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Title (CCTTT)n repeat polymorphism in the NOS2 gene promoter is assosiated with atopy
Author(s) Konno, Satoshi
Issue Date 2002-03-25
DOI 10.14943/doctoral.r5913
Doc URL http://hdl.handle.net/2115/32666
Type theses (doctoral)
Note 共著者あり。共著者名:Hizawa Nobuyuki, Yamaguchi Etsuro, Jinushi Eisei, Nishimura Masaharu.
Additional Information There are other files related to this item in HUSCAP. Check the above URL.
File Information 5913.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
(CCTTT)n repeat polymorphism in the NOS2 gene promoter is associated . with atopy
Konno S - I -
(CCTTT)n repeat polymorphism in the NOS2 gene
promoter is associated with atopy
Satoshi Konno, i\ID; Nobuyuki Hizawa, i\ID; Etsuro Yamaguchi, :\ID;
Eisei Jinushi, i\ID; and i\Iasaharu Nishimura, i\ID
First Department of ;\Ierucine, Hokkaido Cni,-ersity School of i'-Iedicine, Japan
Supported in part by a Grant-in-_-lid for Scientific Research 10307013 from the ;\Iinistry of
Education, Science, and Culture of Japan.
Correspondence to: N. Hizawa at the First Department of ;\Iedicine,
Hokkaido l'ninrsity School of l\Ierucine,
I'-ita-15 Nishi-7 I-.::ita-Ku, Sapporo 060-8638, Japan
Tel.: +81-11-716-1161 (Ext. 5911), Fax: +81-11-706-7899
E-mail: [email protected]
Konno S - 2-
ABSTRACT
Background: Se,-eral studies ha,-e shown that lUtnc oxide (NO) plays a role 111 the
regulation of the T helped (Th 1) /Th2 balance, indicating the potential for NO to
contribute to the de,-elopment of atopy and se,-eral other allergic diseases including
bronchial asthma. Nitric oxide synthase :2 (NOS:2) is critically uwoh-ed in the synthesis of
NO during se,-eral inflammatorl" states, and the gene encoding NOS2 IS located at
chromosome 17ql1.:2-ql:2, where t\\-O genome scans ha,-e identified a candidate locus for
atopy and asthma.
Objective: The l-+-repeat allele of (CCTTT)n repeat polymorphism in the S052 promoter
region is a powerful enhancer of promoter acti,-ity in reporter constructs ill I'ilro. \\'e tested
whether this potentially functional allele in the "\'052 gene influences the de,-elopment of
atopy and asthma.
Methods: We studied a total of -+97 unrelated Japanese subjects (1-+1 non-atopic healthy
controls, 102 atopic healthy controls, 56 non-atopic asthmatics, and 198 atopic asthmatics).
The odds ratio was calculated for atopy and asthma in carriers of the l-+-repeat allele using
logistic regression models. _\topy ,vas defined as ha,-ing a positin specific IgE leyel to at
least one of 10 COlID110n inhaled allergens.
Results: The 14-repeat allele was im-ersely associated with atopy (OR=O.-t2, p<O.Ol). The
Konno S " - j -
assoClatlon remained significant when the model \vas controlled for asthmatic status
(OR :::0.36, p<O.Ol). This allele, howe\-er, was neither associated \vith the de,-elopment of
asthma nor with total serum IgE le\-els.
Conclusion: Our f111clings suggest that the (CCTTT)n repeat polymorphism 111 the
promoter of the ,"\'OS2 gene that affects the promoter acti,-irr IS a risk factor for the
de,-elopment of atopy, and tllls genetic effect seems independent of asthma.
