Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Single Nucleotide Polymorphisms (SNPs) of the TNF- a
(- 2 3 8 / - 3 0 8 ) g ene among TB and non TB patients:
Susceptibility markers of TB occurrence?*
MARTHA MARIA DE OLIVEIRA, JOCILEA C. S. DA SILVA, JOSEANE F. COSTA, LÚCIA HELENA AMIM, CARLA C. S. LOREDO, HEDI MELO, LUIZ F. QUEIROZ, FERNANDA C. Q. MELLO,
JOSÉ ROBERTO LAPA E SILVA, AFRÂNIO LINEU KRITSKI(TE SBPT), ADALBERTO REZENDE SANTOS
Backg ro u n d: Ho st g e n e t ic f a ct o rs m a y p la y a ro le in t h e su sce p t ib ilit y t o a ct ive t u b e rcu lo sis (TB), a n d se ve ra l p o lym o rp h ism s in d iffe re n t cyt o kin e co d in g g e n e s h a ve b e e n d e scrib e d a n d a sso cia t e d wit h d ise a se s t o d a t e .
Objectives: To investigate whether polymorphisms within the promoter region of the TNF-α (- 238/- 308) coding genes are associated to the occurrence of active TB.
Methods: SNPs within the TNF-α gene were analyzed by PCR- RFLP among two groups of individuals: patients with TB (n = 234, and patients non TB (n = 113).
Results: In t his st u dy, t he presen ce of t he - 238A allele was associat ed wit h suscept ibilit y t o TB disease occu rren ce an d severit y (p = 0,00002; OR = 0,15; IC = 0,06- 0,36. On t he con t rary, t he - 308A allele was associat ed wit h prot ect ion t o t he occu rren ce of an ot her pu lmon ary diseases.
Conclusions: These results suggest the importance of genetics studies on TB occurrence. Further studies are needed pursuing a better understanding of the human pathogenesis of M. tb.
Key words: uberculosis/genetics. Polymorphism, single nucleotide/genetics. Tumor necrosis factor/genetics. Alleles. Genotype. Lung diseases.
*St u d y ca rried o u t in t h e La b o ra t o ry o f Gen et ics a n d Mo lecu la r Bio lo g y o f t h e Tu b ercu lo sis Resea rch Un it Ho sp it a l Est a d u a l Sa n t a Ma ria (Sa n t a Ma ria St a t e Ho sp it a l), Lep ro sy La b o ra t o ry - Fio cru z- Rio d e J a n eiro (RJ ). Fin a n cia l su p p o rt : FAPERJ / Pro jet o Milen iu n /Grant no. 6 2 .0 0 5 5 / 01 - 4 PADCT
Fo g a rt y Co rn ell - 3 D4 3 TW0 0 0 01 8 - 1 6 S3
Co rre sp o n d e n ce t o : Dra . Ma rt h a Ma ria d e Olive ira . Av Brig a d e iro Tro m p o wsky s/ n º. Fa x: 5 5 2 1 2 5 5 0 6 9 0 3 . CEP 2 1 9 41 - 5 9 0 Ilh a d o Fu n d ã o . Rio d e J a n e iro . Bra z il. E- m a il: m a rt h o live ira @ ya h o o .co m .b r
fro m 5 % t o 1 0 %. In su ch in d ivid u a ls, t h e d eg ree o f d isea se risk d ep en d s o n t h e a b ilit y o f t h eir im m u n e syst em t o p reven t t h e d o rm a n t Mt b fro m m u lt iplyin g(2 ,3 ).
