Persistence of elevated deamidated gliadin peptide antibodies on a gluten-free diet indicates nonresponsive coeliac disease

Abstract

Background: Histologically nonresponsive coeliac disease (NRCD) is a potentially serious condition diagnosed during the follow-up of coeliac disease (CD) when patients have persistent villous atrophy despite following a gluten-free diet (GFD).

Aim: As current assessments of recovery are limited to invasive and costly serial duodenal biopsies, we sought to identify antibody biomarkers for CD patients that do not respond to traditional therapy.

Methods: Bacterial display peptide libraries were screened by flow cytometry to identify epitopes specifically recognised by antibodies from patients with NRCD, but not by antibodies from responsive CD patients. Deamidated gliadin was confirmed to be the antigen mimicked by library peptides using ELISA with sera from NRCD (n = 15) and responsive CD (n = 45) patients on a strict GFD for at least 1 year.

Results: The dominant consensus epitope sequence identified by unbiased library screening QPxx(A/P)FP(E/D) was highly similar to reported deamidated gliadin peptide (dGP) B-cell epitopes. Measurement of anti-dGP IgG titre by ELISA discriminated between NRCD and responsive CD patients with 87% sensitivity and 89% specificity. Importantly, dGP antibody titre correlated with the severity of mucosal damage indicating that IgG dGP titres may be useful to monitor small intestinal mucosal recovery on a GFD.

Conclusions: The finding of increased levels of anti-dGP IgG antibodies in CD patients on strict GFDs effectively identifies patients with NRCD. Finally, anti-dGP IgG assays may be useful to monitor mucosal damage and histological improvement in CD patients on a strict GFD.

Figure 1

Consensus B-cell epitope identified using bacterial display random peptide libraries. (a) The enrichment of library members containing the FPE motif during the final five rounds of sorting. Alternating subtraction (s) and enrichment (e) steps were used with unique pools of three to five patients’ sera. (b) Web logo plot of the frequency of an amino acid appearing in peptides displayed by 17 single colonies of E. coli from the final round of FACS. Larger font size indicates a higher probability of appearance. (c) Comparison of six library isolated peptide sequences to an expanded epitope (DGP3) and B-cell epitopes from γ- and α-gliadin.

Figure 2

Reactivity of peptides with non-responsive celiac disease (NRCD) and responsive CD patients measured by flow cytometry. Clones were from the random peptide library screened with NRCD patient sera. (a) Response of serum IgG from NRCD (n = 15) and responsive CD patients (n = 45) to peptides DGP3, C83, and C139. Patients that reacted at or below background levels are represented here as 0.1% of the maximum signal so that they appear on the logarithmic plot. Horizontal lines represent the mean. (b) Antibody reactivity with DGP3 after one year gluten-free diet (GFD) for responsive patients that had serum taken at diagnosis and after one year of GFD (n = 24).

Figure 3

Confirmation of deamidated gliadin as the primary antigen mimicked by library isolated peptides and a marker of celiac disease (CD) patient recovery. (a) Anti-deamidated gliadin peptide (dGP) IgG antibody ELISA discriminates non-responsive CD (NRCD) (n = 15) and responsive CD patients (n = 45) (P < 0.0001). An adjusted ELISA cutoff of 12 units yielded 87% sensitivity and 89% specificity. (b) Anti-dGP titer increases with the severity of mucosal damage as described by Marsh staging (see Supplementary Table S2 for binary significance comparisons). (c) Correlation between flow cytometry assays with bacterial display peptides and anti-dGP IgG ELISA (P < 0.0001 for C139 and P = 0.0002 for DGP3). Each point represents an individual patient’s reactivity with C139 (▲) and DGP3 (●). (d) Receiver-operating characteristic (ROC) curve for the anti-dGP IgG antibody ELISA test. The area under the curve (AUC) was 0.94 (95% confidence interval, 0.88 to 0.99).

Figure 4

Proposed diagnostic algorithm for clinical follow-up of celiac disease (CD) patients on a gluten-free diet (GFD).