HLA-DQ2 HETERODIMER IN THE DIAGNOSIS OF CELIAC DISEASE

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1 R. Žunec et al. HLA-DQ2 heterodimer in the diagnosis of celiac disease Original scientific paper Izvorni znanstveni članak HLA-DQ2 HETERODIMER IN THE DIAGNOSIS OF CELIAC DISEASE Renata Žunec 1, Zorana Grubić 1, Zvonimir Jurčić 2, Mladen Peršić 3, Andrija Kaštelan 1, Vesna Kerhin-Brkljačić 1 1 Tissue Typing Centre, Department of Urology, Zagreb Clinical Hospital Center 2 Department of Pediatrics, Sestre milosrdnice Clinical Hospital, Zagreb, 3 Kantrida Childrens Hospital, Rijeka ABSTRACT Objective: Genetic predisposition to CD is strongly associated with HLA-DQ alleles. The majority of patients carry HLA-DQ2 heterodimer (HLA-DQ(α1*0501,β1*02) heterodimer) encoded by DQA1*0501 and DQB1*02 genes, either in cis or trans. The remaining patients carry either DQ8 heterodimer (HLA-DQ (α1*0301,β1*0302) heterodimer) or part of the DQ2 heterodimer. The aim of the present study was to investigate these findings in Croatian CD patients. Methods: Sixty-three unrelated children diagnosed with CD and 119 unrelated healthy controls were typed for HLA-DQA1 and DQB1 alleles. HLA typing was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method. Results: The statistically highly significant (p<10-5 ) difference between patients and controls was found for DQA1*0501 allele (64.3% in patients vs. 24.8% in controls; =5.5), for DQB1*02 allele (61.9% in patients vs 13.4% in controls; =10.5) and for DQA1*0501-DQB1*02 haplotype (51.6% in patients vs. 8.1% in controls; =12.9). The analysis for HLA-DQ heterodimers revealed the presence of HLA-DQ2 heterodimer in 59 (93.7%) patients and in 21 (17.6%) controls. In patients, an increase in homozygotes for DQ2 heterodimer (20.6% in patients vs. 1.7% in controls, =15.2), as well as an increase in heterozygotes for DQ2 heterodimer in cis (61.9% in patients vs. 11.8% in controls; =12.2) was observed. Among 4 patients negative for DQ2 heterodimer, 3 were positive for DQ8 heterodimer, and 1 patient carried only DQB1*02 allele. Conclusion: The value of this typing in the diagnosis of CD lies in its ability to exclude disease in the case of a negative result for the presence of DQ2 heterodimer. Key words: Celiac Disease+diagnosis + immunology + genetics; HLA-DQ Antigens+genetics+immunology Correspondence to: INTRODUCTION Celiac disease (CD (MIM212750) ) is defined as a permanent enteropathy due to intolerance to wheat gliadins and related proteins in rye and barley. Immune response results in villous atrophy and crypt hyperplasia in the small intestine, while symptomatology may vary widely from absolute absence of symptoms to pronounced clinical gastrointestinal or extradigestive manifestations (1). CD affects up to 1 in people in western European and Northern American populations (2-5), being one of the commonest life-long immunological disorders affecting Caucasians. Renata Žunec, PhD, Tissue Typing Centre, Department of Urology, Zagreb Clinical Hospital Center, Kišpatićeva 12; HR Zagreb, Tel: , renata.zunec@kbc-zagreb.hr The genesis and development of CD involves both genetic and environmental factors. The only definitive genetic risk locus, recently assigned as CELIAC1 ( is HLA-DQ, belonging to the HLA class II region situated on the short arm of chromosome 6 (6p21.3). The gene product is HLA-DQ molecule consisting of a heterodimer with a heavy chain (α) and a light chain (β) while the genes encoding HLA-DQα and β chains are DQA1 and DQB1, respectively. For HLA-DQ molecules, both α and β chains contribute to the polymorphism and their corresponding genes are highly polymorphic. In European Caucasian populations, more than 90% of patients with CD carry the HLA- DQ2 molecule (6). Usually, the DQ2 het- BIOCHEMIA MEDICA god. 14, br. 3-4,

