Chromosomal aberrations in peripheral blood lymphocytes in patients with newly diagnosed celiac and Crohn’s disease : European Journal of Gastroenterology & Hepatology

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Original Articles: Coeliac Disease

Chromosomal aberrations in peripheral blood lymphocytes in patients with newly diagnosed celiac and Crohn’s disease

Hojsak, Ivaa; Gagro, Alenkab,d; Petković, Iskrac; Mišak, Zrinjkaa; Kolaček, Sanjaa

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European Journal of Gastroenterology & Hepatology 25(1):p 22-27, January 2013. | DOI: 10.1097/MEG.0b013e328359526c
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Abstract

Introduction

Celiac disease is an immunologically mediated disorder that results in lifelong intolerance to dietary gluten in genetically predisposed individuals 1. Celiac disease shares several similarities to other chronic digestive diseases, such as Crohn’s disease; both are chronic inflammatory diseases of the digestive tract and both have complex genetic traits with multiple genetic and environmental risk factors 2. A recently carried out meta-analysis of genomewide association studies identified four shared risk loci for celiac and Crohn’s disease: PTPN2, IL18RAP, TAGAP, and PUS10 3. Moreover, both diseases are, to some degree, associated with increased potential for malignancy 4–6. A hallmark cancer for celiac disease is enteropathy-associated intestinal T-cell lymphoma (EATL), which originates from intraepithelial lymphocytes (IELs) 7,8. Increased number of IELs is one of the key criteria for the diagnosis of celiac disease 9. However, it can be also found in some other diseases, such as infectious entities, in postenteritic syndrome, intolerance to cow’s milk protein, tumors, and in other immune-mediated intestinal inflammations 10. The IELs phenotype in celiac disease expresses surface CD3 associated with surface CD8 and CD103 integrin, which binds to its ligand, E-cadherin, on intestinal epithelial cells 11. These IELs play an important role in refractory celiac disease, a disease that is unresponsive to a gluten-free diet and that, in 50% of patients, can progress to EATL 7,12. IELs in refractory celiac disease and EATL have an aberrant immunophenotype, generally lacking CD8, and surface CD3, but, similar to their normal counterparts, express CD103 integrin 8,13–15. These aberrant T cells have the ability to disseminate in the other tissues such as different levels of the gastrointestinal tract, cutaneous tissue, and peripheral blood still expressing CD103, which indicates their intestinal origin 16–18. Interestingly, it has been shown that the presence of an aberrant immunophenotype and monoclonality of IELs is not specific to refractory celiac disease as they are also observed during the uncomplicated celiac disease, although often being transient and associated with noncompliance with a gluten-free diet 19.

The pathogenetic mechanism of malignant alteration is not clear. The first cytogenetic study of patients with celiac disease found an increased number of spontaneous chromosome aberrations and suggested that this state may be inherited 20–22. However, subsequent studies found similar chromosomal instability in other chronic enteropathies 23, which raised the possibility that chromosome instability is a secondary phenomenon, possibly caused by chronic inflammation. This was further confirmed by the finding that a gluten-free diet can reduce the rate of chromosomal aberrations 24.

Furthermore, increased chromosome instability was found also in Crohn’s disease 25–27 and seems to be secondary, as found in celiac disease. This was suggested by the Danalioglu’s 27 study, which found that infliximab therapy lowers Crohn’s disease activity and frequency of chromosomal instability, implying that chromosomal instability is associated with chronic inflammation in Crohn’s disease. Moreover, chronic inflammation is a common underlying condition in human tumor development, accounting for ∼20% of human cancers 28. Both celiac disease and Crohn’s disease are associated with chronic inflammation, where the subsequent inflammatory response may be directly linked to increased production of proinflammatory cytokines such as IFN-γ and TNF-α 29. This can cause oxidative stress and damage DNA, which may be associated with an increased risk of malignant neoplasms 30. It has been shown that patients with celiac disease have significantly higher oxidative damage to DNA than controls, irrespective of the diet, providing a new insight in the pathogenesis of this phenomenon 31.

