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Promoter polymorphisms of the CD14 gene in Italian patients suffering from Celiac Disease

CD14. CD14 is a multifunctional receptor involved in innate immune response. In fact it is thought to be one of the most important receptor for LPS and other bacterial wall-derived components. However it is well known that CD14 has several other functions, including the clearance of apoptotic cells.

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Promoter polymorphisms of the CD14 gene in Italian patients suffering from Celiac Disease

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    1. Promoter polymorphisms of the CD14 gene in Italian patients suffering from Celiac Disease Michele Boniotto, Laura Braida, Alessandro Ventura, Luigi Greco, Antonio Amoroso and Sergio Crovella Dipartimento di Scienze della Riproduzione e dello Sviluppo, Universita' di Trieste, Via dell’Istria 65/1, 34137 Trieste, Italy Dipartimento di Pediatria, Universita' di Napoli Federico II, Via Pansini 5, 80131 Napoli, Italy. Servizio di Genetica, IRCCS Burlo Garofolo, Via dell’Istria 65/1, 34137 Trieste, Italy

    2. CD14 CD14 is a multifunctional receptor involved in innate immune response. In fact it is thought to be one of the most important receptor for LPS and other bacterial wall-derived components. However it is well known that CD14 has several other functions, including the clearance of apoptotic cells. It is constitutively expressed primarily on the surface of monocytes, macrophages and neutrophils (mCD14); a soluble form, sCD14, was isolated from serum and is derived both from secretion of CD14 and from enzymatically cleaved glycosyl-phosphatidylinositol- anchored mCD14.

    3. CD14 genetic polymorphisms The serum levels of CD14, and thus the levels of mCD14, are genetically regulated. Four SNPs have been identified in the promoter region of the gene, which are thought to regulate the CD14 gene expression 22. Recently, different studies have correlated a C to T transition at position -159 in the promoter region of CD14 gene, with an increased risk of developing Crohn disease and Ulcerative Colitis ; furthermore, a study by Holla et al. demonstrated an association between a G to T transversion at position –1359 and the severity of periodontitis.

    4. AIMS OF OUR STUDY: CD14 AND CELIAC DISEASE In our study the frequency of the two promoter polymorphisms was assessed in three selected populations in order to explore the influence of CD14 in celiac disease; in fact CD14 gene is located in a "hotbed" region for CD and is involved in the clearance of apoptotic cells, which could be important in CD as already demonstrated by the findings of Boniotto et al.

    5. Patients and Controls For this study 115 Italian celiac patients with typical HLA haplotypes (either DQ2 or DQ8), 37 Italian celiac patients with atypical HLA haplotypes and 180 pan-ethnic controls (healthy blood donors with no history of either gastrointestinal or autoimmune diseases) were enrolled. Diagnosis of celiac disease was performed following the ESPGHAN indications. The study was approved by the Ethical Committee at the Burlo Garofolo Children’s Hospital, Trieste. The importance of a positive result was explained to all participants and verbal consent was obtained from the patients.

    6. CD14 genotyping DNA was extracted from peripheral whole blood using standard laboratory protocols 28. The detection of the bi- allelic polymorphism at position –159 was performed following the PCR- RFLP technique already described by Baldini et al. 29 The detection of the bi-allelic polymorphism at position –1359 was performed with a modified version of the PCR-RFLP technique described by Holla et al. 25 Briefly, PCR was performed with the forward primer 5'- AGCACTTTGAGAGGCCAAGG- 3' and the reverse primer 5'– CCACATCCCTAGACCTCTGGG- 3' , both designed on the basis of the published CD14 human sequence with the software Primer Express 1.5 (Applied Biosystems, Foster City, CA).

    7. RESULTS Allelic frequencies and genotype frequencies for the position -159 in the three populations studied are shown in Table 1. The frequency of the (-159)C allele was 53%, 46% and 50% in HLA typical celiac patients, HLA atypical celiac patients and healthy controls, respectively. Neither were any differences observed in genotype frequencies among the three populations.

    9. Table 2 shows the allelic and genotype frequencies for the G to T transversion at position –1359. The frequency of the (-1359)G allele did not differ in the three populations; in fact it was 80% in HLA typical celiac patients, 77% in HLA atypical celiac patents and 84% in healthy controls (Fisher's exact test, p= 0.631 for typical HLA celiac patients compared to atypical HLA celiac patients; p= 0.251 for typical HLA celiac patients compared to healthy controls and p= 0.172 for atypical HLA celiac patients compared to healthy controls). No differences were observed in genotype frequencies when healthy controls and HLA typical celiac patients were compared (?2 = 4.19, p= 0.123).

