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Susceptibility to develop celiac disease is primarily associated with HLA-DQ alleles
Susceptibility to Develop Celiac Disease Is Primarily Associated with HLA-DQ Alleles Anne Spurkland, Ludvig M. Sollid, Kjersti S. R0nningen, Vidar Bosnes, Johan Ek, Frode Vartdal, and Erik Thorsby
ABSTRACT: We have recently reported that the susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular combination of an HLA-DQA1 (DQAI*0501) and an HLA-DQB I (DQB 1 "0201) allele; i.e., a particular DQ ~//3 heterodimer. To investigate whether certain DP alleles might also contribute to the genetic susceptibility, DPAI and DPB1 genes of 94 CD patients and 132 healthy controls were examined by probing in vitro amplified D N A with sequence-specificoligonucleotideprobes corresponding to all hitherto known DPA 1 and DPB 1 alleles. The frequencies of the DPAI*0201 and of the DPBI*OIO1 alleles were increased in CD patients compared to healthy controls (0.31 versus 0.14 and 0.25 versus 0.08, respectively). However, these DP alleles were in linkage disequilibrium with CD-associated DQ alleles in the normal population, and the difference in frequency of these DP alleles was no longer significant when CD patients and healthy controls carrying the CD-associated DQAI*0501 and DQB 1"0201 alleles were compared. DQB 1"0201 homozygous individuals were overrepresented among DQBl*O2Ol-positive patients compared to controls. When DQBI*0201 heterozygous patients and controls were compared, nearly identical frequencies of the DPAI*0201 and the DPB I *OI O1 alleles werefound. Thus, the observedincrease of the DPA I *0201 and DPB I *Ol Ol alleles among CD patients seems mainly to be caused by linkage disequilibrium to the CDassociated DQ alleles.
ABBREVIATIONS CD celiac disease HTC homozygous typing cell HLA human leukocyte antigen PCR polymerase chain reaction RR relative risk RFLP restriction fragment length polymorphism
SSO 10th IHWS
sequence-specific oligonucleotide Tenth International Histocompatibility Workshop
INTRODUCTION H u m a n leukocyte antigen (HLA) class II genes encode o~/~ heterodimers, which present antigenic peptides to CD4 + T cells [1]. Three closely linked H L A class II genic subregions exist: DR, D Q , and DP. Celiac disease (CD) is strongly
From the Institute of Transplantation Immunology, the National Hospital, Oslo (A.S., L.M.S., K.S.R., V.B., F.V., E.T.), and the Departmentof Pediatrics, Buskerud Central Hospital, Drammen (I.E.), Norway. Address reprint requeststo Dr. Anne Spurkland, Institute of Transplantation Immunology, the National Hospital, 0027 Oslo 1, Norway. ReceivedJanuary 8, 1990; acceptedApril 20, 1990.
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A. Spurkland et al. associated with a combination of a DQA1 (DQAI*0501) [2-4] and a DQB1 (DQBI*0201) [4] allele, which are located in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygotes. The corresponding DQ ~//~ heterodimer is functionally expressed in both cases [5]. Celiac disease has also been reported to be associated with certain DP alleles as defined by analysis of restriction fragment length polymorphisms (RFLP) [6,7] or by sequence-specific oligonucleotide (SSO) probes [8,9]. Thus, Bugawan et al. [8] recently reported an increase of the DPBI*0402 and DPBI*0301 alleles among Italian CD patients, while Kagnoff et al. [9] found an increase of the DPBI*0101 and DPBI*0301 alleles among American CD patients. Here we report the results of genomic DPA1 and DPB1 typing of 94 Norwegian CD patients and 132 Norwegian controls. Our data indicate that DQ and not DP genes confer the primary HLA-associated genetic susceptibility to develop CD.
MATERIALS A N D M E T H O D S
Subjects. The 94 CD patients were unrelated Norwegian Caucasian children fulfilling the European Society for Pediatric Gastroenterology and Nutrition criteria [10]. These patients were included in our previous study on CD [4]. Healthy control subjects were 132 randomly selected blood donors. Serological DR typing was carried out on immunomagnetically isolated cells using highly selected antisera, as described previously [11].
Homozygous typing cell (HTC) lines. Twenty-four Tenth International Histocompatibility Workshop (10th IHWS) homozygous typing cell (HTC) lines served as positive and negative controls in the hybridization assays. They have previously been DP typed by primed lymphocyte typing [ 12 ] and genomically by SSO probes [13].
Oligonucleotide probes. The SSO probes were purchased from Genetic Designs, Houston, TX. The DPB1 probes were identical to those previously employed for DPB 1 typing [ 13,14]. DPA 1 probes (DPAO 1: 5-AAG ATG AGA TGT TCT ATG T-3; DPA02: 5-AAG ATG AGC AGT TCT ATG T-3) were selected to distinguish between the two known DPA1 alleles [14,15]. The DQA1 and the DQB1 probes are identical to those previously employed [4].
In vitro DNA amplification. In vitro DNA amplification was performed on genomic DNA essentially as described by Saiki et al. [16]. One microgram of DNA was amplified during 35 cycles by 0.5 t~M of the specific oligonucleotide primers and 1U Taq polymerase (Perkin Elmer, Emeryville, CT) using a Perkin Elmer Cetus DNA thermal cycler. The DPB 1- and the DPA 1-specific primers are given in ref. 14. The DQB1 primers are described in ref. 17. The DQAl-specific primers DQA01 (5-GAC CAT GTT GCC TCT TAT GGT GTA-3) and DQA02 (5-GGT AGC AGC AT5T AGA GTT GGA G-3) amplified a 240-bp segmerit.
