Aim: An increased prevalence of celiac disease (CD) in cystic fibrosis (CF) has been discussed. The aim of this survey was to determine the prevalence of a genetic predisposition to CD in CF-patients compared to the general population.

Methods: Celiac serology and HLA-DQ2/ -DQ8 screening was performed in 190 CF-patients at the CF-Center Cologne, Germany. Informed consent was obtained from all patients or caregivers.

Results: 62 (32.6 %) carried HLA-DQ2 and 41 (21.6 %) HLA-DQ8 compared to 32% HLA-DQ2 and 17 % HLA-DQ8 in the German population. 5 (2.6%) carried both and 82 (43.2 %) carried neither HLA-DQ2 nor –DQ8. Six CF-patients (3.1%) showed elevated anti-tTG-IgA and two were diagnosed with CD (1.1 %) (both HLADQ2 positive)

Conclusion: There is a higher prevalence of elevated CD serology in CFpatients (3.1% with elevated anti-tTG-IgA compared to 0.8 % in the general German population). The frequency of CD in our CF-population is 1.1 %, which is elevated to the general population. However, the frequency of HLA-DQ2 and –DQ8 is similar to the general German population. Therefore, CF could be a risk factor for elevated celiac serology and CD and could contribute to the development of CD.

Broekaert IJ, Radojska S, Gathof B, Rietschel E, van Koningsbruggen-Rietschel S (2016) Cystic Fibrosis and Celiac Disease: Mere Coincidence? JSM Gastroenterol Hepatol 4(1): 1055.

Celiac disease , Celiac serology , Cystic fibrosis , HLA-DQ2 , HLA-DQ8

In a CF-population 3.1 % had elevated anti-tTG-IgA compared to 0.8 % in the general German population and 1.1 % of the CF patients were diagnosed with CD. The prevalence of HLA-DQ2 and –DQ8 is similar in CF-patients compared to the general population. Therefore, CF could be a risk factor for elevated celiac serology and HLA-typing helps to discriminate mere positive serology from celiac disease.

CD: Celiac Disease; CF: Cystic Fibrosis; Anti-DGP: AntiDeamidated Gliadin Peptide; EMA: Endomysial Antibodies; HLA: Human Leukocyte Antigen; Anti-Ttg: Anti-Tissue Transglutaminase

Cystic Fibrosis (CF) is the most common chronic genetic disease in Caucasians with a global prevalence of 0.74/10000 [1]. Current life expectancy has risen to an average of 38 years [2] and continues to rise.

Celiac disease (CD) is one of the most common lifelong disorders on a worldwide basis, and it is still underdiagnosed [3]. A high prevalence in the general population (1 %) has been reported in Finland thought to be due to nutritional and environmental factors [4]. Using celiac screening with anti-TGIgA Anderson et al. could even increase the prevalence of CD in an Australian population to 1.2 % in men and 1.9 % in women [5]. In the USA studies have shown that CD has a prevalence of approximately 0.8 % [5,6]. In the KiGGS study in Germany (12751 children between 1 and 17 years) 0.8 % showed elevated tGTIgA and/ or tTG-IgG and the prevalence of CD (clinically and serologically, but no histologically) is reported to be 0.9 % [7]. It has also been shown that the prevalence of CD is substantially increasing [4].

Both CF and CD cause intestinal malabsorption in the majority of cases making it difficult to identify CD in CF patients [8]. The co-existence of both CF and CD has been published numerously [9,10]. Valletta et al. calculated a prevalence of 0.45 % of CD in 1100 Italian CF patients [11]. Fluge et al. performed systematic screening for CD in a Scandinavian cohort of 790 CF patients, calculating a prevalence of 1.2 % [12]. A recent study reported an incidence of even 2.13 % of CD among 230 Polish CF patients [8].

The diagnosis of CD is based on the presence of glutendependent symptoms, CD specific antibodies, HLA-DQ2 or -DQ8 haplotypes and histological features [13]. Screening serum antibody tests, such as the anti-tissue transglutaminase (anti-tTG)-IgA, endomysial antibody (EMA)-IgA or the antideamidated gliadin peptide (anti-DGP)-IgA or -IgG tests, have excellent sensitivity and specificity rates. However, duodenal biopsies classified according to the Marsh criteria (intraepithelial lymphocyte infiltration, crypt cell hyperplasia and villous atrophy) are still the gold standard to confirm the diagnosis [13,14].