Key words: _-\topy, asthma, citric oxide syntllase 2 (NOS2)
Konno S - 4-
INTRODUCTION
Nitric oxide (NO) 1S a short-liyed molecule that plays se,-eral important
pathophysiological roles ill the de,-elopment of ,-anous diseases. Nitric oxide sn1thase :2
(NOS2) is induced in a nriety of cell types by proinflammatory cytokines, including IL-l~,
TNF-cx, IFN-y, or microbial products such as LPSs 1. 2 and is critic all,' im-oh-ed ill the
synthesis of NO during infection and other inflanU11atory states. Genetic polymorphisms
at the S052 gene are associated ,vith susceptibility to malaria and parasitic diseases' 4
Se,-eral recent studies ha,-e also indicated that NO 1S not on1\" a potent CYtotoxlC and
antimicrobial agent, but also exhibits significant immunoregulatory acti,-ity, regulating the T
helper1 (Th 1) /Th2 balance particularly 5 - 111
The "\-052 gene lies within the C-C chemokine cluster region, on chromosome
17ql1.2-q12, where a linkage with atopy and asthma has been reported 11. 12 \\'ithin the
5' -flanking DNA, a (CCTTT)n repeat sequence is located ~2.5 kb upstream of the main
1" T\ TA-directed transcription initiation site ' The 14-repeat allele 1S linked to increased
luciferase acti,-in" with III lilro reporter constructs 14 and this allele confers selecti,"e
adnntages in diabetic indi,-iduals by pre,-enting micrm-ascular complications of diabetes 1-1,
15 ."\ccordingly, we postulated that polymorphisms ill the S052 gene influence the
denlopment of atopy and asthma, and examined the prenlence of this 14-repeat allele in
Konno S - 5 -
unrelated Japanese healthy controls and asthmatic subjects.
Konno S - 6-
METHODS
Study Subjects
Cnrelated Japanese subjects \vere recruited to this study (1-1-1 non-atopic health~-
controls, 102 atopic healthy controls, 56 non-atopic asthmatics, and 198 atopic asthmatics;
Table I ). Indi'i-iduals \'i-ho ,-isited our clinic for annual, routine physical examinations were
recruited as healdl'- controls if dle,- had no histon of bronchial asthma or am- other
allergic disease .. \sthmatic subjects were recruited from dle pulmonary clinic at the First
Department of r-Iedicine, Hokkaido Cni,-ersity Hospital. .-\.sthma was defined on the basis
of recurrent episodes of at least two of three symptoms (cough, \'i-heeze, or dyspnea) that
are associated with a demonstrable re,-ersible airflow limitation, and/or increased au-waY
responsi'i-eness as described pre'i-iously 1(, .• \topy was defined as ha'i-ing a positi,-e specific
IgE le\-el (IgE C\P RAST equal to or more than 0.35 CA/m1 to at least one of 10
common inhaled allergens, including DenilatopbagoideJ ImilIae [Del" jJ, grass pollens, animal
danders, and moulds). Total serum IgE le'i-els of all participants \'i-ere also measured using a
radio-immunosorbent test (IgE RIST).
TIllS study was apprm-ed by the Edllcs Conunittee of Hokkaido C ni\-ersity School
of r-Iedicine and informed \vritten consent was obtained from each subject.
Konno S - 7 -
Analysis of a (CCTTT)n repeat polymorphism of the NOS2promoter region
Laboratory staff blinded to clinical data performed genotyping. Genomic D:0L\
was amplified by PCR using a E\i\I-labeled sense prim.er (5'-_-\CC CCT GG_-\ _-\GC CT-\
C-\_-\ CTG C-\ T-3') and an anti-sense primer (5' -GCC _-\CT GC-\ CCC T-\G CCT GTC
TC-\-3') as described pre,-iously D. The PCR products were separated by electrophoresis
through a POP_4TM gel using an _-\BI 310 (PE _-\pplied Bios!-stems, Foster Ci0-, C-\) "-1th
T-\i\Il'R. -\ -500nl as the internal Slze standard. _-\Uele slzes \,-ere calculated USl11g the
Genescan _-\nalysisTl\1 computer program (PE _-\pplied Biosystems).
Statistical analysis
The Hardy-Weinberg equilibrium was examined using the H\\'E program 1.05 in
the LINKl'TIL package 1-. Pearson chi-square analysis compared the distribution of the
14-repeat allele and genotype between non-atopic and atopic subjects, and bet\,-een healthy
controls and asthmatics. \\le then used logistic regression models to calculate odds ratios
(and 95°0 confidence internls) of atopy associated \,-ith the l-1--repeat allele. To control for
potential confounding, we adjusted for age (as a continuous nriable), sex (male or female),
and smoking status (ne\-er smoked, past smoking, or current smoking) in the mlllti\-ariate
models (Stat Yiew 5.0, 1998 SAS Institute Inc.). Genetic influence of the 1-1--repeat allele
Konno S - 8 -
on the de,-elopment of atopy was also examined controlling for asthmatic stanIS (asthma or
healthy controls) in addition to age, sex and smoking stanIs.
