Immunogenetics and Clinical Phenotype of Sympathetic Ophthalmia in British and Irish Patients

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  Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irishpatients Dara J Kilmartin, David Wilson, Janet Liversidge, Andrew D Dick, Julia Bruce,Robert W Acheson, Stanislaw J Urbaniak, John V Forrester Abstract  Background/aims  —Sympathetic ophthal-mia (SO) is a classic example of autoim-mune disease where human leucocyteantigen(HLA)genomicassociationscouldprovide further understanding of mecha-nisms of disease. This study sought toassess HLA genetic polymorphism inBritish and Irish patients with SO, and toassess whether HLA gene variants areassociated with clinical phenotype ordisease severity.  Methods  —High resolution DNA basedHLA typing using polymerase chain reac-tion sequence specific primers was per-formed in 27 patients with SO and 51matched healthy controls. Clinical pheno-type and markers of disease severity weredetermined prospectively in 17 newlydiagnosed patients and from medicalrecord review and repeat clinical exam-ination in 10 previously diagnosed pa-tients.  Results  —HLA-Cw*03 (p=0.008), DRB1*04(p=0.017), and DQA1*03 (p=0.014) weresignificantly associated with SO. For classII alleles at higher resolution, onlyHLA-DRB1*0404 (relative risk (RR) = 5.6,p = 0.045) was significantly associatedwith SO. The highest relative risk for anyof the associated haplotypes was withHLA-DRB1*0404-DQA1*0301 (RR=10.9,p=0.019). Patients with the DRB1*04-DQA1*03 associated haplotype were sig-nificantlymorelikelytodevelopSOearlier,with fewer inciting ocular trauma events,and to require more systemic steroidtherapy to control inflammatory activity. Conclusions—  Sympathetic ophthalmia isassociated with HLA-DRB1*04 andDQA1*03 genotypes in white patients,similar to Japanese patients. Di V  erencesin DRB1*04 gene variant associations(−0404 in Britain and Ireland and −0405 in Japan) may have implications for HLApeptide binding in disease initiation. TheDRB1*04–DQA1*03 haplotype is a markerof increased SO susceptibility and sever-ity, as in Vogt-Koyanagi-Harada disease,which also has similar clinicopathologicaland HLA associations. ( Br J Ophthalmol   2001; 85 :281–286) Sympathetic ophthalmia (SO) is an uncom-moncauseofbilateralgranulomatousposterioruveitis which follows injury to one eye, fromocular surgery or penetrating ocular trauma.Although rare, with a recent reported inci-dence of 0.03/100 000, 1 it remains a seriouseye disease because of its potentially blindinge V  ects on both the exciting injured eye and thecontralateral sympathising eye and usually thepatient is committed to receiving chronicimmunosuppressive therapy to preserve goodvision. 1 2 SO is one of the most feared compli-cations of ocular surgery, and recent evidenceshows that the proportion of SO casesfollowing retinal surgery is increasing. 1 2 SO isalso of particular interest to immunologists asit is regarded as the prototype for autoimmunedisease, where breaching of systemic ocularbarriers compromises the relative immuneprivilege of the eye, and causes sensitisation topreviously sequestered uveoretinal antigens.Although many candidate ocular antigens havebeen implicated, 3–5 the precise autoantigen, ordisease inducing peptide, has yet to bedetermined.Endogenous posterior uveitis (EPU) is aCD4+ T (helper) cell mediated disease 3 and,inSO, both peripheral blood and vitreous T lym-phocytes have been shown to be reactive toretinal antigen stimulation. 5 Immunohisto-chemistry of enucleated exciting eyes in SO hasdemonstrated the pathogenic role of bothCD4+ T cells in the initial stages of disease,and CD8+ T (suppressor) cells in later stages. 