The HLA-DRB1 locus as a genetic component in
giant cell arteritis. Mapping of a disease-linked
sequence motif to the antigen binding site of
the HLA-DR molecule.
C M Weyand, … , G G Hunder, J J Goronzy
J Clin Invest. 1992;90(6):2355-2361. https://doi.org/10.1172/JCI116125.
Giant cell arteritis (GCA) is a granulomatous vasculitis affecting persons over 50 years of age. The inflammatory infiltrate, which is targeted at the aorta and its proximal branches, includes activated CD4+ helper T cells, histiocytes, and giant cells. To investigate whether the genetic polymorphism of the HLA-DRB1 genes contributes to the local accumulation of activated T cells, we have analyzed both HLA-DRB1 alleles in a cohort of 42 patients with biopsy-proven GCA. The majority of patients (60%) expressed the B1*0401 or B1*0404/8 variant of the HLA-DR4 haplotype, both of which also represent the major genetic factors underlying the disease association in RA. GCA patients negative for the disease-linked HLA-DR4 alleles were characterized by a nonrandom distribution of HLA-DRB1 alleles. Sequence comparison among the allelic products identified in the GCA cohort
demonstrated heterogeneity for the sequence polymorphism of the third hypervariable region (HVR), but homology for the polymorphic residues within the HVR2 of the HLA-DRB1 gene. The GCA patients shared a sequence motif spanning amino acid positions 28-31 of the HLA-DR beta 1 chain. In the structural model for HLA-DR molecules, this
sequence motif can be mapped to the antigen-binding site of the HLA complex, suggesting a crucial role of antigen selection and presentation in GCA. In contrast, the sequence polymorphism linked to RA has been mapped to the HVR3 of […]
Research Article
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The
HLA-DRB1 Locus
as a
Genetic
Component
in
Giant Cell
Arteritis
Mapping of aDisease-linked Sequence Motif tothe AntigenBinding Site of the HLA-DR Molecule
CorneliaM.Weyand, Kevin C.Hicok, Gene G.Hunder,andJorgJ. Goronzy
DepartmentofInternalMedicine, Division ofRheumatology, Mayo ClinicandFoundation, Rochester,Minnesota 55905
Abstract
Giant cellarteritis (GCA)isagranulomatousvasculitis affect-ing persons over50yearsofage. Theinflammatoryinfiltrate, which is targeted attheaortaand its proximal branches,
in-cludes activated CD4' helper T cells, histiocytes, and giant
cells.Toinvestigatewhether thegeneticpolymorphismof the HLA-DRB1 genes contributestothe local accumulation of
ac-tivatedTcells,wehaveanalyzedboth HLA-DRB1 alleles ina
cohortof42patientswithbiopsy-provenGCA. Themajorityof
patients(60%) expressedthe B1 *0401orB1*0404/8variant
ofthe HLA-DR4haplotype,both of which also represent the
major geneticfactorsunderlyingthedisease associationin RA.
GCApatientsnegative forthedisease-linkedHLA-DR4 alleles werecharacterized by a nonrandomdistribution ofHLA-DRB1 alleles.Sequencecomparisonamong theallelic products
identi-fiedin theGCAcohort demonstratedheterogeneity forthe
se-quence polymorphism of the third hypervariable region
(HVR), buthomology forthepolymorphic residues withinthe HVR2of the HLA-DRB1 gene. The GCA patients shared a
sequence motif spanning amino acid positions 28-31 of the
HLA-DR,81
chain.Inthe structural model for HLA-DRmole-cules, thissequencemotifcanbemappedtotheantigen-binding siteof the HLA complex, suggestingacrucialroleof antigen selection andpresentation in GCA. In contrast, the sequence
polymorphism linkedtoRAhasbeenmappedtotheHVR3 of
the HLA-DRB1 gene and translates intoadistinct domainof the HLA-DRmolecule,thea-helical loop surroundingthe
anti-gen-binding groove. A consecutive case series study demon-strated that GCA and RA rarelyco-occurred, supporting the interpretation thatdistinctfunctional domains ofthe HLA-DR molecule areimplicatedin thepathomechanisms ofthese two
autoimmune diseases. (J. Clin. Invest. 1992. 90:2355-2361.)
Key words: rheumatoid arthritis * vasculitis * antigen
presenta-tion*HLAcomplex
Introduction
Giant cell arteritis (GCA)' isaclinicopathological syndrome characterized byagranulomatous vasculitis of theextracranial branchesof the proximalaorta(1).In manypopulations GCA
Addresscorrespondenceto CorneliaWeyand, M.D., Mayo Clinic, 200
First Street, Southwest,Rochester,MN 55905.
Receivedfor publication 6 April 1992 and in revised form 7 July 1992.