\Ve used an ANO\T_\ model and compared total serum IgE le,-els according to the
14-repeat genotypes (the 14-repeat allele earners ,-so non-carriers). In this analysis, \\"e
adjusted for asthmatic stanIS and atopic status (atopic or non-atopic) in addition to age, sex
and smoking stanIs.
Konno S - 9-
RESULTS
Characteristics of the 197 non-atopic (141 non-asthmatic, 56 astlU11atic) and 300
atopic (102 non-asthmatic, 198 asthmatic) subjects are presented in Table I . The mean age
\vas highest for non-atopic asthmatics, and females also predominated in this group. o-\topic
subjects had higher total serum 19E le\-els than non-atoplC subjects (unpaired I-test,
p<O.Ol) botl1 in healthy controls and asthmatic subjects. In addition, asthmatic subjects had
higher total serum IgE le,oels than health,- controls (unpaired I-test, p<O.Ol) both In
non-atopic and atopic subjects .
. -\ pentanudeotide repeat 111 the promoter reglOn of the human SOS2 gene
renaled a total of 14 alleles in 994 chromosomes (allele sizes ,-aried from 176bp [8 repeats]
to 241bp [21 repeats]). The (CCTTT)n genotype frequencies did not de\-iate from the
expected nlue b,- Hardy-\\leinberg equilibrium. The m-erall distribution of the allele
frequencies according to atopic status IS shown 111 tl1e Figure. The genotype and allele
distributions for the 14-repeat allele of the 197 non-atopic subjects differed significantl:--
from those of the 300 atopic subjects (genotype; chi-square=6.13, p=O.013, allele;
chi-square=7.48, p=0.0063, Table II .-\). An odds ratio calculated for atopy in carriers of the
14-repeat allele compared with non-carriers was 0.42 (95°oC1 [0.23-0.79]; p=O'(1065, Table
ill.-\). The odds ratio remained significant when the model was also adjusted for asthmatic
Konno S - 10-
status (OR=0.36, 95°oCI [0.18-0.71], p=0.0032; TablelIIB). This repeat polymorphism.,
hO\vever, was not associated with asthma (Table IT B). The mean adjusted total serum IgE
le\-el in the 14-repeat carriers (2.20 [SD 0.55]) was not different from those in non-carriers
(2.13 [0.54]).
Konno S - 11 -
DISCUSSION
In the present study, we identified a significantly increased pre\-alence of the
I-J.-repeat allele of the (CCTTT)n repeat at the "\'052 promoter reg10n 111 non-atoplC
subjects. The mechanism by which tl1e pentanucleotide repeat polymorphism, or a related
regulatory polymorphism in tl1e "\-052 gene promoter region, influences the de\-elopment
of atopy 1S as \-et unclear. Significant aSSOClatlOnS between the 1-t-repeat allele and the
absence of tl1e diabetic retinopathy or nephropathy, especially in insulin-dependent (type
1) diabetic patients, ha\-e been reported 14. 15 It 1S suggested that clinicalh- distinct
inflammatory immune diseases including insulin-dependent diabetes mellitus and atopy are
controlled by a common set of susceptibility genes IH Therefore, the 1-t-repeat allele 1S
potentially a susceptibility or disease-modifying allele of inflammaton- immune diseases
such as atopy and diabetes mellitus .. Polypyrimidine/polypurine repeats 1n the promoter
region may affect transcription by forming the unusual triplex DNA. structure I'). The I-t
repeat allele also displa;;ed a greater 111crease 111 reporter gene expresslOn under both
conditions of normal and high glucose concentrations 14, suggest111g that, 111 indi\-iduals
possessing this potentially functional allele, NOS2 ma:- be expressed to a greater extent in
response to exogenous stimuli including se\-eral pathogens such as nruses or bacteria,
leading to exaggerated NO production. Alternati,-el:-, a true susceptibility allele at the
Konno S - 12-
J\-052 gene or another nearby gene that is ill linkage disequilibrium with the l-l--repeat
allele may explain our fInding of a signifIcant association between atopy and the l-l--repeat
allele.
