Different dose effect of HLA-DQαβ heterodimers in insulin-dependent diabetes mellitus and celiac disease susceptibility

Different Dose Effect of HLA-DQa/3 Heterodimers in Insulin-Dependent Diabetes Mellitus and Celiac Disease Susceptibility Fiorella Petronzelli, Giuseppe Multari, Paola Ferrante, Margherita Bonamico, Gabriella Rabuffo, Liliana Campea, and Maria Cristina Mazzilli

To compare the quantitative effect of the DQc~/3 heterodimers D Q a 5 2 A r g + , 3 5 7 A s p and DQa 1*0501,31"0201 on susceptibility to IDDM and CD, we characterized, at the genomic level, the DQce 52 and DQ3 57 residues of 50 IDDM Italian patients observed in Rome. The results were compared with those of a previous study concerning the oligotyping of DQ dimers in a group of CD children belonging to the same population. Our data confirm that both diseases are primarily ABSTRACT:

associated with HLA-DQoq3 heterodimers, but the distributions of the respective susceptible DQA 1 and DQB 1 alleles in the two diseases were different. In fact, the highest risk of IDDM is for subjects ceSS,/3SS that could express, by either cis- or trans-association, four susceptible heterodimers and decreases in proportion to the number of these; in regard to CD, the highest risk was found for individuals who carried only one predisposing heretodimer. Human Immunology 36. 156-162 ¢1993~

ABBREVIATIONS

bp CD IDDM PCR

base pair celiac disease insulin-dependent diabetes mellitus polymerase chain reaction

P RR S SSO

protective relative risk susceptible sequence-specific oligonucleotide

INTRODUCTION Insulin-dependent diabetes mellitus (IDDM) and celiac disease (CD) are two autoimmune diseases strongly associated with the H L A system [1, 2]. The histories of the study of their associations are quite similar. Initial studies pointed out a predominance of class I human leukocyte antigen H L A - B 8 in both diseases [3, 4]. These were later followed by studies of class II specificities that onstrated two D R alleles, DR3 and DR4 in I D D M [5, 6] and DR3 and DR7 in C D [ 7 - 9 ] , to be more significantly increased in these patients. Later, analysis of restriction

From the Department of Experimental Medicine, Institute of Clinical Pediatrics, Center and Servicefor Pediatric Diabetes, La Sapienza University. Rome. Italy. Address reprint requestJ to Dr. F. Petronzelli, Dipartimento di M edicina Sperimentale, Viale Regina Elena 324, 00161 Rome, Italy. ReceivedJul~ 20, 1992; accepted December 12, 1992. 156 0198-8859/93/$6,00

fragment length polymorphism in I D D M patients [ 10] and radioimmunoassay typing of CD patients [ 1 I] demonstrated that molecules encoded by the D Q locus rather than by the DR locus were more strongly associated with disease development. More recently, both diseases have been demonstrated to be primarily associated with DQc~/3 heterodimers. According to recent molecular data, susceptibility to I D D M is associated with a combination of D Q A 1 and D Q B 1 alleles that encode, respectively, D Q ~ Arg52-positive and a DQ/3 Asp57negative chains [ 1 2 - 1 5 ] , while susceptibility to CD is strongly associated with a DQc~/3 heterodimer encoded by D Q A I * 0 5 0 1 and D Q B I * 0 2 0 1 alleles [16, 17]. We previously reported that in a g r o u p of Italian CD patients observed in Rome, 989~. of DR4-negative subjects carried the DQ(~1"0501,/31"0201) heterodimer, which was inherited in cis- with DR3 haplotype or in transHuman Immunology 36, 156-162 <1993} ~) American Society for Histocompatibilky and lmmunogenetics, 1993

Susceptible DQ Dimers in IDDM and CD

157

association with DR5 and DR7 haplotypes [18]. The present study compared the distributions of the DQA 1/B 1 genotypes that code the respective susceptible (S) chains in IDDM and CD patients. For this purpose, HLA-DRB1 alleles and codons 52 of DQA1 and 57 of DQB 1 genes were characterized by hybridization with sequence-specific oligonucleotide (SSO) probes in 50 Italian IDDM patients and 50 healthy controls. Results obtained in IDDM patients were then compared with previous data concerning the oligotyping of DQA 1 and B1 alleles of CD children [18]. MATERIALS A N D M E T H O D S

Patients and health), controls. Fifty unrelated IDDM patients, observed in Rome, and 50 blood donors were studied. The CD patients were previously described [18]. The healthy controls used in our past study were also used for the present study.