1.Abbreviations usedin this paper: GCA, giant cell arthritis; HVR, hypervariableregion; PMR,polymyalgiarheumatica.
is the most common vasculitic syndrome in persons over 50 yr ofage and if not treated promptly may represent a life-threaten-ing vascular disease (2, 3). The pathogenesis of the disease is essentially unknown. Clues to the understanding of the under-lying pathomechanisms havecome from theanalysis of the cellular infiltrate damaging the wall of the involved vessels. A granulomatous inflammatory reaction with dense infiltrates of mononuclear leukocytes, usually including multinucleated giantcells, is typical for GCA (4). Immunohistochemical
stud-ieshavedemonstrated thatCD4+ helperTlymphocytes repre-sentthedominant cell population (5). Subsequent studies of these cells have shown that markers of T cell activation are
expressedlocally, supporting the notion thataThelper cell-driven immune response is thecauseof the vascular damage. Ingeneral, T cells are activated when they specifically recog-nize a complex formed by afragmentofaforeign antigenand anautologous HLA molecule (6). No disease causative antigen has been identified in GCA. The characteristic histological find-ings of the inflammatory infiltrate accumulating around the
internalelastic lamina have ledtospeculations that degener-ated elastic fibers represent the autoantigen initiating and
per-petuatingthe chronic immune response. Thefrequent acute
onsetof clinical symptomsaccompanied byanintense acute
phasereaction has also raised questions ofaninfectiousagent
playingatleastaprecipitatingrole.
Inthisreport, wehavestudiedallelic HLA polymorphisms that areimportant in determining immunoresponsivenessto exogenous antigens. An association ofthe disease with the HLA-DR4 haplotype has beendescribed but reports on the distribution of HLAhaplotypesinGCApatientshave not been conclusive (7-1 1 ). To address the question whether gene
prod-uctsof the HLA class IIcomplex mightcontribute to GCA, we have analyzed the functionally most important locus, the HLA-DRB1 locus, inacohortof patients with biopsy-proven GCA.Three allelicvariants ofthe HLA-DRB 1 *04 family were found to be overrepresented among GCA patients.
Interest-ingly,GCA islinked tothe very same allelic variants as RA.
However,despitethe overrepresentation of HLA-DRB 1 *04 in GCAandRA,therisktodevelopbothdiseasesis low,
suggest-ing that distinct pathomechanismsunderlie the immune
abnor-malities in both diseases. More importantly, we found a se-quence motifspanning the aminoacid positions 28-31 that is sharedbyallGCApatients and that is different from that found in RA. This sequence stretch translates into a polymorphic site in theantigen-bindinggrooveof the HLA-DR molecule,
sug-gestingacrucialrolein the selection and presentation of anti-gentoTlymphocytes.
Methods
The JournalofClinical Investigation,Inc. Volume90, December 1992, 2355-2361
Patients. 42patientswho wereseen in the Division of Rheumatology,
the study. 22patients were newly diagnosed, and 20 patients were diag-nosedbefore 1991. All patients had characteristic histological findings onbiopsy.41 of thepatientswereCaucasian and 1 was Asian Indian. 32 patients were from a local geographic area. To assess the
demo-graphicandclinical characteristics of the studypopulation,thefiles of
all 42patientswerereviewed. Datafor thefollowingparameters were collected: age at onsetofthedisease;sex; presence ofmusculoskeletal
pain confinedtotheshoulder andpelvic girdle, indicatingsymptomsof
polymyalgia rheumatica (PMR);and permanentvisionloss. To docu-ment permanentvision loss,atrophyoftheopticalnerveasdiagnosed
by anophthalmologistwasrequired.GCApatients diagnosed before
1989 were evaluatedforrelapsesofthedisease orrequirement for long-termcorticosteroid therapy. Patientswereidentifiedwhorequired corti-costeroidtherapyfor>2 yr.
Toidentify patients who had biopsy-proven GCA as well as seropos-itive RA, we used acomputerizedsearch of the Mayo Clinic data base for theperiod1976-1990. 835patientscarried thediagnosisof
biopsy-provenGCA. Within that cohort, 22patientswerediagnosedwith poly-arthritis. The charts of these 22patientswerereviewedtoidentifythe
underlyingdiseasecausingthepolyarthritis. Thediagnosis of
polyar-thritis secondarytoGCAwasverifiedinpatientswhowerenegative for rheumatoid factorandfulfilledtheclinicalandradiological character-istics describedrecently (12). RAandsystemic lupus erythematosus
werediagnosedaccordingtothe criteria of the AmericanCollege of Rheumatology (13, 14).Toestimate theexpectednumbersofcasesof
RAwithinacohortof835patients,data of Linosetal.(15)wereused. Theprevalence of definiteandclassical RAin 72-yr-oldindividuals wascalculated from the accumulated incidenceratesfor acohortof
835 individuals.