Recently NO has emerged as an immunoregulatory molecule that is im-oh-ed in
the regulation of Thl/Th2 subset balance 5·111 NO has an inhibitory effect on T cell
actintion, and cytokine production in Th2 cells is more susceptible to this effect than that
in Th 1 cells -. . -\t the inducti,-e phase of Th 1 cells, NO e,-en shO\\-ed an enhancing effect
on the induction and differentiation of Th 1 cells, with little or no effect on fully con1.mitted
T cells ~. In tl1e fIrst few years of life, children are considered to be highly susceptible to
immunomodulation with long-lasting effects 2" and tl1e ,-ery fIrst period of life is indeed
important ill the establishment of atoplc status 21 Se,-eral [illdings also indicate that
childhood exposure to infectious agents, including mycobacteria, pre,-em allergic
sensitization through the preferential induction of Th 1-type cytokines 22.21. Taken together,
in&i-iduals carryillg the l-l--repeat allele ma\- hm-e higher induction of :\0 b," senral
infectious agents in their early childhood, acquiring a Th 1-polarized immune system, and
hm-e decreased susceptibility to allergic sensitization when the:.- are subseguentl}- exposed
to conunon em-ironmental allergens.
C onwrs ely, some other studies reported tl1at S052 defIcient mice de,-eloped an
Konno S - 13 -
enhanced Thl response following infections and antigenic stimulation ('. ", suggesting that
NO modulates the Th 1 /Th2 balance by fa,-oring Th2 responses. Indeed, a significant
correlation was found between exhaled NO le,-els in adults \\'ith established asthma and
" atopy, measured b,' total serum IgE le,-els and a poslm-e skin-prick test - .. -\ possible
explanation for the discrepanc:.' between this findings and ours is that NO has different
selecti,-e roles 111 the regulation of the Th 1 /Th2 balance depending upon the types of
target cells im-oked, states of mnva,' inflmnmation or the concentratlon of NO locall\'
released in the airways s, 111
.-\ recent longitudinal prospecm-e stud" suggested that asthmatic subjects haw
increased susceptibility to hm-e higher IgE responsi\-eness 25 •• -\sthmatic subjects also ha\-e
increased leyels of NO and NOS2 expression in their ainvays 2(,. Gi\-en the possibility that
asthmatic status modulates the genetic manifestation of the 1 -l--repeat allele, \w also
calculated an odds ratio for the denlopment of atopy by controlling for asthm.atic status in
addition to age, sex and smoking status, and found that the genetic effect of the 1 -l--repeat
allele on atopy was independent of asthma. Our stud" also failed to shO\\' an association
bet\veen asthma and the j\'-OS2 promoter polymorphism. i\foreo\-er, murine models with
targeted deletions of each SOS gene ha\-e shO\vn that SOS! but not SOS2 is important
for the de\-elopment of the airway hyperresponsi\-eness T.2S. Collecti\-ely, it appears that the
Konno S - 14-
14-repeat allele at the ,\'052 gene 1S one of the distinct genetic factors that affect the
,~
de\'elopment of atopy, but not asthma - .
In the SOS2 gene, a bi-allelic tetranucleotide repeat sequence \\'ithin the
5' -flanking region at ~ 750bp from the transcription initiation site in promoter region has
also been identified 311 although the functional consequence of the polymorphism 1S
unclear at this time. Gao and colleagues found that tlus bi-allelic polymorphism is equall)'
disu-ibuted between asthmatics and healtl1Y controls, and bet\\'een atopic and non-atopic
indi,-iduals 31 \\/e did not, howe,'er, detect polymorphisms at this locus 111 -tOO
chromosomes from the 100 Japanese asthmatics and 100 Japanese healthy controls (all
subjects had an identical genotype) in our study population.
In conclusion, a potentiall:- functional 14-repeat allele at the SOS2 gene promoter
reglOn 1S im'erseh- associated with atopy. Tlus result tends to support the notlon that
enhanced expression of NOS:? modulates the Thl/Th:? balance towards Thl responses III
lil'O, which nught result in inhibition of allergic sensitization ",hen exposed to common
allergens. The .1.\'052 gene lies witllin tl1e C-C chemokine cluster region on chromosome
17ql1.2-q12, 11. 12 and the 14-repeat allele Inay contribute to a si~uficant linkage bem'een
tlus region and atopy. Confirmation of our preliminary finding using a second, larger and
independent population sample, and a detailed functional analysis would proyide a better
Konno S - 15 -
understanding of the genetic significance of the 14-repeat allele and thc protectiyc effect
of NO on the de,-elopment of atopy.