To test the hypothesis that chromosomal aberrations are a consequence of chronic inflammation, we compared the number of chromosomal aberrations in patients with Crohn’s disease with patients with celiac disease and healthy controls. Our second aim was to evaluate whether the patients with newly diagnosed celiac and Crohn’s disease have increased number of peripheral blood lymphocytes expressing CD103, indicating their intraepithelial origin.

Materials and methods

Patients

Children with newly diagnosed celiac and Crohn’s disease who presented to our Center from June 2006 to December 2008 and whose parents signed an inform consent were recruited prospectively into the study. Hospitalized patients without acute or chronic gastrointestinal disease served as a control group.

The celiac disease group included 19 newly diagnosed patients. The diagnosis was made on the basis of the following: (a) typical abnormality on small bowel biopsy (all patients had Marsh–Oberhuber 3 score); (b) positive serological tests (antigliadin and antiendomysial antibodies); and (c) improvement on a gluten-free diet.

Crohn’s disease group comprised 13 untreated pediatric patients. Patients were diagnosed following the Porto criteria 32.

The control group included 12 children who underwent investigations for various symptoms (including short stature, recurrent abdominal pain, constipation, and anemia) and who were subsequently not found to have organic disease. All patients had negative serological tests for celiac disease.

The mean age in celiac, Crohn’s disease, and control group was 7.6, 12.6, and 8.3 years, respectively, and the female/male ratios were 12/7, 9/4, and 7/5, respectively. There was no difference between the groups in terms of age (P=0.06) and sex distribution (P=0.88).

Methods

Chromosome aberrations

Chromosome aberrations were analyzed in peripheral blood lymphocytes. Cells were grown in Eagle’s minimal essential medium supplemented with 20% human AB serum and phytohemagglutinin and incubated for 72 h at 37°C. Chromosome preparations were obtained using a standard method including colchicine treatment and hypotonic treatment with 0.075 mol/l KCl, followed by fixation with methanol : acetic acid (3 : 1). Air-dried slides were Giemsa stained. For each patient, 100 metaphases were analyzed for chromosome-type and chromatid-type aberrations (breaks, gaps, and acentric fragments). A single cytogeneticist, who was blinded to the origin of the cells, and was not involved in the treatment of the patients, carried out the analyses manually.

Flow cytometry analysis

The following murine antibodies to human lymphocyte surface antigens were used: anti-CD4 [peridinin chlorophyll protein (PerCP)-conjugated], anti-CD8 (PerCP-conjugated), anti-CD3 (fluorescein isothiocyanate-conjugated), and anti-CD103 (phycoerythrin-conjugated) (all from Becton Dickinson, Heidelberg, Germany). In each experiment, fluorescein isothiocyanate-conjugated, phycoerythrin-conjugated, and PerCP-conjugated isotype controls for the determination of nonspecific binding were included. Briefly, 50 µl of heparinized blood was incubated in 12×75 mm polystyrene round-bottomed tubes (Becton Dickinson, Heidelberg, Germany) with 5 µl of murine antihuman monoclonal antibodies to CD3, CD4, and CD8 for 15 min in the dark at room temperature. Red blood cells were lysed by adding 2 ml of 10% FACS lysing solution (Becton Dickinson, San Jose, California, USA) for 10 min at room temperature in the dark. After washing, the cells were resuspended in 0.5 ml of the staining buffer. Correlated analysis of forward-angle and right-angle scatter was used to establish a lymphocyte gate. A minimum of 10 000 events for three-color immunofluorescence analysis in lymphocyte gate was analyzed by a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, California, USA). The nonspecific staining, assessed by an isotype control, was adjusted to less than 1%. The data collected were analyzed using CELLQuest software (Becton Dickinson, Mountain View, California, USA) and presented as a percentage.

