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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.

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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: nhizawa@med.hokudai.ac.jp

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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Konno S - 18 -

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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