Normal controls.Agroup of 63 individualswasanalyzedfor HLA-DRB1 alleles between 1990 and 1991. None of the normals had any
current orpriorautoimmune diseases. Individuals withafamily his-tory ofaninflammatory rheumaticdiseasewere excluded. 62of the normal controlswereCaucasianand 1wasAsian Indian. 50 of the 63 controlswerefrom thesamepopulationbaseasthe localpatients.
DNA analysis. Peripheralblood mononuclear cellswereusedas a sourceforgenomicDNA.Allele-specificoligonucleotideprimers
spe-cific forthe sequencepolymorphismwithin the firsthypervariable
re-gion (HVR)andforanonpolymorphicstretch from amino acid
posi-tions 87-94were usedto amplify HLA-DRB1 genes. Apolymerase
chain reactionwith 30cycleswasused under thefollowingconditions:
denaturation for 2 min at940C, annealing for 2 minat550C, and
extension for 2 min at 720C. To identifysequence polymorphisms encodedbythesecond and third HVR of the HLA-DRBIalleles,
am-plified productswereblotted ontonylon membranes andhybridized
with oligonucleotide probes specificfor sequence polymorphismsof
the different DRB1 alleles. Theprimer setsand theoligonucleotide
probes used for theidentification ofthe HLA-DRBIalleles have been recently described (16, 17). Sequencepolymorphism atposition 57 within the HLA-DRB1 *04alleles,which isinfrequentinaCaucasian
population,wasnotdetermined.Thus,the approachdidnotallow a
distinctionbetween DRB1*0404and *0405. Forhybridization,
oligo-nucleotide probes were labeled with digoxygenin UTP and
subse-quentlydevelopedwith anantidigoxygeninalkaline phosphatase-la-beledantibody.
Statisticalanalysis.Demographicand clinical characteristics ofthe
patientsubsets and thefrequenciesofHLA-DRBI alleles inpatients
andnormalswere compared byttest,Chi-squaretest, or aFisher's
exactprobabilitytest asappropriate.Accumulated incidencerateswere
i=72
calculatedusingtheequation1-e- z i;wherei=yearlyincidence
rates. i-O
Results
Overrepresentation
of
two allelic variantsof
theHLA-DRBI*04haplotypeinGCApatients.42
patients
withbiopsy-provenGCAwereenrolled into this
study.
The groupincluded9 males and 33females.Allele-specific amplification of
geno-micDNA was used to identify both HLA-DRB I alleles in each
patient. As shown inTableI,thedistribution ofHLA-DRBl
allelesamongGCA patientswasdifferent fromthat in normals. HLA-DRB1*04was present in 59.5% of the patients. Three
allelic variants,B1*0401,B 1*0404,and B 1 *0408, were
identi-fied in the patient cohort.B 1*0404and*0408are distinct only atposition 86 (V-- G) andwere grouped together. Three of sevenpatientstypedHLA-DRB 1*0408. Interestingly,noneof
theHLA-DRB 1*04+patientsused the B 1*0402and B 1*0403 allele.Thesetwoallelesare rareinthe normalpopulation.The
second most frequentallele was HLA-DRB1*03.
Overrepre-sentationofHLA-DRB 1*03,however,didnot reachstatistical significance. Theoverexpression ofHLA-DRB 1*04within the
GCA cohort was accompanied by a reduction of HLA-DRB1*01. A reduced frequency was also seen for HLA-DRB I*l1, B 1*07, and BI *15/16. An equal representation within patientsandnormalswascharacteristicforB1*08 and
B 1*13. We subsequently analyzed the role of allelic
combina-tions in the patientgroup. Fig. 1 summarizes the haplotype combinationsatthe Bl locus for the 25 HLA-DRB1 *04+
pa-tients. In Fig. 2, both HLA-DRBl alleles ofthe 17 HLA-DRB 1*04-patientsareshown. TheHLA-DRB 1*04- patient subset wascharacterized by a nonrandom distribution of al-leles. ThreeHLA-DRB 1variantswereoverrepresented: HLA-DRB 1*03,B 1*08, andB I*13.HLA-DRB 1*03wasfound in 8 of17patientscompared with 11 of 48 DRB 1*04-controls (P = 0.06), DRB 1*08 wasfound in 6 of 17 patients compared with 6 of 48 controls (P=0.04). Onlyfour patientswere
nega-tivefor all three alleles. These fourpatients shared the HLA-DRB 1*15 /16haplotype.HLA-DRB 1*01 and*07wereclearly underrepresented inHLA-DRB 1*04-patients (5.8vs.27%,P = 0.06; and 5.8vs. 29%,P=0.04,respectively) (Fig. 2).The
HLA-DRB 1*0301 allelewas notonlyfrequentlyseenin non-DR4patients,butwasalsopreferentiallyexpressed in
combina-TableI.FrequenciesofHLA-DRBJAllelesinGCAPatients
and Normals
GCApatients Normals
DRBlallele (n =42) (n =63) P Pcorr*
01(DR1) 11.9 28.6 0.043 NS 15/16(DR2) 26.2 33.3 NS NS
03(DR3) 28.6 19.0 NS NS
04(DR4) 59.5 23.8 0.0002 0.003
0401 42.9 15.9 0.002 0.03 0402 0.0 1.6 NS NS 0403 0.0 4.8 NS NS
0404/8 16.7 3.2 0.016 NS
11(DR5) 9.5 22.2 NS NS
07(DR7) 14.3 22.2 NS NS
08(DRw8) 14.3 11.1 NS NS
09(DR9) 0.0 3.2 NS NS
10(DRwlO) 0.0 3.2 NS NS
13(DRw13) 21.4 19.0 NS NS
14(DRw14) 2.4 3.2 NS NS
*Corrected for number of variables tested.