Statistics

The significance of the differences between the groups was analyzed by one-way analysis of variance. If the difference between groups was significant, post-hoc analysis using the Bonferroni test was carried out. P levels of less than 0.05 were considered as significant. To calculate the correlation between numbers of chromosomal aberrations with the number of CD103+ lymphocytes, Pearson’s correlation was used. Statistical analysis was carried out using SPSS 17.0 (SPSS Inc., Chicago, Illinois, USA) statistical software.

The ethical committee of the Children’s Hospital Zagreb had approved the study protocol.

Results

The mean number of chromosomal aberrations/100 metaphases for all three groups is shown in Table 1 and Fig. 1. There was a significantly higher number of aberrations in the celiac disease group compared with the controls (post-hoc analysis; mean 6.8 vs. 4.0, P=0.003) and also in Crohn’s disease vs. the control group (post-hoc analysis; mean 6.2 vs. 4.0, P=0.04). However, there was no difference between the celiac disease group and Crohn’s disease group (post-hoc analysis; mean 6.8 vs. 6.2, P=0.49). A similar significant difference was found in the percentage of aberrant cells for celiac disease and Crohn’s disease group compared with the controls (post-hoc analysis; mean 6.4 vs. 3.7, P=0.001; mean 5.8 vs. 3.7, P=0.016, respectively), but not between the celiac disease group and Crohn’s disease group (post-hoc analysis; mean 6.4 vs. 5.8, P=0.48) (Table 1, Fig. 2). Chromosomal-type aberrations were increased only in the celiac disease group compared with the control group (post-hoc analysis; P=0.02), but not in the Crohn’s disease group compared with the control group and the celiac disease group (P=0.071 and 0.234, respectively). With respect to chromatide-type aberrations, there was no statistical difference (P=0.17). Types of aberrations included the following: (a) in celiac disease patients, there were altogether 63 breaks and 60 gaps, four chromosomal exchanges, six acentric fragments (ace), two dicentric chromosomes (dic), and one premature centromere division (pcd); (b) in Crohn’s disease patients, there were 43 breaks and 32 gaps, one chromatide exchange, two chromosomal exchange, one ace, and one ring chromosome; and (c) in the control group, there were 13 breaks, 21 gaps, and one pcd.

T1-4
Table 1:
Mean (range) number of chromosomal aberrations, aberrant cells, chromatide, and chromosome-type aberrations in every investigated group
F1-4
Fig. 1:
Individual values (with means and 95% confidence interval of means) for chromosomal aberrations/100 metaphases.
F2-4
Fig. 2:
Individual values (with means and 95% confidence interval of means) for aberrant cells.

The mean percentages of CD103+ and CD8+CD103+ cells for all three groups are presented in Table 2 and Fig. 3. There was no statistically significant difference between the groups with respect to the percentage of CD103+ and CD8+CD103+ cells (P=0.16 and 0.41, respectively). There was no correlation between age and percentage of CD103+ and CD8+CD103+ cells (P=0.23 and 0.08, respectively).

T2-4
Table 2:
Distribution of cells expressing CD103. CD103 expressing total T cells (CD3+), helper T cells (CD3+CD4+), and cytotoxic T cells (CD3+CD8+)
F3-4
Fig. 3:
Bar chart showing the distribution of cells expressing CD103 (mean % and SD of means). CD103 expressing total T cells (CD3+), helper T cells (CD3+CD4+), and cytotoxic T cells (CD3+CD8+).

We found no correlation between the total number of chromosomal aberrations and the percentage of CD103+ and CD8+CD103+ cells (P=0.06 and 0.06, respectively).