Probing with SSO. Probing was carried out essentially as described by Angelini et al. [ 13]. Amplified DNA were slot blotted onto nylon filters and then hybridized with end-labeled SSO probe in 6 x saline-sodium citrat buffer, 5 x Denhardt, 0.5% sodium-dodecyl-sulfat for 1 hr a t T a (no. o f G + C x 4 + no. o f A + T x 2 in °C). Autoradiography was carried out for 1 to 4 hr at - 70°C.
Determination of HLA-DPBI alleles. DPB1 allele assignments were based on patterns of reactivity with the SSO probes taking the known sequences of DPB1 alleles into account. A computer program, which reports if any pair of DPB1
HLA Association in Celiac Disease
159
alleles is compatible with the observed set of hybridization bands, was designed to simplify the analysis. (The program is available from the authors on request.)
RFLP analysis. D N A from 46 of the CD patients was analyzed mainly by standard 10th IHWS RFLP techniques [18]. D N A was digested with the enzymes Bgl II and Rsa I (Amersham, UK), separated on a 0.6% agarose gel, and hybridized with DPB and DPA probes provided by the 10th IHWS.
Assignment of DQB 1*020 i-positive haplotypes. Assignment of DQB 1"0201-positive haplotypes was based on data obtained by serological DR typing, genomic typing of D Q alleles using SSO, and RFLP [4] or polymerase chain reaction (PCR) techniques [19] and reports on association between DR and D Q alleles [20].
Statistics. The frequencies of HLA-DPA1 and -DPB1 alleles in patients and healthy controls were compared by the chi-square test. The correlation coefficient, r, was calculated by the formula (x2/N) l/~. The p values were adjusted for number of comparisons. Level of significance was set to 0.05. Relative risk (RR) was estimated according to Wolf with Haldane's continuity correction [21]. Linkage disequilibrium in the normal population was evaluated by the A-value [22].
RESULTS
Specificity of the hybridization assays. The specificity of the DPB1 SSO probes were determined on amplified D N A from 24 10th IHWS HTC representing 12 of the 19 hitherto known DPB 1 alleles. The hybridization patterns obtained were with one exception similar to those previously reported [13]. The exception was the cell line 9021 which displayed a heterozygous pattern in our investigations (DPB 1" 0402-DPB 1"0101). Recently the DP genes of this cell line was sequenced and it was found heterozygous for both DPA1 and DPB1 [23]. The HTC lines reacted with the DPA1 probes as expected from two-dimensional gel analysis [24] with one exception: the HTC line 9006 was found to be DPAI*0201 in our investigations in contrast to DPA 1.1 by two-dimensional analysis.
Frequency of HLA-DPA1 and -DPB1 alleles in CD patients and controls. Samples from CD patients hybridized more frequently to the probe DB12 (reactive with the DPBI*0101 as well as the DPBl*1501 and the DPB1*1801 alleles) and the probe DPA02 (reactive with the DPAI*0201 allele) compared to controls (Fig. 1). A summary of all results is given in Table 1. There was a significantly increased frequency of the DPAI*0201 and the DPBI*0101 alleles in CD patients (0.31 and 0.25, respectively) as compared to controls (0.14 and 0.08, respectively). Except for one blood donor, all DPBl*0101-positive individuals were also DPAl*0201-positive. No other DPB1 alleles showed a significant increase in frequency among CD patients. RFLP analysis of genomic DP polymorphisms in CD patients. RFLP analysis of DP polymorphisms in CD have revealed an increase ofa DPA Bgl II 3.5-kb fragment, and o f a DPB Rsa 1 4.0-kb fragment [6,7]. We therefore carried out RFLP analysis of D N A from 46 of the CD patients using Rsa I and Bgl II restriction enzymes and DPA and DPB probes obtained from the 10th IHWS. A strong correlation between the presence of a DPA Bgl II 3.5-kb fragment [6] and reactivity with the DPA02 probe, which define the DPAI*0201 allele, was found. All 31 patients having this RFLP fragment were also positive with the DPA02 probe (r -- 0.97, p < 0.00001). Fifty-three out of 94 patients (56%) and
A. Spurkland et al.
160
CD patients
Controls
SSO probes DB12 DPB1
DB01
SSO probes
DPA02
types
DB12
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"0101/'0202
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• 0401/*0401 m
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• 0301/'0401 • 0101/'0101
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FIGURE 1 Hybridization to DNA from CD patients and controls, using the DPA02, DB01, and DB12 probes.
34 out of 132 controls (26%) were positive for the DPA02 probe. The presence of the polymorphism defined by the DPB Rsa I 4.0-kb fragment [7] correlated closely to hybridization with the DB01 probe (reactive with D N A of the DPBI*0101, DPBI*0301, DPBI*0501, and some rare DP alleles) as 34 of 36 patients carrying this fragment hybridized with the DB01 probe (r -- 0.81, p < 0.00001). We found that D N A from 67 of 94 patients (71%) and 69 of 132 controls (52%) hybridized with the DP01 probe. No significant difference in hybridization frequency to the DB01 probe was found when DR3DQw2-positive CD patients and controls were compared (67 of 90 patients and 25 of 34 controls; not significant).
