Association study of functional genetic variants of innate immunity related genes in celiac disease

In the present work we have analysed a panel of 105 celiac families characterised by the presence of an affected child with CD. The study participants were recruited at "Hospital Materno-Infantil" and "Hospital Clinico Universitario", Granda, (Spain) and were of Spanish Caucasian origin. All patients were diagnosed following the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) criteria for CD [25]. Their age at study was 7.1 ± 3.9 years and the mean age for disease diagnosis was 2.7 ± 2.72. A 60% were women and 40 % men, showing an anthropometry at diagnosis (weight and height) of P 3–100 percentile. The mean age of gluten introduction was 6.4 ± 1.5 months. Typical symptoms were observed in 72.2 % of patients and 27.8 % showed atypical symptoms. All family members were genotyped in DRB1 and DQB1. DQA1 typing was deduced from DQB1 and DRB1 typing on the basis of the strong linkage disequilibrium among HLA class II alleles.

DNA from patients and controls was obtained from peripheral blood using standard methods. For all of the considered SNPs, except IL-1RN and RANTES -403, samples were genotyped using a Taqman 5' allelic discrimination assay. Table 1 shows the Taqman MGB probes sequences used for each polymorphism provided by the Custom-Taqman-SNP-Genotyping-Assay (Applied Biosystems, Foster City, CA, USA). PCR reaction was carried in a total reaction volume of 5 μl with the following amplification protocol: denaturation at 92°C for 10 min, followed by 50 cycles of denaturation at 92°C for 15 sec and annealing and extension at 58°C for 1 min. Post-PCR, the genotype of each sample was attributed automatically by measuring the allelic specific fluorescence on the ABI PRIM 7900 Sequence Detection Systems using the SDS 2.2.1 software for allelic discrimination (Applied Biosystems, Foster City, CA, USA). RANTES -403 genotyping was performed using a TaqMan SNP-Genotyping-Assay (part number: C__15874407_10, Applied Biosystems, Foster City, CA, USA).

The IL-1RN polymorphism was genotyped by PCR as previously described [26]. Briefly, we used a froward primer 5'- CTC AGC AAC ACCT CCT AT and reverse primer 5'- TCC TGG TCT GCA GGT AA, two amplify five possible alleles with different PCR fragment size: 410 bp (allele 1: 4 repeats), 240 bp (allele 2: two repeats), 325 bp (allele 3: 3 repeats), 500 bp (allele 4: 5 repeats), and 595 bp (allele 5: 6 repeats).

We used the UNPHASED software created for TDT and case-control analysis [27]. We performed a Transmission Disequilibrium Test (TDT), which assesses allele transmission rates in simplex families and tests for deviation from expected 50% transmission. For the haplotype analysis, pair-wise linkage disequilibrium measures were investigated and haplotypes constructed using the expectation-maximization (EM) algorithm implemented in UNPHASED software. The power of the study to detect an effect of a polymorphism in disease susceptibility was estimated using the Quanto v 0.5 software (Department of Preventive Medicine University of Southern California, California, USA) [28].

The transmission pattern for IL-1α -889, IL-1β -511 and IL-1RN VNTR polymorphisms is shown in table 2. When transmission of these genetic variants was analysed, none of the alleles showed statistically significant skewing. IL-1α -889 T allele was slightly more transmitted to the affected children (58% transmission for allele T vs 47% for allele C), however the p value failed to reach statistically significant level (Table 2). With regard to IL-1RN we observed that alleles IL-1RN*1 and IL-1RN*2 were the most frequent in our population (71.6% and 26.8% respectively), accordingly with previously studies in Caucasian populations [26].

The TDT analysis for -607 A/C and -137 G/C IL-18 promoter genetic variants did not reveal biased transmission of any of the alleles to the affected offspring (Table 2).

The haplotype estimation for the -607 A/C and -137 IL-18 promoter variants revealed complete linkage disequilibrium between the two variants (D' = 1). We observed three out of the four possible haplotypic combinations in CD families (Table 3). The transmission pattern of IL-18 promoter haplotypes did not show any statistically significant skewing (Table 3).

After analyzing the MCP-1 -2518 G/A alleles transmission we observed that none of the alleles was preferentially transmitted from heterozygous parents to the affected offspring (Table 2). Regarding to the RANTES promoter genetic variants the mutant alleles -403 A and -28 G showed an overall allele frequency similar to that expected for Caucasian populations (84.1% and 96.5% respectively in our population) [29, 30]. The transmission of both -403G/A and -28 C/G SNPs showed a slightly deviation from the 50% expected transmission pattern (Table 2). Alleles -403 G and -28 C were more transmitted to the affected offspring with borderline significance (P = 0.04 and P = 0.06 respectively) (Table 2). In addition, we estimated haplotypes for both genetic variants. Three out of the four haplotypic combinations were observed, being the -403G/-28C and -403A/-28C haplotypes the most common in CD families. No significant distorted transmission pattern for RANTES promoter haplotypes was observed (Table 3).