Th e im m u n e resp o n se t o Mt b d ep en d s o n a respon se m ediat ed by cells, whose in t eract ion wit h lym p h o cyt es a n d m o n o cyt es cu lm in a t es in t h e p r o d u c t io n o f p r o - a n d a n t i- in f la m m a t o r y m ed iat o rs. No t ab le am o n g t h e cyt o kin es in vo lved , t he t u m or n ecrosis fact or- alpha (TNF-
α
) has been ch a ra ct e riz e d a s o n e o f t h e m o st im p o rt a n t m o lecu les in in fla m m a t o ry resp o n se p ro m o t io n . Smith et al.(4) demonstrated that the local role playedb y TNF-
α
in t h e p u lm o n a ry resp o n se t o Mt b in f e c t io n , e s p e c ia lly in t h e p r o d u c t io n o f g ra n u lo m a s, is co m p lex. In a d d it io n t o a id in g bact erial clearan ce, TNF-α
part icipat es act ively in t h e m o d u la t io n o f p u lm o n a ry in f la m m a t io n . In h ib it in g t h e a ct ivit y o f TNF-α
in t h e lu n g s in t h e in it ia l p h a se o f in fect io n ca u ses p ersist en t in fla m m a t io n . Va ria t io n s in cyt o kin e p ro d u ct io n a m o n g in d ivid u a ls m a y b e re la t e d t o va rio u s g en et ic p o lym o rp h ism s, t h ereb y d et erm in in g t h e im m u n e resp o n se t o t h e d isea se. Differen t a llelic f o rm s o f va rio u s c yt o kin e g e n e s h a ve b e e n i d e n t i f i e d , i n c l u d i n g s i n g l e n u c l e o t i d e p o lym o rp h ism s (SNPs) a t p o sit io n s - 2 3 8(5 ) an d-3 0 8( 6 ) i n t h e TN F p r o m o t e r r e g i o n . A GA
su b st it u t io n o ccu rs in t h e se p o lym o rp h ism s, resu lt in g in m u t a t io n s t h a t seem t o b e rela t ed t o d if f e r e n c e s in g e n e e xp r e s s io n a n d p r o t e in secret ion(7 ,8 ), alt hou gh t his is st ill con t roversial(9 ,10,11).
Th e p resen ce o f - 3 0 8 SNP h a s b een rela t ed t o a n in crea se in TNF-
α
g en e t ra n scrip t io n(1 2 ,1 3 ). Th epresen ce of t he - 308 A allele TNF2 has been relat ed t o clin ical su scep t ib ilit y t o vario u s d iseases, su ch as cerebral malaria(14), mu cocu t an eou s leishman iasis (1 5 ), an d lepromat ou s leprosy(1 6 ). All of t hese diseases
h ave in co m m o n h ig h levels o f TNF-
α
circu lat in g in t h e p erip h era l b lo o d . On t h e o t h er h a n d , t h e p resen ce o f t h is m u t at io n h as also b een relat ed t o p ro t ect io n a g a in st so m e d isea ses, su ch a s severe fo rm s o f lep ro sy(1 7 ,1 8 ,1 9 ). Th e SNP a t p o sit io n - 2 3 8h a s b e e n a s s o c ia t e d w it h r e d u c e d TNF-
α
t ra n scrip t io n(1 7 ,1 9 ). Th e p resen ce o f t h e m u t a n t
-238A allele in pat ien t s wit h rheu mat oid art hrit is(1 3 ),
d en g u e wit h HIV(2 0 ), o r can cer(2 1 ) h a s b een rela t ed
t o p ro t ect io n a g a in st a n d su scep t ib ilit y t o so m e d isea ses, su ch a s ch ro n ic h ep a t it is B a n d C(2 2 ),
m u lt ip le sclero sis(2 3 ) a n d p so ria sis in m a les(2 4 ).
Th e o b ject ive o f t h e p resen t st u d y wa s t o det ermin e whet her promot er region polymorphisms o f t h e g en e en co d in g TNF-
α
(- 2 3 8 a n d - 3 0 8 ) a re rela t ed t o t h e in cid en ce o f TB in p a t ien t s t rea t ed in h o sp it a ls in t h e st a t e o f Rio d e J a n e iro . M o r e o ve r, w e e va l u a t e d w h e t h e r t h e s e p o lym o rp h ism s co rrela t e wit h t h e va rio u s clin ica l fo rm s o f TB a n d wit h HIV co - in fect io n .Patients, Materials and Method
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
qu est ion n aire on dem ographic dat a an d hist ory of d isea ses t h a t co u ld b e rela t ed t o t h e o ccu rren ce o f t h e p o lym o rp h ism s a n a lyzed in t h e p resen t st u d y. Clin ical sam p les were co llect ed an d st o red a t - 2 0 °C fo r la t er u se.