2 HLA-DQ2 heterodimer in the diagnosis of celiac disease R. Žunec et al. erodimer is encoded by the HLA-DQA1*05 and DQB1*02 alleles in cis (on one chromosome) on a DRB1*03 (DR3) haplotype. However, it may also be encoded in trans (across both chromosomes) with DQA1*05 allele on DRB1*11, DRB1*12 or DRB1*13 haplotype and DQB1*02 on DRB1*07 haplotype (DR11/DR7, DR12/DR7 and DR13/DR7 genotypes), (7,8). On the other hand, the HLA-DQ8 heterodimer coded by DQA1*03 and DQB1*0302 alleles in cis on a DRB1*04 (DR4) haplotype is commonly present in HLA-DQ2 negative patients with CD (9). The patients negative for both, DQ2 and DQ8 heterodimer, usually carry either DQA1*05 or DQB1*02 alleles alone (10). Nevertheless, the fact that HLA-DQ2 heterodimer has a relatively high prevalence in the general population (20-30%), together with the existence of a small number of CD patients without HLA-DQ risk heterodimers, indicates that HLA genes alone are not enough for overall genetic predisposition to the disease. In the present study, we investigated the prevalence of DQA1 and DQB1 alleles and DQA1- DQB1 haplotypes in Croatian patients with CD, and compared the data with the existing data for Croatian general population. The frequencies of DQ2 and DQ8 heterodimers were studied in patients and controls, which will provide further data for population comparison purposes. SUBJECTS The present report is based on a set of 63 unrelated patients with CD from two centers: Sestre Milosrdnice Clinical Hospital, Zagreb (48 patients) and Kantrida Childrens Hospital, Rijeka (15 patients). Children aged from 8 months to 17 years (median age 3.8 yrs) have been diagnosed following the criteria recommended by European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) (11). Allele and haplotype frequencies for HLA-DQA1 and -DQB1 in a sample representing the Croatian general population were taken into account for comparison purposes (12). METHODS DNA was prepared from peripheral blood lymphocytes using the standard salting out protocol (13). and patients were typed for HLA class II alleles by a high-resolution polymerase chain reaction sequence specific oligonucleotide probes (PCR-SSOP) method, as described previously (14). PCR amplification was performed with a set of specific primers for the second exon of DRB1, DQA1 and DQB1 genes. PCR products were blotted on nylon membranes and hybridized with a set of 125 biotinylated SSOP, as specified at the 12th International Histocompatibility Workshop- IHW. Forty-two DRB1 alleles, 8 DQA1 alleles and 13 DQB1 alleles were tested, representing the most common Caucasoid HLA class II alleles. The nomenclature used was that of the XIIth International Histocompatibility Workshop (14). The frequency of HLA-DQA1 and DQB1 alleles and haplotypes was estimated by direct counting. The HLA class II haplotypes were assigned on the basis of known linkage disequilibrium, as described in previous studies (15). It was presumed that the investigated loci had no blanks and, based on this presumption, when a single HLA class II allele was observed, the individual was considered homozygous for that allele. The significance of differences in allele and haplotype frequencies was evaluated using chi-square while Fisher s exact test was used if any value in a 2x2 table was lower than five (16). Relative risk () was calculated according to Woolf (17). To estimate the value of the HLA-DQ typing for the presence of DQ(α1*0501-β1*02) heterodimer in order to identify individuals who are most likely to have CD, we also calculated sensitivity, specificity and positive and negative predictive values of the HLA-DQ2 heterodimer typing test. RESULTS The HLA-DQA1 and DQB1 allele frequencies of 63 CD examined patients are listed in Table 1 and 2, and also compared with the frequencies in the control group. A total of 7 DQA1 and 10 DQB1 alleles were identified in patients with CD. Within DQA1 alleles, two alleles had frequency higher than 10%: DQA1*0501 (64.3%) and DQA1*0201 (10.3%), representing jointly 74.6% of the total. At the DQB1 locus, two alleles exceeded the frequency of 10%: DQB1*02 (61.9%) and DQB1*0301 (12.7%) which represented 74.6% of the total. Overall, three alleles were found with statistically significant difference in comparison with controls: DQA1*0501 and DQB1*02 were 120 BIOCHEMIA MEDICA god. 14, br. 3-4, 2004.

