Novel assay for detecting celiac disease–associated autoantibodies in dermatitis herpetiformis using deamidated gliadin-analogous fusion peptides

Novel assay for detecting celiac diseaseeassociated autoantibodies in dermatitis herpetiformis using deamidated gliadin-analogous fusion peptides Michael Kasperkiewicz, MD,a Cornelia D€ahnrich, PhD,b Christian Probst, PhD,b Lars Komorowski, PhD,b Winfried St€ ocker, MD,b Wolfgang Schlumberger, PhD,b Detlef Zillikens, MD,a and Christian Rose, MDa L€ ubeck, Germany Background: Dermatitis herpetiformis (DH) is a cutaneous manifestation of celiac disease (CD), the latter being identified by circulating autoantibodies against native gliadin (nGli), tissue transglutaminase (tTG), endomysium, or a combination of these. Recently, a novel serologic assay using deamidated gliadinanalogous fusion peptides (GAF3X) showed high diagnostic sensitivity in patients with CD. Objective: The aim of this study was to explore the anti-GAF3X enzyme-linked immunosorbent assay (ELISA) and to compare it with a panel of classic CD-related serologic tests in patients with DH. Methods: Antibodies against nGli, GAF3X, and tTG were determined by separate ELISA tests and against endomysium by indirect immunofluorescence microscopy in 45 patients with DH and 52 control patients (30 patients with bullous pemphigoid and 22 patients with linear IgA disease). A total of 24 patients with DH underwent intestinal biopsies to confirm underlying CD. Results: Antibodies to nGli were detected in 26 (58%) (IgA) and 24 (53%) (IgG), to GAF3X in 38 (84%) (IgA) and 36 (80%) (IgG), to tTG in 35 (78%) (IgA), and to endomysium in 32 (71%) (IgA) patients with DH. Combined testing of IgA and IgG antibodies to GAF3X detected 7 of 10 (70%) IgA-anti-tTG-negative patients with DH and together with the IgA-anti-tTG ELISA showed the highest serologic hit rate (93%) for CD. Limitations: Intestinal biopsies were not performed in all patients with DH. Conclusion: The novel anti-GAF3X ELISA shows a higher sensitivity to detect CD-associated autoantibodies in patients with DH compared with tests using nGli, tTG, or endomysium as substrates. ( J Am Acad Dermatol 2012;66:583-8.) Key words: celiac disease; deamidated gliadin; dermatitis herpetiformis; GAF3X; tissue transglutaminase.

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ermatitis herpetiformis (DH) is a pruritic subepidermal blistering autoimmune disease characterized by granular IgA deposits in the papillary dermis. It is considered a specific cutaneous manifestation of celiac disease (CD), also known as gluten-sensitive enteropathy, a state of increased immunologic responsiveness to ingested

From the Department of Dermatology, University of L€ ubeck,a and b Euroimmun Medizinische Labordiagnostika AG. Funding sources: None. Conflicts of interest: Drs D€ahnrich, Probst, St€ ocker, and Schlumberger are stock owners of Euroimmun AG. Drs Kasperkiewicz, Komorowski, Zillikens, and Rose have no conflicts of interest to declare. Accepted for publication February 9, 2011.

Abbreviations used: CD: DGP: DH: ELISA: nGli: tTG:

celiac disease deamidated gliadin peptides dermatitis herpetiformis enzyme-linked immunosorbent assay native gliadin tissue transglutaminase

Reprints not available from the authors. Correspondence to: Michael Kasperkiewicz, MD, Department of Dermatology, University of L€ ubeck, Ratzeburger Allee 160, 23538 L€ ubeck, Germany. E-mail: [email protected]. Published online August 16, 2011. 0190-9622/$36.00 Ó 2011 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2011.02.025

