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Tissue Antigens ISSN 0001-2815

Study of KIR genes in tuberculosis patients

A. Mendez', H. Grandaz, A. Meenagh3, S. Contreras', R. Zavaleta', M. F_Mendoza',

L Izquierdoz, M. E. SarmientoZ, A. AcostaZ & D. Middleton3

'"

1 t..abofatoty of Expermer1t Pathdog,t. Faculty of Medicine. l.JoNersidad Vwaauzana. Mexico

2 Instituto Finlay. Centro de Investigaci6n - PrOOucx::i6nde Vao..nas y $oems. CAIdadHabana. Cuba

3 Histooornparibi'ty and Immunogenetics Laboratofy. City Hospir.al. Belfast. UK

4 Faculty of BK>medicalScience. University of Uistef. Coleraine. UK

Keywords

KIAgenes; Mexico; tuberculosis

Correspondence

Derek Middleton

Histocx:rnpatibility and Immunogenetics

laboratory

City Hosp tal

Belfast

UK

Tel: +442890263676

Fax; +44 2890263881

e-mail: derek.middletonOblI.n-i.nhs.uk

Received 11 May 2006; revised 28 July 2006;

accepted 14Au~s: 2006

dol; 10.1111/j.1J99.{)Q39.2006.ocaJ5.x

Abstract

A totalof97patients with tuberculosis(fB) and 51 controls from Xalapa. Veracru~

Mexico. W(X studied for the prcscno: and absence of killer ce1 l immunoglobulin-

like rca:pto< (KIR) genes. The number of patients with either KlR2DLI or

KlR2DL3 differed signil">cantlycompared with the controls. However. only the

difference ia KIR2DL3 remained significant after correction for the number or

faclors anal]5Cd. We also found KlR2DS2 with its presumed CI group ligand lessprevalent in TB patients than in the control group. but this result Jost signif1C3ncc

after correction.

Introduction

Tuberculosis (TR) is the leading cause of deata from

a curable infectious disease. In 2004. there was an eStKnaled

8.9 milJion new cases. and nearly 2 million people die each

year. Relatively little is known on genetic predisposition to

the disease. The purpose of the present study was to

ascertain whether the frequency of receptors on aatural

~iller (NK)cells differed in patients compared with conlrols.

Killer cell immunoglobulin-like receptors (KIRs) are

members of a group of regulatory molecules foond on

subsets oflymphoid cells. They Wete flfst identified by their

ability to impart some spccificilyon NKcytolysis(I,2). The

KlR gene cluster, containing a family of polymorpllic and

highly homologous genes, maps 10 chrom05Ome 19q13.4within the I-Mb Ieu~ocyte receptor complex (LRC). The

LRC also encodes the leu~ocyte immunogloboIin.li~e

receplor family. the leukocyte-associated inhibitory recep-

tor family and the Fca. receptor. KJR genes an: arnagcd in

tandem over ahout 150 ~h. with the remarkable fcatare that

gene content varies between haplotypes.

386

Although KI R haplotypes vary in the number and type of

genes present (3-{j), the genes KlR2DL4, KlRJDPI.

KIR3DL2 and KlR3DLJ are present on virtually aD

haplotypes and have therefore been termed framewor~ loci

(7).All other KlR genes exisl on only a fraction ofthe tota!

haplotypic pool. The number of putatively expressed KIR

genes present on a single haplotype ranges from about 7 to

12. depending primarily on the presence or absence of

activating KJR.

Two distinct haplotypes (A and B) have heen designated

according to the numba and type of genes present. but

perhaps the most functionaUy relevant distinction between

these is the number of stimulatory receptors present, only

one(KIR2DS4)in haplotypcA.A limited numhcrofstudiesaddressing genetic associations between KIR genes aDd

spccifocdiscascs have beeDreporled to date, primarily due to

the very recent and ongoing characterization of the genes

and their haplotypes. NK cells have been implicated in the

defence against infectious diseases, particularly viral in-

fections. through mechanisms involving cytotoxicity and

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A.M~et81.

cytokine production, presumably mediated in part by

stimulatory KlR molecules (8). Given the RlCCptor-ligand

relationship between certain combinations of KJR and

human leukocyte antigen (HlA) class J molecules, it is

reasonable to hypothesise a synergistic reJatiooship between

these polymorphic loci that may ultimately regulate NK-

cell-mediated immunity against infectious pathogens.