3 R. Žunec et al. HLA-DQ2 heterodimer in the diagnosis of celiac disease T a b l e 1. DQA1 HLA-DQA1 allelic distribution in celiac patients and controls T a b l i c a 1. Raspodjela alela HLA-DQA1 u bolesnika s GE i zdravih ispitanika (N=141) n % n % * * < * * * * * < N number of tested individuals; n number of observed alleles; relative risk p c p value was corrected for the number of DQA1 alleles N broj ispitivanih osoba; n broj uočenih alela; relativni rizik; p c p vrijednost ispravljena prema broju alela DQA1 T a b l e 2. DQB1 HLA-DQB1 allelic distribution in celiac patients and controls T a b l i c a 2. Raspodjela alela HLA-DQB1 u bolesnika s GE i zdravih ispitanika (N=119) n % n % * * * * * * *02 # < * * * N number of tested individuals; n number of observed alleles; relative risk p c p value was corrected for the number of DQB1 alleles; # subtyping for DQB1 *0201, *0202, *0203 was not performed N broj ispitivanih osoba; n broj uočenih alela; relativni rizik; p c p vrijednost ispravljena prema broju alela DQB1; # podtipizacija za alele DQB1 *0201, *0202, *0203 nije provedena significantly increased in patients (p c < , =5.45; p c < , =10.46 respectively) while DQA1*0102 was decreased in patients (p c < , =0.19). HLA-DQA1-DQB1 haplotypes found in celiac patients and corresponding frequencies in controls are listed in Table 3. Eleven different DQA1- DQB1 haplotypes were found in CD patients. Statistically significant difference was found only for the haplotype DQA1*0501-DQB1*02 which was increased in patients (51% in patients vs 8.05% in controls, p c < , =13.02). In our CD patients, we did not find an increased frequency of DQA1*0201-DQB1*02 haplotype, as reported in several studies (18). The analysis of patients and controls for the presence of DQ2 heterodimer (defined as the presence of both DQA1*0501 and DQB1*02 alleles) revealed that 13 patients (20.6%) carried DQ(α1*0501,β1*02) heterodimer in a double dose (homozygotes) and 39 patients (61.9%) possesed it in a single dose in cis. The observed homozygosity for DQ(α1*0501,β1*02) heterodimer in CD patients was significantly increased in comparison with controls (20.6% vs 1.7%; p c < ; =15.2), as well as the frequency of patients possesing a single dose of DQ(α1*0501,β1*02) heterodimer in cis (61.9% vs 11.8%, p c < ; =12.2). Seven patients had a DQ(α1*0501,β1*02) heterodimer in trans which was not significantly different from controls. Six patients had a DRB1*07/DRB1*11 genotype and one patient had a DRB1*07/DRB1*1303 genotype. In total, 59 (93.7%) patients carried genes encoding the DQ(α1*0501,β1*02) heterodimer whereas 21 out of 119 (17.6%) healthy individuals were also carriers of DQ2 heterodimer. BIOCHEMIA MEDICA god. 14, br. 3-4,

4 HLA-DQ2 heterodimer in the diagnosis of celiac disease R. Žunec et al. T a b l e 3. DQA1 - DQB1 HLA-DQA1-DQB1 haplotypes in celiac patients and controls T a b l i c a 3. Raspodjela haplotipova HLA-DQA1-DQB1 u bolesnika s GE i zdravih ispitanika (N=119) n % n % * * * * * * * * * * * * * * * * * * * * < * * N number of tested individuals; n number of observed haplotypes; relative risk p c p value was corrected for the number of DQA1-DQB1 haplotypes N broj ispitivanih osoba; n broj uočenih haplotipova; relativni rizik; p c p vrijednost ispravljena prema broju haplotipova DQA1-DQB1 T a b l e 4. HETERODIMER DQ(α1*0501,ß1*02) and DQ(α1*0301,ß1*0302) heterodimers in celiac patients and controls T a b l i c a 4. Heterodimeri DQ(α1*0501,ß1*02) i DQ(α1*0301,ß1*0302) u bolesnika s GE i zdravih ispitanika (N=119) % % DQ(α1*0501,ß1*02) < HOMOZYGOTES DQ(α1*0501,ß1*02) < CIS DQ(α1*0501,ß1*02) TRANS DQ(α1*0301,ß1*0302) CIS # < N number of tested individuals; n number of