In this study of children at increased genetic risk for CD we found that antibodies to Glo-3A are higher in children who go on to develop CD than in matched controls. There was no difference between CD cases and controls in the age when antibodies to Glo-3A peak, but the peak Glo-3A antibody levels as well as Glo-3A levels over time are higher in children who developed CD. While the mean age of seroconversion to TTG positivity in cases was just under 5 years of age, the peak response to Glo-3A was evident, on average, before age 3 years.
The observation of increased levels of antibodies to proteins found in food may be interpreted several ways. The first possible interpretation is that the antibody response is a reflection of increased diversity in the child’s diet. The second is that Glo-3A antibodies are a biomarker of impaired immune tolerance and increased gut permeability. Finally, these results may indicate that the immune pathology and subsequent damage that are characteristic of CD start early in life.
Glo-3A is a salt-soluble globulin, and can remain trapped within the wheat gluten complex upon the processing of wheat for human consumption (10
). Because storage proteins can become trapped with the gluten complex, the reaction to Glo-3A may be a marker of dietary exposure to gluten and it becomes evident around the time the typical western diet becomes varied. The finding that the mean age at which Glo-3A levels peak does not differ between cases and controls supports this explanation.
It is possible that these higher antibody titers may reflect a higher antigen intake in cases that increased the risk for CD. We do not have a direct measure of wheat intake in these children. We did, though, estimate daily servings of foods containing gluten, obtained from an annual Willett Food Frequency Questionnaire. This questionnaire is a semi-quantitative food survey and therefore does not enable calculation of the weight of wheat proteins ingested. Data are available on approximately 50% of the study visits, so it was not considered as a covariate in these analyses. However, the mean number of daily servings of foods containing gluten for all visits in cases was 4.2, while mean daily servings of foods containing gluten for all visits in controls was 4.8. These results suggest that there is probably no difference in wheat intake between the two groups, but if a difference exists, wheat intake is lower in cases. Therefore, higher antigen intake does not explain higher Glo-3A antibody levels.
Another interpretation may be that the Glo-3A immune response provides an early marker of impaired oral tolerance and increased gut permeability. Glo-3A has also been observed to have sequence homology with the tight junction proteins participating in regulation of intestinal permeability(10
) and another food antigen, the highly immunogenic peanut allergen, Ara-h1(10
). Intestinal permeability has been implicated in the autoimmune cascade leading to T1D(23
). Indeed, several studies report that intestinal permeability is increased in CD, and that zonulin, at least in part, is a mediator of this effect(25
). Zonulin is an intestinal peptide that regulates the opening of gut epithelial cell tight junctions(28
). Our finding that CD cases have higher Glo-3A antibody responses throughout childhood could also be interpreted as evidence that impaired oral tolerance and increased gut permeability have an etiologic role in CD. Measuring Glo-3A antibodies along with biomarkers of intestinal permeability over time would help to elucidate how well they are correlated in an individual. Our preliminary data suggest that Glo-3A antibodies in a large proportion of cases peak in the pre-celiac autoimmunity period, a time which has not been previously characterized. (, S1
We interpret the pattern of Glo3A responses, whereby the peak occurs on average at 2.9 years and subsequently wanes, to possibly reflect changes in mucosal exposure to wheat proteins with the evolution of the infant and childhood diet. It is quite possible that the interaction between mucosal host factors (eg. intestinal permeability) and dietary antigen load produce the responses we found, and that this early period in the development of mucosal immunity and tolerance is critical in determining the subsequent risk for celiac disease.
Finally, ongoing exposure to the gluten macromolecular complex in wheat, rye and barley, exposes the patient to a sustained immune insult(4
). Classic indicators of this process include elevated TTG autoantibodies and villous atrophy(15
). Our finding that the Glo-3A antibody response is higher in cases compared to controls prior to the identification of clinical disease may be evidence that these processes are under way long before diagnosis of CD.
Children with diabetes-related autoimmunity have been previously shown to be more reactive to the Glo-3A antigen(12
) and in diabetes-prone rats higher Glo-3A reactivity was associated with pancreatic damage(10
). CD and T1D share high risk HLA haplotypes(2
), and so it is possible that an antigen that provokes an immune response in diabetic individuals could also be involved in the immunopathology of CD.
This study was performed in a group of children at increased genetic risk for CD and therefore the applicability to the general population is unknown. We cannot determine whether Glo-3A antibodies in our control population reflect responses in the population with a lower genetic risk for CD, because no data exist on antibody responses to Glo-3A in the general population. The control population in this study is at increased genetic risk compared to the general population based on the enrollment criteria of the CEDAR study. Further studies are required to examine responses to Glo-3A in a population that reflects the genetic distribution of the general population.
In addition, we cannot exclude the possibility that some of the control subjects will go on to develop celiac disease. Followup of this cohort will enable further analysis of Glo-3A antibodies and celiac disease in this population.
We cannot establish, based on these data, sensitivity, specificity, positive or negative predictive values for Glo-3A antibodies in CD. In order to identify possible diagnostic utility, it is necessary to measure Glo-3A antibodies in a larger sample of children from the general population to establish normal and abnormal levels. In order to rule out the possibility that elevated Glo-3A antibodies in early childhood are a predictor of later development of CD, a larger cohort of children needs to be followed from an early age for development of TTG and CD.
This study shows that higher levels of Glo-3A antibodies are associated with CD both at the time of clinical diagnosis as well as prior to that point. Further studies are warranted exploring this marker of mucosal immunity in relationship to development of CD and T1D.