While gluten is a well known environmental factor, inherited genetic factors are also known for affecting disease susceptibility as seen in family and twin studies [15]. Among the genetic factors, the HLA genes have the greatest impact on the development of the disease [15]. Results from genetic linkage studies have shown that CD is strongly associated with HLA-DQ genes located on chromosome 6p21. The presence of genes coding for HLA-DQ2 (DQA1*0501/DQB1*0201) and -DQ8 (DQA1*0301/DQB1*0302) molecules of the HLA complex class II explains up to 40 % of the occurrence of CD in European populations [16]. It is hypothesized that HLA-DQ2 is more efficient than HLA-DQ8 in presenting gliadin to the immune system [17]. Therefore, the more severe forms of CD are marked by the HLA-DQ2 allele, especially in the homozygous condition [17]. The absence of theses alleles is important for their high negative predictive value [18].

The aim of this survey was to evaluate the prevalence of elevated celiac serology and CD as well as the genotypes HLADQ2 and -DQ8 and to compare them to the general population in order to assess if CF is a risk factor for CD.

Patients

HLA typing was performed and celiac serology was obtained in 190 CF patients aged between 0 and 68 years. The diagnosis CF was defined according to the CF guidelines [19]. The diagnosis of CD was established according to the guidelines of the ESPGHAN [13]. Informed consent was obtained from all patients or their caregivers.

HLA-DQ2 and -DQ8 genotyping

All CF patients were genotyped for the expression of the HLADQ2 and –DQ8 heterodimers by DNA tests for the HLA-DQB1 and –DQA1*0501 loci coded for the alpha and beta chain of the DQ molecule. Genomic DNA was extracted from whole blood (EZ1 DNA Blood 350 µl Kit, Qiagen Group). HLA genotyping was performed by the PCR-SSP-method (HLA-DQB1*/DQA1* Low Resolution CTS-PCR-SSP Tray Kit, Department of Transplantation Immunology, University Clinic Heidelberg, Germany).

Serological tests

In all CF patients anti-tTG-IgA, anti-DGP-IgA and anti-DGP-IgG were measured by ELISA (anti-human-anti-tTG-IgA, anti-humanGliadin (GAF-3X)-IgA and anti-human-Gliadin (GAF-3X)-IgG (Euroimmun AG, Luebeck, Germany). EMA-IgA was determined by indirect immunofluorescence using monkey liver as substrate (Euroimmun AG, Luebeck, Germany). Sera were diluted 1:10 in PBS-Tween, and the antigen–antibody complex was determined by fluorescein tagged anti-human-EMA-IgA (Euroimmun AG, Luebeck, Germany) using fluorescence microscopy. Total IgA was determined by immunoturbidimetry using anti-humanIgA antibodies (Tinaquant IgA-2 test; Roche Diagnostics GmbH, Mannheim, Germany). The cut-off for anti-tTG-IgA was 20 RU/ ml, for EMA-IgA >1:10, for anti-DGP-IgA and anti-DGP-IgG both 25 RU/ml. In case of an IgA-deficiency (≤0.3 g/l below age 15, ≤0.85 g/l for women, and ≤1 g/l for men) anti-DGP-IgG testing was performed [13].

In patients with elevated anti-tTG-IgA an esophagogastroduodenoscopy was suggested. The histology of the duodenal biopsies (including the duodenal bulb) was classified according to the Marsh criteria [14].

190 CF patients aged between 0 and 68 years (median 17 +/- 12 years) were examined. 100 (53 %) CF patients were female, 159 (84 %) were pancreatic insufficient. 62 (32,6 %) carry HLA-DQ2; 41 (21.6 %) HLA-DQ8 (cf. table 1). 82 (43,2%) carry neither HLA-DQ2 nor -DQ8 and 5 (2.6 %) carry both HLADQ2 and -DQ8.

14 CF patients (7.4 %) showed an abnormal celiac serology. Positive tTG-IgA was found in 6 CF patients (3.2 %); in 2 of them HLA-DQ2 and –DQ8 were negative and in 2 other patients duodenal histology was normal (Table 2). 2 CF patients were diagnosed with CD (Table 2); the frequency of CD in our CF population was 1:90 (1.1 %).

