Nonpathogenic Common Variants of IFNGR1 and
IFNGR2 in Association with Total Serum IgE Levels
Pei-Song Gao,* Xiao-Quan Mao,* Emmanuelle J ouanguy,† Annaick Pallier,† Rainer Do ¨ffinger,†
Yosuke Tanaka,‡ Hitoshi Nakashima,‡ Takeshi Otsuka,‡ Mark H. Roberts,* Tadao Enomoto,§
Yasuhiro Dake,§ Mitsuru Kawai,¶Sei Sasaki,? Sarah R. Shaldon,** Phillip Coull,**
Chaker N. Adra,†† Yoshiyuki Niho,‡ J ean-Laurent Casanova,†
Taro Shirakawa,*,1and J ulian M. Hopkin*
*Experimental Medicine Unit, University of Wales Swansea, Swansea, United Kingdom; †INSERM U429, Hospital Necker-
Enfanta Malades, Paris, France; ‡1st Department of Medicine, Faculty of Medicine, Kyushu University, Fukuoka, J apan;
§Department of Otolaryngology, J apanese Red Cross Society, Wakayama Medical Center, Wakayama, J apan;
Preventive Medical Center, Kyoto, J apan; ?Department of Pediatrics, Osaka Medical College, Takatsuki, J apan; **Osler
Chest Unit and Immunology Section, Churchill Hospital, University of Oxford, Oxford, United Kingdom; and
††Haematology/Oncology Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
Received August 5, 1999
Atopy is an immune disorder in which a T h2 domi-
nant mechanism leads to high IgE levels and the clin-
ical disorder asthma. It has been postulated that the
T h1 cytokine IF N?, acting through its heterodimeric
receptors, IF N?R 1 and IF N?R 2, in the induction/
proliferation of T h1 cells, might suppress the T h2 re-
sponses that may underlie atopic asthma. However,
neither murine nor human variants of IF N? associate
with atopy. Several dysfunctional mutations have
been identified in IF N? receptor genes (IF NGR 1 and
IF NGR 2) in relation to severe and selective infections
with poorly pathogenic organisms. However, little is
known about common polymorphisms and their func-
tional role in atopy. T o test whether such variants of
IF NGR 1 and IF NGR 2 relate to atopic asthma, we con-
ducted a genetic association study in both British (n ?
300) and J apanese (n ? 200) populations. An intronic
variant of IF NGR 1 showed marginal association with
total serum IgE levels in the British population com-
pared with those with total IgE levels < 30 IU/ml and
those with > 120-500 IU/ml [odds ratio ? 2.00 (95% CI
1.00–4.07), P ? 0.048]. A coding variant, Gln64Arg of
the IF NGR 2, also associated with total serum IgE lev-
els in the British population [?2? 5.08, P ? 0.024].
F urther genetic and functional analyses are needed to
clarify the role of variants of IF N? receptor genes in
© 1999 Academic Press
Key Words: human; T h1/T h2; allergy; IF N? receptors;
Atopy is characterised by raised IgE levels, and it
underlies the clinical disorders asthma, eczema, and
rhinitis (1). IL-4, IL-13, and IL-5 from Th2 cells play a
crucial role in induction of atopic asthma (2). Interfer-
ons are species-specific glycoproteins produced by va-
riety of immune cells (3). At least three different spe-
cies of IFNs, IFN?, IFN?, and IFN?, are produced in
humans (4). IFN? from Th1 cells inhibits Th2 re-
sponses though IFN? receptors (IFN?R1, IFN?R2) on
Th2 cells, in addition to regulation of HLA class II
molecules (5). The peripheral blood mononuclear cells
from atopic asthmatics exhibit a decreased capacity to
produce IFN? (6–8) and IFN levels show an inverse
association with total serum IgE levels (1, 9–11).