Relationship of the CD-associated DQAI*0501 and DQBI*0201 alleles to the DPAI*0201 and DPBI*OIO1 alleles. To evaluate whether the DPAI*0201 and DPBI*0101 alleles might contribute to the HLA-associated CD susceptibility independent of the DQAI*0501 and DQBI*0201 alleles, we first examined whether the former alleles were nonrandomly distributed compared to the HLAD Q alleles among the controls (Table 2). We found significant associations be-
HLA Association in Celiac Disease TABLE 1
161
Frequencies of DPA1 and DPB1 alleles among celiac disease patients and healthy controls Patients (n = 94)
Controls (n = 132)
No.
Frequency
No.
Frequency
128 59
0.68 0.31
227 37
0.86 0.14
Total DPB1 "0401 *0402 "0201 *0202 "0101 "0301 "0501 "0601 "0801 "0901 "1001 "1101 Other
188
0.99
264
1.00
67 21 16 2 46 22 0 0 1 2 0 5 6
0.36 0.11 0.09 0.01 0.25 0.12 0.00 0.00 0.01 0.01 0.00 0.03 0.03
113 42 24 1 21 29 7 3 3 3 3 7 4
0.43 0.16 0.09 0.01 0.08 0.11 0.03 0.01 0.01 0.01 0.01 0.03 0.02
Torn
188
1.02
264
1.00
DPA1 "0101 "0201
<0.0001
<0.0001
tween DQBI*0201 and DPAI*0201, between DQBI*0201 and DPBI*0101, and between DQAI*0501 and DPBI*0101. We next compared the DQAl*0501,DQBl*0201-positive patients and controls as to the distribution of the DPA 1"0201 and DPB 1"0101 alleles. The results are given in Table 3. It can be seen that the frequency of DPAI*0201 and DPBI*0101 is slightly increased among the DQAl*0501,DQBl*0201-positive CD patients compared to the controls, but the differences did not reach statistical significance. DQBI*0201 homozygous individuals were overrepresented among the patients (47 / 93; 51%) compared to the co ntrols ( 1/ 37; 3 % ). We therefore compared
TABLE 2
Association between the CD-associated DQ alleles (DQA 1"0501, DQBI*0201) and the DPBI*0101 and the DPAI*0201 alleles in healthy individuals
n = 132
+ +
+ _
_ +
DQA1*0501-DPA1*0201 D Q B 1* 0 2 0 1 - D P A 1*0201 DQAI*0501-DPBI*0101 D Q B 1*0201-DPB 1"0101 D P A I * 0 2 0 1 - D P B 1"0101
16 23 12 15 19
18 11 8 5 15
36 37 40 45 1
a ~-values are given in 10 s. bp values only given when <0.05.
62 61 72 67 97
~_value ~
pb
15 43 21 32 64
<0.01 <0.05 <0.01 <0.00001
162
A. Spurkland et al. TABLE 3
D Q A l * 0 5 0 1 , D Q B l * 0 2 0 1 - p o s i t i v e CD patients and controls carrying ( + ) or not carrying ( - ) the DPAI*0201 or the D P B I * 0 1 0 1 allele DPAI*0201
+
CD patients n = 93 Controls n = 37 X2
DPBI*0101
--
53 14
+
40 23
--
40 12
3.83
53 25 1.19
the frequency o f the DPA 1"0201 and DPB 1"0101 alleles among D Q B 1"0201/X heterozygous patients and controls, where X is any DQB1 allele other than D Q B I * 0 2 0 1 . Nearly identical allele frequencies were found in the two groups (Table 4).
Relative risk of various CD-associated HLA class II alleles. The RR to develop CD for individuals carrying both the D Q A 1"0501 and D Q B 1"0201 alleles was found to be 165. In contrast the RR for the DPAI*0201 and the D P B I * 0 1 0 1 alleles were both 4. The RR for DR3 was 55. DISCUSSION We have examined whether certain DP alleles might add to the D Q A I * 0 5 0 1 and DQBl*0201-associated susceptibility to develop CD. We found that the D P A I * 0 2 0 1 and D P B I * 0 1 0 1 alleles were significantly increased among the patients. H o w e v e r , this increase seemed mainly to be due to linkage of these alleles to CD-associated D Q alleles. The increase of the DPAI*0201 allele among Norwegian CD patients is in accordance with a previous RFLP study of British CD patients [6]. The DPA Bgl II 3.5-kb fragment, reported by Niven et al. [6] to be increased in CD patients, correlated completely to the presence of the DPAI*0201 allele. Howell et al. [7] reported that a DPB Rsa 1 4.0-kb fragment was more frequent in D R 3 D Q w 2 patients than in DR3DQw2 matched controls. The presence of this fragment correlated closely to reactivity with the DB01 probe. However, in contrast to the results of Howell et al. [7], we did not find a significant difference in the frequency of reactivity with the DB01 probe in DR3DQw2 patients and controls (67/90 versus 25/34). Interestingly, a recent report of Latin American Caucasian individuals revealed an equal frequency of the DPB Rsa I 4.0-kb fragment among D R 3 D Q w 2 CD patients and healthy controls [25].