6 Human leucocyte antigen (HLA) associationswith autoimmune disease are central to under-standing disease mechanisms in ocular inflam-matory disease,including SO.HLA gene prod-ucts, through peptide-HLA binding a Y nity,can uniquely influence the presentation of dis-ease inducing peptides, or adjust the develop-ment of the T cell repertoire in the thymus bydeleting autoreactive lymphocytes leading toself tolerance. 7 8 Certain types of clinical andexperimental uveoretinal inflammatory dis-eases are HLA or major histocompatibilitycomplex (MHC) restricted—for example,birdshot retinochoroidopathy, Behçet’s dis-ease, Vogt-Koyanagi-Harada’s disease (VKH),and experimental autoimmune uveoretinitis,and individuals possessing the HLA/MHCsusceptibility gene have increased diseaseseverity in both human and animal disease. 9–13 A precise HLA association with SO could pro-vide further understanding of mechanisms of disease pathogenesis, help reveal a diseaseinducing or tolerogenic peptide, and explainvariations in clinical phenotype seen in patientswith SO. Br J Ophthalmol   2001; 85 :281–286 281 Department of Ophthalmology,University of Aberdeen, Scotland,UK  D J Kilmartin J LiversidgeA D Dick J V Forrester Tissue TypingLaboratory, Aberdeenand NortheastScotland BloodTransfusion Service,Scotland, UK  D Wilson J BruceS J Urbaniak Department of Ophthalmology, MaterMisericordiaeHospital, Dublin,Ireland R W Acheson Correspondence to:Dara J Kilmartin,Department of Ophthalmology, Royal PerthHospital, Wellington Street,Perth WA 6000, for publication8 November 2000  group.bmj.comon October 22, 2014 - Published by bjo.bmj.comDownloaded from   Both SO and Vogt-Koyanagi-Harada(VKH) disease have been shown to have simi-lar clinical and immunohistopathological fea-tures, and identical associations with HLAclass II genes (HLA-DRB1*0405 andDQB1*0401) in Japanese patients. 12 14–16 Inaddition, Japanese VKH patients with theHLA-DRB1*0405 subtype were shown tohave significantly increased disease severity. 11 12 However, HLA-disease associations are de-pendent on allelic frequency which varies dra-matically from one ethnic group to another. 17 18 In western SO patients, no DNA based typinghas been performed but two older serologybased studies found an increase in frequency of HLA-DR4 19 20 and HLA-DQ3. 20 In this study,we sought to assess HLA genetic polymor-phism and the development of SO, and toassess whether HLA gene variants were associ-ated with clinical phenotype or disease severity. Methods PATIENTS AND CONTROLS Twenty seven patients (14 male and 13 female)with newly diagnosed and established SO wererecruited from throughout the UK and Ireland.Seventeen newly diagnosed patients were re-cruited in 1997 and 1998 through the BritishOphthalmological Surveillance Unit of theRoyal College of Ophthalmologists, as previ-ously reported. 1 Ten established cases wererecruited from uveitis clinics at GrampianUniversity Hospitals, Aberdeen, and the MaterHospital, Dublin. All patients were white andwere native ethnic English (14 patients), Scot-tish (eight patients), Irish (four patients), andNorthern Irish (one patient). All patients hadbilateral posterior or panuveitis with a definitehistory of ocular trauma (accidental or surgical)followed by contralateral posterior uveitis withfeatures clinically consistent with SO. Nineteenpatients were diagnosed with SO by a uveitissubspecialist and eight had been diagnosed by aretina subspecialist. Although the diagnosis of SO was determined clinically, enucleated excit-ing eyes from five patients demonstrated histo-logical features consistent