Copyr ight I? Miinkrgrrnrd 1994

T I S S U E A N T I G E N S ISSN OOOI-2RIS

Molecular characterization of HLA class I1 genes I in celiac disease . patients . . 4mencan Caucasian ongm M. Herrera, G. Theiler, F. Augustovski, L. Chertkoff, L. Fainboim, S . DeRosa, E. P. Cowan, M. L. Satz. Molecular characterization of HLA class I1 genes in celiac disease patients of Latin American Caucasian origin. Tissue Antigens 1994: 43: 83-87. 0 Munksgaard, 1994

Abstract: In the present study, the polymorphic domain of HLA class I1 genes present in a pediatric population of Argentinian celiac disease patients was analyzed by hybridization to sequence-specific oligonucleotides and DNA sequencing. Sixteen out of 16 DR5/7 heterozygous patients bore the DQA1*0501 and DQB1*0201 alleles implicated in the DQ2 risk speci- ficity. The second exon of DQAI, DQBl and DRBl genes from 2 DR5/7 patients was characterized by DNA sequencing. The following alleles were found in both patients: DRB1*1101 and DRB1*0701; DQB1*0301 and DQBl*0201; DQA1*0501 and DQA1*0201. Previous serological analysis in this population had shown the presence of DQ2 in 95% of the patients (40% in controls) and a negative association with DQ1 haplo- types, suggesting the presence of other “permissive” or neutral alleles. The following HLA-DQB 1 alleles, besides DQBl*O201, were identified in 3 1 CD patients: DQB1*0301, 0302, 0401 and 0402. All these alleles share a common pattern of residues between positions 84 and 90, and distinct from that present in DQI-related alleles.

Introduction

Celiac disease (CD) is a gluten-induced hypersensi- tivity that produces damage in the small intestine (1). The disease develops preferentially in individ- uals inheriting certain HLA class I1 alleles, es- pecially those in the HLA-DR3,DQ2 haplotype (2). In all populations studied, HLA-DQ2 is pres- ent in about 95% of the patients and this specificity, either encoded in cis (as in the DR3 haplotype) or in trans (as in DR5/7 heterozygotes), seems to be responsible for the primary association (2-4). However, other HLA-DR, -DQ and even -DP alle- les have been regarded as being involved in differ- ent populations (5-8). HLA-DP associations seem to be caused mainly by linkage disequilibrium to HLA-DQ alleles (9, 10). On the other hand, the association with certain HLA-DR haplotypes is more difficult to explain. For example, in most populations, an excess of heterozygous DR3/ 7 phenotypes over homozygous DR3/DR3 or DR7/

of Latin

Mariana Herrera, Graciela Theiler, Federico Augustovski, M i e n Chertkoff, Leonardo Fainboirn, Susana DeRosa’, Elliot P. Cowan‘ and M. Leonardo Satz Laboratorio de Inmunogenetica, Hospital de Clinicas and Departamento de Microbiologia, h- cultad de Medicina, Universidad de Buenos Aires. ‘Servicio de Gastroenterologia, Hospital Nacional de Pediatria J. P. Garraham, Buenos Aires, Ar- gentina, and 2Neuroimmunoiogy Branch, NINDS, NlH, Bethesda. MD, U.S.A.

Key words: HLA class I I genes - celiac disease

Received 23 June, revised, accepted for publi- cation 13 October 1993

DR7 phenotypes is observed in CD patients (1 1). An increased number of HLA-DR5 / 7 hetero- zygotes is mostly observed in various Mediterrane- an and Latin CD populations but not among other Caucasians (12-14). This is not due to increased frequencies of these alleles in normal Latin popula- tions: for example, both DR5 and DR7 are alleles with similar frequency among British and Spanish populations (DR5: 10% and DR7: 20%, ref. 15) but only the Spanish population shows the association with DR5/7 (12). As the primary association of CD seems to be with HLA-DQ, it is possible that, in addition to DQ2, other HLA-DQ alleles (rather than HLA-DR alleles) play an important role in CD.

In order to characterize the HLA class I1 alleles present in a pediatric population of Latin Amer- ican Caucasian CD patients, we have studied the polymorphic domains fo HLA-DQA1, DQB 1 and DRBl genes of these patients by DNA oligotyping and sequencing.

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Herrera et al.

Material and methods Patients

A previously described population of 62 CD children of Latin American background was studied (7, 14). In all cases, the diagnosis of CD was made according to ESPGAN criteria, which included three intestinal biopsies.