TABLE 4
Frequencies of the DPAI*0201 and DPBI*0101 alleles among D Q B I * 0 2 0 1 / X heterozygous CD patients and controls
Subjects
No. of individuals
DPB 1"0101
DPA 1"0201
Patients Controls
46 57
0.14 0.13
0.24 0.22
HLA Association in Celiac Disease
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Our results do not support the findings made by Bugawan et al. [8], who reported an increase of the DPB 1"0402 and the DPB 1"0301 alleles among Italian CD patients. An increase of the DPBI*0101 allele (as well as the DPBI*0301 allele) is more recently reported among American CD patients [9]. Also among English CD patients the frequency of the DPBI*0101 allele has been found to be increased [26] but not more than expected from the linkage disequilibrium to DR3. Linkage disequilibrium of DPw 1 (the PLT defined counterpart of DPB 1*0101 ) to DR3 has repeatedly been reported [27-30]. Since we found that the DPAI*0201 allele is associated with DQBI*0201, and the DPBI*0101 allele associated both with DQBI*0201 and DQAI*0501 among healthy individuals, an increase of the two DP alleles might merely be a secondary effect. Consistent with this observation, the difference in frequency of the DPAI*0201 and DPBI*0101 alleles was no longer significant when CD patients and healthy controls carrying the CD-associated DQAI*0501 and DQBI*0201 alleles were compared. Furthermore, we found nearly identical frequencies of DPAI*0201 and of DPBI*0101 alleles among DQBI*0201 heterozygous CD patients compared to DQBI*0201 heterozygous controls. The RR to develop CD of individuals carrying both the DQAI*0501 and DQBI*0201 alleles (RR = 165) is far higher than that of individuals carrying DPAI*0201 or DPBI*0101 (RR = 4). It is also higher than the RR for DR3 (RR = 55). This is intriguing as the D Q subregion is located between the DR and the DP subregions and both the CD-associated DP and DR alleles are in linkage disequilibrium with DQAI*0501 and/or DQBI*0201. Our results suggest that CD is primarily associated with an HLA D Q oe//3 heterodimer encoded by the DQAI*0501 and DQBI*0201 either in cis or in trans position [4], and that DP alleles do not independently add to this HLA association in CD. This is further strengthened by the observation that CD seems to be associated to various DP alleles in different ethnic populations, while the association with D Q seems to be the same [3,4,8,25,26].
ACKNOWLEDGMENTS
We wish to thank Hallvard Gjerde and Kari Lise Forset for excellent technical assistance and Gunnar Markussen and P~tl M¢ller for helpful discussions. This work was supported by Norwegian Research Council for Sciences and the Humanities and Medinnova.
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5. Lundin KEA, Sollid LM, Qvigstad E, Markussen G, Ek J, Thorsby E: T lymphocyte recognition of a celiac disease associated cis or trans encoded HLA-DQ c~//3 heterodimer. J Immunol, in press. 6. Niven MJ, Caffrey C, Sachs JA, Cassel PG, Gallagher RB, Kumar P, Hitman GA: Susceptibility to coeliac disease involves genes in HLA-DP region. Lancet 2 3:805, 1987. 7. Howell MD, Smith JR, Austin RK, Kelleher D, Nepom GT, Volk B, Kagnoff MF: An extended HLA-D region haplotype associated with celiac disease. Proc Natl Acad Sci USA 85:222, 1988. 8. Bugawan TL, Angelini G, Larrick J, Auricchio S, Ferrara GB, Erlich HA: A combination of a particular HLA-DP/3 allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease. Nature 339:470, 1989. 9. Kagnoff MF, Harwood JI, Bugawan TL, Erlich HA: Structural analysis of the HLADR, -DQ, and -DP alleles on the celiac disease-associated HLA-DR3 (DRw 17) haplotype. Proc Natl Acad Sci USA 86:6274, 1989. 10. Meeuwisse GW: Diagnostic criteria in coeliac disease. Acta Paediatr Scand 49:461, 1970. 11. Vartdal F, Gaudernack G, Funderud S, Bratlie A, Lea T, Ugelstad J, Thorsby E: HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation--a fast and reliable technique. Tissue Antigens 28:301, 1986. 12. Mickelson E, Reinsmoen N, Robbins FM, Hartzman R, Odum N, Svejgaard A, Farrell C, Honeyman M, Flomberg N, Sell T, Eckels D: HLA-Dw and HLA-DP typing of the reference panel of B-lymphoblastoid cell lines. In Dupont B (ed): Immunobiology of HLA, vol 1. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989. 13. Angelini G, Bugawan TL, Erlich HA, Ferrara GB: HLA-DP typing by DNA amplification and hybridization with allele-specific oligonucleotides. Hum Immunol 26:169, 1989. 14. Bugawan TL, Horn GT, Long CM, Mickelson E, HansenJA, Ferrara GB, Angelini G, Erlich HA: Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DP-A and DP-B loci. J Immunol 141:4024, 1988. 15. Trowsdale J, Young JAT, Kelly AP, Austin PJ, Carson S, Meunier H, So A, Erlich HA, Spielman RS, BodmerJ, Bodmer WF: Structure, sequence and polymorphism in the HLA-D region. Immunol Rev 85:5, 1986. 16. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis B, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988. 17. Todd JA, Bell JI, McDevitt HO: HLA-DQ~ gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329:599, 1987. 18. Marcadet A, O'Connel P, Cohen D: Standardized southern blot workshop technique. In Dupont B (ed): Immunobiology of HLA, vol 1. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989. 19. Rcnnilagen KS, Iwe T, Halstensen TS, Spurkland A, Thorsby E: The amino acid at position 57 of the HLA-DQ/3 chain and susceptibility to develop insulin-dependent diabetes mellitus. Hum Immunol 26:215, 1989. 20. Horn GT, Bugawan TL, Long CM, Erlich HA: Allelic sequence variation of the HLA-DQ loci: Relationship to serology and to insulin-dependent diabetes mellitus susceptibility. Proc Natl Acad Sci USA 85:6012, 1988.