FirstDRB1 Allele
Second DRBI Allele 401 402 403 404/8
GCA-1 /( -
-GCA-2 0 - - - 01
GCA-3 - - - 01
GCA-4 01
GCA-5 - 15/16
GCA-6 03
GCA-7 - - - 03
GCA-8 - - - 03
GCA-9 . . - 03
GCA-10 - - - 03
GCA-11 - - - 11
GCA-12 - - - 07
GCA-13 - 07
GCA-14 - * - 07
GCA-15 - - - 08
GCA-16 - - - 13
GCA-17 - - - 13
GCA-18 - - - 13
GCA-19 . . . 0 01
GCA-20 - - - 0 15/16
GCA-21 - - - 0 15/16
GCA-22 - - - 0 15/16
GCA-23 - . . 0 03
GCA-24 - - - 0 07
GCA-25 - - . 0 07
Figure 1.Alleliccombinationsatthe HLA-DRB1 locus in
HLA-DRB1*04-positive patients.
tions with one of the HLA-DRB 1*04 variants (Fig. 1).
Al-thoughnosingleHLA-DRB 1 allelic variant can bepinpointed as aprimary geneticcomponentinGCA, the nonrandomness
of HLA-DRB 1 genes present inGCApatients suggests thata
geneticelement linked to the HLA class IIcomplex is relevant
in thepathogenesis of the disease.
Clinical presentation in HLA-DRBJ*04+ and
HLA-DRBJ
*04- GCA patients. Genotyping for the HLA-DRBllocus indicated that individuals expressing one oftwoallelic variants, B1*0401 and B1*0404/8, hada higher risk to de-velopGCA.However, 40.5% ofall patientswerenegative for thetwodisease-associatedhaplotypes.Wethereforeaddressed
thequestionwhether theclinicalpresentationof thevasculitic syndrome was different in HLA-DRB1*04+ and HLA-DRB1*04- patients. The patientgroup was analyzed for the followingparameters: age atdisease onset, sex,symptoms of PMR, relapses ofsymptoms in patientswho werediagnosed before 1989, and permanent visual impairment. As demon-strated in TableII, expression oftheHLA-DRB1*04allelesdid
notaffectthe age atdiseaseonset and symptomsofPMR were
foundinsimilar frequencies inbothpatientsubsets. In general, allpatients responded well to theinitial high-dose corticoste-roidtherapy. Both patient subsets included individuals who relapsed and required the resumption of steroid treatment. Among the 17 HLA-DRB1*04- patients, 3 received metho-trexate orazathioprineinadditionto prednisone. None of the HLA-DRB1*04+patients was treated with an additional im-munosuppressive agent. Interestingly, we identified four
pa-Patient
GCA-26 GCA-27 GCA-28 GCA-29 GCA-30 GCA-31 GCA-32 GCA-33 GCA-34 GCA-35 GCA-36 GCA-37 GCA-38 GCA-39 GCA-40 GCA-41 GCA-42
FirstDRB1 Allele
03
03 03 03 03 03 03 03 08 08 08
13
15/16 15/16 15/16 15/16
Second DRB1 Allele
LI
111
15/16 13 13 13 08
08
08 07 13
14 11
11
01
15/16
Figure 2.AlleliccombinationsattheHLA-DRB1 locus in HLA-DRB1*04-negative patients.
tients withpermanentvision loss duetoischemic
opticus
neuri-tis.Allfourpatientscarried theB 1*0401orB 1*0404/8 allele.
Thisfindingindicates that theHLA-DRB 1*04+patients might developmoreextended disease withmorerapid progression.