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gliadin proteins from wheat, barley, and rye, leading deposits of IgA in the dermal papillae and/or along to malabsorption. Patients with DH and CD usually the dermoepidermal junction in all cases. All serum have mild enteropathy presenting without clinical samples were collected before initiation of treatment symptoms.1 and a gluten-free diet. Sera from 52 patients, includPatients with DH and CD demonstrate IgA antiing 30 patients with bullous pemphigoid and 22 endomysium antibodies in their sera, which are patients with linear IgA disease served as control. directed against tissue transglutaminase (tTG).2-5 The diagnosis of underlying CD was confirmed by Although serum levels of IgA small intestinal biopsy speciantibodies to tTG reflect the mens in 24 patients with DH CAPSULE SUMMARY extent of histopathologic and classified according to changes of the small bowel Marsh.20 The study was apA novel celiac disease (CD) serologic and decrease under a glutenproved by the local ethics assay using deamidated gliadinfree diet,5-7 antibodies against committee and informed analogous fusion peptides (GAF3X) has epidermal transglutaminase consent was obtained from shown high diagnostic sensitivity in CD. are believed to play a central all patients. role in the pathogenesis and We found that compared with maintenance of the cutaneous conventional serologic tests, this antiAntibody detection disease in patients with DH, GAF3X enzyme-linked immunosorbent systems independently of a glutenassay yielded a higher detection rate for IgA or IgG reactivity to free diet.8-10 However, antiCD-associated autoantibodies in GAF3X was determined bodies against epidermal dermatitis herpetiformis. using enzyme-linked immutransglutaminase are not a nosorbent assay (ELISA) apThis novel assay facilitates identification suitable and evaluated tool plying the two nonapeptides of CD-related autoantibodies in to detect CD in these patients. PLQPEYPFP and PEQLPQFEE dermatitis herpetiformis. As in patients with DH the in a 3-fold repetition within associated CD is often mild, one recombinant polypepdetection rate of CD-associated antibodies in these tide as described previously (Euroimmun patients is lower than in patients with CD alone. In Labordiagnostika AG, L€ ubeck, Germany).14,15,19 both CD and DH, antibodies against native gliadin The first peptide was identified as the main epitope (nGli) have been considered to be of little diagnostic of deamidated gliadin and is targeted by autoantihelp.11 In contrast, deamidated gliadin fragments, bodies in the majority of patients with CD, whereas which are considered to be produced in vivo by the the second peptide sequence increases sensitivity. action of tTG under inflammatory conditions preFor comparison, IgA or IgG reactivity to nGli and tTG vailing in the small intestinal mucosa of patients with was determined by ELISA (both from Euroimmun CD,12-15 have recently been described as a more Labordiagnostika AG) using recombinant forms of suitable target for the detection of autoantibodies these proteins as target antigens. Each sample for in patients with CD.14-19 These reports used ELISA was tested in duplicate and antibody concensynthetic glutamine-glutamate substituted gliadin trations were expressed in arbitrary units. Cut-off homologous peptides (deamidated gliadin peptides values of 25 U/mL for anti-nGli ELISA and 20 U/mL [DGP])16-18 and, more recently, deamidated gliadinfor anti-tTG ELISA were established by the mean of analogous fusion peptides (GAF3X)14,15,19 as the 164 healthy blood donors 1 5 SD, whereas the cut-off antigenic target. value of 25 U/mL for anti-GAF3X ELISA was deterIn this study, we explored the performance of the mined by calculating the percentiles of 400 healthy novel GAF3X-based assay to detect circulating IgA blood donors and equates to the 98th percentile. IgAand IgG autoantibodies in a large cohort of patients anti-endomysium antibodies were detected by indiwith DH and compared it with conventional rect immunofluorescence microscopy on monkey CD-related antibody detection systems. esophagus. In addition, total serum IgA levels were determined in selected patients by nephelometric immunoassay (Nephelometer BN Prospec, Siemens METHODS Healthcare Diagnostics, Marburg, Germany). Patients Serum samples were obtained from 45 patients RESULTS with DH, whose demographic data are summarized A total of 45 sera samples from patients with DH in Table I. The diagnosis of DH was confirmed by and 52 control sera samples were studied for both direct immunofluorescence microscopy of perileIgA and IgG reactivity against nGli and GAF3X and sional skin biopsy specimens, showing granular d

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Table I. Comparison of different celiac diseaseerelated antibody assays and intestinal biopsy specimen findings in patients with dermatitis herpetiformis Patient No.