heterodimer positive individuals; relative risk; # individuals negative for both DQ(α1*0501-ß1*02) and DQ(α1*0301-ß1*0302) heterodimers N broj ispitivanih osoba; n broj ispitanika pozitivnih za heterodimer; relativni rizik; # ispitanici negativni za heterodimer DQ(α1*0501-ß1*02) i DQ(α1*0301-ß1*0302) The remaining 4 patients negative for DQ2 heterodimer were analyzed for the presence of DQ8 heterodimer (defined as the presence of both DQA1*0301 and DQB1*0302 alleles) which is known to be the second susceptible heterodimer for CD. Three patients were positive for a DQ(α1*0301-β1*0302) heterodimer and only 1 patient was negative for both susceptible heterodimers. The HLA type of this patient was DQA1*0101-DQB1*0501/DQA1*0201- DQB1*02, thus he possessed only one susceptible DQB1 allele (DQB1*02). Test sensitivity, i.e. the proportion of patients with CD who were correctly identified by the presence of DQ(α1*0501,β1*02) heterodimer was 93.6% (59 out of 63). Specificity of the test, i.e. the probability that subjects were not patients, was 82.4% (98 out of 119) given that there was a negative result for the presence of DQ (α1*0501,β1*02) heterodimer. Positive predictive value, i.e. the proportion of DQ(α1*0501,β1*02) heterodimer typings that correctly predicted CD, was 73.75%. Finally, negative predictive value, i.e. the likelihood that an individual who was negative for the presence of DQ(α1*0501-β1*02) heterodimer was actually unaffected, was 96.1%. DISCUSSION Involvement of HLA polymorphism in CD susceptibility in Croatia has so far been studied by serology techniques (19). The present study 122 BIOCHEMIA MEDICA god. 14, br. 3-4, 2004.

5 R. Žunec et al. HLA-DQ2 heterodimer in the diagnosis of celiac disease provided the data on distribution of HLA-DQA1 and DQB1 alleles and DQA1-DQB1 haplotypes in a sample of 63 Croatian celiac disease patients typed by the DNA technique. The results confirmed a highly significant increase in DQA1*0501 and DQB1*02 alleles separately, as well as of the DQA1*0501-DQB1*02 haplotype in patients in comparison to the controls. The previous population study revealed 28 different DQA1-DQB1 haplotypes, whereas 11 haplotypes found in this sample of CD patients represented 83% of the total patient number (12). This is in concordance with the results obtained for patients from all European populations studied in the project European Genetics Cluster on Coeliac Disease (20). The calculated relative risks for those HLA markers were =5.45 for DQA1*0501, =10.46 for DQB1*02, and =12.9 for haplotype DQA1*0501-DQB1*02. A large number of studies have substantially illustrated the fact that DQ(α1*0501,β1*02) and DQ(α1*0301,β1*0302) heterodimers are involved in the susceptibility to celiac disease. The results were reported that % of European celiac patients carry the haplotype which encode the DQ(α1*0501,β1*0201) molecule, while the remaining patients usually carry DQ(α1*0301,β1*0302) molecule (10, 21). The results from the present study show that, among 63 Croatian patients with CD, 59 (93.7%) patients were carriers of DQ(α1*0501,β1*02) molecule, either in homozygosis (20.6%) or in cis (61.9%) and trans (11.1%) heterozygosis. In addition, 3 patients (4.8%) were carriers of DQ(α1*0301,β1*0302) molecule. The only patient who was negative for both DQ(α1*0501,β1*02) and DQ(α1*0301,β1*0302) heterodimers was a carrier of the DQB1*02 allele alone, which is in agreement with findings reported in recent investigation (20). The association between HLA and CD is well described and results clearly suggest that the presence of one of two susceptible heterodimers is necessary for the development of the disease. But as not all of the patients with CD