with SO.All referringophthalmologists returned questionnaire dataon clinical features, and 12 (two newly diag-nosed and 10 previously diagnosed) patientshad SO clinical features confirmed on examina-tion by three study investigators (DJK, ADD, JVF).Clinicalphenotypeandmarkersofdiseaseseverity assessed included visual acuity, timeintervalbetweenSOonsetandlastocularinjury,number of preceding inciting injuries, ophthal-moscopy features, such as degree of vitreouscellular infiltrate or haze (assessed by slit lampbiomicroscopy and binocular indirect ophthal-moscopy (BIO)), 21 macular and disc oedema,Dalen-Fuchs nodules and choroidal neovascu-larisation, and therapeutic interventions such asenucleation and immunosuppressive therapy.Recurrence of uveitis was defined as an increasein disease activity, with worsening of visual acu-ity and/or BIO score, requiring an increase inimmunosuppressive therapy from previouslystable maintenance therapy. Median follow upwas 12 months (range 6–354 months).Controlsfor HLA typing were age, sex, and regionmatched. Two controls were obtained for eachpatient and these were normal unrelated volun-teers without any history of uveitis or otherautoimmune disease attending the same hospi-tal or primary care clinic as the patient. Thisyielded 51 controls for HLA class I and class II(HLA-DRB1,HLA-DQB1) typing,as only onematching control could be obtained for threepatients, and 49 controls for HLA-DQA typingas insufficient DNA was available in twocontrols. Informed consent was obtained fromboth patients and controls and the studyadhered to the tenets of the Declaration of Hel-sinki. Local institutional review board approvalwas obtained. HLA TYPING Ten ml of peripheral blood was obtained fromeach patient and control and DNA wasextracted using the salt extraction method. 22 HLA class I (A, B, and C) and II (DRB1,DRQA1, and DRQB1) typing was performedusing polymerase chain reaction sequence spe-cific primer sets (PCR-SSP) (AllSet and Clas-sic PCR-SSP, Dynal Ltd, Liverpool) based onthe method of Olerup. 23 Initially low resolutiontyping was performed for class I and II, andthen higher resolution typing was repeated forclass II subtypes. HLA-DPB1 typing was notperformed as the only previous DNA basedHLA study of sympathetic ophthalmia foundno HLA-DPB1 association. 16 Briefly, DNAsamples were amplified by PCR with 0.38units per µl of Taq polymerase (AppligeneOncor Lifescreen Ltd, Watford) and nucle-otides (final concentration 0.2 mM of each)(Amersham Pharmacia Biotech Ltd, Bucks).All other reagents for PCR were provided inthe PCR-SSP sets and used according tomanufacturer recommendations. The reactionmixture was processed in an automatedthermocycler (Perkin Elmer 9600,Warrington,UK) and subjected to 94 ° C for 2 minutes fol-lowed by 10 cycles at 94 ° C for 10 seconds and65 ° C for 60 seconds, 20 cycles at 94 ° C and61 ° C for 50 seconds, and 72 ° C for 30 secondsfor denaturation, annealing, and extension toallow DNA amplification. After amplification,10 µl of PCR products were electrophoresed ina 2% agarose gel containing ethidium bromidewith 0.5% TRIS-borate-EDTA bu V  er. HLAtypes were assigned on the basis of PCR-SSPpatterns using WHO nomenclature. 24 STATISTICS The   2 test with the continuity correction orFisher exact test were used to compare patientand control groups.Significance was attributedwhen the p value was less than 0.05. The pvalue was not corrected by the number of com-parisons made to correct for the number of alleles. Relative risk (RR) was calculated usingthe Woolf formula (a  ×  d)/(b  ×  c) where a, b, c,and d represented the numbers of patients withthe marker, patients without the marker,controls with the marker and controls withoutthe marker, respectively. 