HLA-DQB1*02 dose effect on RIA anti-tissue transglutaminase autoantibody levels and clinicopathological expressivity of celiac disease
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Journal of Pediatric Gastroenterology and Nutrition 47:288–292 # 2008 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition
HLA-DQB102 Dose Effect on RIA Anti-tissue Transglutaminase Autoantibody Levels and Clinicopathological Expressivity of Celiac Disease Raffaella Nenna, yBarbara Mora, yFrancesca Megiorni, yMaria Cristina Mazzilli, yFabio Massimo Magliocca, zClaudio Tiberti, and Margherita Bonamico Departments
of Paediatrics, {Experimental Medicine, and {Clinical Sciences, ‘‘Sapienza’’ University, Rome, Italy
ABSTRACT Objectives: Celiac disease is an autoimmune enteropathy caused by gluten ingestion in genetically susceptible individuals. Antitransglutaminase autoantibody (tTGAb) assay is useful to detect candidates undergoing intestinal biopsy. Our aim was to investigate whether the DQB102 allele could influence tTGAb titers and the clinicopathological expressivity of the disease. Methods: A total of 124 patients with celiac disease, tested for RIA tTGAb at diagnosis, were typed for HLA-DRB1, -DQA1, and -DQB1 genes and divided according to the number of DQB102 alleles: group 1, homozygous; group 2, heterozygous; group 3, negative. Results: The mean of tTGAb indexes was significantly higher in group 1 patients than in group 2 (P < 0.02) and group
3 patients (P < 0.01). Patients with at least 1 DQB102 allele showed more often a typical CD and diffuse histological lesions than did patients in the other groups. Conclusions: The study demonstrates that tTGAb titers are HLA-DQB102 dose dependent, with significantly higher levels in homozygous individuals. Moreover, individuals with at least 1 HLA-DQB102 allele tend to have a more expressed clinical and histological form of celiac disease. JPGN 47:288– 292, 2008. Key Words: Anti-transglutaminase autoantibodies— Celiac disease—Clinical data—Histology—HLA. # 2008 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition
Celiac disease (CD) is a chronic autoimmune enteropathy caused by the ingestion of gluten and related prolamins, occurring in genetically susceptible individuals, characterized by villous atrophy and crypt hyperplasia of the small bowel mucosa. These histological lesions can be patchily distributed or can present only on the bulb mucosa (1). With respect to other multifactorial diseases, CD is unique, inasmuch as the critical environmental factor has been identified. In fact, patients with CD experience complete remission while receiving a gluten-free diet and experience a relapse when gluten is reintroduced. Celiac disease may appear either in a typical presentation, with gastrointestinal complaints, or in a subclinical form (2), including atypical symptoms and signs (3) such as anemia, hypertransaminasemia (4,5), and short
stature (6,7), or silent symptoms. The prevalence of CD is increased in some autoimmune diseases (8,9) and chromosomal aberrations (10,11). There is evidence of longlasting CD complications, which may include osteopathy (12), endocrinopathy (13), infertility, low birth weight (14,15), cancer (16), dilated cardiomyopathy, and other forms of heart failure (17). Patients with CD who receive a gluten-containing diet have increased levels of anti-gliadin, anti-endomysium (18), and anti-tissue transglutaminase antibodies (tTGAb) (19). The detection of these serum antibodies is useful in selecting candidates for intestinal biopsy. The identification of tTG as the main autoantigen recognized by anti-endomysium (20) has promoted various studies whose results suggest an important role of this enzyme in the etiopathogenesis of CD (19,21). The availability of human recombinant tTG has improved the performance of this analysis (22,23), and a novel radioimmunological assay using human recombinant tTG seems to be the most sensitive method (24–26). Celiac disease develops mostly in individuals carrying the HLA-DQ2 and HLA-DQ8 high-risk heterodimers.
Received May 31, 2007; accepted November 2, 2007. Address correspondence and reprint requests to Prof Margherita Bonamico, Department of Paediatrics, Sapienza University, V.le Regina Elena 324, 00161, Rome, Italy (e-mail: margherita.bonamico@ uniroma1.it). The authors report no conflicts of interest.