Primers and probes. The primers used were identical to DRBAMP-AN, DRBAMP-B; DQAAMP-A, DQAAMP-B; DQBAMP-A and DQBAMP-B distributed during the l l t h International Histocompatibility Workshop. The SSO probes employed are listed in Table 1. DRB 1 probes were selected from the reagents of

the workshop to determine sequences corresponding to DR 1-10 serologic specificities on DRBl-amplified DNA. Instead, DQA1 and DQB1 probes used to define the DQc~ 52 and DQfl 57 residues were constructed according to the published nucleotide sequences [19]. SSO-2, LSO, and SSO-1 were used as positive controls for DRB 1, DQA 1, and DQB 1 genes, respectively.

Polymerase chain reaction (PCR) amplification of genomic DNA. A salt-chloroform DNA extraction was performed on whole blood cells according to the method reported by Mullembach et al. [20]. The second exons of the DRB 1, DQA 1, and DQB 1 genes were amplified as described by the 11th Workshop protocol. Specifically, 0.5/xg ofgenomic DNA was amplified during 30 cycles by 1 unit of cloned Taq polymerase (Perkin Elmer Cetus) and 25 pM of the specific primers in a programmable thermocycler. Each cycle consisted of a 96°C denaturation (30 seconds), a 58°C annealing (1 minute), and a 72°C extension (2 minutes). Final extension was performed at 72°C for 10 minutes. The extent of amplification was controlled by submitting 5% of the PCR products to electrophoresis in an ethidium-bromidestained gel. The DRB 1-specific primers amplified a 274bp (base pair) segment. The DQA1 and DQB1 fragments were 229 bp and 214 bp long, respectively.

TABLE 1 List of oligonucleotide probes used in the present study Gene

DRB 1

DQA 1

DQB 1

Probe

Codons

Alleles

SSO-2 1001 1002 7004 1004 5703 8602 1003

11-17 11-16 73-78 9-14 55-61 82-87 9-15

1006 1005 1007 1008

9-14 10-16 8-14 9-14

All DRB 1 DRBI*0101,0102,0103 DRB 1" 1501,1502,1601,1602 DRB 1"0301,0302 DRB 1"0401-0411 DRBI*1101,1102,1103,1104 DRBl*1201,1202 DRB1*1301-1305,1401-1405, 0301,0302,1101-1104 DRBl*0701,0702 DRBl*0801-0804 DRB 1"0901 DRBI*1001

LSO 52-SER 52-HIS 52-ARG 1 52-ARG2

51-57 51-57 51-57 51 -57

All DQA 1 DQAI*0101,0102,0103 DQA 1*0201 DQA 1"03011,030 l 2,0302 DQA 1*0401,05011-05013,0601

SSO- 1 57-ASP l 57-ASP2 57-ASP3 57-VAL 57-SER 57-ALA

55 -61 55-61 55-61 55-61 55-61 55-61

All DQB 1 DQB 1*05031 ,*0601 ,'0301 ,*0303 DQB 1"05032,*0602,*0603 DQB 1*0401,0402 DQB 1"0501,*0604,*0605 DQB 1*0502,*0504 DQB 1*0201 ,*0302

Serologic equivalent All DR DRI DR2 DR3 DR4 DR11(5) DR12(5) DR6 + DR3,DR11 DR7 DR8 + 12 DR9 DRI0