25 When a value was 0,the Haldane modification was used by adding0.5 to every number. 282  Kilmartin,Wilson,Liversidge,et al  group.bmj.comon October 22, 2014 - Published by bjo.bmj.comDownloaded from   Results HLA GENOTYPE FREQUENCIES For British and Irish patients with SO andhealthy controls, the genotype frequencies of HLA class I and class II alleles in are shown inTables 1 and 2 respectively. For class I alleles,only HLA-Cw*03 showed a significantly in-creased frequency (RR=5.2, p=0.008) in SOpatients. For class II alleles, both HLA-DRB1*04 (RR=3.7, p=0.017) and DQA1*03(RR=3.9, p=0.014) were significantly in-creased in SO patients. No significant decreasein allele frequency was observed in SO patientscompared with controls.Higher resolution genotype frequencies of HLA-DRB1 and DQ alleles are shown in Table3.OnlyDRB1*0404wassignificantlyincreased(RR=5.6, p=0.045) in SO patients. WhenDQA1*03 positive patients had higher resolu-tion typing, no significant increase in thefrequency of any DQA1*03 subtype was found.When all significantly increased HLA allelefrequencies were assessed for haplotype fre-quency, the haplotype frequency Cw*03-DRB1*04-DQA1*03 was significantly in-creased (RR=6.7, p=0.013) in SO patientscompared with controls (Table 4). A high rela-tive risk for the associated haplotype Cw*03-DQA1*03 (RR=9.9, p=0.003) was alsoobserved. For the DRB1*04-DQA1*03 haplo-type, the relative risk was weaker (RR=3.5,p=0.024).However,at higher resolution of thishaplotype,DRB1*0404-DQA1*0301,the high-est relative risk was found for any of the associ-ated haplotypes (RR=10.9,p=0.019).AlthoughtheDQA1*0301frequencywasnotsignificantlyincreased alone,when in the significantly associ-ated haplotype DRB1*0404-DQA1*0301, allDQA1*0301 positive patients possessed theDQA*03011 subtype.The haplotype frequencyof C3-DRB1*0404-DQA1*0301 was notsignificantly increased in SO patients. HLA HAPLOTYPE AND CLINICAL PHENOTYPE To assess the influence of HLA gene variants/haplotype on clinical phenotype and diseaseseverity in SO,clinical features were assessed inSO patients with the significantly associatedhaplotype DRB1*04-DQA1*03 (Table 5).Patients with SO and the DRB1*04-DQA1*03haplotype were significantly more likely todevelop SO within 3 months of last ocularinjury (p=0.001),require less inciting events todevelop SO (p=0.038), or require more than10 mg oral prednisolone daily as maintenancetherapy (p=0.031) to control inflammatoryactivity. Although not reaching statistical sig-nificance, there was an increased frequency inSO patients with this haplotype of moderate tosevere vitritis and recurrence of uveitis activitywhile on systemic therapy. In addition, therewas an increased trend in patients without thishaplotype to develop disease more than 1 yearafter last injury. As all DRB1*04 positivepatients also possessed the genotypeDQA1*03, it was not possible to determinewhether the DRB1*04 or DQA1*03 genotypewas associated with this clinical phenotype of increased disease severity.No significant di V  er-ence was found in recognised clinical featuresof SO,such as Dalen-Fuchs nodules or choroi-dal neovascularisation, between patients withand without the DRB1*04-DQA1*03 haplo-type. In addition, there was no di V  erencefound in clinical phenotype or markers of  Table 1 Frequencies of HLA class I alleles in patients with SO  AllelesControls (n=51) Patients (n=27)Relative risk p Value No (%) No %() A*01 20 (39.2) 8 (29.6)A*02 18 (35.3) 13 (48.1)A*03 15 (29.4) 7 (25.9)A*11 8 (15.7) 2 (7.4)A*23 1 (2.0) 0A*24 12 (23.5) 4 (14.8)A*25 