Genome-wide searches have identified several candi-
date loci for atopy and/or asthma (12–14), including
chromosome 12q where the IFNG is localized. These
functional and genetic characteristics suggest the pos-
sibility that IFN? genes may act as an atopy or asthma
locus. However, variants of neither human (15) nor
murine (16) IFNG associated with any phenotype of
IFN?R consists of heterodimeric chains—the IFN?R1
(? chain) and IFN?R2 (? chain) (17). Experimental
analysis shows that IFN? unresponsiveness is due to
lack of cellular expression of IFN?R2 chain though
down-regulation of this chain (17); Th1 cells which
1To whom correspondence and reprint requests should be ad-
dressed at Experimental Medicine Unit, University of Wales
Swansea, Singleton Park, Swansea SA2 8PP, UK. Fax: ?44-1792-
513054. E-mail: T.Shirakawa@Swansea.ac.uk.
Biochemical and Biophysical Research Communications 263, 425–429 (1999)
Article ID bbrc.1999.1368, available online at http://www.idealibrary.com on
Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
produce IFN? but lack the IFN?R2 chain are not re-
sponsive toIFN?, while Th2 cells which donot produce
IFN? but express this chain are responsive to IFN?.
IFN? therefore might regulate expression of its
IFN?R2 chain and thereby determine the ability of Th
cells to respond to IFN? (17). In contrast, uncoupled
IFN?R1 chains enter a large intracellular pool and
there does not appear to be direct correlation between
the expression levels of IFN?R1 chain and the magni-
tude of responsiveness toIFN? (17). However, IFN?R1
deficient mice are unable to augment IFN?-induced
biological reaction, suggesting that both IFN?R1 and
IFN?R2 chains are essential for IFN? signalling. The
loci are located on the different chromosomes, 6q23-24
and 21q22 (18, 19), respectively; this is of particular
interest because these loci have been linked toatopy in
genome-wide searches (12–14).
The aim of this study was to identify biallelic vari-
ants of the IFNGR1 and IFNGR2 genes and to test
whether these variants associated with atopy and
asthma in British (n ? 300) and J apanese (n ? 200)
MATERIALS AND METHODS
scribed elsewhere for the British study (20) and the J apanese study
All the asthmatic subjects had been diagnosed with asthma by a
specialist physician and had (i) recurrent breathlessness and chest
tightness requiring on-going treatment, (ii) physician documented
wheeze, and (iii) documented labile airflow obstruction with variabil-
ity in serial peak expiratory flow rates ?30%. They showed positive
skin prick test of ?5 mm against any antigens or a RAST score of
more than 2. Marked asthma was designated as chronic rather than
episodic asthma and needing multidrug therapy. The disease of
atopy was determined by IgE serology (see below). There were no
heavy smokers (?20 cigarettes per day) among these subjects.
Details of the subject selection have been de-
dust mite) and GX (grass mix) was detected by the CAP immunoas-
say system (Pharmacia, Uppsala, Sweden). Thecriteria for a positive
ASE were as described previously (20, 21). A high total IgE by CAP
system was taken to be greater than published normal values for
children and adults (20, 21). Atopy was defined as a high concentra-
tion of total serum IgE, a positive specific IgE titre against one or
more of aero-allergens, or a combination of these two features.
Specific IgE (ASE) against HDM (house
(IsoQuick, Microprobe Corporation, Garden Grove, USA). A PCR in a
mixture including 1.5 mmol/L of magnesium chloride was performed
in a Perkin Elmer Cetus thermal cycler using a preliminary dena-
turing at 95°C for 10 min and then 45 cycles (95°C for 30 s, 60°C for
30 s) with AmpliTaq Gold (Perkin Elmer Cetus). Primers were
5?GACGGAAGTGACGTAAGG and 5?CCATCTCAGCCCTGGTCA
for Val14Met variant of the IFNGR1 (22), 5?CGGGGTTGGAGC-
CAGCGAC and CCTCCCTCCCTCTCGT for thesecond intronic vari-
ant of the IFNGR1, and 5?CAGCTGCCCGCTCCTCAG and 5?GGC-
TTACTATTTAAACTGGACT for the Arg64Gln variant of the
IFNGR2. The underlined sequences were exchanged to incorporate
the polymorphic site. PCR products were digested with TspRI for
Val14Met of IFNGR1, and Cac8 I for the second intronic variant of
IFNGR1, and with HinfI for Arg64Gln of IFNGR2.
DNA samples were extracted using a commercial kit
Sequencing was conducted with the “big-dye system” (ABI, UK)
using downstream primers for IFNGR1 and IFNGR2 genes, and the
image was visualized in the commercial POP-6 gel using an auto-
mated sequencer (ABI Prism 310 genetic analyzer).
dence intervals, and significance values were estimated by comput-
erized methods (SPSS program version 8.0). If the numbers in the
column was less 10, Fisher’s exact test was done. ANOVA analysis
was also performed using this programme.