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21. Tiwari JL, Terasaki PI: The data and statistical analysis. In TiwariJL, Terasaki PI (eds): HLA and Disease Associations. New York, Springer-Verlag, 1985. 22. Svejgaard A, Hauge M, Jersild C, Platz P, Ryder LP, Staub L, Nielsen, Thomsen M: The HLA system. An introductory survey. Monogr Hum Genet 7, 1979. 23. LeeJS, Sartoris S, Briata P, Choi E, Cullen S, Lepalslier E, Yunis I: Sequence polymorphism of HLA-DP/~ chains. Immunogenetics 29:346, 1989. 24. Knowles RW: Structural polymorphism of the HLA class IIce and/3 chains: Summary of the 10th Workshop 2-D gel analysis. In Dupont B (ed): Immunobiology of HLA, vol 1. Histocompatibility Testing 1987. New York, Springer-Verlag, 1989. 25. Herrera M, Chertkoff L, Palavecino E, Mota A, del Carmen Guala M, Fainboim L, Satz ML: Restriction fragment length polymorphism in HLA class II genes of Latin American Caucasian celiac disease patients. Hum Immunol 26:272, 1989. 26. Rosenberg WMC, Wordsworth BP, Jewell DP, Bell JI: A locus telomeric to HLADPB encodes susceptibility to coeliac disease. Immunogenetics 30:307, 1989. 27. Shaw S, Duquesnoy RJ, Smith PL: Population studies of the HLA linked SB antigens. Immunogenetics 14:153, 1981. 28. Moen T, Bratlie A, Kiss E, Bruserud O, Thorsby E: Identification of four SB antigens by cloned cells. Tissue Antigens 22:298, 1983. 29. Matsui Y, Alosco SM, Awdeh Z, Duquesnoy RJ, Page PL, Hartzman RJ, Alper CA, Yunis EJ: Linkage disequilibrium of HLA-SB1 with the HLA-A1, B8, DR3, SCO 1 and of HLA-A26, Bw38, Dwl0, DR4, SC21 extended haplotypes. Immunogenetics 20:623, 1984. 30. Rodriguez de Cordoba S, Marshall P, Rubinstein P: Molecular characterization by high resolution isoelectric focusing of the products encoded by the class II region loci of the MHC in humans. II. DP-c~ and DP-/3 gene variants. J Immunol 142:836, 1989.
ABSTRACT: We have recently reported that the susceptibility to develop celiac disease (CD) seems to be primarily associated to a particular combination of an HLA-DQA1 (DQAI*0501) and an HLA-DQB I (DQB 1 "0201) allele; i.e., a particular DQ ~//3 heterodimer. To investigate whether certain DP alleles might also contribute to the genetic susceptibility, DPAI and DPB1 genes of 94 CD patients and 132 healthy controls were examined by probing in vitro amplified D N A with sequence-specificoligonucleotideprobes corresponding to all hitherto known DPA 1 and DPB 1 alleles. The frequencies of the DPAI*0201 and of the DPBI*OIO1 alleles were increased in CD patients compared to healthy controls (0.31 versus 0.14 and 0.25 versus 0.08, respectively). However, these DP alleles were in linkage disequilibrium with CD-associated DQ alleles in the normal population, and the difference in frequency of these DP alleles was no longer significant when CD patients and healthy controls carrying the CD-associated DQAI*0501 and DQB 1"0201 alleles were compared. DQB 1"0201 homozygous individuals were overrepresented among DQBl*O2Ol-positive patients compared to controls. When DQBI*0201 heterozygous patients and controls were compared, nearly identical frequencies of the DPAI*0201 and the DPB I *OI O1 alleles werefound. Thus, the observedincrease of the DPA I *0201 and DPB I *Ol Ol alleles among CD patients seems mainly to be caused by linkage disequilibrium to the CDassociated DQ alleles.
ABBREVIATIONS CD celiac disease HTC homozygous typing cell HLA human leukocyte antigen PCR polymerase chain reaction RR relative risk RFLP restriction fragment length polymorphism
SSO 10th IHWS
sequence-specific oligonucleotide Tenth International Histocompatibility Workshop
INTRODUCTION H u m a n leukocyte antigen (HLA) class II genes encode o~/~ heterodimers, which present antigenic peptides to CD4 + T cells [1]. Three closely linked H L A class II genic subregions exist: DR, D Q , and DP. Celiac disease (CD) is strongly
From the Institute of Transplantation Immunology, the National Hospital, Oslo (A.S., L.M.S., K.S.R., V.B., F.V., E.T.), and the Departmentof Pediatrics, Buskerud Central Hospital, Drammen (I.E.), Norway. Address reprint requeststo Dr. Anne Spurkland, Institute of Transplantation Immunology, the National Hospital, 0027 Oslo 1, Norway. ReceivedJanuary 8, 1990; acceptedApril 20, 1990.