Discussion

The results of this study have clearly shown that both groups of patients – those with celiac disease and those with Crohn’s disease, before treatment initiation, had significantly increased the number of chromosomal aberrations in the peripheral blood lymphocytes. Increased chromosomal instability in celiac disease and Crohn’s disease has been found in other studies, both in adults 20–22,25–27 and in children 23. However, a recent study carried out by Martin-Arruti et al.33 failed to confirm genomic instability in patients with celiac disease, but the study used a different technique that measured the frequency of two translocations: t(14;18) and t(11;14). The correlation between chromosomal instability and malignancy has been published previously; there are genetic disorders, such as ataxia teleangiectasia and Fanconi anemia, with increased chromosomal instability, associated also with various cancers 34. Moreover, a recently published study showed evidence of increased chromosomal damage in lymphocytes of incident cancer patients compared with healthy controls 35. It has been confirmed that patients with celiac disease have a 1.3-fold higher risk of malignancies compared with the general population, with the non-Hodgkin’s T-cell lymphoma being the most commonly reported, especially EATL 4,36,37. Although the genetic and epigenetic abnormalities associated with EATL have not been fully characterized, consistent chromosomal imbalances were found indicating a link between refractory celiac disease and EATL 38. Cytogenetic analysis of the immunophenotypically abnormal T-cell clones isolated from patients with refractory celiac disease showed partial trisomy 1q22–44 39, indicating that gain at chromosome 1q might be an early event during the multistage process of EATL development 38. These EATLs are believed to arise from the IELs compartment, and share immunophenotypic characteristics to the aberrant IELs in refractory celiac disease 40. These aberrant T cells have the ability to disseminate in the other tissues such as peripheral blood, still expressing CD103 17. However, our results found no elevation in circulating CD103 positive cells in newly diagnosed celiac disease patients compared with the healthy controls. Moreover, follow-up of these patients indicated a good response to a gluten-free diet, with no refractory celiac disease. On the basis of these results, we can speculate that dissemination of cells expressing CD103 is limited to premalignant lesions, meaning refractory celiac disease. Overall, the number of cells expressing CD103 in peripheral blood was low in patients and in healthy children. Our study found that the total number of cells expressing CD103 in peripheral blood of healthy children is comparable with the number previously described in adults, 1–2% and for CD3+CD103+ up to 1% 41,42.

Nevertheless, IELs are not exclusively involved in the pathogenesis of celiac disease; they can also be found in other diseases such as Crohn’s disease, which also has a higher risk for malignancies; however, the link between these characteristics is not known. The pooled relative risk for developing lymphoma in patients with Crohn’s disease is 1.42 43. Further, in Crohn’s disease, there is a question of whether the increased risk is caused by the disease itself or by the immunosuppressive drugs commonly used for treatment. Two studies have investigated the risk in patients who were not treated with immunosuppressive drugs and have also found a two times higher risk for malignant tumors than in the general population 6,7. Our study confirmed an elevation in chromosomal aberrations in treatment-naive Crohn’s disease patients, indicating that chronic inflammation in Crohn’s disease patients, per se, causes chromosomal instability.

We are aware of the limitations of our study; the number of patients included was rather small and the overall frequency of chromosomal aberrations was low, which can also be attributed to the patients’ young age. This may explain why the difference in the separately analyzed chromatide and chromosomal aberrations was not statistically significant for Crohn’s disease compared with the controls.

Conclusion

Both patients with active celiac disease and newly diagnosed Crohn’s disease, before treatment initiation, have a significantly increased number of chromosomal aberrations in peripheral blood lymphocytes. We found no dissemination of intraepithelial cells in the blood and no correlation to the chromosomal aberration. We can therefore speculate that chronic inflammation that links both diseases can cause chromosomal instability, not associated with the IELs.

Acknowledgements

This research was supported by the Ministry of Science, Education and Sports of the Republic of Croatia projects: Celiac disease in children: prevention and pathogenesis of chromosome instability (072-1083107-2054); Modulation of human regulatory T-cell function (072-1080229-0337).

Conflicts of interest

There are no conflicts of interest.

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Keywords:

celiac disease; children; chromosomal aberrations; Crohn’s disease; intraepithelial lymphocytes

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