DNA typing

DNA from peripheral blood leukocytes was avail- able for extraction from 53 patients. The polymor- phic domain (exon 2) from DRB1, DQBl and DQAl genes was PCR-amplified with reagents and protocols from the 1 lth International Histocom- patibility Workshop (1 IHW). Class I1 alleles were characterized by DNA typing using 32P-labeled sequence-specific oligonucleotides provided by the 11HW.

DNA sequencing

After amplification, one-half of the PCR reaction was processed for subcloning into the SmaI site of pUC18, as previously described (16). Plasmid templates were sequenced using the Sequenase 2.0 kit (USB Corp.).

Results and discussion Molecular characterization of HLA class II genes in DRY7 patients

Previous serological analysis of the HLA speci- ficities present in this CD population had shown that 29% of the patients were HLA-DR5/7, in contrast to 7% in the general Argentinian popula- tion (7, 14). Evidence from previous RFLP analysis in these patients (3) led us to suggest the presence of DQA and DQB genes able to encode -in trans- the HLA-DQ2 risk specificity (DQA1*0501 from the DR5 haplotype and DQB 1 *0201 from the DR7 haplotype). In order to confirm the presence of these risk DQ genes, the polymorphic domains of DQA and DQB genes in our DR5/7 CD patients were first studied by PCR-amplification and hy- bridization to specific oligonucleotides. Sixteen of 16 DR5/7 patients had the risk heterodimer DQB1*0201 /DQA1*0501, similar to that present in DR3 individuals (Table 1 ) . The same DQ2 heterodimer was found in 3 DR5/7 Norwegian CD children and in DR5/7 CD children of Italian origin (4, 17, 18). The association of CD with DR5/7 phenotypes is only observed in certain Me- diterranean populations (12-14). In order to inves- tigate whether this association is due to the pres- ence of unusual HLA class I1 genes, the polymor-

Table 1. HLA-DQB1 and DPA1 alleles present in 53 Argentinian CD patients

Patient HLA-OR typing HLA-DQB1 HLA-DQA1 -

1 to 16 OR37 0301 10201 0501 10201 17 to 20 OR36 0301 10201 0501 21 to 23 DR3/4 0302/0201 0501/03 24 to 38 DR3/7 0201 0501/0201 39 to 44 DR3/x 0201 0501 45 to 47 DR3/8a 0302/0201 NO 48 DR3/8 0301 /0201 ND 49 DR3/8 0402/0201 ND 50 DR3/1 0501 10201 NO 51 DR4/7b 0302/0201 0201/03 52' DR4/xb 0302 03 53 DR5/8 0402/0301

HLA-DQBl alleles were established by PCR of genomic DNA and hybridization to sequence-specific oligonucleotides. Other patients not available for DNA analysis included: one DR5/7, two OR311 2, one DR3/1, one DR3/4, one DR3/ 9, two DR3/7, and one DR4/5. a The HLA-DR specificity of these patients was confirmed by DNA oligotyping

of sequencing. HLA-OR4 subtypes were established by oligotyping: Patient 51 typed as DRB1'0404 while patient 52 was DRB1'0401/0402 heterozygous. Only one DPAl allele (DQA1'03) and one DQB1 allele could be detected in this patient after the sequencing of 12 recombinant clones derived from each locus, suggesting that both OR4 haplotypes carry the same DQ alleles.

0501/0401

NO: not done

phic second exon of DQA1, DQBl, and DRBl genes from 2 DR5/7 CD patients was character- ized by DNA sequencing. The following alleles were found in both patients:

DRB 1 locus: DRB 1 * 1 10 1 and DRB 1 *0701 alleles DQB 1 locus: DQB 1 *0301 and DQB 1 *020 1 alleles DQAl locus: DQAl*0501 and DQA1*0201 alleles

In all cases, the sequences were identical to those reported previously for the same specificities in other cells (19). Therefore, our DR5/7 CD patients have DQA, DQB and DRB molecules with poly- morphic domains and present in extended haplo- types, identical to those present in other caucas- ians.

What, then, could explain the high prevalence of DR5/7 among CD patients of Latin origin? One possibility is that, in certain haplotypes, genes other than HLA-DQ but in close linkage to it play an important role. For example, in Italian CD patients from the Bologna area, a significant num- ber of DR7 patients who are neither DR3 nor DR5 are found, which speaks against the DQ2 heterodimer hypothesis and suggests the additional role of other HLA-linked genes (1 1, 20). If the HLA-DR genes themselves are involved in this association, i t would be worth analyzing the contri- bution of the different DRl 1 variants in each popu- lation and their prevalence among CD patients.