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HLA-DQB102 DOSE EFFECT ON RIA tTGAb LEVELS AND CELIAC DISEASE The a and b chains of the DQ2 molecule are encoded by the DQA105 and DQB102 alleles, present in cis in DR3 individuals or in trans position in DR5/DR7 heterozygous patients, whereas the DQA103 and DQB10302 alleles, on the DR4 haplotype, encode for the DQ8 molecule. In a recent study, it has been proposed that also the presence of 1 chain of the DQ2 heterodimer (ie, DQA105 or DQB102 but not both) determines a moderate risk for CD (27). Some studies have indicated that DR3-DQ2/DR7-DQ2 and DR3-DQ2/DR3-DQ2 individuals have an increased risk for development of the disease (28–30). This could be explained by a gene dosage effect of DQB102 allele in CD susceptibility, as suggested by Ploski et al (31). Several studies have investigated the relation among HLA alleles, serological markers (antigliadin) (32,33), and clinicopathological expressivity of CD (31,34–37). The aim of our study was to investigate whether the number of the DQB102 alleles could influence the tTGAb titers, clinical manifestations, and distribution and severity of histological lesions of the duodenum. MATERIALS AND METHODS Study Design We studied 124 patients with CD (47 male, 77 female, age ranges 1–45 years, median age 6.8 years) who attended the Pediatric Department at ‘‘Sapienza’’ University of Rome. The personal data of each patient were recorded on an ad hoc clinical questionnaire. Asymptomatic patients were enrolled during CD screening in first-degree relatives of patients with CD. Serum samples from all of the patients receiving a gluten-containing diet were tested for RIA tTGAb. No patient with IgA deficiency participated in the study. All of the patients were typed for HLA-DRB1, -DQA1, and DQB1 genes; patients were divided into 3 groups according to the presence of DQB102 allele, as follows: group 1 ¼ DQB102 homozygous (0201/0201 or 0201/0202 or 0202/0202), group 2 ¼ DQB102 heterozygous (0201 or 0202), group 3 ¼ DQB102 negative. The number of patients, age at diagnosis, and sex in the 3 groups are shown in Table 1. All of the patients, fasting overnight, underwent upper endoscopy and the performance of multiple biopsies (1 sample from bulbous mucosa and at least 4 samples from the distal duodenum) after narcosis. The histological lesions of the intestinal mucosa were evaluated according to the Marsh classification as modified by Oberhuber et al (38). TABLE 1. Characteristics of 3 groups of patients with celiac disease
No. of patients Age, y (mean SD) Sex, M:F
Not significant.
Group 1
Group 2
Group 3
26 6.5 8.65 10:16
85 7.8 5.9 31:54
13 10.9 9.5 6:7
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The diagnosis of CD was made according to the modified European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) (39) and North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) (40) criteria.
Autoantibody Radioimmunoassay Human full-length tTG cDNA construct (a.a.1-687) used in the present study (kind gift of Dr G.S. Eisenbarth, Barbara Davis Center for Childhood Diabetes, University of Colorado, Health Sciences Center, CO, USA) was transcribed and translated in vitro in the presence of [35S]-methionine (NENTM Life Science Products Inc) using the TNT-coupled rabbit reticulocyte system (Promega) with Sp6 RNA polymerase. Autoantibodies against tTG construct were detected according to a previously described quantitative radioimmunoprecipitation assay procedure (26). Briefly, each [35S]-methionine human tTG construct (50,000 cpm/tube) was incubated with serum at 1:25 dilution overnight at 48C. Then, 25 mL of goat anti-human IgA-Agarose (Sigma) was added to this solution to separate antibody-bound labeled products from free ones and left to incubate at 48C for 1 hour. After elimination of the aspecific radioactivity by washing with buffer solution and aspiration of the supernatant, 100 mL of scintillation liquid (Packard) was added to each tube to resuspend the pellet. Subsequently, the suspension was carefully transferred to a scintillation vial and counted in a b counter. Autoantibody levels were expressed as an index defined as follows: (sample cpm–negative standard control cpm)/(positive standard control cpm–negative standard control cpm). The threshold of positivity, established by use of a ROC plot curve, corresponded to the 99th percentile of 112 healthy control sera (67 females, 45 males, mean age 16.0 14.2 years, range 1.1–50 years) and was 0.050 (26).