KlR haplolypes might suggest the possibility of pleio-

tropic KJR effecls on different diseases. '-"iacrebya KIR

gene conferring protection against one disease may pre-

dispose 10another perhaps less deadly disease (9-11).

Materials and methods

A 10lal of97 patients wilh TB and 51 blood donor controls

from Xalapa. Veracruz. Mexico. were studied for the

presence and absence of KIR genes. Patients and controls.

none of whom were related. were of Mestizo ethnicity and

came from the same region. Informed consent from patients

and controls was obtained. In the Veracruz rez;oo. there are

relatively sma)) communities that arc ethnicaHy homo.

geneous and have a higher incidence of TO than the average

in Mexico. The mean age of patients and controls was

similar (38.3 ".f 34.4 years). Whereas patients had a gender

ratio of males (54.6) to females (45.4), there were more

males in the control group (78.5 '.f 21.5). ConflTtl1ation of

TB was by sputum smear and culture.

The KIR genes were studied using a polymerase chain

reaction.scquence specific oligonucJeotide probes KIR gene

identification system (12. J3). To this system.,..-e have added

a new probe that enables us to distinguish whelher a person

positive for KlR2DS4 is carrying the nondekled version.

the deleted version, or both. The oligonucleotide sequence

for this probe is CGGAGCTCCTATGACATG(I J II I).

GTGCGCAGCATCMACGG. The first section is found inexon 5 nucleotide position 84-101. with the scoond inexon 5

position 15(H 66, and the intermediate 48-hp area is covered

by five nonspecific thymidines in the probe sequence. which

act as a spacer between the two sections of the probe. Thus.

two separate polymorphic sites some distance apan within

eXOD5 of the amplifJcation were combined 10 produce a

probe. This pmbe will only hybridise to KIR2DS4 alleles

carrying the nondeletion.

HLA-el and -e2 gmups were defined usin~ a modifICa-

tion of the melhod used for full HlA-C allele typing (14) in

that only two pmbes fmm this system, which differentiated

Ducleotides at positions 77 and 80. were used. These probes

were C293 for CI gmup and C291 for C2 group (14).Similarly, to define HLA.Bw4, we used four pmbes (BL20,

BL21, B122 and BL23) with the same polymerase chain

reaction conditions as the previously reported typing system

for HlA-B (15). These probes are also ahle 10distinguish

whether or not threonine or isoleucine is presc:at at position

80 of the HlA-Bw4 molecule.

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..Ioum. oompMllOfl &8086-3891 c 2006 8lactwel Mtri:. ud

KIR genes in tuberculosis

Fishers exact test was used for statistical oomparisons.

Probabilities were corrected for the number of gene

frequencies compared (n=20) according to the Bonfcrroniprincipal.

Results and diKussion

Table I shows theRSults and the statistical significance. We

found thaI the presence of KlR2DLl (P=0.013) and

KIR2DL3 (P = 0JI0l) in the TB patients was significantly

higher in eclatio. to the control group. The statistical

difference for KlR2DLI was Jost after Bonferroni correc-

tion, but that for K:JR2DL3 remain significant (P=0.02).

Kl R2DL3 is nOI1ll3llyfound on the A haplotype, and

KlR2DLl is morooften found on the A haplotype than on

the B haplotype. KJR2DL2 and KlR2DS2 normaUy found

on the B haplotype were co

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KIRgenes in tuberculosis

patients compared with the contrals. This is not surprising

as KIR2DL2 and KIR2DLJ are.,w believed to be alleles

of the same gene. and KIR2DL2 - . J KIR2DS2 are in suchhigh linkage disequilibrium that !hey are seldom discor-

dant in their presence/absence. Incited, in all the subjects of

the present -study (pati ents and control s), only one

individual was discordant for K1I2DS2 and KIR2DL2.

this individual. a patient. beiDJ: KIR2DL2 posilive/KIR2DS2 negalive.

We investi gated t he presence of t he genes with andwithout their liLA ligand. The CI group alleles are much

more prevalent than the C2 gro. alleles, and t he vast

majority of p atients and controls with KIR2DL2 or

KIR2DL3 were also positive for !heir CI group ligand.