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HLA class I1 genes in celiac disease

children with the following phenotypes: 16 DR5/ 7 patients, 4 DR3/5,3 DR314, 15 DR317, 6 DR3/ x, 5 DR3/8 patients and 3 patients not expected to encode the DQw2 risk molecule (Table 1). As can be seen, several DQ3- and DQ4-related DQBl alleles were present with DQ2 in heterozygotes. Perhaps the most surprising finding was the pres- ence in 3 patients of the DR8,DQ8 haplotype, a rare combination only found in 1 out of 161 North American Caucasians (2 1). The frequency of this haplotype among Agentinian Caucasians has not yet been established, although it seems to be very rare (0/53 individuals studied) among other Latin American Caucasians living in Texas (mostly of Mexican origin, ref. 21 and M. Fernandez-Viiia, personal communication). Interestingly, this haplo- type is associated with type I diabetes (IDDM) among Japanese (22, 23) and it was also found in 1 out of 69 North American IDDM patients (24).

Three patients lacked the DQ2 heterodimer: 1 had the DQA1*0501 allele, another had the DQB1*0201 allele and the 3rd was negative for both markers. It is of interest that 2 of these pa- tients were HLA-DR4, similar to what has been previously described in other populations (25-27). These DR4 patients, as well as those DR3/4 het- erozygotes (Table l), carried the DR4/DQ8 haplo- type. Recently, Spurkland et al. (28) and Mantor- ani et al. (29) suggested that this could be a second HLA haplotype associated with CD. Various HLA- DR4 subtypes were found in C D patients bearing this haplotype (Table 1, and ref. 28); these are frequent subtypes among Caucasians and the prevalence of any of them will have to be investi- gated with data collected from different popula- tions. It is clear from this and other studies (28), that certain CD patients do not bear the risk dimer DQB1*0201 /DQA1*0501, thereby implicating other factors besides HLA in C D susceptibility (1).

On the other hand, different populations might exhibit polymorphism at the level of T-cell receptor variable genes, that would affect the fine specificity of the cells involved in the pathogenesis of the disease.

Molecular characterization of HLA class II genes in heterozygous HLA-DQ2/DQ other CD patients

Previous analysis of the serological specificities present in our pediatric patients (7, 14) had shown a negative association with DQ1 haplotypes (3.2% in patients vs. 53.8% in healthy controls). In order to analyze if the lack of DQ1 is due to a displace- ment by DQ2 haplotypes, the frequency of DQ1 haplotypes was compared among our C D patients (n=62) and normal controls (n=197), after re- moving the DQ2 haplotypes in both groups. HLA- DQ1 was present in two haplotypes out of 42 that remained in 39 CD patients, compared to 114 DQ1 haplotypes out of 273 present in 176 normal con- trols (relative risk: 0.07, 2: 21.4, p < 0.00005). This negative association to DQ 1 remained significant even when, besides DQ2, patients with the HLA- DR517 phenotypic combination (very frequent in our patients) were removed from the comparison: DQ1 was present in two of 25 haplotypes of 22 patients, and in 114 of 264 haplotypes of 167 nor- mal controls (relative risk: 0.1 1 , ~ ' : 11.8, p < 0.001). These results suggest that HLA-DQ1 might have a dominant "protective" effect over DQ2. Con- versely, it also suggests the presence of other "per- missive" or neutral alleles.

About one-third of our patients have been typed as homozygous HLA-DQ2 (either because they are DR3/x or DR3/7) and two-thirds were found to be heterozygous. In order to characterize the HLA- DQ alleles present with HLA-DQ2 in heterozygous patients, DNA typing was performed in C D

antno acid sequence ma2 specificity

0503 0601 0602 0603 0604 0605 OM3

W - D R associated spedficity

DM, 7 DR4,11, lZ,14,8 DR4 DR4,8 DR4,8

Mu DRl5 DRl4 DR8,15 DI(15

Dr(13 DRB DRI

1x13

FO;itrc, I , Alignment or amino acid residues or DQBl ~illeles present ( A ) and absent (B) in Argentinian C D palienls.

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The clinical history of 3 such patients (numbers 51 to 53 in Table 1 and a 4th patient who typed as DR415 but whose DNA was not available) was reviewed: their symptoms, diagnostic tests and re- sponse to gluten-free died was indistinguishable from that of DQ2 CD patients.