HLA Typing HLA-DRB1, -DQA1, and -DQB1 typing was performed by polymerase chain reaction-sequence specific primers (PCR-SSP) by use of commercial kits (Dynal, UK).
Ethical Issues The study was performed according to the Declaration of Helsinki. All of the patients were informed of the objectives of the study. Informed consent was obtained from patients or from parents for their children.
Statistical Analysis The Mann-Whitney nonparametric U test was used to determine differences between groups. P < 0.05 was considered statistically significant. Statistical analysis was performed by use of True Epistat (Round Rock, Richardson, TX, USA).
RESULTS As shown in Table 1, group 1 (DQB102 homozygous) and group 2 (DQB102 heterozygous) patients with CD were younger at diagnosis than were group 3 (DQB102
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NENNA ET AL. TABLE 2. Characteristics of patients with celiac disease in Group 3 Serology HLA typing DRB1
Patient 1 2 3 4 5 6 7 8 9 10 11 12 13
Age, y
Sex
tTGAb
EMA
Clinical form
1.5 11 3.8 8.2 10 17.3 15.1 4.5 2.4 11.4 40 10.6 8.5
M F M M F M M F F F M M F
Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.
Neg. Pos. Pos. Pos. Neg. Nd. Pos. Pos. Pos. Pos. Pos. Neg. Neg.
T S T S T S A T T T S T T
Histology
-DQA1-DQB1
3A 3B 3C patchily 3C only bulb 3C patchily 3C 3C 3B 3B 3C only bulb 3A 3B 2
11-05-03/11-05-03 04-03-0302/11-05-03 04-03-0302/11-05-03 04-03-0302/11-05-03 04-03-0302/11-05-03 04-03-0302/14-05-03 04-03-0302/13-01-06 04-03-0302/11-05-03 04-03-0302/04-03-0302 01-01-05/04-03-0302 04-03-0302/13-01-06 04-03-0302/04-03-0302 15-01-05/04-03-0302
EMA ¼ antiendomysium; T ¼ typical; S ¼ silent; A ¼ atypical.
negative) patients and had a male:female ratio of 1:1.7, whereas in group 3 no predominance of females was found (Table 2). All group 1 (26/26) patients, 98.8% (84/85) of group 2 patients, and 100% (13/13) of group 3 patients were found to be tTGAb positive. As shown in Figure 1, the mean of tTGAb levels was significantly higher in group 1 patients with CD (0.96 0.49 Ab index) with respect to group 2 (0.75 0.35 Ab index) (P < 0.02) and group 3 (0.58 0.37 Ab index) patients with CD (P < 0.01) (group 2 vs group 3 P < 0.07). Group 1 included 10 DR3/3 (DRB103-DQA10501DQB10201/DRB103-DQA10501-DQB10201) and 15 DR3/7 (DRB103-DQA10501-DQB10201/DRB1 07-DQA10201-DQB10202) patients carrying 2 DQB102 and at least 1 DQA105 alleles and hence the DQ2 heterodimer. The remaining patient was DR7-
DQB102 homozygous, lacking the a chain of the DQ2 heterodimer. Interestingly, this patient showed a tTG value near the mean of this group. Of the 85 patients in Group 2, 41 carried the DQA105 and DQB102 alleles in cis and 31 in trans combination. All of the 13 remaining patients were DR7-DQB102, with a second haplotype negative for the DQA105 allele. Of these 13 patients, 5 were also DR4-DQ8 positive, and the remaining patient was only DQA105 positive. The hemoglobin values (mean standard deviation) of patients in the 3 groups did not differ significantly (group 1, 12 2 g/dL; group 2, 12.4 1.2 g/dL; group 3, 12.7 1 g/dL). The percentages of patients with typical forms, compared with subclinical forms (atypical and silent), were higher in group 1 and group 2 (80% and 76%, respectively) with respect to group 3 (61.5%) (not significant). Figure 2 shows the percentages of the different histological lesions (type 2, type 3a, type 3b, and type 3c, according to the Marsh modified classification) (38) in the duodenal mucosa in the 3 groups. The most frequent lesion observed in all of the groups was the most severe: type 3c (total villous atrophy). The histological lesions were seen to be diffuse in 91.7%, 92.3%, and 69.2% of group 1, 2, and 3 patients with CD, respectively. In 1 group 2 (1.2%) and in 2 group 3 (15.4%) patients with CD, histological lesions were detected only in the duodenal bulb mucosa. DISCUSSION
FIG. 1. tTGAb titers in group 1, group 2, and group 3 patients with celiac disease. Group 1 ¼ DQB102 homozygous (0201/0201 or 0201/0202 or 0202/0202); group 2 ¼ DQB102 heterozygous (0201 or 0202); group 3 ¼ DQB102 negative. Group 1 vs group 2 ¼ P < 0.02; group 1 vs group 3 ¼ P < 0.01; group 2 vs group 3 ¼ P < 0.07.