In 6 CF patients isolated elevated anti-DGP-IgA was detected (1 of them also had elevated anti-DGP-IgG). Since all patients were asymptomatic and 2 of them were HLA-DQ2/-DQ8 negative no duodenal biopsies were obtained (Table 2). One patient had isolated elevated EMA-IgA, but he remained asymptomatic and was HLA-DQ2/-DQ8 negative.

We could demonstrate a higher prevalence of elevated celiac serology as well as a higher prevalence of CD in our CF-patient population compared to the general German population. 7.4 % of CF patients had abnormal celiac serology and 3.2 % of the CF-patients had elevated anti-tTG-IgA compared to 0.8 % in the general German population. 1.1 % or 1:90 of our CF patients were diagnosed with CD as the remaining CF patients did not show histological features of CD, were asymptomatic or HLA-DQ2/ -DQ8 negative.

Prolonged delays in the diagnosis of CD are common as the symptoms of CD are difficult to differentiate from CF related malabsorption [3]. Therefore, we emphasize that serological screening for CD should be included in the initial diagnostic work-up of CF patients older than nine months of age or about three months after gluten has been introduced in the diet as well as in CF patients with persistent gastrointestinal symptoms [12]. As the prevalence of CD is steadily increasing [4] and as CD can develop later in life, it has been discussed that celiac serology should be tested on a regular basis. Anti-tTG-IgA is a sensitive screening test [20-22] and significantly correlates with histological a finding [23], which remains the gold standard to diagnose CD.

It has been discussed whether the co-existence of CF and CD is more than a mere coincidence. Walkowiak et al. and Fluge et al. could show that the incidence of CD in CF patients is significantly higher than in the general Polish and Scandinavian population, respectively [8,12]. The CD prevalence in this CF patient survey is 1.1 % and thus mildy elevated compared to the serological and clinical prevalence of CD in a German population of 0.9 % [7].

HLA typing is suggested to be a useful tool to exclude CD or make the diagnosis unlikely in the case of a negative test result for both HLA-DQ2 and -DQ8 [13]. In children with a strong clinical suspicion of CD and high specific celiac antibodies it is suggested to perform HLA typing to strengthen the diagnosis if duodenal biopsies are not performed [11]. Furthermore, HLA typing may be offered in asymptomatic individuals with CD associated conditions to select them for further CD specific antibody testing [13]. We could show that the frequency of HLA-DQ2 and –DQ8 in our CF population is similar to the general German population (Table 1) (32,6% carry HLA-DQ2 and 21,6 % carry HLA-DQ8 versus 32 % and 17 %, respectively, in the general German population) (www.allelefrequencies.net). Walkowiak et al. could show similar results with the prevalence of HLA-DQ2 being 24.8 % in the CF patient cohort compared to 28.5 % in the general Polish population and a prevalence of 9.6 % HLA-DQ8 compared to 10,0 % in the general population [8] confirming the ethnic influence on HLA-DQ frequencies.

Interestingly, CF is not listed as CD associated condition such as type 1 diabetes, Down syndrome, Turner Syndrome, Williams Syndrome, autoimmune thyroid disease, autoimmune liver disease, selective IgA deficiency and first-degree relatives with CD [2,13]. Clinical signs of CD might be incorrectly attributed to CF making it difficult to identify CD in CF patients, especially when they have significant pulmonary or gastrointestinal disease associated with malabsorption or in case of nonadherence to enzyme replacement therapy. Therefore, it is helpful to identify CF patients at risk for developing CD, because early diagnosis and treatment of CD may positively influence the course of CF. Regular celiac serology testing is a very reliable method to screen for CD, however, as the absence of HLA-DQ2 or -DQ8 has a very high negative predictive value, 43,2 % of our CF patients do not need regular testing.

The complications of CD typically occur after many years of disease and usually are observed in adults not adhering to a gluten free diet. There is an increased risk for the classic CD associated lymphoma (enteropathy associated T-cell lymphoma [EATL]), for adenocarcinoma of the small intestine and for gastrointestinal carcinoma [3]. Therefore, it is of further importance having identified CF patients at risk for CD being able to screen regularly and treat early in order to reduce the risk of developing malignancies.