Contingency table analysis, odds ratios, 95% confi-
We identified variants at Val18 and Met18 in
IFNGR1 encoded by GTG (22) and ATG (23). We also
identified a substitution in the second intron of this
gene. In IFNGR2, we identified only the Arg64 and
Gln64 variants as suggested in the original cloning
literature (24). These were confirmed by sequencing 20
random samples (data not shown).
No Val18Met variant of IFNGR1 was identified in
the 50 British control samples; we therefore did not
perform further analysis on this variant in the British
population. The frequency of the Val18Met variant in
the J apanese population was slightly higher than that
reported previously (22). Thegenotypefrequency of the
second intronic polymorphism in IFNGR1 in the Brit-
ish controls was concordant with Hardy–Weiburg
(H-W) equilibrium. The Arg64Gln variant of IFNGR2
was also matched with H-W equilibrium in both con-
trols (Table 1). However, the allele frequencies were
significantly different between Brititsh and J apanese
controls; P(Gln) ? 0.16, P(Arg) ? 0.84 vs P(Gln) ?
0.49, P(Arg) ? 0.51[?2? 63.79, df ? 2, P ? 0.000001].
Therewas nosignificant association of IFNGR1 vari-
ants with atopy in the J apanese population . A weakly
significant [odds ? 2.00, 95% CI: 1.00–4.07, P ? 0.049]
difference in IFNGR1 genotype frequencies between
those with very low IgE levels (?30 IU/ml) and those
with mild IgE levels (120–500 IU/ml) was shown in the
British population. No association was found between
variants of IFNGR1 and asthma in both populations.
Homozygosity for Gln64 of IFNGR2 associated with
asthma andlower IgE levels, but this effect was marginal
[P ? 0.02 and P ? 0.04] in the small numbers of such
homozygotes (Table 1). No association, however, was
found between this variant and any phenotype of atopy
and asthma in the J apanese population (Table 2).
ANOVA demonstrated that there was no significant
interaction between IFNGR1 and IFNGR2 variants on
log (total IgE levels) in the two populations (data not
In this study we found an association between vari-
ants of IFNGR1 and IFNGR2 and total serum IgE
levels in a British population. The data support the
Vol. 263, No. 2, 1999BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hypothesis that Th1/Th2 immune imbalance might un-
derlie atopy (1, 25).
IFN? operates through its heterodimeric receptors,
members of the class II cytokine receptor family,
IFN?R1 and IFN?R2 chains (17). Binding between
IFN? ligand and receptor, the IFN?R1 chain is associ-
ated with J AK1, while IFN?R2 associates with J AK2
(17). These transactivations of J AK1 and J AK2 allow
docking to Stat1, and thereby Stat1 activation (17).
IFN? thus shows a pleiotropy in the regulation of MHC
class I and class II molecules on immune cells by J AK–
Stat activation through IFN?Rs (5, 25, 26). IFN? is
produced by T cells and NK cells and promotes Th0
development towards a Th1 phenotype in coordination
with IL-12, and it regulates immunoglobulin switching
in B cells away from IgE synthesis induced by IL-4 (6,
7, 11). These functional actions of IFN? question
whether dysfunction or deficiency of IFN? function
might lead to atopic asthma.