    11. However an increased frequency of (-1359)TT homozygous was observed in the latter population (relative risk TT vs. GT and GG genotypes: 3.65; 95% confidence interval (CI) 0.96 to 13.84). In fact the frequencies of (–1359)GG, (–1359)GT and (–1359)TT genotypes were 70%, 28% and 2% in controls and 67%, 27% and 6% in HLA typical celiac patients, respectively. The genotype frequencies in the HLA atypical celiac patients were 67%, 19% and 14% for the genotype (–1359)GG, (–1359)GT and (–1359)TT, respectively. This distribution did not differ when compared to that of HLA typical celiac patients (?2 =2.680, p= 0.262), but it is significantly different from that of the Italian healthy controls (?2 = 12.640, p= 0.0018, pcorr< 0.05). This difference is mainly due to the difference in the frequency of (-1359)TT homozygous individuals. The relative risk for TT genotype in HLA atypical celiac patients is 8.11 (95% CI 2.03 to 32.45).

    12. DISCUSSION Celiac disease is one of the most common multifactorial autoimmune disorders in the Caucasian population (1 in 200 individuals). The principal genetic (HLA-linked genes) and environmental (wheat gluten) factors responsible for CD are well known, but little data is available for non-HLA-linked genes.

    13. CD14 GENETIC POLYMORPHISMS AND THEIR EFFECTS OF CD14 SERUM LEVELS In this case-control study we have assessed the frequency of two polymorphisms in the promoter region of the CD14 gene that regulate the expression of the protein in three selected populations. LeVan et al. demonstrated that (-159)T allele promotes CD14 gene transcription and homozygous subjects for this allele have increased CD14 serum levels . In our study the allelic and genotype frequencies of C(-159)T did not differ among the 115 celiac patients carrying the typical HLA haplotype (DQ2 or DQ8), the 37 celiac patients with an atypical HLA haplotype and the 180 selected healthy controls of the same ethnic origin.

    14. (-1359) TT POLYMORPHISM Interestingly, there was a significant skewing in celiac patients with atypical HLA for the genotype frequencies at position –1359 when compared to healthy controls. This difference is mainly due to the higher frequency of (-1359)TT subjects in celiac patients carrying the atypical HLA haplotype. The frquency of (-1359)TT patients is also greater in celiac patients with typical HLA, even though our results don't reach the statistical significance. As demonstrated by Vercelli et al. 32 (-1359)TT subjects have lower serum levels of CD14. HLA atypical celiac patients (-1359)TT have a Relative Risk of 8.11 (95% CI 2.03 to 32.45) of developing celiac disease.

    15. CD14 AND CELIAC DISEASE Several hypotheses could be made to explain the involvement of CD14 in celiac disease. Firstly, individuals with low levels of sCD14, and thus mCD14, might be more prone to Gram- negative bacterial infections. These infections may disrupt the permeability of intestinal epithelia leading to an increased concentration of gluten in the lamina propria, where most of the gluten-reactive lymphocytes are localised. Such individuals may be more susceptible to developing celiac disease. Unfortunately, no studies linking the G(-1359)T polymorphism to a risk of infections have been carried out yet. Moreover a recent study by Agnese et al. evidenced a significant correlation between TLR4 polymorphisms and an augmented risk of Gram-negative bacterial infections: no association was found between CD14 C(-159)T polymorphism and the incidence of infections or patients' outcome.

    16. CD14, CELIAC DISEASE AND APOPTOSIS Another possibility, and in our opinion the most interesting, may derive from the role of CD14 in apoptosis regulation. The defective clearance and removal of apoptotic cells has been closely linked to autoimmune and persistent inflammatory diseases. Several studies demonstrate that CD14 is able to bind several apoptotic-cell-associated molecular patterns (ACAMPs), such as ICAM-3 on apoptotic leukocytes, and may either act as a phagocyte pattern-recognition receptor or bind directly ACAMPs to initiate engulfment of apoptotic cells.

    17. CONCLUSIONS At the moment, linkage disequilibrium of the G(-1359)T allele with the real causative gene for CD in the genomic region 5q31-33 can't be excluded. In conclusion, a significant association between (-1359)TT genotype and an increased risk for CD was shown in celiac patients with atypical HLA haplotype. There was a positive trend for a higher frequency of (-1359)TT individuals in HLA typical celiac patients, even though this study doesn't reach the statistical significance. However, to our knowledge, this is the first study which has found a significant association between a polymorphism in a gene located in the candidate genomic region 5q31-33 and celiac disease. Finally, our findings demonstrated the importance of apoptosis regulation. They indicate that the genes encoding proteins, which play a key role in this process, are potential candidates for CD. A larger cohort of HLA typical celiac patients should probably be studied, and functional studies carried out, to clarify the role of CD14 in celiac disease.

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