Two HLA-DRBJ *04-associated diseases, GCA and RA, arenotlinked.Theallelicvariants of HLA-DRB 1 *04 that are
overrepresented in GCApatientsaretherisk-conferringalleles
for yet another autoimmune disease, RA. The seropositive form ofRAistightlylinkedto asequence motif encodedbythe
HLA-DRB1 locus, which has beenmappedtotheHVR3 ofthe HLA-DRB I locus. The RA-associatedsequencemotif ismost
frequently found integrated into the HLA-DRB1 *04
haplo-type, butitcanalso beintegratedinto the HLA-DRB 1*0101 and*1402 alleles. The commonassociation of GCA and RA
with the allelic subtypesDRB1*0401 and*0404/8 raises the
question whether there is an increased risk for HLA-DRB1*04+ individuals to develop both diseases. Both
syn-dromes are morecommon infemales, but theage of disease onsetmay be decades apart. To investigate whether both
HLA-TableII.ClinicalCharacteristics of GCA Patients
HLA-DRB1*04+ HLA-DRB1*04-patients patients (n=25) (n=17)
Age atdiseaseonset(yr) 71.2±8.4 72.9±7.9 Sex (%females) 76.0 82.4 Symptoms ofpolymyalgia
rheumatica (%) 56.0 64.7 Permanentvisualimpairment (%) 16.7 0.0
Numberofpatientsrequiring
DRB 1*04-linked diseasesarefoundtofrequentlyco-occur, we
analyzedacohort of 835consecutivepatients with tissue-con-firmed diagnosis of GCA. This cohort included all patients with GCA from 1976 to 1990 who had a positive temporal arterybiopsy within this institution. Among the 835 patients, 22werediagnosedtosuffer from polyarthritis before,
concomi-tantwith,orafter theonsetofGCA. On review ofthe records of these 22patients,wefoundthat1patientwasfalsely enteredas
GCA. The 21patientswereanalyzedtoidentifytheunderlying
conditionleadingtothediagnosis of polyarthritis. The results
oftheanalysisaregivenin TableIII. Weonly foundonepatient
who hadseropositiveRAandwas diagnosedtohave GCA.He
haddevelopedRAin 1937 andreceivedsalicylates,
corticoste-roids, andfinally parental gold. The disease tookadestructive
and nodularcourse.Over the severalyearsbefore thediagnosis
ofGCA hisjointdiseasewasstable andnotexplicitly active.In
1976, he was admitted with bitemporal headaches, malaise, and weightloss. Temporal artery biopsyshowed
granuloma-tousarteritis. Within the cohort of 835patients,therewere no
femalepatients withahistoryofseropositiveRA.Themajority ofpatients in the cohortwiththecooccurrence of GCA and
polyarthritishadaseronegativevariant ofasymmetric polyar-thritis,which insome casespresented withjointspace
narrow-ing and erosions. Thistype ofseronegative polyarthritis has beendescribedas amanifestationofjointdisease in GCA pa-tients.Theaccumulated incidencerateforRAinapopulation
withan ageof72yr was19of606 females and 3.4of229 males ( 15).These data indicate that the risk ofan HLA-DRB 1*04+ individual todevelopseropositiveRAandGCAisverylow.
A sequence motifsharedbyHLA-DRBI *04+ and HLA-DRBI *04- GCApatients. RA has been linked to a set of HLA-DRB1 genes,allofwhich share thedisease-associatedsequence motifin the HVR3ofthe
HLA-DR#
I chain. Themostimpor-tantmembersofthegroupof alleles
conferring
the riskto de-velop RA are the HLA-DRB1*0401, HLA-DRB1*0404/8,
and HLA-DRB 1*0101 alleles. Data presented in Table I dem-onstratethatthe HLA-DRBI *0101 allele is not enriched in
GCApatients. Thisalleleisrather diminished, in particular, in
theHLA-DRB1*04-GCA population (Fig. 2), indicating that
thesequencepolymorphismof the HVR3 found in RA is not relevantfortheriskof GCA patients to develop the vasculitis. Thegenotypingfor the HLA-DRB 1 genes in GCA revealed an
overrepresentation of HLA-DRB1*04 alleles but also a
nonrandomspectrum of non-DRB 1 *04 haplotypes. This
find-ing raisedthe possibility that a region of sequence homology
integrated into HLA-DRB 1*04, but also into some of the DRB1*04- gene products, was shared by GCA patients. To
identifya common denominator among the GCA patients, we
compared thesequence polymorphism of the HLA-DRB 1 al-leles B 1*0301,BI * 1301 /2, and B 1 *0801 frequently found in DRB1*04- GCA patients with B 1*0401 and *0404/8. The disease-associated alleles share a sequence motif encoded by
the HVR2; in particular, the polymorphic amino acid
posi-tions 28, 30, and 31 are identical (Fig. 3). The amino acid sequence motifDRYF, whichwas expressed by all 42 GCA
patients, isnot presentinthe HLA-DRB 1 alleles*01and *07,
whichwerefoundto be decreased in the GCA population. The recentresolution ofthe three-dimensionalstructure of an HLA molecule has allowed correlation of polymorphic sites with functional regions ofthemolecule(18). Human HLA-DR
mol-ecules are expressedon the cell surface as aheterodimer.