DNA extraction. We used a protocol based on a
commercial extraction kit (Invitrogen, Carlsbad, CA, USA) and a DNA isolation reagent (DNAzol, Gibco BRL/ Life Technologies, Gaithersburg, MD, USA), adapted for use on a small scale directly from total blood (fresh or frozen ) in ou r laborat ory. In su mmat ion , aft er thawing the samples, 300
µ
L of blood were transferred to a fresh test tube, and 1 mL of 0.9% NaCl solution were a d d ed . Aft er cen t rifu g a t io n , t h e resu lt in g sediment was resuspended in a hypotonic TE solution (20 mM Tris- HCl; 10 mM EDTA) at 4°C. After another centrifugation, DNAzol was added in order to break down the sediment and liberate the DNA, which was then precipitated with the addition of absolute ethanol. The precipitate was dried at room temperature and redissolved in 50 mL of alkaline solution (8 mM NaOH). After having been redissolved, the DNA sample was electrophoresed on a 1- % agarose gel, stained with e t h id iu m b ro m id e t o d e t e rm in e in t e g rit y a n d concentration, and then stored at - 20°C.DNA amplification using polymerase chain reaction and g enotyping . Th e g en o t yp in g o f t h e p o lym o rp h ism wit h in t h e TNF-
α
p ro m o t er g en e wa s ca rried o u t a s fo llo ws: fo r t h e - 3 0 8 p o sit io n , aft er amplificat ion of t he region of in t erest (a 107-b p fra g m en t ) a s d escri107-b ed 107-b y Wilso n et a l.(5 ), Nco Ien zym e wa s u sed fo r d ig est io n ; fo r t h e - 2 3 8 posit ion , aft er amplificat ion of a 165- bp fragmen t , Ba m HI en zym e was u sed fo r d ig est io n . In sh o rt , a p p ro xim a t e ly 1 0 0 n g o f t h e DNA e xt ra ct e d , con sist in g of 50 m M of KCl, 10 m M of Tris- HCl pH 8 .3 , 1 .5 m M o f Mg Cl2, 2 0 0 µ M o f d NTP a n d 1 .2 5 U of AmpliTaq Gold DNA Polymerase (Perkin - Elmer Cet u s, No rwa lk, CT, USA), wa s a d d ed t o ea ch p o lym e ra se ch a in re a ct io n . Mu t a t io n - sp e cific o lig o n u cleo t id es (1 µ M fo r - 3 0 8 a n d 1 2 .5 p m o l fo r TNF - 2 3 8 ) were a lso a d d ed , resu lt in g in fin a l vo lu m es o f 5 0 µ l (- 3 0 8 ) a n d 4 0 µ l (TNF - 2 3 8 ). All m ixt u res were in cu b a t ed a t 9 5 °C fo r 1 0 m in u t es a n d s u b s e q u e n t l y s u b m i t t e d t o 3 8 - c y c l e amplificat ion s: (for TNF - 308) at 94°C for 1 min u t e, a t 5 7 °C fo r 1 m in u t e a n d a t 7 2 ºC fo r 1 m in u t e; (fo r TNF - 2 3 8 ) a t 6 0 °C fo r 3 0 seco n d s, 7 2 °C fo r 3 0 seco n d s, 9 4 °C fo r 3 0 seco n d s a n d 7 2 °C fo r 7 min u t es.
Statistical analysis: Statistical significance between p ro p o rt io n s o f so cio d em o g ra p h ic d a t a a n d t h e differences between genotypic and allelic frequencies was determined using the chi- square test or, when appropriate, Fisher’s exact test (Epi Info, version 6, Cen t ers for Disease Con t rol an d Preven t ion ). The magnitude of each correlation was estimated as an odds ratio (OR), with its respective confidence interval (CI). The level of significance adopted was 5%.