The presence of KIR2DLI with it>ligand (C2 group) was

higher in patients compared with the contrnls, but this

difference was not significant. This is to be expected when

the frequencies of KIR2DLI are different in patients VJ'

controls, but it is interesting to notelhat of the controls with

KIR2DLI.44.7% (21/47) had IheCZgroup, whereas 54.6':1.

(53/97) of patients had thiscombinaJion. The comparison of

the frequency of the twoaetivating ~es that may usc HLA-

C as their ligand showed a signiHca.t decreased frequencyof KIR2DS2 with CI group in the tients compared with

the controls. AU these results poi~ to the patients being

mOre Jikely to have functional inhibilory KIR genes and less

likely to have functional activatingUR genes. a silUation

which may be detrimental to dealill&with infection.

It has previously been reported thai isoleucine at position

BO(lIe BO)of the Bw4 molecule was a.sociated with grealer

NK inhibition than threonine (Thr f1 J ) at the same position

(16). In the present study, the percentage of individuals

having Bw4 was not significantly ~erent in patients V .f

controls (44.3 .31.4':1.). A higher n.-nber ofhoth palients

and controls had lie BOrather than ThBO(2B.9 , .B.2% for

patients; 25.5 V .f 3.9% for controls; 72% patients and 2.0%

of controls had both amino acids at position 80). These

differences did not translate into patimts and controls being

significantly different in Ihe use of Bw4lie BOor Bw4Thr BO

as ligands for 3DLl or 3DSI.

We calculated the number of adivaling genes in each

group. The introduction of a new probe to our typing system

allowed us to only count KI R2DS4 as acti vati ng if the

nonde)cted version was present. Ho_ver, one could spee-

ulale of the possibility lhat soluble IOR2DS4 could play

a role. All patients and controls had IOR2DU. which ac-

cording to its structure could have an inhibiting and an

activating role. We did nOlconsiderKIR2DL4 in !hisanalysis.

No SigniflCaOI differences were found m the number of acti-vating or inhibitory genes in patients

. . tcontrols (Table I).

II has been reported lhal the stren~ of inhibition varies

according to receplor and ligand. KJR2DLl with its C2

group ligand gives stronger inhibitioalhan KIR2DL2 with

CI group, which gives stronger inhihilion than KIR2DL3

388

A M8nde.z IJt sl.

with CJ group (17). [n an investigalion inlD the possible

association of KIR receplOrs and clearance of hepalitis C

viru~ Kha!f.oo et al. used this variation in inhibitory

strengtll to show thai individuals who were able 10 clear

the vir.; were more likely to be KlR2Dl3 positive,

KJRlIJL2 negative and CI group homoznous (IB). In

thaI sceaario. even ifKIR2DLI was presenl!he lack of ilS

C2 group ligand would mean Ihal KIR2DU would be the

main inJllibitory group. We appJied this theory to the results

in the present study but did not find uy signiflcanl

difTcrencr in frequency between the patieDls. 25.8% of

whom " " ' T e KIR2DL3 positive. KIR2DL2 negalive. CI

group homozygous, oompared with 31.4'-0 ofcontrols.

NK cdIs are a very important part of the iJmate system,

helping 10 limil the dissemination of a path~D infection

before the adaptive immune system is initiated. The NK cell

is invoJvaJ in the control of cytokine release. aad it has been

shown tllat tumour necrosis factor is critical in preventing

est abli slment of mycobacterial infect ion and i n t he

mai nt enance of l at ent TB (J9). Thus, a rol e for NK cel l

receptors in prolection againsl TB is feasible. As recently

rcvicwedby Parham and colleague. activating receptors are

selected for their role in resistance to infection as well as

reprodutor of the innale immune respon'C, are highJy

susceptible to Mycobacterium tuberculosLr infettion (21, 22).

Other recquors including vitamin D receptor and inter leu-

kin-12 ra:zptor have had dubious associatic.s reported

with clinical TB (23).

AlthouYt our results suggesl greater inhibitioo in patients

relative to controls, the ac(ual differences in percentages is

not vast and t he possibil it y of chance fi ndings due t o

mult iple compari sons must be considered- It woul d

therefore be very important that. these findings are verified

in an indq>endent population. It will also be of utmost

importanCE to consider KIRexpression levels inthis disease.

Acknowledgment

We thank lIelen Durkin for excellent secretarial assistance.

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