#-pleated sheets encoded by theHVR 1 and HVR2ofthe a and/ chain form the bottom ofagroovethatissurrounded by two
a-helical formationsbuilding the walls ofthe cleft (Fig. 4).
Thereis overwhelming evidencethat the groove represents the
binding siteforantigenic peptidesthat are embedded between
thea-helical walls. The complex formedby theantigenic
pep-tide bound in thegrooveof theHLAmolecules represents the
ligandfor theTcell receptor andis requiredto initiate T cell activation.Thus,ourdataindicate thatapolymorphic region
TableIII. Co-occurrenceofGCAandRA
Found Expected
Total Female Male Female Male
n
Biopsy-provenGCA 835 606 229
Patients withpolyarthritis 22 19 3
Misentry 1 1 0
Patients with GCA andpolyarthritis 21 18 3 Diseaseentitycausingpolyarthritis
Polyarthritisin GCA 11 9 2
Osteoarthritis 3 3 0
Gout 1 1 0
Seronegativemonoarthritis/oligoarthritis 2 2 0
Systemic lupus erythematosus 1 1 0
Rheumatoid arthritis 3 2 1 19/606 3.4/229
Seronegative,nonerosive 2 2 0
Seropositive 1 0 1
The Mayo Clinic data bankwasusedtoestimate howfrequentlypatientswithbiopsy-provenGCA carriedthediagnosisofRA.835patientswith GCAwereregisteredbetween 1976 and 1990,22werediagnosedtohavepolyarthritis.Charts of these 22patientswerereviewed.Expected prevalenceratesin72-yr-oldpatientswerecalculatedastheaccumulated incidenceratesforacohort of 606 female and 229 male individuals from the datapublished byLinosetal.(15).
HVRI AAposftlon 4 910 11 12 13 14 DRB1allele
DIseasesociated
401 R E Q V K H E 404/8 R E Q V K H E
0301 R E Y S T S E 1301 R E Y S T S E 0801 R E Y S T R E 1501 RW QP K R E
Diseas non-associated
0101 RW QL K F E 0701 QW Q G K Y K
HVR2 26 28 30 31 32 33 37
F D Y F Y H Y F D Y F Y H Y
Y D Y F H N N F D Y F H N N F D Y F Y N Y F D Y F Y H S
L E C I Y N S F E L F Y N F
HVR3
57 5860 67 70 71 73 74 78 86
D A Y LQ K A A Y G D A Y LQ R A A Y V
D A Y LQ K G R Y V D A Y ID E A A Y V S A Y FD R A L Y G D A Y IQ A A A Y V
Figure3.Conservedregion ofsequence
polymor-D A Y L 0 R A A Y G phism inHLA-DRBl allelesexpressedbyGCA
pa-V A S ID R G aV G tients.
on the floor of theantigen bindingsite of the HLA-DR
mole-cule is sharedbypatientswhodevelopGCA
(Fig.
4).Discussion
A lymphocytic infiltrate in the walls of medium- and large-sized arteries is thecharacteristic findingin GCA (4, 5).
Im-portantclinical features associated withthevasculitisarethe frequent acute onset of symptoms,abriskproduction ofacute
phase reactants, and the prompt response of symptoms and
acutephaseproteinstocorticosteroids. Evidence foracrucial roleof Tlymphocytescomesfromthe demonstration ofCD4+
Thelper cellsconstitutingthedominating cellpopulation in theinflammatory infiltrate and reportsdescribinga selective depletion and activation of CD8+ T cells in the peripheral
blood (5, 19). Thegoal ofthis studywas toinvestigateHLA
molecules that exert a considerable control of T cell function. CD4+ T cells recognize foreign antigen only when displayed in association with self-HLA-DR molecules (6). Thus, HLA mol-eculesare anabsolute requirement for selection and binding of antigenic determinants and the subsequent triggering of the T
cellreceptor.
The polymorphism of HLA-DR molecules is mainly deter-minedby the highly polymorphic HLA-DRBI locus for which
an estimated 80 alleles have been described (20). Here, we
report that the expression of HLA-DRB1 alleles bypatients with GCA is nonrandom. 60% ofthe patients carried one ofthe closely related allelic variants, HLA-DRB1 *0401 and *0404/ 8. The difference between these three alleles is limited to a
polymorphism in residue positions 71 and 86 of the B1 gene product. Two otherallelic products ofthe HLA-DRB 1 *04 fam-ily, B 1 *0402 and *0403, were not identified within the patient population.
k Figure 4. Mapping ofthe GCA-linked sequence motif totheantigen-bindingsite ofthe HLA-DR molecule.
The HLA-DR sequencewasmodeled on the crystal structureof the HLA-A2asdescribed by Brown et al.