It was apparent from this analysis that other alleles besides DQ2 were present in our CD pa- tients, yet it was also apparent that some DQ al- leles were conspicuously absent in these patients. In order to determine if there was a common se- quence shared between the DQ alleles in the CD patients that was different to that of the “protec- tive” alleles, the amino acid residues from the polymorphic domain of DQBl alleles present in our CD patients were aligned and compared with those of alleles not found in our patients [Fig. 1). As can be seen, there is an evident common pattern of residues between positions 84 and 90, characteristic of DQ2, DQ7, DQ8 and DQ4 alle- les, and distinct from that present in DQI-related alleles, the latter behaving as “protective” in our population. Some of these substitutions introduce residues with significant differences in size and or charge; moreover, positions 85, 86, 88 and 89 in HLA-DRBl*OlOl were recently shown to be in- volved in peptide binding (30). It is of interest that functional studies have also shown that polymorphism in this region of DRBl molecules affects T-cell recognition (3 1). Therefore, certain HLA-DQB chains paired with susceptibility DQA1*0501 chains (28) may bind peptides in- volved in triggering and/or effector mechanisms in tissue damage in CD.

Other HLA-class I1 sequence motifs have also been implicated in alleles associated with rheu- matoid arthritis and multiple sclerosis (32, 33).

In summary, Latin American CD patients bear DQBl alleles which carry shared polymorphic amino acid sequences. It would be interesting to compare these DQBl genes with those present in CD patients from other ethnic groups.

Acknowledgments

This work was supported in part by a Biotech- nology Career Fellowship Grant from the Rocke- feller Foundation to M. L. Satz, and by grants from the University of Buenos Aires. M. Herrera and L. P. Chertkoff are Research Fellows of CONICET, F. Augustovski is a Research Fellow of the Univ. of Buenos Aires; and L. Fainboim and M. L. Satz are members of research career of CONICET. M. L. Satz is especially grateful to Dinah Singer and Jocelyn Weissman for constant support and encouragement.

References

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

1 5 .

16.

17

18

19

Trier JS. Celiac sprue. N Engl J Med 1991: 325: 1709- 19. Tosi R, Vismara D, Tanigaki N, et al. Evidence that celiac disease is primarily associated with a DC locus allelic speci- ficity. Clin Immunol Immunopathol 1983: 28: 395404. Palavecino E, Mota A, Awad J, et al. HLA and celiac disease in Argentina: involvement of the DQ subregion. 10th International Histocompatibility Conference and 13th Annual Meeting of the American Society for Histocompat- ibility and Immunogenetics. 1987, Abstract No. 257. Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. Evidence for primary association of celiac dis- ease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med 1989: 169: 345-50. Niven MJ, Caffrey C, Sachs JA, et al. Susceptibility to celiac disease involves genes in the HLA-DP region. Lancet 1985: 2: 805. Howell MD, Smith JR, Austin RK, et al. An extended HLA-D region haplotype associated with celiac disease. Proc Natl Acad Sci USA 1988: 85: 222-6. Herrera M, Chertkoff L, Palavecino E. et al. Restriction fragment length polymorphism in HLA class I1 genes of Latin American Caucasian celiac disease patients. Hwn Immunol 1989: 26: 272-80. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA. A combination of a particular HLA-DPB allele and HLA-DQ heterodimet confers susceptibility to celiac disease. Nature 1989: 339: 470-3. Spurkland A, Sollid LM, Ronningen KS, Bosnes V, Ek J, Vartdall F, Thorsby E. Susceptibility to develop celiac dis- ease is primarily associated with HLA-DQ alleles. Hum Immunol 1990: 29: 157-65. Bolsover WJ, Hall MA, Vaughan RW, Welsh KI, Ciclitira PJ. A family study confirms that the HLA-DP associations with celiac disease are the result of an extended HLA-DR3 haplotype. Hum Immunof 1991: 31: 100-8. Scholz S, Albert E. HLA and diseases: involvement of more than one HLA-linked determinant of disease susceptibility. Imrniinol Rev 1983: 70: 77-88. Mearin ML, Biemond I, Pena AS. et al. HLA-DR pheno- types in Spanish coeliac children: their contribution to the understanding of the genetics of the disease. Guf 1983: 24: 532-7. Morellini M, Trabace S, Mazzilli MC, et al. A study of HLA class I1 antigens in an Italian paediatric population with coeliac disease. Dis Markers 1988: 6: 23-8. Palavecino E, Mota A, Awad J, et al. HLA and celiac disease in Argentina: involvement of the DQ subregion. Dis Markers 1990: 8: 5-10. The Data Book. 11 th International Histocompatibility Workshop, Yokohama, 199 I . Central Data Analysis Com- mittee. Allele frequencies of HLA-A,B,C,DR and DQ loci. pp. 793. Cowan EP, Pierce ML, McFarland HF, McFarlin DE. HLA-DR and DQ allelic sequences in multiple sclerosis patients are identical to those found in the general popula- tion. Hum Immunol 1991: 32: 203-10. Ferrante P, Petronzelli F, Mariani P, Bonamico M, Mazzilli MC. Oligotyping of Italian celiac patients with the 11th International Histocompatibility Workshop reagents. Tissue Antigens 1992: 39: 38-9. Tighe MR, Hall MA, Barbado M, Cardi E, Welsh KI. Ciclitira PJ. HLA class I1 alleles associated with celiac disease susceptibility in a Southern European population. Tissue Antigens 1992: 40: 90-7. Mnrsh SGE. Bodmer JG. HLA class I 1 nucleotide se- quences I99 1 . Hion /nimmo/ 199 I : 31: 107- I 1 .

