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', 3 L Izquierdoz, M. E. SarmientoZ, A. AcostaZ & D. Middleton '"
1 t..abofatoty of Expermer1t Pathdog,t. Faculty of Medicine. l.JoNersidad Vwaauzana. Mexico 2 Instituto Finlay. Centro de Investigaci6n - PrOOucx::i6n Vao..nas y $oems. CAIdad Habana. Cuba de 3 Histooornparibi'ty and Immunogenetics Laboratofy. City Hospir.al. Belfast. UK 4 Faculty of BK>medicalScience. University of Uistef. Coleraine. UK

Keywords

Abstract A totalof97patients with tuberculosis(fB) and 51 controls from Xalapa. Veracru~ Mexico. W(X studied for the prcscno: and absence of killer ce1l immunoglobulinlike 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 less prevalent in TB patients than in the control group. but this result Jost signif1C3ncc after correction.

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

14 Au~s:

2006

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

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 19q13.4 within the I-Mb Ieu~ocyte receptor complex (LRC). The LRC also encodes the leu~ocyte immunogloboIin.li~e receplor family. the leukocyte-associated inhibitory receptor 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.

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 numhcrofstudies addressing 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 infections. through mechanisms involving cytotoxicity and
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A.M~et81. KIR genes in tuberculosis

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

Fishers exact test was used for statistical oomparisons. Probabilities were corrected for the number of gene frequencies compared (n = 20) according to the Bonfcrroni

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

these polymorphic loci that may ultimately regulate NKcell-mediated immunity against infectious pathogens.

KlR haplolypes might suggest the possibility of pleiotropic KJR effecls on different diseases. '-"iacreby a 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

Kl R2DL3 is nOI1ll3lly found 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<rcspondingly reduced in the
T.b1. 1 Frequency arC percentage of patients positive fcI" KIRgenes. in

tuberctJosis CTB)pattac:s from Xa!ape. Veracruz. MeJOCo. comparedwith healthy controls from _ same population

18
Genettc markers 20Ll In=97%)

Controls (n=51%)

P
uncorrected 0.013

p
oorreaed 0.26

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 in exon 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 eXOD 5 of the amplifJcation were combined 10 produce a

2Dl2 20L3 20LS 3011 3051 2051 2052 2053 2DS4 2055 2Dl4. 3Dl2. 30L3
2011 with C2 2011 without C2 2D1.2 with Cl 2DL2 without C1 2DL3with 3011 3011 20S1 20S2 Cl 20L3 without Cl with Bw4 without Bw4 with C2 with C1

9711001 J5 (36.11 9711001 50 151.51

i17(89.7)

47192.2) 26 (510) 45188.2) 31 160.8) 46 (90.2) 26 (51.01

26 (51.0)

o.{Xn

002

49150.51 47 (4851 34135.11 9(931 07189.71 (45.41

97 (100)

26 (51.01 7(13.n 46 (90.21 2514901

51 (lOO) 21 (41.2)

53 (54.61 (45.41 31 (32.01 414.11

89 (91.8)

26151.01 25149.01 1 (2.01

42 (82.4)

probe. This pmbe will only hybridise to KIR2DS4 alleles

carrying the nondeletion.

20S 1 without C2

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

8(8.2) 38 (39.21 49(50.51 24 (24.n 23123.n 30 (30.91

4 (4.1)

31591
15 (29.4)

2DS2 w;thoot C1
30S 1 with Bw4 3051 without Bw4

31 (60.8) 11 (21.6) 15 (29.41 25 (49.01 1 (2.0) 7113.n 1907.31 16(31.41

0.034

0.68

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

2DL3 w;thoot 2012,

C1 hanozygous Activating KIR ganes.

n(22.n 27127.81 25(25.8)

>1 1

10(61.91 34 0511 30.1)

37(n.51 13 (25.51 1 (201

80 of the HlA-Bw4 molecule.

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have one or have no ~ating

> 1. 1 and Oref8f to tbI number of incividuBls 'Nhohave more than one. genes. rFtSpectiV8ly.

ud

387

KIRgenes in tuberculosis A M8nde.z

IJt

sl.

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 KIR2DS2 are in such high linkage disequilibrium that !hey are seldom discordant in their presence/absence. the present -study (patients Incited, in all the subjects of and controls), only one

-.J

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,

individual was discordant for K1I2DS2 and KIR2DL2. this individual. a patient. beiDJ: KIR2DL2 posilive/ KIR2DS2 negalive.
We investigated the presence of the genes with and without their liLA ligand. The CI group alleles are much more prevalent than the C2 gro. alleles, and the vast majority of patients and controls with KIR2DL2 or

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 ""'Te

KIR2DL3 positive. KIR2DL2 negalive. CI

KIR2DL3 were also positive for !heir CI group ligand. The presence of KIR2DLI with it>ligand (C2 group) was
higher in patients compared difference was not significant. the frequencies of KIR2DLI controls, but it is interesting to with the contrnls, but this This is to be expected when are different in patients VJ' notelhat of the controls with

KIR2DLI.44.7% (21/47) had IheCZgroup, whereas 54.6':1. (53/97) of patients had this combinaJion. The comparison of
the frequency of the twoaetivating ~es that may usc HLA-

C as their ligand showed a signiHca.t decreased frequency of 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

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 establislment of mycobacterial infection and in the maintenance of latent TB (J9). Thus, a role for NK cell 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 reprodu<Jion sua:ess and autoimmunity susceptibility (20).

As only a small percentage of patients

Mycohaclerium

iafecled wilh

tuherculosi.f develop clinical TIl,. it is feasible

lhat genetic factors play a role. Recently. it has been

demonslrated thai mice deficient in the ToIl-likE- receptor 2. another m:q>tor of the innale immune respon'C, are highJy susceptible to Mycobacterium tuberculosLr infettion (21, 22). Other recquors including vitamin D receptor and inter leukin-12 ra:zptor have had dubious associatic.s reported

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

NK inhibition than threonine (Thr f1J) 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 Th BO(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

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 the possibility of chance findings due to multiple comparisons must be consideredIt would 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.

significantly different in Ihe use of Bw4lie BOor Bw4 Thr 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 activating if the nonde)cted version was present. Ho_ver, one could spee-

Acknowledgment
We thank lIelen Durkin for excellent secretarial assistance.

ulale of the possibility lhat soluble IOR2DS4 could play a role. All patients and controls had IOR2DU. which according to its structure could have an inhibiting and an

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