Abstract

Anti-tissue transglutaminase (tTg) IgA are considered the most frequently used serological marker for celiac disease diagnosis. Despite its recommended leading position by the 2012 ESPGHAN diagnostic criteria, it exposes multiple false positive and negative titers. In view of the critical opinions expressed lately in the literature against the application of those criteria, the bias in the central place occupied by tTg-IgA in the new ESPGHAN CD Diagnostic Guidelines and the emergence of newer serological marker for celiac disease, it is hoped that the revised guidelines will open up the limited, problematic and single Tg2-IgA antibody for other or additional single or combined serological diagnostic markers.

1. Introduction

Celiac disease (CD) is increasing in incidence and despite powerful serological markers its diagnosis present a great challenge ^{1, 2}. The rise in worldwide gluten consumption, the evolutionary growth in wheat gluten content and toxicity, changing phenotype toward older age, a/hypo symptomatic presentation, multi-extraintestinal organs affected, non-gluten associated environmental factors involved and the sequential changes in the diagnostic criteria, make the suspicion, awareness and diagnosis more complex ^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14}. This is the raison why Villanueva et al, should be congratulated for tackling the topic of IgA and IgG anti-tissue transglutaminase (tTg) antibodies place in CD diagnosis ¹⁵. Based on up-to-date literature, the authors concluded that: 1. it is insufficient to understand the difference of classes and subclasses detected in CD and other autoimmune conditions, 2. Data does not support the use of anti-tTg IgG for diagnosing CD in IgA-sufficient individuals and therefore 3. IgG should not be used in the routine diagnostic process of CD. Personally, I fully support the last two conclusions, but would like to expand on the first one. The present editorial will widen on the diagnostic reliability of the anti-tTg IgA, on the problematic new ESPGHAN diagnostic flow chart based primarily on anti-tTg IgA ¹⁶, the newer serological markers in CD diagnostics ^{17, 18, 19} and the potential functions of anti-tTg IgA- in CD induction or progression.

2. False Positive and Negative of Anti-tTg IgA

Anti-tTg IgA- antibodies are quite sensitive and specific non-invasive celiac diagnostic markers. It is the most frequently used to screen for CD and it is recommended by ESPGHAN ^{16, 19}. Several drawbacks still exist since its reflection of the intestinal damage and monitoring of disease activity, is not good enough. Its performance in children is better than in adulthood and elderly. It has multiple limitations and Table 1 summarizes its false positivity and negativity, as reflected in the current literature.

It is worthwhile mentioning that the antibody is directed against the enzyme tTg. The enzyme wide abundance and corporal distribution, its specialized structural conformation, vast substrate specificity, and indispensable cellular functions, explain its involvement in multiple physiological, as well as pathological conditions ^{21, 22}. tTg is involved in genetic, metabolic, senescence, rheumatic, cancerous, nephrogenic, pulmonary, endocrine, hepatic, neurodegenerative and multiple autoimmune diseases ^{4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 21, 23}. The enzyme pleiotrophism, ubiquitous expression and pivotal biological functionality might explain some of false result of the anti-tTg IgA antibodies directed against it (Table 1).

3. Bias in the Central Place Occupied by anti-tTg IgA in the 2012 ESPGHAN CD Diagnostic Guidelines

According to the new ESPGHAN CD diagnostic criteria, additionally to other fulfilled parameters, if the titer of anti-tTg IgA is above 10 times the upper limit of the normal, the diagnosis of CD can be established without a duodenal biopsy ¹⁶. Since then, several CD specialized groups expressed discomfort, and even criticized those guidelines ^{24, 25, 26, 27, 28, 29, 30}.

The main criticisms of the central place occupied by anti-tTg IgA autoantibodies in the 2012 ESPGHAN guidelines, to omit, in certain circumstances, intestinal biopsy in a symptomatic child are:

1. Lack of serological markers standardization and relative definitions of the upper limit of normal cut-off levels ^{19, 20, 24, 25, 26, 27}.

2. Lack of more extended, multicenter data on the optimal multiplication times of the upper limit of normal cut-off, to be used ^{26, 27}.

3. The subjectivity and inter-observer variability of the anti-endomysial antibodies that confirm anti-tTg IgA positivity ³¹.