( 18). TheconservedtetrapeptideDRYF
encompass-ingamino acid positions28-31 maps to the
antigen-bindinggroove.Amino acid positions 28 and
30arepredictedtopointupand are highlighted as
AlthoughanHLA-DR association of thediseasehasbeen reported, data have been controversial. Several studies have attempted toidentifyaGCA-associated haplotype (7-1 1 ). All available data usedconventionaltechniques and could not
dis-tinguishamongallelicvariants of the DRB 1 *04 family. In all reports, the frequency of HLA-DR4 was increased, although thedifferenceamong the patientpopulationand normal con-trolsdid not always reach statistical significance. Interestingly, the HLA-DR4 association of GCA is supported by differences in the incidence of the disease in distinct ethnic groups. An increased frequency of the vasculitis in Caucasians compared with black individuals has been described. The highest inci-dence has been reported in Scandinavia and Minnesota, mainly including a population of Northern European descent (2, 3).Studiesof Italian and Israeli populations have demon-strated lowfrequencies(21, 22). These ethnic differences corre-late to the frequency of HLA-DR4 expressed in the different
populations.
The finding that 40% of the patients were negative for B1*0401 and B 1*0404/ 8 indicated that the contribution of the HLA-DR molecule in GCA is probably notrelated to a
unique function oftheDRB 1*04 allele.Wehypothesized that the disease is linkedto asequencepolymorphismpresent in the
twofrequentHLA-DRB 1*04 alleles but also expressed in
non-DRB 1*04 alleles. Thishypothesiswassupported by our find-ing that the distribution ofthe DRB1 genes in HLA-DRB1*04-patientswasnonrandom.HLA-DRB1*03,B1*13, andB 1*08were morefrequentin the HLA-DRB1*04-patient
populationthan in the controlpopulation. Sequence
compari-sondemonstratedthat all allelesoverrepresentedin thepatient populationhadadiscreteregion ofsequenceidentity stretching
from residues 28 to 31. This sequence stretch includes one nonpolymorphic residue inposition 29. The sequence motif DRYF wasfound in all42 GCApatients. Interestingly, two
haplotypesfrequently encounteredin the normalpopulation,
HLA-DRl
and HLA-DR7,carry verydifferentamino acid resi-duesatpositions 28, 30,and 31. The HLA-DR 1haplotypewasunderrepresentedin the GCA
population.
ExpressionofHLA-DR7waslimitedtoGCApatientswhoexpressedthe disease-linked sequence cassette DRYF on their second haplotype.
These data suggested that a single copy of the disease-asso-ciatedsequence stretch is sufficienttoconfer disease
susceptibil-ity and that sequence variations in residues28, 30,and31 on
the second haplotype appear not to be protective. The
se-quence stretch DRYF is also found in the DRBI*11 and
DRBI*15116alleles, and in the 0101 and0301 alleles of the HLA-DRB3 locus. HLA-DRB1 * 11wasinfrequentinthe total GCA
population;
HLA-DRB1*15/16 was found in the four patientswhotypednegativefor HLA-DRBI alleles03, 04, 08,and 13. Thefindingthat the presence ofthemotifDRYFby
itselfdoes not necessarily confersusceptibilityto GCA
indi-catesthatotherregionsoftheHLA-DRf I chainhavea
modu-latoryeffectonthebiological function ofthe HLA-DR mole-cule,resultinginanincreased risktodevelopGCA. This
inter-pretation is supported by the finding that there is a clear hierarchy ofHLA-DRB1 allelespredisposing for GCA, with
HLA-DRB1 *0401 being the strongest risk factor. The
exis-tence of distinct roles for different disease-associated alleles sharingaparticularsequence stretch isnotuniquefor GCAbut
isalsoobserved forRA. The HLA-DRBI alleles*0101, *0401,
*0404/8,
and*1402,whichareassociated withRAand shareasequence stretch in theHVR3,clearly have distinctimpactsin
susceptibilityand severity of the disease (17).
Itcould be argued that theHLA-DRBI locus does not repre-sent the primary genetic factor linked to GCA. HLA haplo-typesincludemultiple loci that arecharacterizedby a linkage
disequilibrium.NoHLA-DQAor B allele can beidentifiedthat could represent a common denominator among the DR4, DR3, DRw8, and DR 13 haplotypes. In line with this finding, previousstudieshave failed to establishanHLA-DQ associa-tionofthe disease (8).