RESULTS
Genotypic and allelic distribution of 238G/A and -308G/A polymorphisms in the population studied
Patients with TB. Th e p resen ce o r a b sen ce o f SNPs in t he gen e en codin g TNF-
α
was det ermin ed. This det ermin at ion was made for t he - 238 posit ion in 2 0 0 TB p a t ien t s a n d fo r t h e - 3 0 8 p o sit io n in 2 0 5 TB p a t ien t s. In t h e a n a lysis o f m u t a t io n a t posit ion s - 238 an d - 308, n o sign ifican t differen ces re la t e d t o g e n d e r o r HIV c o - in f e c t io n w e re d et ect ed in t h e in cid en ce o f t h e m u t a n t a llele o r in an y of t he possible gen ot ypes (dat a n ot shown ). Pat ien t s were st rat ified by clin ical presen t at ion o f t h e d isea se (p u lm o n a ry versu s ext ra p u lm o n a ry o r d issem in a t ed ). Fo r t h e m u t a t io n a t t h e - 2 3 8 p o sit io n , a sig n ifica n t ly h ig h er in cid en ce o f t h e wild - t yp e GG h o m o zyg o t e g en o t yp e wa s seen in t h e g ro u p o f p at ien t s wit h p u lm o n ary fo rm s o f TB t han in t hose wit h ext rapu lmon ary or dissemin at ed fo rms (p = 0 .0 0 3 ; OR = 5 .5 4 ; CI = 2 .01 - 1 5 .0 0 ). In co n t ra st , in cid en ce o f t h e m u t a n t GA a n d AA gen ot ypes was sign ifican t ly lower in t he grou p wit h pulmonary forms than in those with extrapulmonary o r d issem in a t ed fo rm s (p = 0 .0 4 ; OR = 0 .3 3 ; CI = 0 .11 - 1 .0 7 a n d p = 0 .0 01 ; OR = 0 .0 5 ; CI = 0 - 0 .4 4 , respect ively). A sign ifican t in crease in t he in ciden ce o f t h e - 2 3 8 A allele was also o b served in t h e g ro u p o f p a t ien t s wit h t h e m o re severe (ext ra p u lm o n a ry o r d issem in a t ed ) fo rm s (p = 0 .0 0 0 0 2 ; OR = 0 .1 5 ; CI = 0 .0 6 - 0 .3 6 ). Reg a rd in g t h e - 3 0 8 p o sit io n , n o sig n ifica n t d ifferen ces in g en o t yp ic a n d a llelic f r e q u e n c ie s w e r e s e e n a m o n g t h e c lin ic a l p resen t a t io n fo rm s o f TB (d a t a n o t sh o wn ).TABLE 1
Genotypic distribution of SNPs at the – 2 3 8 and - 3 0 8 positions in theTNF-α
promoter reg ion in tuberculosis and non- tuberculosis patients
TB patients NTB patients
- 2 3 8 n = 2 0 0 n = 1 0 0 p va lu e OR CI
GG 173 (86.5%) 97 (97%) 0.004 0.20 0.05- 0.71
GA 20 (10%) 3 (3%) 0.031 3.59 0.98- 15
AA 7 (3.5 %) 0 (0%)
f (A) 0.085 0.015 0.0007 6.10 1.77- 25
- 3 0 8 n = 2 0 5 n = 11 3
n (%) n (%)
GG 174 (84.8%) 102 (90.3%) 0.17 0.61 0.27- 1.32
GA 06 (2.9%) 05(4.4%) 0.34 0.65 0.93
AA 25 (12.1%) 06 (5.3%) 0.04 2.48 0.93
f (A) 0.13 0.084 0.02 1.94 1.07- 3.58
TB: t u b ercu lo sis; NTB: n o n - t u b ercu lo sis; n: n u m b er o f p a t ien t s st u d ied ; OR: o d d s ra t io ; CI: co n fid en ce in t erva l; GG: wild - t yp e h o m o zyg o t e g en o t yp e; GA: h et ero zyg o t e g en o t yp e; AA: m u t a n t h o m o zyg o t e g en o t yp e; f: freq u en cy HIV (d a t a n o t sh o wn ).
