No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population

Begoña Diosdado,1,* Dariusz T. Stepniak,2,* Alienke J. Monsuur,1,* Lude Franke,1 Martin C. Wapenaar,1 Maria Luisa Mearin,3 Frits Koning,2 and Cisca Wijmenga1

1Complex Genetics Section, Department of Biomedical Genetics, University Medical Centre, Utrecht, and Departments of 2Immunohematology and Blood Transfusion and 3Paediatrics, Paediatrician Unit of Paediatric Gastroenterology, Leiden University Medical Centre, Leiden, the Netherlands

Submitted 8 February 2005 ; accepted in final form 28 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain ~40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21–22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.


CELIAC DISEASE (CD) is a chronic autoimmune disorder caused by the ingestion of dietary gluten. Gluten toxicity in CD patients is, in part, determined by the proline- and glutamine-rich gliadins, secalins, and hordeins present in wheat, rye, and barley, respectively. This toxicity results from the presence of a repertoire of T-cells in the lamina propria of the intestines of CD individuals that are able to recognize many different gluten peptides and provoke an erroneous immune response in the small intestine. This leads to specific tissue damage characterized by lymphocytic infiltration of the mucosa (Marsh I), a Marsh II stage presenting crypt hyperplasia together with the Marsh I features, and Marsh III (MIII) stage in which, in addition to Marsh II, villous atrophy develops (4, 13, 15).

So far, the only treatment for CD patients is a strict gluten-free diet, but new alternatives have been recently proposed based on an improved understanding of the disease ethiopathogenesis (11, 12, 14). One of the most attractive new approaches consists of an enzymatic therapy using bacterial prolyl-endopeptidase from Flavobacterium meningosepticum, an enzyme that can remove gluten toxicity by cleaving it into small fragments that lack T-cell stimulatory properties (11). This bacterial enzyme has a well-conserved evolutionary homologue in humans (EC 3.4.21.26 [EC] ) (17) that encodes for a cytosolic enzyme that also hydrolyzes amide bonds of very rich proline peptides shorter than 30 amino acids (18). It is tempting to speculate that an impaired function of the human prolyl endopeptidase (PREP) would result in the accumulation of long, immunostimulatory gluten peptides in the lumen or lamina propria, and that this could play a role in breaking down an individual’s tolerance to gluten.

Interestingly, the human PREP gene is located in the chromosomal region 6q21–22 that showed suggestive linkage (lod score 3.10, P = 1.3 x 10–4) to CD in the Dutch population (16). In addition, microarray experiments performed in the same population showed an approximately twofold upregulation of PREP in seven untreated CD patients compared with four treated CD patients, all 11 of whom still showed villous atrophy (P < 0.005) (5).

Because these results suggested a role for the human PREP gene as a primary candidate for CD in the Dutch population, we performed a detailed analysis of PREP activity and follow-up expression in biopsies of patients and controls, sequenced the PREP gene in a large group of patients, and carried out genetic association studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Seven CD patients from seven not related sibpairs who contributed to the linkage peak on chromosome 6p21–22 and showed two alleles identical by descent for this region were selected for resequencing the PREP gene to define new variants in exon and exon-intron boundaries.

We collected 47 biopsies for the enzyme activity studies (Table 1) from 24 CD patients with an MIII biopsy proven lesion, and 23 controls who had a biopsy examination for other reasons, such as abdominal pain or failure to thrive. The diagnosis of the CD patients was done according to the ESPGHAN criteria (20). DNA material was available for 37 of these samples [18 CD patients (individuals 24-41, Table 1) and 19 controls (individuals 1-19, Table 1)], which allowed us to assess both genotype and activity data.


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Table 1. Data on individuals (CD patients and controls) included in the study

 
The genetic study comprised a group of 311 independent CD cases and 180 independent age- and sex-matched random hospital controls, all of Dutch Caucasian origin. Only CD patients with a biopsy-proven MIII lesion were included in this study. We collected blood samples and isolated DNA according to standard laboratory procedures (16).