Sex/age, y

1 M/58 2 F/72 3 M/58 4 F/41 5 F/26 6 M/72 7 M/68 8 F/55 9 F/53 10 M/46 11 M/36 12 M/18 13 M/42 14 F/29 15 M/73 16 F/59 17 M/45 18 M/61 19 F/46 20 F/85 21 M/66 22 M/71 23 M/33 24 F/10 25 M/86 26 F/63 27 F/65 28 F/69 29 F/67 30 F/71 31 F/35 32 M/38 33 M/50 34 F/75 35 F/65 36 M/43 37 M/28 38 M/54 39 F/35 40 F/53 41 M/42 42 M/39 43 M/67 44 M/63 45 M/44 Total detection rates

IgA-anti-nGli ELISA, U/mL

IgG-anti-nGli IgA-anti-GAF3X IgG-anti-GAF3X IgA-anti-tTG ELISA, U/mL ELISA, U/mL ELISA, U/mL ELISA, U/mL

10 2 52 30 33 124 55 124 168 24 2 48 52 26 [200 45 195 166 [200 65 143 167 60 [200 20 3 24 4 6 8 42 13 148 [200 199 [200 79 15 [200 97 83 188 93 49 [200 [200 89 [200 21 19 83 73 55 [200 3 2 30 8 13 36 122 59 103 17 2 54 20 28 20 50 62 128 14 10 78 5 11 24 28 15 51 77 13 179 59 49 [200 2 5 2 61 154 84 87 89 94 35 43 105 114 59 [200 30 50 [200 9 4 100 27 96 57 13 14 61 13 14 22 29 54 69 17 168 12 23 42 110 12 10 76 62 3 100 3 8 96 26/45 (58%) 24/45 (53%) 38/45 (84%)

18 98 130 2 46 35 143 53 44 22 36 [200 127 35 70 49 52 58 29 [200 38 9 30 143 158 55 36 48 103 6 65 27 74 172 [200 41 54 35 6 13 2 141 55 3 48 36/45 (80%)

16 8 [200 [200 [200 [200 [200 [200 5 24 [200 [200 [200 [200 [200 [200 [200 [200 80 66 15 72 [200 [200 13 \2 38 [200 155 8 199 199 [200 [200 [200 [200 150 93 11 139 5 101 129 7 [200 35/45 (78%)

IgA-anti-endomysium Marsh type of intestinal (IIF on monkey biopsy esophagus)

Negative Negative Positive Positive Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative Positive Positive Positive Negative Negative Negative Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Positive Negative Positive 32/45 (71%)

I I IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIa IIIb IIIb IIIb IIIb IIIb IIIb IIIc IIIc IIIc IIIc n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.a.

ELISA, Enzyme-linked immunosorbent assay; F, female; GAF3X, gliadin-analogous fusion peptides; IIF, indirect immunofluorescence microscopy; M, male; n.a., not applicable; n.d., not done; nGli, native gliadin; tTG, tissue transglutaminase. Cut off for IgA- and IgG-GAF3X ELISA along with IgA- and IgG-anti-nGli ELISA: 25 U/mL; cut off for IgA-anti-tTG ELISA: 20 U/mL.

for IgA reactivity against tTG and endomysium. The results of the analysis of the 45 patients with DH are summarized in Table I.

No single serologic test was able to detect autoantibodies in all patients with DH. In contrast, although only two patients had negative findings