158

Analysis of PCR products using SSO probes. The samples (3 >1 of the PCR product, 47 tzl of denaturing solution, 0.4 N N a O H , 25 mM EDTA) were slot blotted onto nylon membranes (Gene Screen Plus) using a Minifold II apparatus/Schleicher and Schuell, N e w Hampshire). D N A was fixed with ultraviolet irradiation (5 minutes). The filters were then hybridized with digoxigeninddUTP-3'-end-labeled SSO probes (5 pM) and the signal detected with 3-(2'-spiroadamantane)-4-methoxy-4(3"-phosphoryloxy)-phenyl-l,2-dioxetane (AMPPD) as chemiluminescent substrate for alkaline phosphatase conjugated to antidigoxigenin antibody Fab fragments. Filters were autoradiographed on Fuji x-ray film for 20 minutes at room temperature.

Nomenclature. The 1991 recommendation from the W H O Nomenclature Committee was used [21]. The D Q combinations were indicated as o~o~,3/3 where the first ~ and the first/3 are encoded by the same haplotype. Therefore the difference between o~SP,/3SP and aPS,/3SP is that the c~- and/3-susceptible chains are carried in cis- and in trans-configurations, respectively.

RESULTS

DRB I oligotyping in IDDM patients and controls. Eleven SSO probes were used to type at the genomic level the D R I - 1 0 serologic specificities. The results are shown in Table 2. As expected, DR3 and DR4 alleles were positively associated with the disease. The relative risks (RRs) were 13.0 and 9.7, respectively. The frequencies of DR5 and DR6 alleles were significantly lower in patients than in controls.

F. Petronzelli et al.

TABLE

2

Frequencies (%) and RRs of DRB1 alleles and of codons D Q A 1 52 and DQB1 57 in I D D M patients and controls Diabetics (n = 50)

Controls (n = 50)

1

8

2 3 4 5 6 7 8 9 10

8 68 52 14 16 8 0 0 0

20 22 14 10 46 46 26 2 0 6

28 8 50 78

80 24 10 56

0.1

5.8 x 10 7

9.0

4.0 x 10-5

100

60

67.9

7.6 x 10 14

DQB 1 57-ASP1 57-ASP2 57-ASP3 57-VAL 57-SER 57-ALA

28 0 4 18 8 80

60 28 0 36 8 38

0.3 0.0

7.0 x 10-9 2.0 x 10-8

6.5

1.1 x 10 4

Non-Asp

98

66

25.2

1.7 x 10 5

RR

Pc

DR

DQA1 52-SER 52-HIS 52-ARG1 52-ARG2 Arg

13.0 9.7 0.2 0.2

2.6 4.3 4.4 1.1

x × x ×

10 ~ 10 5 10-~ 10 2

by H a r d y - W e i n b e r g equilibrium and their distributions are shown in Fig. 1A.

DQ~ residue 52 in IDDM patients and controls. Four

DQ~ residue 57 in IDDM patients and controls. Six

D Q A 1 SSOs corresponding to codons 5 1 - 5 7 were synthesized and used as probes to define the D Q a residue 52 in I D D M patients and controls. The probes were named to indicate the amino acid 52 encoded by the sequence detected. Their associations with the D Q A 1 alleles are reported in Table 1 . 5 2 - A R G 1 and 52-ARG2 oligonucleotides recognized two different DQA1 sequences coding an Arg52-positive DQol chain, in linkage disequilibrium with DR3 and DR4, respectively. The positivities of both o f these probes were higher in the patients than in controls (see Table 2) even if only 52-ARG1 remained significant after correction (RR = 9.0). On the contrary, the frequency o f the sequence 52-SER was significantly decreased (RR = 0.1). Altogether, 100% of I D D M patients carried an Arg52positive D Q ~ chain as compared with 60% in controls (RR = 67.9). The frequencies of the classes Arg/Arg, Arg/non-Arg, and non-Arg/non-Arg were as expected