Initially, 16 biopsies from eight MIII CD patients and eight M0 CD patients and a pool of 16 RNA samples from control individuals were used to validate the microarray results for the PREP gene using real-time RT-PCR (see Table 1 of supplemental data at http://ajpgi.physiology.org/cgi/content/full/00056.2005/DC1). These samples were not used in the further studies.

The study was approved by the Medical Ethics Committees of the University Medical Centres in Utrecht and Leiden, and informed consent was obtained from all individuals.

Determination of PREP enzyme activity. To measure the PREP activity, we modified the method described by Goossens et al. (8). The duodenal biopsies were washed with PBS, frozen, and stored at –80°C for no longer than 18 mo. The biopsies were thawed on ice and ground with an Ultra Turrax homogenizer (Ika Labortechnik, Staufen, Germany) at 22,000 rpm in the presence of 500 µl of lysis buffer (20 mM Tris·HCl, pH 7.4, 137 mM NaCl, 2 mM EDTA, 10% glycerol, and 1% Triton X-100). The lysates were centrifuged (14,000 rpm, 15 min, 4°C) and the assay was performed in 96-well black plates with a clear bottom (Corning). Every measurement was performed four times. Twenty microliters of lysates were preincubated with 75 µl of incubation buffer (100 mM K3PO4, pH 7.5, 1 mM EDTA, and 1 mM DTT) for 5 min at 37°C. The reaction was started by adding 5 µl of substrate solution [4 mM Z-Gly-Pro-7-amino-4-methylcoumarin (pro-AMC) in 60% methanol]. After 1 h of incubation at 37°C, the reaction was stopped with 50 µl of 1 M acetic acid. The concentration of the released AMC was measured fluorimetrically at an excitation wave-length of 360 nm and an emission wavelength of 460 nm using a CytoFluor multi-well plate reader (PerSeptive Biosciences). One unit of the enzyme was defined as the catalytic activity that releases 1 µmol of AMC per minute. Both Z-Gly-Pro-AMC substrate and standard AMC were purchased from Fluka Chemie (Buchs, Switzerland). Total protein concentration in lysates was determined using a Bradford protein assay (Bio-Rad, Munchen, Germany) and a BCA protein assay (Pierce, Rockford, IL), with BSA (Pierce) as the standard in both cases.

Quantitative real-time RT-PCR. Quantification of PREP transcriptional activity was performed by real-time RT-PCR on RNA from biopsies as previously described (19). We used an Assay-on-Demand Gene Expression product for the PREP gene (ABI Hs.00267576), and the GUSB gene (detected by PARD 4326320E) as an endogenous reference to correct for expression-independent sample-to-sample variability (Applied Biosystems, Foster City, CA). To quantify the relative expression by the 2{Delta}{Delta}Ct method (19), equimolar amounts of total RNA from 16 control individuals were pooled and used for normalization of the expression data. Both genes were tested in duplicate for all the individual patient samples and the control pool on an ABI 7900 HT (Applied Biosystems).

Sequence analysis. PCR amplification was performed on all 15 exons and exon-intron boundaries of the PREP gene. Details about the primer sequences and the PCR conditions can be found in Table 2 of the supplementary data. The PCR products were examined on a 2% agarose gel and purified with the Millipore Vacuum Manifold (Billerica, MA), according to the manufacturer’s protocol. Samples were prepared with the ABI PRISM BigDye terminator cycle sequencing ready kit (Applied Biosystems) according to the manufacturer’s protocol. PCR and sequencing amplification were performed on a GeneAmp PCR system 9700 (Perkin-Elmer, Wellesley, MA). Sequencing was performed on a 3730 DNA sequencer (Applied Biosystems). Analysis and alignment was carried out with the Sequence Navigator (Applied Biosystems) and Vector NTI (InforMax).