Human Immunology29, 157-165 (1990) © AmericanSocietyfor Histocompatibilityand lmmunogenetics,1990
157 0198-8859/90/$~.50
158
A. Spurkland et al. associated with a combination of a DQA1 (DQAI*0501) [2-4] and a DQB1 (DQBI*0201) [4] allele, which are located in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygotes. The corresponding DQ ~//~ heterodimer is functionally expressed in both cases [5]. Celiac disease has also been reported to be associated with certain DP alleles as defined by analysis of restriction fragment length polymorphisms (RFLP) [6,7] or by sequence-specific oligonucleotide (SSO) probes [8,9]. Thus, Bugawan et al. [8] recently reported an increase of the DPBI*0402 and DPBI*0301 alleles among Italian CD patients, while Kagnoff et al. [9] found an increase of the DPBI*0101 and DPBI*0301 alleles among American CD patients. Here we report the results of genomic DPA1 and DPB1 typing of 94 Norwegian CD patients and 132 Norwegian controls. Our data indicate that DQ and not DP genes confer the primary HLA-associated genetic susceptibility to develop CD.
MATERIALS A N D M E T H O D S
Subjects. The 94 CD patients were unrelated Norwegian Caucasian children fulfilling the European Society for Pediatric Gastroenterology and Nutrition criteria [10]. These patients were included in our previous study on CD [4]. Healthy control subjects were 132 randomly selected blood donors. Serological DR typing was carried out on immunomagnetically isolated cells using highly selected antisera, as described previously [11].
Homozygous typing cell (HTC) lines. Twenty-four Tenth International Histocompatibility Workshop (10th IHWS) homozygous typing cell (HTC) lines served as positive and negative controls in the hybridization assays. They have previously been DP typed by primed lymphocyte typing [ 12 ] and genomically by SSO probes [13].
Oligonucleotide probes. The SSO probes were purchased from Genetic Designs, Houston, TX. The DPB1 probes were identical to those previously employed for DPB 1 typing [ 13,14]. DPA 1 probes (DPAO 1: 5-AAG ATG AGA TGT TCT ATG T-3; DPA02: 5-AAG ATG AGC AGT TCT ATG T-3) were selected to distinguish between the two known DPA1 alleles [14,15]. The DQA1 and the DQB1 probes are identical to those previously employed [4].
In vitro DNA amplification. In vitro DNA amplification was performed on genomic DNA essentially as described by Saiki et al. [16]. One microgram of DNA was amplified during 35 cycles by 0.5 t~M of the specific oligonucleotide primers and 1U Taq polymerase (Perkin Elmer, Emeryville, CT) using a Perkin Elmer Cetus DNA thermal cycler. The DPB 1- and the DPA 1-specific primers are given in ref. 14. The DQB1 primers are described in ref. 17. The DQAl-specific primers DQA01 (5-GAC CAT GTT GCC TCT TAT GGT GTA-3) and DQA02 (5-GGT AGC AGC AT5T AGA GTT GGA G-3) amplified a 240-bp segmerit.
Probing with SSO. Probing was carried out essentially as described by Angelini et al. [ 13]. Amplified DNA were slot blotted onto nylon filters and then hybridized with end-labeled SSO probe in 6 x saline-sodium citrat buffer, 5 x Denhardt, 0.5% sodium-dodecyl-sulfat for 1 hr a t T a (no. o f G + C x 4 + no. o f A + T x 2 in °C). Autoradiography was carried out for 1 to 4 hr at - 70°C.
Determination of HLA-DPBI alleles. DPB1 allele assignments were based on patterns of reactivity with the SSO probes taking the known sequences of DPB1 alleles into account. A computer program, which reports if any pair of DPB1
HLA Association in Celiac Disease
159
alleles is compatible with the observed set of hybridization bands, was designed to simplify the analysis. (The program is available from the authors on request.)
RFLP analysis. D N A from 46 of the CD patients was analyzed mainly by standard 10th IHWS RFLP techniques [18]. D N A was digested with the enzymes Bgl II and Rsa I (Amersham, UK), separated on a 0.6% agarose gel, and hybridized with DPB and DPA probes provided by the 10th IHWS.
Assignment of DQB 1*020 i-positive haplotypes. Assignment of DQB 1"0201-positive haplotypes was based on data obtained by serological DR typing, genomic typing of D Q alleles using SSO, and RFLP [4] or polymerase chain reaction (PCR) techniques [19] and reports on association between DR and D Q alleles [20].
Statistics. The frequencies of HLA-DPA1 and -DPB1 alleles in patients and healthy controls were compared by the chi-square test. The correlation coefficient, r, was calculated by the formula (x2/N) l/~. The p values were adjusted for number of comparisons. Level of significance was set to 0.05. Relative risk (RR) was estimated according to Wolf with Haldane's continuity correction [21]. Linkage disequilibrium in the normal population was evaluated by the A-value [22].
RESULTS
Specificity of the hybridization assays. The specificity of the DPB1 SSO probes were determined on amplified D N A from 24 10th IHWS HTC representing 12 of the 19 hitherto known DPB 1 alleles. The hybridization patterns obtained were with one exception similar to those previously reported [13]. The exception was the cell line 9021 which displayed a heterozygous pattern in our investigations (DPB 1" 0402-DPB 1"0101). Recently the DP genes of this cell line was sequenced and it was found heterozygous for both DPA1 and DPB1 [23]. The HTC lines reacted with the DPA1 probes as expected from two-dimensional gel analysis [24] with one exception: the HTC line 9006 was found to be DPAI*0201 in our investigations in contrast to DPA 1.1 by two-dimensional analysis.