4. Insufficient understanding by the clinicians of the performance of their celiac serological tests ³⁰.

5. Lack of adherence to and/or understanding of the guidelines, even by subspecialists ³².

6. Even the gold diagnostic standard of mucosal histology is debatable in several aspects: grading ³², intraepithelial lymphocyte count ³³, and pathologist’s reproducibility ³⁴. Notably, the decision of anti-tTg IgA antibody leading position was based on this gold criteria.

7. Geographical and national variability in CD diagnostic tools applicability, due to lack of resources ³⁵.

8. The choice of the anti- tTg IgA as the main serological diagnostic marker for CD was challenged by multiple clinical and laboratory professionals, advocating combined serology ^{19, 26, 36, 37, 38, 39, 40, 41, 42, 43, 44}. And finally, to set up the stage for the next topic:

9. Lack of comparison of CD additional specific autoantibodies to challenge anti-tTg IgA premiership in the guidelines, for example with the tTg neo-epitope ^{17, 38, 40, 45, 46, 47, 48, 49}. Recent observations show that the tTg neo-epitope outperforms tTg ^{17, 39, 49} and also a combination test including IgA and IgG isoforms ¹⁸. Adding an additional autoantibody can detect Marsh 3 intestinal damage among subjects with moderate anti-tTg levels ⁵⁰.

4. Newer Serological Markers in CD Diagnostics

The CD associated serological markers are continuously expanding. After more than 30 year, since the early 80th when EMA IgA was found ^{51, 52}, we witnessed the tTg, DGP IgG or IgA isotypes and combined IgA and IgG isotype emergence, in CD screening and diagnosis ^{18, 19}. Recently, two additional CD associated markers emerged. But this family of markers represents a new concept in CD pathogenesis. tTg and its family member, the microbial Tg (mTg) are well known to deamidate gliadin, but they can also cross-link gliadin, resulting in a new 3-dimensional complex where new immunogenic epitopes are exposed, namely: tTg-neo and mTg-neo-epitope complexes. The CD patients, in face of new immunogenic molecules, mount specific antibodies, namely: anti-tTg neo-epitope and anti-mTg neo-epitope antibodies of IgG and IgA isotypes. The anti-tTg-neo-epitope IgA and IgA+IgG were shown, in multiple studies to be very reliable in CD diagnosis ^{17, 18, 19, 26, 39, 40, 45, 45, 47, 48, 49, 53, 54, 55, 56, 57, 58}. In fact, anti-tTg neo-epitope was shown most recentlyto be more reliable than the anti-tTg IgA antibody ⁴⁹.

mTg is a secreted microbial transglutaminase that is very important for bacterial survival. Its sequence homology to tTg is poor, but at their active site, the homology is much higher, resulting in shared functions, mainly in cross-linking gliadin peptides ^{17, 23, 59, 60, 61, 62, 63}. Most recently, mTg-neo-epitope was shown to induce specific IgG antibodies in CD children, compared to controls. It was shown, for the first time that mTg-neo-epitope is immunogenic in a CD population ⁶². Not less important, in the last ESPGHAN 2017 meeting in Prague, the pathogenicity of the mTg and gluten was shown on CD duodenal biopsies and human originating intestinal cell line. (Sebastian S, Zimmer K-P, Giessen, Germany-personal unpublished data). The authors showed that when incubated with CD intestinal biopsy or with the cell line, mTg may influence the intracellular localization of gliadin in the intestinal mucosa of CD patients. Much more, mTg reaches the lamina propria, indicating an antigenic interaction with cells of the immune system.

5. The Potential Functions of Anti-tTg IgA in CD Induction or Progression

The specific autoantibody against tTg is multifunctional, affecting many of the enzyme activities. Notably, many of them induce the loss of enzyme function, while fewer functions are associated with gain of function of the tTg. It should be stressed that until today, no beneficial protective effects, but only pathogenic ones, were assigned to those CD associated autoantibodies. In summary, the anti-tTg antibodies potentially promote small bowel intestinal or extra-intestinal damage in CD patients. A word of caution: The majority of the anti-tTg autoantibody activities where studied in vitro and ex-vivo, very few though in animals but none in vivo, in human. Its differential role in CD induction or progression is far from being discovered. Unraveling them might open some new therapeutic strategies for CD.

References