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Table 2. P values obtained from testing the case-control cohort for three coding and three intronic SNPs

 
Genetic association studies and data analysis. Five of the selected single nucleotide polymorphism (SNPs) were typed using assay-on-demand probes from the PREP gene: hCV1963751 (ABI no. C___1963751_10), rs9486069 (ABI no. C__11638424_10), rs1078725 (ABI no. C___8304693_10), rs2793389 (ABI no. C__11635753_10), and rs1051484 (ABI no. C___8304751_20). The sixth selected SNP, rs12192054, was typed by using an assay-by-design probe from Applied Biosystems. These SNPs were tested in a case-control study (311 cases and 180 controls) and analyzed on an ABI Prism 7900 HT system (Applied Biosystems). Hardy-Weinberg equilibrium was evaluated separately in cases and control, for all SNPs tested (data not shown). Differences in allele frequencies and genotype distributions were compared between cases and controls using the {chi}2-test.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We compiled our earlier microarray (5) and linkage (16) data from Dutch CD patients using TEAM, a bioinformatics tool developed in house (6), which allowed us to define the physical location of the differentially expressed genes under the genetic linkage peaks. Integration and analysis of these two data sets revealed that PREP was one of the differentially expressed genes located under the linkage peak on chromosome 6q21–22 in the Dutch genome screen (Fig. 1, A and B). The 6q21–22 region encompasses 22 megabases and contains 111 genes. The relative risk in the Dutch CD population attributed to this locus is 2.3 (16). Quantitative expression studies by real-time RT-PCR on a set of eight RNA samples from treated CD patients in complete remission (M0), eight untreated CD patients with total villus atrophy (MIII), and a pool of normal controls validated these findings. The experiments showed that PREP was significantly downregulated in treated M0 patients compared with MIII patients ingesting gluten (1.3-fold, P < 0.05; Table 1 of supplementary data), although to a lesser extent than previously described (5).



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Fig. 1. A: linkage data of 101 sibpairs [Dutch celiac disease (CD) patients] on chromosome 6. The dashed line indicates the linkage graph before fine mapping, whereas the continuous line is after fine mapping. B: 95% confidence interval containing 111 genes; the dashed square indicates the position of the prolyl endopeptide (PREP) gene. C: exonic-intronic view of PREP. The dashed line represents the catalytic domains of the protein and the continuous line the {beta}-propeller domain. D: table includes the 6 exonic single nucleotide polymorphisms (SNPs) identified by sequencing in 44 individuals and the 3 intronic SNPs. *SNPs selected for the genetic studies. SNP ID, SNP number; UTR, untranslated region; ND, not determined.

 
Sequence analysis. To investigate whether the enzymatic properties of this gene product or its expression levels were different in CD patients due to an underlying genetic variation, we performed sequence analysis on the entire coding region and exon-intron boundaries of the PREP gene to identify putative mutations or variants in the CD population. The human PREP gene is fully annotated in the public databases and consists of 2,905 nucleotides distributed over 15 exons that encode 710 amino acid residues (17). Because the tertiary structure of the human PREP has not yet been described, we used the tertiary structure of its porcine homologue as reference in defining which exons were encoded by which domains. The human and porcine enzymes are 97% homologous at the amino acid level, and in the porcine PREP, exons 1–3 and 10–15 encode the catalytic domain and exons 3–10, the characteristic {beta}-propeller domain that regulates its proteolitic activity (Fig. 1C) (7).

Sequence analysis of all 15 exons and exon-intron boundaries in 44 individuals revealed six SNPs in the coding region of PREP. These SNPs were present in exon 1, exon 5, exon 9 (two SNPs), and exon 15 (two SNPs) (Fig. 1B). The SNP in exon 1 and one of the two SNPs in exon 15 have not yet been annotated in public databases. The published allele frequencies and the frequency of occurrence of these SNPs in the sequenced individuals are shown in Fig. 1D.

Only two of the identified SNPs lead to an amino acid change in the PREP protein. A SNP found in exon 9, 1050T->G, gives rise to a leucine to valine substitution at position 351 while a SNP in exon 15, 2118 G->A, gives rise to a valine to isoleucine substitution at position 706. This latter substitution is not expected to have any impact on the function of the PREP protein because the amino acid at position 706 is not conserved (valine in man, isoleucine in pigs, bovines, rats and mice). The leucine-to-valine substitution at position 351 is a conservative one and, therefore, we cannot rule out that this substitution may impact PREP function.