CD is a complex disease in which genetic factors interact with environmental factors inducing the disease [24]. It has been shown that the introduction of complimentary foods containing gluten before the age of four months and after the age of seven months increases the risk of developing CD in a population at risk [25]. Various hypotheses for the co-morbidity of CF and CD have been discussed in the literature, from mere coincidence to a higher antigen load of undigested gluten proteins in the intestinal mucosa due to exocrine pancreatic insufficiency [26]. In addition, malnutrition might contribute to additional mucosal damage causing increased intestinal permeability, which leads to increased exposure to gluten in the diet [8]. Furthermore, intestinal inflammation could influence intestinal permeability causing higher gluten exposition [27]. It has been shown that CF patients have increased intestinal inflammatory markers, including eosinophilic cationic protein and neutrophil elastase [28]. Viscous mucus production [29] as well as small intestinal bacterial overgrowth [30] could also play an important role for augmenting permeability for antigen presentation. All mechanisms mentioned above can induce an inappropriate immunological response to dietary gluten, which may explain elevated celiac serology without manifestation of CD in 12 of our patients. In these cases HLA typing is a very useful tool to discriminate CD patients from mere positive serology. Further studies are needed to characterize risk factors in genetically predisposed CF patients, which are involved in the development of CD.

The prevalence of HLA-DQ2/-DQ8 in CF patients does not differ from the general German population. However, the prevalence of CD in CF patients (1.1 %) is higher than in the general German population (0.9 %). The coexistence of CF and CD may have a great impact on morbidity, quality of life, and treatment outcome. Therefore, all CF patients should be screened with celiac serology early in the course of disease. Furthermore, CF patients - especially those with positive celiac serology - could be assessed for HLA-DQ2 and –DQ8 to identify those at risk for developing CD. CF patients at risk should be screened with celiac serology on a regular basis. In conclusion, CF might be a risk factor for CD despite a similar genetic predisposition compared to the general population.

1. Farrell PM. The prevalence of cystic fibrosis in the European Union. J Cyst Fibros. 2008; 7: 450-453.

2. Cystic Fibrosis Foundation. Facts about cystic fibrosis. 2013.

3. [No authors listed]. NIH Consensus Development Conference on Celiac Disease. NIH Consens State Sci Statements. 2004; 21: 1-23.

4. Lohi S, Mustalahti K, Kaukinen K, Laurila K, Collin P, Rissanen H, et al. Increasing prevalence of coeliac disease over time. Aliment Pharmacol Ther. 2007; 26: 1217-1225.

5. Not T, Horvath K, Hill ID, Partanen J, Hammed A, Magazzu G, et al. Celiac disease risk in the USA: high prevalence of antiendomysium antibodies in healthy blood donors. Scand J Gastroenterol. 1998; 33: 494-498.

6. Fasano A, Berti I, Gerarduzzi T, Not T, Colletti RB, Drago S, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med. 2003; 163: 286-292.

7. Laass MW, Schmitz R, Uhlig HH, Zimmer KP, Thamm M, Koletzko S. The prevalence of celiac disease in children and adolescents in Germany. Dtsch Arztebl Int. 2015; 112: 553-560.

8. Walkowiak J, Blask-Osipa A, Lisowska A, Oralewska B, Pogorzelski A, Cichy W, et al. Cystic fibrosis is a risk factor for celiac disease. Acta Biochim Pol. 2010; 57: 115-118.

9. Cohen-Cymberknoh M, Wilschanski M. Concomitant cystic fibrosis and coeliac disease: reminder of an important clinical lesson. BMJ Case Rep. 2009; 2009.

10. Venuta A, Bertolani P, Casarini R, Ferrari F, Guaraldi N, Garetti E. [Coexistence of cystic fibrosis and celiac disease. Description of a clinical case and review of the literature]. Pediatr Med Chir. 1999; 21: 223-226.

11. Valleta EA, Mastella G. Incidence of celiac disease in a cystic fibrosis population. Acta Paediatr Scand. 1989; 78: 784-785.

12. Fluge G, Olesen HV, Gilljam M, Meyer P, Pressler T, Storrösten OT, et al. Co-morbidity of cystic fibrosis and celiac disease in Scandinavian cystic fibrosis patients. J Cyst Fibros. 2009; 8: 198-202.