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for all tests, 43 of 45 (96%) had positive results for at least one assay. Antibodies to nGli were detected in 26 (58%) (IgA) and 24 (53%) (IgG), to GAF3X in 38 (84%) (IgA) and 36 (80%) (IgG), to tTG in 35 (78%) (IgA), and to endomysium in 32 (71%) (IgA) patients with DH. The results of the performance of the antiGAF3X ELISA are detailed in Fig 1. In the group of 24 patients with DH, whose CD was confirmed by small intestinal biopsy specimens, antibodies to nGli were detected in 15 (63%) (IgA) and 14 (58%) (IgG), to GAF3X in 21 (88%) (IgA) and 20 (83%) (IgG), to tTG in 20 (83%) (IgA), and to endomysium in 19 (79%) (IgA) patients with DH. Combined determination of IgA and IgG antibodies to GAF3X detected 7 of 10 (70%) IgA-anti-tTG-negative patients with DH. In contrast, there was one case of positive IgA antibodies to tTG, whereas no IgA or IgG reactivity against GAF3X was found. Combined results of the IgA- and IgG-anti-GAF3X ELISA resulted in a 91% detection rate for circulating autoantibodies in patients with DH and outperformed the corresponding anti-nGli ELISA by 27%. The serologic hit rate slightly increased to 93% when the IgA-anti-tTG assay was performed in addition to IgA- and IgG-anti-GAF3X ELISA. In two patients who tested negative for all assays and in 3 patients who tested positive in the IgG-anti-GAF3X ELISA but negative in the IgAanti-GAF3X ELISA the total serum IgA levels were determined to rule out relative IgA deficiency and false-negative results.21 In none of these patients total serum IgA values below the normal range were found. Combined results of the 3 ELISA tests are shown in Fig 2. Although only one patient from the control group with bullous pemphigoid showed positive results in the IgA-anti-nGli and IgA-antitTG ELISA, none of the remaining control sera were tested positive in any of the applied test systems. In the 24 patients in whom intestinal histology was available, the concurrent determination of IgA and IgG reactivity to GAF3X identified 19 of 20 (95%) patients with mild enteropathy showing at most partial villous atrophy (Marsh I-IIIb) and 4 of 4 (100%) patients with total villous atrophy (Marsh IIIc). Analyzing the individual remaining tests shown in Table I, positive results were obtained in less than or equal to 17 of 20 ( # 85%) cases with Marsh I to IIIb and in 3 of 4 (75%) patients with Marsh IIIc lesions, respectively.

DISCUSSION Recently, a new generation of promising assays detecting the presence of antibodies against deamidated gliadin fragments either by the anti-DGP or anti-GAF3X ELISA has shown high diagnostic performance in patients with CD.13-19 However, the

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Fig 1. Scatter plot of IgA (A) or IgG (B) autoimmune reactivity against deamidated gliadin-analogous fusion peptides (GAF3X ) in patients with dermatitis herpetiformis (DH ) and control patients with bullous pemphigoid (BP) and linear IgA disease (LAD) as determined by enzyme-linked immunosorbent assay (ELISA). Interrupted line corresponds to assay cut off (25 U/mL).

anti-GAF3X ELISA has not been used in patients with DH so far. On the other hand, conflicting results were obtained by the use of the anti-DGP ELISA for detecting gluten sensitivity in patients with DH.22,23 Although Sugai et al22 found IgA or IgG-anti-DGP antibodies in 78% of 18 patients with untreated DH, Jaskowski et al23 reported sensitivities of only 46% and 61% by the IgA- and IgG-DGP screen in 80 patients with DH on a normal diet, respectively. The reasons for these discrepant outcomes in patients with DH are unclear, as both investigations used the same ELISA system. However, a potential bias as a

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Fig 2. Venn diagram of concurrent determination of IgA/IgG reactivity against deamidated gliadin-analogous fusion peptides (GAF3X ) and IgA reactivity against tissue transglutaminase (tTG) in patients with dermatitis herpetiformis (DH ) as determined by enzyme-linked immunosorbent assay (ELISA).

result of difference in sample size cannot be excluded. In the current study, we used the anti-GAF3X ELISA to detect CD-associated circulating autoantibodies in patients with DH and compared the results with those obtained by conventional CD assays. We detected IgA- and IgG-anti GAF3X antibodies in 84% and 80% of DH sera, respectively. The sensitivity of the new anti-GAF3X test exceeded that of the conventional anti-nGli assay by 27% and was even higher than that of the previously reported antiDGP test in patients with DH.22,23 Our results in patients with DH are comparable with those recently carried out in a large cohort of patients with childhood CD, where sensitivities of 84% and 96% were found by the IgA- and IgG-anti-GAF3X ELISA, respectively.15 Importantly, IgA- and IgG-anti-GAF3X ELISA detected 7 of 10 IgA-anti-tTG-negative patients with DH, whereas only one patient showed tTG- but not GAF3X-specific antibodies. In total, the highest serologic hit rate of 93% was achieved by the combined use of the IgA- and IgG-anti-GAF3X ELISA with the IgA-anti-tTG assay. These findings in DH are in accordance with previous reports for CD, showing that deamidated gliadin assays can enhance the sensitivity for detecting gluten