are carriers of susceptible HLA markers, this also suggests the involvement of additional genetic risk factors in disease susceptibility. Recent linkage studies revealed genetic risk factor for CD on chromosomes 5q, 11p11 and 2q33 (22). Diagnosis of CD requires a small intestinal biopsy which is neither an easy nor cheap method. Methods that include blood levels of antigliadin and antiendomysial antibodies have been developed in an attempt to help identify individuals who are most likely to have CD and who should consequently undergo diagnostic biopsy. However, current antibody tests do not constitute an absolute guide to the presence or absence of CD (23, 24). Moreover, many patients still remain undiagnosed due to poor clinical manifestations and the lack of digestive symptoms. These facts suggest that additional blood test would be a valuable tool for specialists to detect or to exclude the disease. The results obtained on sensitivity (93.6%), specificity (82.4%) and positive (73.75%) and negative predictive values (96.1 %) of the test that include HLA typing for the presence of the DQ(α1*0501,β1*02) heterodimer in Croatian CD patients, were in agreement with findings reported in a recent investigation from Spain (25). These results suggest that the analysis of the HLA-DQ polymorphism by DNA typing techniques could become an additional test in the diagnosis of CD. The value of the test lies particularly in its 96% ability to exclude the disease when a negative result is recorded. HETERODIMER HLA-DQ2 U DIJAGNOSTICI GLUTENSKE ENTEROPATIJE SAŽETAK Cilj: Aleli HLA-DQ igraju važnu ulogu u genetskoj predispoziciji za glutensku enteropatiju (GE). Većina bolesnika s GE nositelji su heterodimera HLA-DQ2 (HLADQ(α1*0501-β1*02)) kodiranog genima HLA- DQA1*0501 i HLA-DQB1*02, koji su raspoređeni u položaju cis ili trans. Preostali bolesnici nositelji su ili heterodimera HLA-DQ8 (HLADQ(α1*0301,β1*0302)) ili dijela heterodimera HLA-DQ2. Cilj ovog istraživanja bio je analizirati heterodimere HLA-DQ kod bolesnika s GE u hrvatskoj populaciji. Metode: Šezdesettri nesrodna djeteta s dijagnozom GE i 119 zdravih nesrodnih ispitanika testirano je za alele lokusa HLA-DQA1 i -DQB1. Za molekularnu tipizaciju HLA korištena je metoda lančane reakcije polimerazom i probama specifičnih sekvenci (PCR-SSOP). Rezultati: Statistički značajna razlika (p<10-5 ) između bolesnika i zdravih ispitanika uočena je za alel DQA1*0501 (64,3% kod bolesnika nasuprot 24,8% kod kontrole; =5,5), alel DQB1*02 (61,9% kod bolesnika nasuprot 13,4% kod kontrole; =10,5), kao i haplotip DQA1*0501-DQB1*02 (51,6% kod bolesnika nasuprot 8,1% kod kontrole; =12,9). BIOCHEMIA MEDICA god. 14, br. 3-4,

6 HLA-DQ2 heterodimer in the diagnosis of celiac disease R. Žunec et al. Analiza heterodimera HLA-DQ pokazala je prisutnost heterodimera HLA-DQ2 kod 59 (93,7%) bolesnika i kod 21 (17,6%) zdravog ispitanika. Uočena je i povećana učestalost homozigota za heterodimer HLA-DQ2 među bolesnicima (20,6%) u odnosu na kontrolu (1.7%); =15,2. Također je otkrivena i povećana učestalost heterozigota za heterodimer HLA-DQ2 u položaju cis (61,9% među bolesnicima prema 11,8% među zdravim ispitanicima; =12,2). Od četiri bolesnika koji nisu imali heterodimer HLA-DQ2, tri su bila pozitivna za heterodimer DQ8, dok je jedan bolesnik imao samo alel DQB1*02. Zaključak: Dobiveni rezultati ponajprije ukazuju na potrebu preispitivanja dijagnoze GE u slučaju kada je bolesnik negativan za heterodimer HLA-DQ2. Ključne riječi: Celijakija+dijagnoza+imunologija+genetika, HLA-DQ antigeni+genetika+imunologija REFERENCES 11. Troncone R, Greco L, Auricchio S. Gluten sensitive enteropathy. 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