13. Husby S, Koletzko S, Korponay-Szabó IR, Mearin ML, Phillips A, Shamir R, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr. 2012; 54: 136-160.

14. Oberhuber G, Caspary WF, Kirchner T, Borchard F, Stolte M; German Society for Pathology Task Force on Gastroenterologic Pathology. [Diagnosis of celiac disease and sprue. Recommendations of the German Society for Pathology Task Force on Gastroenterologic Pathology]. Pathologe. 2001; 22: 72-81.

15. Greco L, Romino R, Coto I, Di Cosmo N, Percopo S, Maglio M, et al. The first large population based twin study of coeliac disease. Gut. 2002; 50: 624-628.

16. Bevan S, Popat S, Braegger CP, Busch A, O’Donoghue D, FalthMagnusson K, et al. Contribution of the MHC region to the familial risk of coeliac disease. J Med Genet. 1999; 36: 687-690.

17. Biagi F, Bianchi PI, Vattiato C, Marchese A, Trotta L, Badulli C, et al. Influence of HLA-DQ2 and DQ8 on severity in celiac Disease. J Clin Gastroenterol. 2012; 46: 46-50.

18. Kaukinen K, Partanen J, Mäki M, Collin P. HLA-DQ typing in the diagnosis of celiac disease. Am J Gastroenterol. 2002; 97: 695-699.

19. Bombieri C, Claustres M, De Boeck K, Derichs N, Dodge J, Girodon E, et al. Recommendations for the classification of diseases as CFTR-related disorders. J Cyst Fibros. 2011; 10 Suppl 2: S86-102.

20. Basso D, Guariso G, Fogar P, Meneghel A, Zambon CF, Navaglia F, et al. Antibodies against synthetic deamidated gliadin peptides for celiac disease diagnosis and follow-up in children. Clin Chem. 2009; 55: 150- 157.

21. Lurz E, Scheidegger U, Spalinger J, Schöni M, Schibli S. Clinical presentation of celiac disease and the diagnostic accuracy of serologic markers in children. Eur J Pediatr. 2009; 168: 839-845.

22. Hopper AD, Hadjivassiliou M, Hurlstone DP, Lobo AJ, McAlindon ME, Egner W, et al. What is the role of serologic testing in celiac disease? A prospective, biopsy-confirmed study with economic analysis. Clin Gastroenterol Hepatol. 2008; 6: 314-320.

23. Donaldson MR, Firth SD, Wimpee H, Leiferman KM, Zone JJ, Horsley W, et al. Correlation of duodenal histology with tissue transglutaminase  and endomysial antibody levels in pediatric celiac disease. Clin Gastroenterol Hepatol. 2007; 5: 567–573.

24. Johnson TC, Diamond B, Memeo L, Negulescu H, Hovhanissyan Z, Verkarre V, et al. Relationship of HLA-DQ8 and severity of celiac disease: comparison of New York and Parisian cohorts. Clin Gastroenterol Hepatol. 2004; 2: 888-894.

25. Ivarsson A, Hernell O, Stenlund H, Persson LA. Breast-feeding protects against celiac disease. Am J Clin Nutr. 2002; 75: 914-921.

26. Borowitz D, Durie PR, Clarke LL, Werlin SL, Taylor CJ, Semler J, et al. Gastrointestinal outcomes and confounders in cystic fibrosis. J Pediatr Gastroenterol Nutr. 2005; 41: 273-285.

27. Hendriks HJ, van Kreel B, Forget PP. Effects of therapy with lansoprazole on intestinal permeability and inflammation in young cystic fibrosis patients. J Pediatr Gastroenterol Nutr. 2001; 33: 260- 265.

28. Smyth RL, Croft NM, O’Hea U, Marshall TG, Ferguson A. Intestinal inflammation in cystic fibrosis. Arch Dis Child. 2000; 82: 394-399.

29. Norkina O, Kaur S, Ziemer D, De Lisle RC. Inflammation of the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol. 2004; 286: G1032-1041.

30. Lisowska A, Wójtowicz J, Walkowiak J. Small intestine bacterial overgrowth is frequent in cystic fibrosis: combined hydrogen and methane measurements are required for its detection. Acta Biochim Pol. 2009; 56: 631-634