Distribution of - 2 3 8 and - 3 0 8 G/ A
polymorphisms in TB and Non- TB patients Th e d ist rib u t io n o f g e n o t yp ic a n d a lle lic freq u en cies o f p o lym o rp h ism s a t t h e 2 3 8 a n d -3 0 8 p o sit io n s in t h e g ro u p o f p a t ien t s d ia g n o sed wit h TB a n d in t h e g ro u p d ia g n o sed wit h lu n g d isea ses o t h er t h a n TB is sh o wn in Ta b le 1 . In t h e grou p of pat ien t s wit h ot her diseases, a sign ifican t in crea se in t h e in cid en ce o f t h e wild - t yp e - 2 3 8 GG g e n o t yp e w a s o b s e r ve d . In ve r s e ly, t h e freq u en cy o f t h e h et ero zyg o t e GA g en o t yp e wa s sig n ifica n t ly h ig h er in t h e g ro u p o f p a t ien t s wit h TB, a n d n o p a t ie n t s fro m t h e n o n - TB g ro u p p resen t ed t h e m u t a n t h o m o zyg o t e AA g en o t yp e. Th e a n a lysis o f a llelic freq u en cy sh o wed t h a t t h e in cid en ce o f t h e - 2 3 8 A a llele wa s sig n ifica n t ly h ig h er in t h e TB g ro u p t h a n in t h o se in t h e n o n -TB g ro u p (p < 0 .01 ).
Comparison of the genotypic distribution of SNP in t h e p o sit io n - 3 0 8 A b et ween t h e g ro u p s sh o wed t h a t , in t h e g ro u p o f p a t ien t s wit h TB, t h ere wa s sig n ifica n t ly h ig h er in cid en ce o f b o t h t h e m u t a n t
b een eleg a n t ly d em o n st ra t ed in va rio u s a n im a l m o d els(2 5 ,2 6 ,2 7 ). Vario u s st u d ies wit h h u m an s h ave
sh o wn a g en et ic co m p o n en t t o h o st suscep t ib ilit y, resist an ce t o TB in fect ion an d con version t o act ive TB(2 8 ). Co n co rd a n ce b et ween m o n o zyg o t ic t win s
in t h e d evelo p m en t o f act ive TB ran g es fro m 6 5 % t o 8 5 %, co m p ared wit h 2 0 % t o 3 5 % fo r d izyg o t ic t win s. St ead et al., in an in n o vat ive t u b ercu lin su rvey co n d u ct ed in a h o m e fo r t h e eld erly in Arka n sa s, rep o rt ed t h a t Afro - Am erica n s b eca m e in fect ed wit h Mt b m o re o ft en t h a n Ca u ca sia n s(2 9 ).
Ot h er st u d ies h a ve sh o wn t h a t p a t ien t s ca rryin g m u t a t io n s in IFN-
γ
a n d IL- 1 2 recep t o rs p resen t in fect ion wit h BCG an d at ypical mycobact eria more f re q u e n t ly a n d d e ve lo p m o re se ve re c lin ic a l m a n ifest a t io ns o f t h e d isea se(3 0 ,31 ). Mo re recen t ly,correlat ion st u dies have been carried ou t in volvin g t h e g e n e s e n co d in g va rio u s f a ct o rs, su ch a s NRAMP1, vitamin - D receptor (VDR), IL- 10, IL- 1 an d IFN-
γ ,
t h a t a re im p o rt a n t in t h e p a t h o g en esis o f TB(3 2 , 3 3 , 3 4 ). Fo u r p o lym o rp h ism s, d e le t io n s o rJornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Guinea(32,33). The SNPs fou n d in the IL- 1 gen e clu ster
a n d in t h e VDR g e n e h a ve b e e n re la t e d t o su scept ibilit y t o TB in pat ien t s in In dia(34,35). Selvaraj
et al.(3 9 ), in a recen t ca se- co n t ro l st u d y, rep o rt ed
n o c o rre la t io n b e t w e e n t h e - 2 3 8 a n d - 3 0 8 p o lym o rp h ic sit es o f TNF-
α
p ro m o t er g en e a n d t h e d evelo p m en t o f p u lm o n ary TB. Ho wever, t h e a u t h o rs d id n o t co m p a re t h e in cid en ce o f t h ese p o lym o rp h ism s wit h t h e va rio u s clin ica l fo rm s o f TB. In t h e p resen t st u d y, we an alyzed t h e p o ssib le correlat ion s bet ween t hese polymorphisms an d t he severit y o f t h e va rio u s fo rm s o f TB. In a d d it io n , we in vest ig a t ed p o t en t ia l co rrela t io n s b et ween t h ese p o lym o rp h ism s a n d va rio u s clin ica l a n d d em o g ra p h ic ch a ra ct erist ics, su ch a s severit y o f clin ical man ifest at ion , gen der an d HIV co- in fect ion .Polymorphisms in the TNF-