Genetic association studies. To further investigate whether genetic polymorphisms in PREP are associated with CD in the Dutch population, we performed genetic association studies. For our linkage peak on chromosome 6p22, with a relative risk of 2.3 and a SNP frequency in the range of 0.1–0.4, our sample size had 80% power to detect a confidence interval of 95%.

Four exonic SNPs [exon 1 (–80), rs9486069, rs12192054, and rs1051484] were selected based on their high heterozygosity in our sequence samples and their possible influence on the protein. Unfortunately, the SNP in the 5'-untranslated region (5'-UTR) could not be designed because of the extreme repetitiveness in the region. None of the three SNPs, however, showed a statistical difference between the cases and controls (Table 2).

To further exclude PREP as a causative gene, we selected three noncoding SNPs for further genetic association studies on the basis of a minor allele frequency of >10% (Table 2). These three SNPs also showed a lack of statistical difference between the cases and controls (Table 2). Haplotype analysis did not change these results (data not shown). Finally, we tested a microsatellite marker located in intron 2 of the PREP gene, which also showed no association with CD (data not shown). Overall, we found no association between any of our genetic markers and CD.

Activity of PREP in biopsy material from patients and controls. To further investigate whether an impaired enzymatic activity of PREP could be responsible for a decreased digestion of gluten peptides in the small intestine of CD patients and, hence, activation of an aberrant immune response, the catalytic activity of the enzyme was measured in 47 biopsies from CD patients and controls. The activity values lay in the range of 1.71 to 8.52 U/g protein with an average of 4.8 U/g protein (SD = 1.61), which is in agreement with the described PREP activities measured in other human tissues (8). First, patients were grouped according their histological status and adhering to the treatment in treated CD (M0) and untreated CD (MIII) and independently of their genotypes. The average PREP activity levels measured in the untreated CD patients were lower than in the treated CD patients (P < 0.05). No significant differences were observed between the treated or untreated CD patients and the controls (Fig. 2). We were not able to correlate PREP activity levels with the age or gender of the studied individuals (data not shown).



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Fig. 2. PREP activity in duodenal biopsies. The activity was measured with Z-Gly-Pro-AMC substrate and corrected for protein concentration determined with BCA assay. TCD 5.35 ± 0.46; UCD 4.12 ± 0.39; P < 0.05; controls 5.03 ± 0.033. TCD, treated celiac disease (gluten-free diet); UCD, untreated celiac disease (normal diet); controls, no celiac disease and normal diet.

 
Activity-genotype correlations. To further detect an influence of the tested genetic variants on the expression and activity results, we calculated whether there was any association between the different genotypes of the SNPs and the enzymatic activity of PREP.

For activity-genotype correlation, the genotypes of four identified coding SNPs of the gene (Fig. 1D) and the activity measurements of 37 individuals were studied (Table 1, individuals 1–19 and 24–41). To do so, individuals were grouped according to their genotypes and the average of the activity for each group was calculated for each of the coding SNPs [except SNP rs6902415, because all individuals were homozygote C/C, and exon 15 (680), because all individuals but one were C/C] (Table 3 of supplementary data). An association t-test was used to find genotype-activity correlations but revealed no significant association for any of the four SNPs (data not shown). We concluded that the activity is not modulated by the sequence of the gene, which further supports the findings of our genetic association studies.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CD is a complex genetic trait in which genetic and environmental factors are the primary causative determinants for the disease. Although gluten has been identified as the major environmental factor (3), only the genetic contribution of the human leucocyte antigen (HLA) region is well understood (13). Recently, a genome-wide screen in our Dutch population has been successful in finding significant linkage to two non-HLA associated regions, one to chromosome 19 and another to chromosome 6q21–22 (16). No causative gene has been identified yet for either of these regions.