sensitivity among IgA-anti-tTG-seronegative patients and further increase the fraction of patients identified as having CD when combined with the conventional IgA-anti-tTG screen.14,15,23-26 As the detection assays for deamidated gliadin fragments show high sensitivity for identification of CD, it remains to be clarified whether these tests are equivalent to histologic investigations of intestinal biopsy specimens. A recent large prospective study showed that it is possible to reach or rule out diagnosis of CD without biopsy in 92% of patients with different pretest probabilities for having the disorder using a combined detection screen for antibodies to tTG and DGP.26 However, it is still unclear whether these findings can be extrapolated to the subgroup of patients with CD presenting with only mild intestinal damage such as in the majority of patients with DH. A previous report with a limited number of patients with DH correlated the outcomes of CD-related serologic assays with the degree of intestinal damage. Although most tests detected patients with total villous atrophy and only 50% of those with normal intestinal histology had positive assays, patients with mild intestinal damage had discordant result. Classic CD-specific test results were positive in only some patients having mild

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enteropathy with signs of only partial villous atrophy, whereas all of them were identified by the anti-DGP assay.22 Although no clear conclusion regarding correlation of serologic results and Marsh grades can be drawn from our study, our results also highlight the advantage of the anti-GAF3X test over the other conventionally used CD assays in patients with DH presenting not only with total villous atrophy (Marsh IIIc), but also with histologically proven mild damage of the intestine (Marsh I-IIIb). However, these results need to be further confirmed by a greater number of patients with CD and mild mucosal damage. In conclusion, we show that in patients with DH, the novel IgA- and IgG-anti-GAF3X ELISA shows higher sensitivity to detect CD-associated autoantibodies compared with conventional CD serology tests and the recently applied anti-DGP assay. As intestinal damage is usually mild in DH, this disease constitutes a very useful model to further clarify whether these findings can be extrapolated to the subgroup of patients with CD having mild enteropathy. We thank Dr Leif Dibbelt for excellent technical assistance. REFERENCES 1. Nicolas ME, Krause PK, Gibson LE, Murray JA. Dermatitis herpetiformis. Int J Dermatol 2003;42:588-600. 2. Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797-801. 3. Dieterich W, Laag E, Bruckner-Tuderman L, Reunala T, Karpati S, Zagoni T, et al. Antibodies to tissue transglutaminase as serologic markers in patients with dermatitis herpetiformis. J Invest Dermatol 1999;113:133-6. 4. Porter WM, Unsworth DJ, Lock RJ, Hardman CM, Baker BS, Fry L. Tissue transglutaminase antibodies in dermatitis herpetiformis. Gastroenterology 1999;117:749-50. 5. Caproni M, Cardinali C, Renzi D, Calabr o A, Fabbri P. Tissue transglutaminase antibody assessment in dermatitis herpetiformis. Br J Dermatol 2001;144:196-7. 6. Rose C, Dieterich W, Br€ ocker EB, Schuppan D, Zillikens D. Circulating autoantibodies to tissue transglutaminase differentiate patients with dermatitis herpetiformis from those with linear IgA disease. J Am Acad Dermatol 1999;41:957-61. 7. Tursi A, Brandimarte G, Giorgetti GM. Prevalence of antitissue transglutaminase antibodies in different degrees of intestinal damage in celiac disease. J Clin Gastroenterol 2003;36:219-21. 8. Sardy M, Karpati S, Merkl B, Paulsson M, Smyth N. Epidermal transglutaminase (TGase 3) is the autoantigen of dermatitis herpetiformis. J Exp Med 2002;195:747-57. 9. Marietta EV, Camilleri MJ, Castro LA, Krause PK, Pittelkow MR, Murray JA. Transglutaminase autoantibodies in dermatitis herpetiformis and celiac sprue. J Invest Dermatol 2008;128: 332-5. 10. Rose C, Armbruster FP, Ruppert J, Igl BW, Zillikens D, Shimanovich I. Autoantibodies against epidermal transglutaminase are a sensitive diagnostic marker in patients with dermatitis

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