Frequency of HLA-DPA1 and -DPB1 alleles in CD patients and controls. Samples from CD patients hybridized more frequently to the probe DB12 (reactive with the DPBI*0101 as well as the DPBl*1501 and the DPB1*1801 alleles) and the probe DPA02 (reactive with the DPAI*0201 allele) compared to controls (Fig. 1). A summary of all results is given in Table 1. There was a significantly increased frequency of the DPAI*0201 and the DPBI*0101 alleles in CD patients (0.31 and 0.25, respectively) as compared to controls (0.14 and 0.08, respectively). Except for one blood donor, all DPBl*0101-positive individuals were also DPAl*0201-positive. No other DPB1 alleles showed a significant increase in frequency among CD patients. RFLP analysis of genomic DP polymorphisms in CD patients. RFLP analysis of DP polymorphisms in CD have revealed an increase ofa DPA Bgl II 3.5-kb fragment, and o f a DPB Rsa 1 4.0-kb fragment [6,7]. We therefore carried out RFLP analysis of D N A from 46 of the CD patients using Rsa I and Bgl II restriction enzymes and DPA and DPB probes obtained from the 10th IHWS. A strong correlation between the presence of a DPA Bgl II 3.5-kb fragment [6] and reactivity with the DPA02 probe, which define the DPAI*0201 allele, was found. All 31 patients having this RFLP fragment were also positive with the DPA02 probe (r -- 0.97, p < 0.00001). Fifty-three out of 94 patients (56%) and
A. Spurkland et al.
160
CD patients
Controls
SSO probes DB12 DPB1
DB01
SSO probes
DPA02
types
DB12
~
,..,.
m
"0801/'0901
"0101/'0202
I "
.,,,-.
mm.
"0401/'0401
"0101/'0301
~
O
~
"0201/'1001
• 0101/*0101
so
~
m
*0401/*0402
• 0401/*0401 m
m
~
~ ~
• 0301/'0401 • 0101/'0101
~
~
am ~
m
i=,,
~
"0101/'0301
~
~
"0101/'0101
~
~
*0101/*0401
Q
~
o
"0101/'0401
~
~-"
m
*0201/*0401
imm
"0401/'0402 *0201/*0401
• 0401/'0801
~
"0201/'0401
• 0401/'0402
• 0301/'0402
e,m
"0301/'0402
• 0401/'0402
• 0101/*0401
,=m
"0401/'0401
"0301/'0401 "0101/'0401
DPA02
DPB1 t y p e s
• 0101/'0401
"0101/'0401
DB01
*0401/*0401 ~=.
~ ~
m
FIGURE 1 Hybridization to DNA from CD patients and controls, using the DPA02, DB01, and DB12 probes.
34 out of 132 controls (26%) were positive for the DPA02 probe. The presence of the polymorphism defined by the DPB Rsa I 4.0-kb fragment [7] correlated closely to hybridization with the DB01 probe (reactive with D N A of the DPBI*0101, DPBI*0301, DPBI*0501, and some rare DP alleles) as 34 of 36 patients carrying this fragment hybridized with the DB01 probe (r -- 0.81, p < 0.00001). We found that D N A from 67 of 94 patients (71%) and 69 of 132 controls (52%) hybridized with the DP01 probe. No significant difference in hybridization frequency to the DB01 probe was found when DR3DQw2-positive CD patients and controls were compared (67 of 90 patients and 25 of 34 controls; not significant).
Relationship of the CD-associated DQAI*0501 and DQBI*0201 alleles to the DPAI*0201 and DPBI*OIO1 alleles. To evaluate whether the DPAI*0201 and DPBI*0101 alleles might contribute to the HLA-associated CD susceptibility independent of the DQAI*0501 and DQBI*0201 alleles, we first examined whether the former alleles were nonrandomly distributed compared to the HLAD Q alleles among the controls (Table 2). We found significant associations be-
HLA Association in Celiac Disease TABLE 1
161
Frequencies of DPA1 and DPB1 alleles among celiac disease patients and healthy controls Patients (n = 94)
Controls (n = 132)
No.
Frequency
No.
Frequency
128 59
0.68 0.31
227 37
0.86 0.14
Total DPB1 "0401 *0402 "0201 *0202 "0101 "0301 "0501 "0601 "0801 "0901 "1001 "1101 Other
188
0.99
264
1.00
67 21 16 2 46 22 0 0 1 2 0 5 6
0.36 0.11 0.09 0.01 0.25 0.12 0.00 0.00 0.01 0.01 0.00 0.03 0.03
113 42 24 1 21 29 7 3 3 3 3 7 4
0.43 0.16 0.09 0.01 0.08 0.11 0.03 0.01 0.01 0.01 0.01 0.03 0.02
Torn
188
1.02
264
1.00
DPA1 "0101 "0201
<0.0001
<0.0001
tween DQBI*0201 and DPAI*0201, between DQBI*0201 and DPBI*0101, and between DQAI*0501 and DPBI*0101. We next compared the DQAl*0501,DQBl*0201-positive patients and controls as to the distribution of the DPA 1"0201 and DPB 1"0101 alleles. The results are given in Table 3. It can be seen that the frequency of DPAI*0201 and DPBI*0101 is slightly increased among the DQAl*0501,DQBl*0201-positive CD patients compared to the controls, but the differences did not reach statistical significance. DQBI*0201 homozygous individuals were overrepresented among the patients (47 / 93; 51%) compared to the co ntrols ( 1/ 37; 3 % ). We therefore compared
TABLE 2
Association between the CD-associated DQ alleles (DQA 1"0501, DQBI*0201) and the DPBI*0101 and the DPAI*0201 alleles in healthy individuals
n = 132
+ +
+ _
_ +
DQA1*0501-DPA1*0201 D Q B 1* 0 2 0 1 - D P A 1*0201 DQAI*0501-DPBI*0101 D Q B 1*0201-DPB 1"0101 D P A I * 0 2 0 1 - D P B 1"0101
16 23 12 15 19
18 11 8 5 15
36 37 40 45 1
a ~-values are given in 10 s. bp values only given when <0.05.