DQB1 SSOs complementary to codons 5 5 - 6 1 were used to characterize DQ,8 residue 57. The allelic specificities of the probes are reported in Table 1. Three probes were used to recognize sequences coding for an Asp57 residue while the remaining three were used to recognize a non-Asp (Val,Ser,Ala) amino acid. 57-ASP 1 and 57-ASP2 SSOs were significantly decreased in patients whereas 57-ALA was significantly increased (RR = 6.5) (see Table 2). In all, 98% of patients versus 66% of controls carried a DQ~-chain non-Asp57 (RR = 25.2). Figure 1B shows the distribution of the genotypes Asp/Asp, Asp/non-Asp, and non-Asp/non-Asp in patients and controls. The frequencies of the combinations were not significantly different from those expected.

Haplotypic combinations in IDDM patients and controls. The haplotypic combinations of DR alleles and D Q A 1 / B 1 sequences coding ~52 and/357 residues, as

Susceptible DQ Dimers in IDDM and CD

159

predicted by the known linkage disequilibria, are presented in Table 3. Only the haplotypes present in the patients are listed. A comparison between diabetics and controls demonstrated that the genotypes DR3,3;DQc~52 Arg,Arg;DQ/357 Ala,Ala and DR3,4;DQa52 Arg,Arg;DQ/357 Ala, Ala were significantly increased in the patients (RRs = 26.2 and 17.2). O f the haplotypic combinations present in controls, 78% were never detected in the patients ("others" in the table, p = 6 × 10 ~3).

A

80 60 40 20 0 IDDM (n-50) Controls (n-50

DQ~/3 heterodimers susceptible to and protective of IDDM. According to Khalil et al. [15], the two DQA1 sequences coding for Arg52-positive DQoe chains were designated as S while the two Arg52-negative sequences were named as protective (P). Similarly, the D Q B 1 sequences coding for Asp57-negative chain DQ/3 and Asp57-positive chain DQ/3 were defined as S and P, respectively. O f the I D D M patients, 98% carried at least one DQce and one DQ/3 S chain compared with 34% of healthy controls (RR = 95.1) (see Fig. 2). As shown in Fig. 3A, the highest risk of IDDM (RR = 53.1) is for subjects cxSS,/3SS, who potentially express, by cisor trans-association, four D Q ~ 5 2 A r g + , / 3 5 7 A s p - susceptible dimers. IDDM frequency decreases in proportion to the number of these dimers at risk. In fact, the frequencies of patients

TABLE 3

100

B

100 80 60 40 20 0

IDDM (n-50) I Controls (n-50

m

IODM (n-50)

~

Controls In-60)

FIGURE 1 Frequencies (%) of DQc~ 52 (A) and DQ/3 5 ~ (B) combinations in IDDM patients and controls.

Presumed haplotypic combinations (%) of DR alleles and DQA1/B1 sequences coding ~52 and/357 substitutions in I D D M patients and controls

DR

DQce52

DQ/357

1DDM (n = 50)

Controls (n = 50)

DR

DQce52

DQ/357

IDDM (n = 50)

1 4

ser arg

val asp

6

0

3 7

arg his

ala ala

4

2

1 5

ser arg

val asp

2

8

4 4

arg arg

ala ala

6

0

2

ser

ser

3

arg

ala

4

arg

ala

6

2

5

arg

2 5

se r arg

ser asp

asp

4

4

2

0

4 6

arg ser

asp asp

2

0

3 3

arg arg

ala ala

20

0

26.2

3 x 10 5

4 6

arg ser

ala val

2

0

3 4

arg arg

ala ala

26

2

17.2

8 x 10 ~

4 6

arg ser

asp val

2

0

3 5

arg arg

ala asp

4

2

4 7

arg his

ala ala

4

0

3 6

arg arg

ala asp

4

0

5 6

arg ser

asp val

2

2

3 6

arg ser

asp val

0

78

4

0

RR

Pc

Others

Controls (n = 50)