By integrating a data set from our microarray experiments with the genetic information of the 6q21–22 region, we identified eight differentially expressed genes located under this linkage peak. Because one of these differentially expressed genes was PREP, we hypothesized that an altered PREP activity in the intestinal mucosa could be responsible for the inefficient breakdown of gluten peptides, which could consequently facilitate the onset of CD. We therefore performed a comprehensive set of complementary studies to investigate the putative role of PREP in the pathogenesis of CD.

Because expression studies showed the existence of altered levels of PREP mRNA in the biopsies of CD patients, we hypothesized that we might identify a DNA polymorphism or a variant that would slightly alter the activity of the enzyme, rather than a major mutation that would fully abolish its function. Sequence analysis did not reveal any major mutations in 25 CD patients, but six SNPs were found in the coding region of this gene. One of the SNPs is in one of the residues of the catalytic triad (His680), but it does not give rise to an amino acid change. A novel SNP was found in the 5'-UTR of PREP. Because the promoter region of PREP is not known, in silico studies using Transfac TF professional version 8.2 were used to define whether putative binding sites and regulatory sequences in the 5'-UTR of PREP reside at the position of this SNP. No putative regulatory sequence was predicted at the site of the SNP (data not shown), suggesting that this SNP may not affect the transcriptional regulation of PREP. SNP rs9486069, located within the first 10 nucleotides of exon 5, was also of potential interest because it has been well established that sequences within the first or last 20 nucleotides of an exon can influence the splicing machinery by enhancing or silencing its effects (1). We therefore looked for a possible influence of this SNP on the splicing machinery using Spring Harbor software (2), but found none (data not shown).

From the sequence and follow-up analysis we concluded that none of the SNPs would directly provoke a change in the structure of the protein. Neither did our later genetic studies support a role for PREP as a primary gene in CD. The microsatellite marker and the six SNPs inside PREP did not show any significant differences nor any trend towards significance. Besides, because the promoter region of the PREP gene is unknown, SNPs in this region could not be totally excluded.

Finally, to further exclude any functional consequence of these coding polymorphisms in PREP activity that could implicate it in the pathogenesis of CD, we determined the catalytic activity of PREP in biopsies from 47 children. As expected from the genetic association studies, we found no significant differences between the treated or untreated CD children and the pediatric controls or when individuals were grouped by their genotypes. Nevertheless, because the biopsies for normal controls came from individuals who might have had altered intestinal mucosa due to diarrhea or abdominal pain, these results could be an underestimation. However, the PREP activity in untreated CD children was slightly decreased compared with treated CD pediatric patients, possibly as the result of intestinal tissue damage associated with the disease. These observations are perfectly in line with findings of Donlon and Stevens (J. Donlon and F. M. Stevens, personal communication) but do not support results published by Matysiak-Budnik et al. (9), who described an increased PREP activity in the intestinal mucosa of eight treated (i.e., following a gluten-free diet) CD patients compared with seven controls. It remains to be established why our results differ from those of Matysiak-Budnik.

In conclusion, these results clearly indicate that no genetic polymorphisms in the PREP gene can be linked to CD. This finding is further supported by the activity determinations in which we found no differences in the enzyme activity between CD patients and controls. Thus PREP does not seem to be implicated in the pathogenesis of CD.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by the Netherlands Organization for Scientific Research Grants 902-22-094 and 912-02-028, the Celiac Disease Consortium, an innovative cluster approved by The Netherlands Genomics Initiative and partially funded by Dutch Government Grant BSIK03009, and Dutch Digestive Disease Foundation Grant WS00-13.


    ACKNOWLEDGMENTS
 
The authors thank Ellen van Koppen and Remi Steens for the help in acquiring patient data; Jackie Senior for editing the text; and Alfons Bardoel, Daniel Chan, and Alexandra Zhernakova for the practical work.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. Wijmenga, Complex Genetics Section, Stratenum 2.117, Dept. of Biomedical Genetics, Univ. Medical Centre Utrecht, PO Box 85060, 3508 AB Utrecht, The Netherlands (e-mail: t.n.wijmenga{at}med.uu.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* B. Diosdado, D. T. Stepniak, and A. J. Monsuur contributed equally to this work. Back


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