2 (3.9) 1 (3.7)A*29 3 (5.9) 5 (18.5)A*30 4 (7.8) 2 (7.4)A*31 5 (9.8) 1 (3.7)A*32 3 (5.9) 1 (3.7)A*34 0 1 (3.7)A*68 1 (2.0) 1 (3.7)A*74 0 1 (3.7)B*07 20 (39.2) 9 (33.3)B*08 15 (29.4) 9 (33.3)B*14 6 (11.8) 3 (11.1)B*15 3 (3.9) 2 (7.4)B18 2 (3.9) 1 (3.7)B*18 2 (3.9) 1 (3.7)B*27 7 (13.7) 1 (3.7)B*35 5 (9.8) 1 (3.7)B*37 1 (2.0) 1 (3.7)B*39 3 (5.9) 0B*40 4 (7.8) 3 (11.1)B*44 16 (31.4) 8 (29.6)B*47 3 (5.9) 0B*49 1 (2.0) 2 (7.4)B*51 1 (2.0) 3 (11.1)B*55 0 2 (7.4)B*57 2 (3.9) 0B*58 0 1 (3.7)Cw*01 4 (7.8) 1 (3.7)Cw*02 5 (9.8) 1 (3.7)Cw*03 6 (11.8) 11 (40.7) 5.2 0.008Cw*04 8 (15.7) 2 (7.4)Cw*05 9 (17.6) 4 (14.8)Cw*06 10 (19.6) 3 (11.1)Cw*07 37 (72.5) 18 (66.7)Cw*08 4 (7.8) 3 (11.1)Cw*12 0 1 (3.7)Cw*14 1 (2.0) 1 (3.7)Cw*15 0 1 (3.7)Cw*16 5 (9.8) 2 (7.4) Table 2 Frequencies of HLA-DRB1 and DQ alleles in patients with SO  AllelesControls (n=51)* Patients (n=27)Relative risk p Value No (%) No (%) DRB1*01 12 (23.5) 3 (11.1)DRB1*03 11 (21.6) 8 (29.6)DRB1*04 13 (28.3) 15 (55.5) 3.7 0.017DRB1*06 1 (2.0) 0DRB1*07 16 (31.4) 6 (22.2)DRB1*08 2 (3.9) 1 (3.7)DRB1*10 1 (2.0) 0DRB1*11 7 (13.7) 1 (3.7)DRB1*12 1 (2.0) 1 (3.7)DRB1*13 8 (15.7) 5 (18.5)DRB1*15 20 (39.2) 6 (22.2))DQA1*01 33 (67.3) 13 (48.1))DQA1*02 17 (34.7) 6 (22.2)DQA1*03 12 (24.5) 15 (55.5) 3.9 0.014DQA1*04 2 (4.1) 1 (3.7)DQA1*05 20 (40.8) 11 (40.7)DQB1*02 23 (45.1) 14 (51.9)DQB1*03 27 (52.9) 18 (66.7)DQB1*04 2 (3.9) 1 (3.7)DQB1*05 16 (31.4) 4 (14.8)DQB1*06 23 (45.1) 9 (33.3)*n=49 for DQA1* alleles due to insu Y cient DNA. Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irish patients  283  group.bmj.comon October 22, 2014 - Published by bjo.bmj.comDownloaded from   disease severity in SO patients with theCw*03-DRB1*04-DQA1*03 haplotype, orwith the individual genotypes Cw*03,DRB1*0404 or DQA1*0301 (data not shown)compared with SO patients without these HLAgene variants. Discussion The most important finding of this DNAtyping study of sympathetic ophthalmia is thatthere is a similar association with HLA-DRB1*04 and DQA1*03 genotypes in whitepatients and Japanese patients with SO. Of equal importance is the finding that theDRB1*04-DQA1*03 haplotype is a marker of more severe clinical phenotype in SO, withincreased disease susceptibility and severity,similar to VKH, which has identical HLAassociations and increased disease severityassociated with DRB1*04 gene variants. 11 12 Previous serological HLA studies of SO havereported an increase in both HLA-DR4 andDQ3 in non-Japanese and Japanese pa-tients. 16 19 20 Previous studies outside of Japanhave been small and control groups were inap-propriate as they were too large, falselyincreasing the power of statistical analysis, 8 20 did not have racial matching,or were not typedin the same laboratory at the same time as thepatients. 20 26 Our SO patient (n=27) andcontrol (n=51) groups are relatively small instatistical terms, and interpretation of the datamust be more cautious with the smaller SOpatient subgroups in phenotypic analysis.However, this is the largest population based(UK and Ireland) cohort of SO patients yetdescribed, with rigorous matching of controlsto each patient, and all DNA based typing wasperformed in the same laboratory at the sametime. With the greater accuracy and resolutionof DNA based typing compared to serology,greatly reducing the chance of a type 1 error,we have not performed Bonferroni’s correctionfor multiple comparison procedures, similar toother recent DNA based HLA studies. 7 12 16 There has been only one previous DNAbased HLA study of SO, on 16 Japanesepatients, which found significant associationswith HLA-DRB1*0405, DQA1*03, andDQB1*0401. 