RR

Pc

6 x

10 -;5

160

F. Petronzelli et al.

(RR = 17.4) is for individuals with ~PS,BSP that express, by trans-association, one predisposing combination of the four potential DQ dimers. All subjects belonging to this class were typed as DR5,7, and all DR5,7 heterozygotes were included in this class except one (aPS,flPP;DQ7,DQ9). The DR3 patients, encoding one dimer in cis-configuration, were distributed in the classes aSS,flSS; aSS,flSP; aSP,flSS; and aSP,flSP in conformity with the second haplotype. The remaining three individuals (c~PP,flSP; ~PS,flPP; aPP,fiPP) were DR4 positive. In all, 92~: of the patients carried at least one predisposing dimer: 62% (42c~; DR5,7 and 20% DR3,x), 28c~ and 2c~ encoding for one, two, and four ~fl SS combinations, respectively.

100 80 60 40

20 0 IODM In-50) Controls (n-50)

I--,O°.,n-50, Con,ro,.,.-60, ] FIGURE 2 Frequencies(%) of IDDM patients and controls carrying susceptible DQc~ and 3 chains.

DISCUSSION with four, two, and one ~S,flS combinations were 52%, 28%, and 18%, respectively. The remaining patient was ~PP,flSP and was typed as DR4,6;DQa52 Arg,Ser;DQ357 Asp,Asp.

We documented here the commonly observed increase of DR3 and DR4 alleles in IDDM patients, while, in contrast to previously reported data [22-24], we observed a higher frequency of DR3,3 than that of DR3,4 combinations. On the other hand, the significant decrease of DR5 and DR6 alleles in patients with respect to controls reflects the high frequencies of these specificities in the normal Italian population. However, DR oligotyping of the patients was mainly performed as help to derive, by the known linkage disequilibria, the DQc~fl cis- and trans-associations. With regard to the DQ genes, we confirmed the important role of both DQc~ 52 and DQfi 57 residues establishing susceptibility to IDDM. In fact, 49 of the 50 IDDM patients examined were found to carry DQA1 and DQB 1 alleles coding DQa Arg52 +/DOff Asp57 predisposing dimers. However, the subject homozygous fi57 Asp/Asp supports the observation that IDDM rarely occurs when none of the DQc~3 heterodimers of susceptibility are present [25]. Furthermore, the

DQ~fl heterodimers susceptible to CD. We previously reported [18] the frequencies of DR, DQA1, and DQB1 genotypes in 50 CD patients observed in Rome and 50 healthy controls. Considering that the highest risk of CD is for subjects carrying a DQ(~l*0501,f11*0201) heterodimer (RR = 52), we named DQAI*0501 and DQBI*0201 alleles as S. Non-S alleles were reported as P by analogy to IDDM, even if protection as such was not documented in CD. Figure 3B shows the distribution of genotypes coding S and P a and/3 chains in patients and controls. It is evident that the highest risk FIGURE 3 Distribution of HLA-DQ genotypes coding different number of susceptible dimers in IDDM (A) and CD (B) patients and controls.