The comparison of theclinical findings in the DR4' and DR4- patient subsets alsosupportedthe view that the patients suffered from a similar diseaseentity. Especially,wecould not confirm the results of a study applyingmetaanalysisto evaluate the roleof HLA-DR4in GCA (10). These authors discussed
that theoverrepresentationofHLA-DR4might be caused by a linkage ofHLA-DR4toPMR.In ourclinically
well-character-ized patient cohort, symptoms ofPMR were encountered as
frequently inthe DR4- as the DR4' patientsubset. Interest-ingly, allpatientswho haddeveloped blindnessdue toischemic opticus neuritis carriedthe HLA-DRB1*0401 or *0404/8 al-lele. In earlystudies ofGCA,blindnesswas a much more fre-quent complication. The increased awareness of physicians probablyhas lead to earlydiagnosisandtreatmentbefore irre-versibleischemicdamage hasevolved. However,thefindingof a higher frequency of blindness in the HLA-DRB1 *0401/4/ 8+ group mayindicate thatthe disease progresses at a faster
rate in these individuals, suggesting a potential role ofthese
allelic variants in disease severity.
Extensive studies on yet another functional region of the
HLA-DRB1*04 alleleshave
demcnstrated
that a discretepor-tion ofthe HLA-DR molecule can function as the primary
genetic unit underlyingHLA anddisease association. A set of
alleles at the DRB1 locus, including HLA-DRB1 *0401, *0404/8, *0101, and*1402, have beendirectly implicatedin the pathogenesis of seropositive RA (23-27). The polymor-phism that is shared by this set ofRA-associated alleles but
distinct from severalcloselyrelatedHLA class II alleles that are notassociated withRA has been mappedto a short sequence
stretchofDRB 1allelesspanningcodons 67-74 (28-30).Here, we present data that a different segment of the DRB1 gene
found inB1*0401 andB 1*0404/8 accountsfortheassociation ofDRB 1*04 alleles with GCA. This interpretation is supported
by theunderrepresentationofHLA-DR I intheGCA popula-tion, indicating that the sequence polymorphism within the
HVR3region ofthe geneisnot relevant for theriskto develop
GCA. Our finding thattwodifferent polymorphicdomains of the HLA-DR moleculearelinkedtotwoseparate disease enti-tiessuggeststhatdistinct biological functions ofthe HLA-DR4
moleculesareimportant inthepathogenesis of GCAand RA. Thishypothesis is strengthenedby
epidemiologic
dataanalyz-ing thecooccurrence ofRAandGCA.Intheconsecutivecase
series of835patients with biopsy-proven GCA,weidentified
only 1 patient who had classicalseropositiveRA,amalewho
developed arthritis before GCA. Therewere nofemalepatients withseropositive RA.This indicatesthat thecooccurrence of
these two syndromes iscertainly notincreased, even though
they share ageneticmarker. Theresults ofthe caseseriesare
consistent withthepaucityofreportsintheliterature
describ-ing GCAinpatientswhopreviouslyorsubsequently developed
RA
(
31).
Suchcaseshavebeenreported
fromthis institutionandinclude the one caseidentified in our current case series
analysis. Somereportshave indicatedanincreased mortalityof RA patients, which, however, is notsufficient toexplain the low cooccurrenceofbothdiseases. There isno reason to believe that cases with both GCA and RA wouldnotbe recognized clinically. Rather, these datasuggestthepossibility that GCA
patientsmayhaveanactually reduced risktodevelop RA.The exactpathological mechanisms leadingtoseropositiveRAare
notunderstood,althoughacrucial role ofTlymphocytesinthe
disease has been established. It could be speculated thatthe
immune abnormalities characteristic for RAprevent an
abnor-mal immune response resulting ingranulomatous vasculitic vessel wallinfiltrates in GCA.
The definition ofasequence cassette linked withdisease
susceptibility is important for ourunderstanding ofimmune
functions in GCA patients. Experimental evidence has been
provideddemonstratingthatadiscrete cluster of polymorphic sites withinan HLA-DR sequencecanfunctionasafunctional unit on different allelic backgrounds. Defining the sequence stretch DRYF as the genetic element predisposingto GCA
raises thequestionontheimportance ofthis unit in immuno-logical recognition. Modeling of the HLA-DR molecule pre-dicts that adiscreteantigen bindinggroove is surroundedon
bothsides by a-helicalloops ( 18).Inthismodel,residues
56-86of the :chain, includingthe sharedepitopelinkedtoRA, form oneof thea-helical loops.In contrast,residues 28-31 of thef chainlinkedtoGCA would contributetothe floor ofthe peptidebinding cleft. Data derived from detailed studies ofthe functional consequences of sequence variations in murine MHC moleculesindicate that the amino acid inposition 28 is of crucialimportancefor the selectivebindingofantigen
frag-ments(
18).
Our datasuggestthatfurtheranalysis
of the role of this residue in the immuneresponseof GCAmayprovide in-formation on the possibility that a specific disease-causativeantigen mightbe boundby the disease-linkedHLA polymor-phismthatinitiates the vasculitic reaction in the arterialwall. Acknowledgments
We thankMiss T. L. Buss for providing us with secretarialassistance.
This work was supported in part by agrant fromthe American HeartAssociation and the Mayo Foundation.
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