IFN?-null cases have not been reported, but a few
cases of dysfunctional IFN?R1 or IFN?R2 chain have
been identified in subtypes of infections (27). Inherited
complete (28–31) or partial (32) IFN?R1 deficiencies
caused by null or missense mutations have been re-
ported; these lead tosevere and selective susceptibility
to infection with atypical mycobacteria and/or Salmo-
nella species (27). Complete IFN?R2 deficiency has
also been identified in association with atypical myco-
bacteriosis (27, 33). Both deficiencies show a similar
clinical and histopothological phenotype, suggesting
Association between Variants of IFNGR1 and IFNGR2 Genes and Atopy, Asthma, ASE,
and Total Serum IgE Levels in the British Population
IFNGR1 genotypeIFNGR2 genotype
Second intronic polymorphismArg64Gln genotype
AA AB ? BB
Low IgE (?120 IU/ml)
High IgE (?120 IU/ml)
Low IgE (?30 IU/ml)
High IgE (120–500 IU/ml)
1.10 (0.66–1.84)P ? 0.696 0.97 (0.93–1.00)P ? 0.024
1.41 (0.85–2.36)P ? 0.160 0.97 (0.94–0.99)P ? 0.044
2.00 (1.00–4.07)P ? 0.0480.98 (0.95–1.01)P ? 0.203
0.91 (0.54–1.54)P ? 0.7300.87 (0.14–5.27)P ? 0.876
1.05 (0.61–1.82)P ? 0.8400.73 (0.12–4.48)P ? 0.737
Association between Variants of IFNGR1 and IFNGR2 Genes and Asthma, Atopy, ASE,
and Total Serum IgE Levels in the J apanese Populations
IFNGR1 genotypeIFNGR2 genotype
Val18Met genotype Arg64Gln genotype
0.64 (0.22–1.89) P ? 0.4210.89 (0.46–1.72)P ? 0.737
0.63 (0.21–1.91)P ? 0.4010.98 (0.50–1.94)P ? 0.957
110.70 (0.21–2.30)P ? 0.5591.26 (0.63–2.50)P ? 0.515
100.92 (0.30–2.82)P ? 0.8881.26 (0.64–2.49)P ? 0.503
aVal/Val ? Val/Met vs Met/Met.
bGln/Gln vs Gln/Arg ? Arg/Arg.
Vol. 263, No. 2, 1999BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that IFN?–IFN?R ligation is essential for immunity to
these ubiquitous but poorly pathogenic organisms (27).
Despite loss of IFN?-induced biological effect, none of
these cases were markedly atopic nor asthmatic (34).
Also, IgE levels are not raised in IFN?R1-null mice
(17). However, earlier onset of fatal infection may not
allow sufficient survival for the development of atopic
asthma phenotypes; it is possible that milder genetic
variants of IFNGR1 might promote atopy.
We demonstrate that an intronic variant of IFN?R1
genes showed a modest association with total serum
IgE levels in the British population, but not with other
atopy and asthma phenotypes. This is greater [odds ?
2.00 (95% CI: 1.00–4.07), P ? 0.049] than that be-
tween those with very low (?30 IU/ml) and those with
moderately high IgE levels (120–500 IU/ml), suggest-
ing the possibility that IFNGR1 variants may modu-
late IgE change in low IgE levels. Since this variant is
intronic and only one missense mutation, I87T, has
been identified to date in two Portugal cases with par-
tial IFN?R1 deficiency (28), further studies are needed
to identify other missense mutations and test if they
might modulate IgE synthesis.
IFN?R2 is a single gene (35) and spans 33 kb on
chromosome 21q (18). This region includes a cluster of
cytokine receptors loci including IFNGR2, IFNAR1,
IFNAR2, and IL10R2, and genome-wide searches have
identified a linkage to atopic asthma (12–14). We have
identified a variant, Gln64Arg, in the IFNGR2 gene
and showed a modest association with total IgE levels
in the British population. All five cases homozygous for
Gln64 showed low IgE levels and were nonasthmatic,
suggesting that this variant might protect a high IgE
levels. Since this variant is in an extracellular domain
and represents charge change, it might alter receptor
affinity for ligand, IFN?. Further studies are needed to
clarify the functional role of this variant in relation to
total serum IgE levels.
In conclusion, we have identified several variants in
IFNGR1 and IFNGR2 in relation to atopic asthma.
However, there donot appear tobe major loci for atopy
on chromosomes 6 and 21, whilst they may play some
role in regulating total IgE levels. Several dysfunc-
tional mutations in both genes have been well docu-
mented in relation to severe infections with nonpatho-
genic organisms. Thus, these common variants alter
IFN-?-induced immunity in a manner completely dif-
ferent from that induced by dysfunctional mutations.
Whilst the exact functional role of these variants re-
mains unsolved, alteration of receptor sensitivity to
ligands or of receptor stability is hypothesised.
This work is supported in part by a research grant from the
Wellcome Trust Collaborative Study and from Ombas Corporation
(Tokyo, J apan).
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