A

B

DOA1B1

|nun

$8.88

~R:IA1,BI 88.8.S

.1

i RR

SS.SP

8S.SP

5P,SS

8P,88

PS.SP

PS.SP

SP.SP

8P.SP

PP.SS

PP.SS

SS.PP

8S,PP

PP, SP

PP, SP

PS,PP

PS.PP

PP.PP

IR

m

PP.PP 60

50

40

30

20

O0

0

10

~n~ IODM psllsnlo (n-SO) DOA~. IS'Aro52* P'A~g52-

20

30

40

50

eO

Conlrols (n-§O) DOBI: S-AIpS}'P-Asp5?*

17

60

50

40

~1CD

30

20

10

O

psllenlu (n-50) DOAI: S-DOA I.O501. P-OOA I.O501-

10

20

30

40

60

80

Conlwols In-50| DQBI: S-DOBI-O201P-DQBI,020 I-

Susceptible DQ Dimers in IDDM and CD

357Asp alleles recognized by probes 57-ASP1, 57-ASP2, and 57-ASP3 apparently have different protective effects. In fact, 57-ASP1 was significantly decreased in the patients in comparison to controls, but was present in the 28% of the cases; 57-ASP2, on the contrary, was never found in I D D M subjects. 57-ASP3 did not show any protective effect. With regard to the non-Asp alleles, only those 57-ALA positives showed a significant association with the disease, whereas the 57-SER alleles were found equally among patients and controls and those 57-VAL were even decreased in the patients. Similarly, the DQA1 alleles positive for 52-ARG L or 52-SER were related to the disease with a significant positive and negative association, respectively, while the 52-HIS or 52-ARG2 were not significantly associated. Recently, different studies reported that IDDM is associated with different DQ~/3 heterodimers conferring a susceptibility of varying strength [22, 24]. Altogether these observations should indicate that the residues ~52 and/357 alone are not the entire story of the H L A / I D D M association. However, very large samples of patients and controls will be required to exclude that these inconsistencies might depend on linkage disequilibrium. Regarding the genotypic combinations of D Q A 1 and B1 alleles, the highest risk was found for SS,SS subjects and decreased in proportion to the number of c~ and 3 S chains encoded [15]. While the apparent risk for I D D M was found directly proportional to the number of susceptible dimers present, no such dependency was observed in CD. In fact, 9 2 ~ of Italian CD children typed as DR3,x or DR5,7, so in most cases they carry only one DQc~ and one DQfi S chain, coded in cis- or in trans-association by D Q A 1"0501 and D Q B 1*0201 alleles. Therefore, it appears that CD develops in most cases in the presence of only one predisposing combination of the four D Q possible, whereas most I D D M subjects carry four D Q ~SS,/3SS molecules. The prevalence of DQA1 SS,SS genotypes in IDDM patients was seen as recessive behavior of the HLA susceptible genes [13]. On the contrary, the development of CD in a number of subjects carrying only one predisposing heterodimer suggests dominant behavior. Neither in I D D M nor in CD, however were these trends absolute. Otherwise, the apparent differences could be explained by a different role of non-S alleles in the two diseases: these alleles show a protective effect in IDDM, whereas this effect is not evident in CD. In this case, the disease develops in most patients in spite of the presence of non-S alleles. Our patients were characterized by the prevalence of DR5,7 heterozygotes and by a frequency of DR3 lower than that reported in the population of Northern Eu-

161

rope. Even if different distributions of the D Q A1/B1 genotypes from those reported here were expected, we believe that the prevalence of CD patients carrying only one predisposing dimer will be confirmed in all populations. The different number of predisposing dimers in IDDM and CD patients and the evidence that D Q confers susceptibility and protection in IDDM but just susceptibility in CD could reflect different functional mechanisms. Most hypotheses regarding the interpretation of the HLA associations and diseases are based on two main concepts: (a) HLA molecules work as restriction elements in the presentation of relevant peptides to T cells [26], and (b) class II dimers are involved in the positive and negative selection of the T-cell repertoire during ontogenesis of the immune system [27]. With respect to the first hypothesis, we must keep in mind that most ofT-cell clones relevant to pathogenesis in HLA-DQ-related diseases are D R restricted and that DQ-restricted clones are rare when compared with DPand DR-restricted clones. Therefore, a role in the thymic selection would explain the associations between D Q alleles and autoimmune disease better than presentation via D Q to pathogenetic T cells. Class II molecules and products of new genes in the HLA region [28] might be involved in different stages of the immune response and their effects could overlap. This fact explains the complex behavior of the HLA-linked susceptibility and makes difficult the identification of the specific role of D Q gene products. Moreover, because complete information regarding the etiopathogenesis of the two diseases are absent, it is impossible at the moment to define on a molecular basis the differences between IDDM and CD. ACKNOWLEDGMENT

This work has been supported by P. F. Ingegneria Genetica, CNR.

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