Konno S - 16-
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TABLE I. Demogl'aphic characteristics of study participants according to the 14-repeat genotype
No. of subjects Age Sex Current smoker TotallgE
yr f/m No. C/o) log lU/ml
Non-atopic subjects
Hclthy Control 141 44.4+ 10.7 Gl/80 54 (38.3%) 1.57±0.55
14-repeat (+) 17 43.4+ 11.3 8/9 5 (29.4(Yt» 1.82±0.28
14-repeat (-) 124 44.6±1O.7 53/71 49 (39.5°;(» 1.53±O.57
Asthma 56 55.4± 11.2 4()/16 8 (14.1<1'0) 1.90±O.55
14-repeat (+) 10 55.4+ 11.2 7/3 () ( 0 %) 1.86±0.38
• 14-repeat (-) 46 55.3± 11.3 33/13 8 (17.4%) 1.91 ±O.58
Atopic subjects
Hclthy Control 102 42.3 ± 11.7 40/62 38 (37.3%) 2.l8±().54
14-repeat (+) 6 34.2± 12.2 3/3 2 (33.3%) 1.87±().G3
14-repeat (-) 96 42.8± 10.7 37/.'19 36 (37.5%) 2.20±O.53
Asthma 198 40.9± 16.6 lOO/98 56 (28.3°/c» 2.S8±O.56
14-repeat (+) IS :').'1.1 ± 1 ·1.·1 8/7 7 (46.7%) 2.64±().70
I-I-repeat (-) 183 41.1 ± 1(1.7 92/91 49 (26.S"") 2.57±O.S5
Data are mean ± SD
TABLE II. Genotype and allele frequencies for the 14-repeat allele
(A)
Genory-pe, No. of subject (%)
14-repeat/ 14-repeat
14-repeat/ orhers
others/ orhers
Allele, No. of subjecrs (%)
14-repeat
others
(B)
Genory-pe, No. of subject (%)
14-repeat/ 14-repeat
14-repeat/ others
orhers/ orhers
Allele, No. of subjects (%)
14-repeat
others
Non-atopic subjects
3 (1.5)
24 (12.2)
170 (86.3)
30 (7.6)
364 (92.4)
Healthy Controls
1 (0.4)
22 (9.1)
220 (90.5)
24 (4.9)
462 (95.1)
Atopic subjects
1 (0.3)
20 (6.7)
279 (93.0)
22 (3.7)
578 (96.3)
Asthmatic subjects
3 (1.2)
22 (8.7)
229 (90.1)
28 (5.5)
480 (94.5)
(A) Significant differences in genotj'pe and allele distributions for the 14-repcat allelc \\'crc obse[\"Cd
(gcnotype; chi-scluarc=6.13, p=0.013, allelc; chi-squarc=7,48, p=0.00(3) bctween non-atopic and atopic subjects.
(B) ~o significant differences wcrc obsc[\'cd (gcnotj'pe; chi-squarc=0.02, p=0.89, allclc; chi-squarc=O.l-. [1=0.(8)
betwcen healthy control and asthmatic subjccts.
TABLE ill. Logistic regression models for the development of atopy*
(A) (li)
Variables OR 95(Y<JC:I p Value Variables OR 95% C1
Age 0.97 0.96-0.98 <0.0001 Age 0.96 0.94-0.97
Sex 1.22 0.82-1.79 0.33 Sex 1.57 1.02-2.42
Smoking status 0.79 0.52-1.20 0.27 Smoking status 1.(l2 0.64-1.67
14-repeat allele 0.42 0.23-0.79 0.0065 14-repeat allele 0.36 0.18-0.71
Asthmatic status 6.61 4.23-10.3
Defillitioll o/ab/mJI'iati(J/1 OR : odds ratio, C1 : confidence interval
* The odds ratio was calculated for the development of atopy in the 14-repeat allele carriers compared with non-carriers.
1n a model (B), the analysis was adjusted for asthmatic status (asthmatic or healthy control) in addition to adjustment: for age, sex, smoking status
(never smoked, past smoking or current: smoking), and the 14-repeat: genotypes (carriers or non-carriers).
P Value
«WOOl
(1.040
0.92
O'(l032
<0.0001
FIGURE
25 I
N = 994 Alleles I
00 Q) 20 ~
Q) I III II D Non-atopic ::::l ~ ~
15 1 II Atopic 0 Q) OJ) ~ ~
Q 10 Q) u ~ Q)
~ 5
o 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Nluuber of (CCTTT) pentanucleotide repeats