α
promoter region andtheir correlation with TB: severity and infection In t he presen t st u dy, on ly 8% of pat ien t s in t he n o n - TB g ro u p ca rried t h e - 2 3 8 A a llele, wh erea s 1 5 % o f p a t ien t s in t h e TB g ro u p p resen t ed t h is m u t a t io n (p d 0 .01 ). Th ese resu lt s co n firm d at a fo u n d in t h e lit era t u re, wh en co m p a rin g p a t ien t s wit h TB t o t h o se wh o h a ve b een in co n t a ct wit h TB p a t ien t s a n d t est p o sit ive o n t u b ercu lin t est s, sin ce t h e p resen ce o f t h is a llele (- 2 3 8 A) ca n b e co n sid ered a m arker o f su scep t ib ilit y t o TB(4 0). Th e co rrela t io n o f t h e - 2 3 8 A a llele in cid en ce wit h ext rapu lmon ary an d dissemin at ed TB, as eviden ced in t he presen t st u dy, has n ever before been report ed in t h e lit era t u re. Fu t u re st u d ies eva lu a t in g t h is co rrela t io n a n d Mt b viru len ce m a y co n firm it s usefu ln ess as a marker of clin ical severit y, especially in recen t ly in fect ed p a t ien t s wh o a re a t risk fo r developin g t he form s of TB t hat presen t high rat es o f m o rb id it y an d m o rt alit y.
On e of t he prin cipal limit at ion s of ou r st u dy was that patients diagnosed with lung diseases other than TB were not submitted to tuberculin tests. Therefore, in our analysis of mutant allele correlations, we could n ot compare n on - TB t o TB pat ien t s.
However, when t he dist ribu t ion of m u t at ion at t he - 308 posit ion was st u died in com bin at ion wit h in creased gen e t ran script ion an d t he con sequ en t higher prot ect ion again st t he in ciden ce of a given disease, we observed sign ifican t ly higher in ciden ce of t his allele an d of AA gen ot ype in TB pat ien t s t han in n on - TB pat ien t s. A possible explan at ion for this discrepancy is that the non- TB group comprised
som e pat ien t s diagn osed wit h diseases relat ed t o high TNF-
α
produ ct ion , which is charact erist ic of carriers of su ch alleles. Therefore, t his grou p can n ot be con sidered a “clean ” con t rol grou p. It is also kn own t hat t he TNF-α
gen e is regu lat ed at variou s t ran script ion al an d post - t ran script ion al levels(3 8 ).Therefore, an ot her possibilit y is t hat some pat ien t s diagn osed wit h TB an d carryin g t he - 308 allele (an d t herefore fu n ct ion ally m ore able t o produ ce
TNF-α )
also carry m u t at ion s in ot her an t i- TB im m u n e-regu lat ory gen es, su ch as t hat en codin g IFN-γ
, or even in t he TNF recept or it self. Therefore, alt hou gh t h ese p a t ien t s wo u ld p resen t a m u t a t io n t h a t in du ced great er TNF -α
produ ct ion , t he absen ce of recept or m alfu n ct ion wou ld preven t t his cyt okin e fro m exert in g it s in fla m m a t o ry a ct io n , t h ereb y favorin g t he developm en t of TB.The immune response, whether protective against TB development (or even TB severity) or not, is related t o a n et work of cyt okin es produ ced over t he cou rse of the infection. Therefore, identifying the molecular mechan isms t hat precede t he produ ct ion of t hese cyt o kin e s m a y re p re se n t a p o we rf u l t o o l f o r researchin g n ew vaccin es an d t herapeu t ic dru gs.
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