean 3.27 × 106 cells/mL, compared with
1.33 × 106 cells/mL (t test,
P = .04). This finding applied both to neutrophils (1.52 × 106 cells/mL compared with
0.60 × 106 cells/mL) and macrophages
(0.32 × 106 cells/mL compared with
0.16 × 106 cells/mL). The mechanism is unknown.
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| Figure 1.
Ratio of GT/GTGT sequence in patients with CD and UC
and 3 parents of CGD patients.
Three ratio populations were apparent, approximating to 2:1, 1:1, and
1:2. There was a significant excess of the 1:1 ratio in CD
(P < .05), implying an excess of the type II
NCF1 pseudogene. Each point represents the mean of
triplicate measurements.
|
|
Three parents of confirmed p47phox CGD patients, presumed
heterozygous for mutant NCF1, had ratios of 3.4, 3.2, and
1.6 (Figure 1). The ratio of 1.6 indicates either that the type
II NCF1 pseudogene was present or that spontaneous
mutation had occurred in NCF1 to cause CGD.
We agree with the conclusion of Heyworth et al that determining
the GT/GTGT ratio cannot always detect NCF1
heterozygosity. As such, we were unable to conclusively refute our
original hypothesis, although it appears unlikely that
p47phox haploinsufficiency is a susceptibility factor for
IBD. However, it is conceivable that the presence of type II
NCF1 exaggerates the inflammatory response and so
predisposes to IBD. This needs to be confirmed in a
replicative association study.
Marcus Harbord, Andrea Hankin, Stuart Bloom, and Hannah Mitchison
Correspondence: Marcus Harbord, Department of Medicine,
University College London, The Rayne Institute, 5 University St,
London, WC1E 6JJ; e-mail: doc{at}dircon.co.uk
Supported by The CGD Research Trust.
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