16 HLA disease associations are dependent onthe prevalence of a specific HLA allele in thepopulation and the evolution of particular link-age disequilibria—for example, betweenHLA-DR and HLA-DQ, is also dependent onethnic background. Japan has a relativelyhomogeneous ethnic genetic island populationand HLA disease associations with particulargenotypes may not be as relevant outside of  Japan. Owing to strong linkage disequilibriumbetween DRB1*0405 and DQB1*0401 in Japan only, 17 18 and lack of serological SOassociation with DQ4 in North Americanpatients, 20 the authors of the Japanese studysuggested that DQB1*0401 was unlikely tocontrol the development of SO but wereunable make a definite conclusion. Our studysupports this finding as DQB1*0401 was notassociated with SO, nor was DRB1*0405,probably because the prevalence of DRB1*0405 is low outside the Far East. 17 18 The subtypes of DRB1*04 are invariablylinked with DQA1*03 across all races. 18 How-ever,DQA1*03 is linked with other types,suchas subtypes of DRB1*08, DRB1*09, andDRB1*12, and these other types were notincreased in the patients with SO in our studywhere only DRB1*04 (0404) was significantlyincreased. Our data support the finding that Table 3 Frequencies of higher resolution subtypes of HLA-DRB1 and DQ alleles in patients with SO  AllelesControls (n=51)* Patients (n=27)Relative risk p Value No (%) No (%) DRB1*0103 1 (2.0) 3 (11.1)DRB1*0401 8 (15.7) 9 (33.3)DRB1*0403 1 (2.0) 0DRB1*0404 2 (3.9) 5 (18.5) 5.6 0.045DRB1*0405 0 1 (3.7)DRB1*0407 0 1 (3.7)DRB1*0408 2 (3.9) 1 (3.7)DRB1*0413 1 (2.0) 0DQA1*0101 12 (24.5) 4 (14.8)DQA1*0102 8 (16.3) 5 (18.5)DQA1*0201 17 (34.7) 6 (11.8)DQA1*0301 7 (14.3) 8 (15.7)DQA1*0302 0 1 (3.7)DQA1*0303 6 (12.2) 8 (29.6)DQA1*0401 2 (4.1) 1 (3.7)DQA1*0501 20 (40.8) 11 (40.7)DQB1*0301 16 (31.3) 10 (37.0)DQB1*0302/08 8 (15.7) 9 (33.3)DQB1*0303 7 (13.7) 0*n=49 for DQA1* alleles due to insu Y cient DNA. Table 4 Frequencies of associated haplotypes in patients with SO  AllelesControls (n=51)* Patients (n=27)Relativerisk p Value No (%) No (%) Cw*03-DRB1*04-DQA1*03 3 (6.1) 8 (29.6) 6.7 0.013DRB1*04-DQA1*03 13 (26.5) 15 (55.5) 3.5 0.024DRB1*0404-DQA1*0301 1 (2.0) 5 (18.5) 10.9 0.019Cw*03-DRB1*04 3 (5.9) 8 (29.6) 6.7 0.007Cw*03-DQA1*03 2 (4.1) 8 (29.6) 9.9 0.003*n=49 for DQA1* alleles due to insu Y cient DNA. Table 5 Influence of HLA-DRB1*04-DQA1*03 haplotype on clinical phenotype in patients with SO Ocular signs* DRB1*04-DQA1*03 positive (n=15)† DRB1*04-DQA1*03negative (n=12)‡  p Value No (%) No (%) VA <0.5§ 10 (67) 9 (75)Onset <3 months \  13 (87) 3 (25) 0.001Onset >12 months \  2 (13) 5 (42)>2 inciting events# 4 (27) 8 (67) 0.038Surgical cause of SO 5 (33) 7 (58)Moderate/severe vitritis** 11 (73) 5 (42)Persistent macular oedema >3months 7 (47) 5 (42)Disc oedema 2 (13) 4 (33)Dalen-Fuchs nodules 10 (67) 8 (67)Choroidal neovascularisation 1 (7) 1 (8)Enucleation 5 (33) 3 (25)Prednisolone >10 mg/day†† 10 (67) 3 (25) 0.031>1 immunosuppressive drug 12 (80) 8 (67)Recurrence uveitis on therapy 12 (80) 6 (50)*Ocular signs during follow up in sympathising eyes only, where applicable.†HLA-DRB1*04-DQA1*03 positive patients possessed both alleles.‡HLA-DRB1*04-DQA1*03 negative patients did not possess either allele.§Worst level of best corrected visual acuity. \  Onset of SO from last ocular injury.#Inciting event defined as penetrating eye trauma or surgery.**Vitritis where vitreal haze/binocular indirect ophthalmoscopy score or cellular infiltrate was 2+or worse. †† Maintenance systemic prednisolone dose. 284  Kilmartin,Wilson,Liversidge,et al  group.bmj.comon October 22, 2014 - Published by bjo.bmj.comDownloaded from 

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