It is well known that genetic background and environmental factors contribute to the clinical expressivity of CD. The HLA CD-related genotype is essential to the presence of the disease, but how it modules CD phenotype is a question open to discussion (31,34–37).
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HLA-DQB102 DOSE EFFECT ON RIA tTGAb LEVELS AND CELIAC DISEASE
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FIG. 2. Histological lesions in group 1, group 2, and group 3 patients with celiac disease. Group 1 ¼ DQB102 homozygous (0201/0201 or 0201/0202 or 0202/0202); group 2 ¼ DQB102 heterozygous (0201 or 0202); group 3 ¼ DQB102 negative.
The results of the study demonstrate significant differences, in terms of tTGAb titers, using a sensitive RIA method, among patients with CD receiving a glutencontaining diet, according to the dose of DQB102 allele. We found that the tTGAb levels depended on the number of DQB102 alleles, without differences between 0201 and 0202, with the highest value in patients with a double dose of this allele (DR3/DR3 and DR3/DR7). A DQA1 gene effect was not evident, inasmuch as 1 patient in group 1 (DR7-DQB102 homozygous) and 13 in group 2 (DR7-DQB102 heterozygous, 5 of them also DR4-DQ8), all negative for DQA105, had tTGAb levels from medium to high. The finding that DQA10201-DQB102 positive patients with CD without DQ2/DQ8 at-risk molecules produce tTGAb, even if at a lower level than DQ2-positive cases, seems to be of interest. Karell et al (27) were the first to show that CD could develop in patients coding for a heterodimer formed by the b2 chain together with an a2 instead of an a5 chain. A recent study gave evidence that these dimers are able to present the gluten epitopes, although less efficiently than the classic diseaseassociated DQ2 dimers (41). The tTGAb data, using a more sensitive method, confirm our previous findings, performed on antigliadin antibody levels, showing lower immune response in DR3 and DR7 negative patients with CD (33). Several studies have been performed with the aim of evaluating the relation between HLA genotype and clinical phenotype in CD (31,34–37). In our study, we observed only in patients positive for HLA-DQB102 allele the well-known predominance of female patients, whereas in DQB102 negative patients the male:female
ratio was 0.8:1. Moreover, homozygous patients, as in other studies (35–37), showed a younger age at diagnosis. In addition, when the clinical appearance of the disease was analyzed, the percentage of the typical form was higher in homozygous patients, with a trend to decrease in heterozygous and negative patients, showing more frequently a subclinical presentation of the disease, as reported by others (35–37). Among data collected from clinical records, we did not find relevant differences for either gastrointestinal or extraintestinal signs and symptoms in the 3 groups. The histological examination of duodenal mucosa showed that total villous atrophy, which was the prevalent lesion in all 3 groups of patients, was more frequent in homozygous patients than in the other groups, as shown by Zubillaga et al (36) and Karinen et al (37), as well. Recently, CD-related histological lesions of the intestinal mucosa, localized only in the bulb, have been reported both in children (1) and in adults (42). In the present series, 2 HLA-DQB102 heterozygous and 2 HLA-DQB102 negative patients showed such lesion distribution, whereas in homozygous patients only diffuse lesions or patchy villous atrophy were found. In conclusion, our study demonstrates, for the first time as far as we are aware, a gene-dose effect of the DQB102 allele on tTGAb titers in CD at diagnosis, with significantly higher levels in homozygous patients.
Acknowledgments: The authors thank Rita Pia Lara Luparia, Antonella Castronovo, Francesca Menasci, and Federica Lucantoni for clinical and technical assistance and Patricia Byrne for help with English style.
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