HLA class I1 genes in celiac disease

28. Spurkland A, Sollid LM, Polanco I. Vartdal F, Thorsby E. HLA-DR and -DQ genotypes of celiac disease patients serologically typed to be non-DR3 or non-DR5/7. Hum Immirnol 1992: 35: 188-92.

29. Mantovani V, Corazza GR, Bragliani M, Frisoni M, Zani- boni MG, Gasbarrini G. Asp57-negative HLA-DQB chain and DQAl*0501 allele are essential for the onset of DQw2- positive and DQw2-negative coeliac disease. Clin Exp Irn- munol 1993: 91: 153-6.

30. Brown JH, Jardetzky TS, Gorga JC, et al. Three dimen- sional structure of the human class I1 histocompatibility antigen HLA-DRI. Nature 1993: 364: 33-39.

31. Lang B, Navarrete C, LoGalbo PR, et al. Further DNA sequence microheterogeneity of the HLA-DR4IDw 13 haplotype group: importance of amino acid position 86 of the DRBl chain for T cell recognition. Hum Imrnunol 1990:

32. Todd JA, Acha-Orbea H, Bell JI, et al. A molecular basis for MHC class I1 autoimmunity. Science 1988: 240: 1003-9.

33. Vartdal F, Sollid LM, Vandvik B, Markussen G, Throsby E. Patients with multiple sclerosis carry DQBl genes which encode shared polymorphic amino acid sequences. Hum Immunol 1989: 25: 103-10.

27: 378-89.

20. Colonna M, Mantovani W, Corazza GR, et al. Reassess- ment of HLA association with celiac disease in special reference to the DP association. Hum Immunol 1990: 29:

21. Fernandez-Vitia MA, Gao X, Moraes ME, et al. Alleles at four HLA class I1 loci determined by oligonucleotide hybridization and their associations in five ethnic groups. Immunogenetics 1991: 34: 299-312.

22. Hamaguchi K, Kimura A, Dong RP, et al. Specific combi- nations of HLA-DRB and DQB alleles confer susceptibil- ity, while DQA genes determines resistance to IDDM in Japanese. In: Tsuji K, Aizawa M, Sasazuki T, eds. HLA 199 I , Proceedings of the Eleventh International Histocom- patibility Workshop and Conference. New York: Oxford University Press Inc., 1992: vol. 2: 488-92.

23. Ikegami H, Kawaguchi Y, Fukuda M, et al. Analysis of DQAl and DQBl alleles in Japanese. 11th International Histocompatibility Workshop and Conference. Yokohama, 199 1. Abstract PS-I 1-20.

24. Morel PA, Dorman JS, Todd JA, McDevitt HO, Trucco M. Aspartic acid at position 57 of HLA-DQ beta chain protects against type I diabetes: a family study. Proc Natl Acad Sci USA 1988: 85: 81 11-5.

25. Tosi R, Tanigaki N, Polanco I, DeMarchi M, Woodrow JC, Hertzel PA. A radioimmunoassay typing study of non- DQw2 associated celiac disease. Clin Irnrnunol Immunopa-

26. DeMarchi M, Carbonara AO, Ansaldi N, et al. HLA-DR3 and HLA-DR7-negative celiac disease. In: Albert ED, ed. Histocompatibility Testing 1984. Berlin: Springer-Verlag, 1984.

27. Hertzel PAS, Bennett GD, Sheldon AB, et al. Genetic markers in Australian Caucasian subjects with coeliac dis- ease. Tissue Antigens 1987: 30: 18-22.

263-74.

Ill01 1986: 39: 168-72. Address: Dr. Leonard0 Sat2 Laboratorio de Inmurnogenetica Hospital de Clinicas Josi de San Martin Av. Cordoba 235 I , piso 3 1120 Buenos Aires Argentina Tel.: (541) 961-9366 Fax: (541) 962-5404.

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