62 61 72 67 97
~_value ~
pb
15 43 21 32 64
<0.01 <0.05 <0.01 <0.00001
162
A. Spurkland et al. TABLE 3
D Q A l * 0 5 0 1 , D Q B l * 0 2 0 1 - p o s i t i v e CD patients and controls carrying ( + ) or not carrying ( - ) the DPAI*0201 or the D P B I * 0 1 0 1 allele DPAI*0201
+
CD patients n = 93 Controls n = 37 X2
DPBI*0101
--
53 14
+
40 23
--
40 12
3.83
53 25 1.19
the frequency o f the DPA 1"0201 and DPB 1"0101 alleles among D Q B 1"0201/X heterozygous patients and controls, where X is any DQB1 allele other than D Q B I * 0 2 0 1 . Nearly identical allele frequencies were found in the two groups (Table 4).
Relative risk of various CD-associated HLA class II alleles. The RR to develop CD for individuals carrying both the D Q A 1"0501 and D Q B 1"0201 alleles was found to be 165. In contrast the RR for the DPAI*0201 and the D P B I * 0 1 0 1 alleles were both 4. The RR for DR3 was 55. DISCUSSION We have examined whether certain DP alleles might add to the D Q A I * 0 5 0 1 and DQBl*0201-associated susceptibility to develop CD. We found that the D P A I * 0 2 0 1 and D P B I * 0 1 0 1 alleles were significantly increased among the patients. H o w e v e r , this increase seemed mainly to be due to linkage of these alleles to CD-associated D Q alleles. The increase of the DPAI*0201 allele among Norwegian CD patients is in accordance with a previous RFLP study of British CD patients [6]. The DPA Bgl II 3.5-kb fragment, reported by Niven et al. [6] to be increased in CD patients, correlated completely to the presence of the DPAI*0201 allele. Howell et al. [7] reported that a DPB Rsa 1 4.0-kb fragment was more frequent in D R 3 D Q w 2 patients than in DR3DQw2 matched controls. The presence of this fragment correlated closely to reactivity with the DB01 probe. However, in contrast to the results of Howell et al. [7], we did not find a significant difference in the frequency of reactivity with the DB01 probe in DR3DQw2 patients and controls (67/90 versus 25/34). Interestingly, a recent report of Latin American Caucasian individuals revealed an equal frequency of the DPB Rsa I 4.0-kb fragment among D R 3 D Q w 2 CD patients and healthy controls [25].
TABLE 4
Frequencies of the DPAI*0201 and DPBI*0101 alleles among D Q B I * 0 2 0 1 / X heterozygous CD patients and controls
Subjects
No. of individuals
DPB 1"0101
DPA 1"0201
Patients Controls
46 57
0.14 0.13
0.24 0.22
HLA Association in Celiac Disease
163
Our results do not support the findings made by Bugawan et al. [8], who reported an increase of the DPB 1"0402 and the DPB 1"0301 alleles among Italian CD patients. An increase of the DPBI*0101 allele (as well as the DPBI*0301 allele) is more recently reported among American CD patients [9]. Also among English CD patients the frequency of the DPBI*0101 allele has been found to be increased [26] but not more than expected from the linkage disequilibrium to DR3. Linkage disequilibrium of DPw 1 (the PLT defined counterpart of DPB 1*0101 ) to DR3 has repeatedly been reported [27-30]. Since we found that the DPAI*0201 allele is associated with DQBI*0201, and the DPBI*0101 allele associated both with DQBI*0201 and DQAI*0501 among healthy individuals, an increase of the two DP alleles might merely be a secondary effect. Consistent with this observation, the difference in frequency of the DPAI*0201 and DPBI*0101 alleles was no longer significant when CD patients and healthy controls carrying the CD-associated DQAI*0501 and DQBI*0201 alleles were compared. Furthermore, we found nearly identical frequencies of DPAI*0201 and of DPBI*0101 alleles among DQBI*0201 heterozygous CD patients compared to DQBI*0201 heterozygous controls. The RR to develop CD of individuals carrying both the DQAI*0501 and DQBI*0201 alleles (RR = 165) is far higher than that of individuals carrying DPAI*0201 or DPBI*0101 (RR = 4). It is also higher than the RR for DR3 (RR = 55). This is intriguing as the D Q subregion is located between the DR and the DP subregions and both the CD-associated DP and DR alleles are in linkage disequilibrium with DQAI*0501 and/or DQBI*0201. Our results suggest that CD is primarily associated with an HLA D Q oe//3 heterodimer encoded by the DQAI*0501 and DQBI*0201 either in cis or in trans position [4], and that DP alleles do not independently add to this HLA association in CD. This is further strengthened by the observation that CD seems to be associated to various DP alleles in different ethnic populations, while the association with D Q seems to be the same [3,4,8,25,26].
ACKNOWLEDGMENTS
We wish to thank Hallvard Gjerde and Kari Lise Forset for excellent technical assistance and Gunnar Markussen and P~tl M¢ller for helpful discussions. This work was supported by Norwegian Research Council for Sciences and the Humanities and Medinnova.
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