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Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0b013e3181afce6c
Short Communications

Association of the CLCA1 p.S357N Variant With Meconium Ileus in European Patients With Cystic Fibrosis

van der Doef, HPJ*; Slieker, MG; Staab, D; Alizadeh, BZ§; Seia, M||; Colombo, C; van der Ent, CK; Nickel, R; Witt, H#; Houwen, RHJ*

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*Departments of Pediatric Gastroenterology, The Netherlands

Pediatric Pulmonology, The Netherlands

§Complex Genetics Section, DBG-Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands

Departments of Pediatric Pulmonology and Immunology, Germany

#Hepatology and Gastroenterology, Charité Universitätsmedizin, Berlin, Germany

||Molecular Genetics Laboratory, Italy

Department of Pediatric Gastroenterology, Fondazione IRCCS, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy. Dr Witt is now with Department of Pediatrics, Klinik und Poliklinik für Kinder- und Jugendmedizin des Klinikums rechts der Isar, Technische Universität München (TUM), Munich, Germany.

Received 25 September, 2008

Accepted 19 May, 2009

Address correspondence and reprint requests to H.P.J. van der Doef, MD, Department of Pediatric Gastroenterology [KE.04.133.1], University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands (e-mail: h.p.j.vanderdoef@umcutrecht.nl).

The study was funded by the Wilhelmina Research Fund, grant number OZF 2005/04, and by a grant from The Netherlands Organization for Health Research and Development (ZonMW), grant number 920-03-295.

The authors report no conflicts of interest.

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Abstract

In Cftr−/− mice that mostly die because of intestinal obstruction, intestinal expression of Clca3 is decreased, whereas upregulation of Clca3 results in amelioration of intestinal disease. The aim of the study was to investigate whether the p.S357N variant in CLCA1, the human orthologue of Clca3, acts as a modifier gene in a cohort of 682 European patients with cystic fibrosis (CF)–99 patients with meconium ileus. The 357SS genotype was significantly overrepresented in both patients with meconium ileus and also with a severe CFTR genotype (P = 0.009) and in p.F508del homozygotes (P = 0.002). This suggests that CLCA1 has similar important functions in CF-related intestinal obstruction in humans as in Cftr−/− mice.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). Of all of the patients with CF, a minority of 10% to 20% will develop meconium ileus, an intestinal obstruction in the neonatal period caused by increased viscosity of luminal secretions (1,2). This gastrointestinal phenotype can be explained partially by variation in the CFTR genotype, such as homozygosity for the delta F508 deletion, the most common CFTR variant in patients with CF that is strongly associated with the presence of meconium ileus. Also, a modifier locus for meconium ileus (Cfm1) on chromosome 7 has been described in a murine CF model (3), and subsequently, several markers on human chromosome 19, the region syntenic to the mouse locus, showed significant linkage with the presence of meconium ileus in 185 CF sibling pairs (4). However, in a genomewide analysis, including more than 1000 patients, this reported linkage between CFM1 and meconium ileus could not be replicated (1). Thus, a role of CFM1 in the development of meconium ileus is likely not possible. Consequently, other genes have to be involved, because meconium ileus is clearly the result of both genetic and environmental factors (1).

A possible candidate gene for influencing intestinal obstruction in CF is CLCA1, the human orthologue of murine Clca3. In human rectal CF epithelium, CLCA1 is strongly expressed. Family-based analysis suggests that CLCA1 interacts with CFTR because the CLCA cluster modifies the electrophysiological properties of rectal epithelium in patients with CF (5). Furthermore, in severely affected CFTR knockout mice that mostly die of intestinal obstruction (6,7), the expression of Clca3 in the intestine is decreased (8,9), whereas upregulation of Clca3 in these mice results in ameliorated intestinal disease and improved survival (9). Therefore, we investigated whether CLCA1 acts as a modifier gene in patients with CF and also with meconium ileus.

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PATIENTS AND METHODS

Patients

The European cohort consisted of pediatric and adult patients with CF treated at the University Medical Center Utrecht, the Charité University Hospital, and the University of Milan. This study was done with the approval of the medical ethical review committees of the 3 universities.

The diagnosis of CF had been established on the basis of characteristic clinical findings (typical pulmonary or gastrointestinal disease) in combination with persistently elevated sweat chloride concentrations (>60 mmol/L quantitative pilocarpine iontophoresis) and/or 2 pathologic CFTR alterations (10,11).

Meconium ileus was defined as lack of passage of stool within 24 hours after birth, evidence of obstruction on abdominal radiography, and subsequent treatment for this obstruction (1).

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CFTR Genotype

The CFTR genotype was subdivided into 5 classes and was classified as severe (class I-III) and mild (class IV-V) (12). Furthermore, patients homozygous for F508del were analyzed as a separate subgroup.

In the association analysis between meconium ileus and CFTR genotype, the frequency of meconium ileus was compared between patients with a severe genotype (class I-III) and a mild genotype (class IV-V), and between F508del homozygotes and patients with a mild genotype. Patients with an unknown CFTR genotype or unknown CFTR genotype class were excluded.

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The p.S357N Variant in CLCA1

The c.1070G>A (p.S357N) (rs2734705) variant was selected because it leads to an alteration in CLCA1 and is a common variant (www.ensembl.org). The p.S357N variant is not in linkage disequilibrium with other common variants.

From all of the study participants, genomic DNA was extracted from peripheral blood leukocytes. The laboratory staff was blinded for the phenotype (meconium ileus) of each DNA sample. The p.S357N variant in CLCA1 was determined in the European patients with CF by melting curve analysis using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics, Mannheim, Germany) (primers 5-TGCTCTACATTAAGGCAgCCACT-3 and 5-CACATCTCACAGTAAATGCCG-3, and the hybridization probes 5-CCCATGTACAAAATGAACTCATAC-FL and LC610-GATAAACAGTGGCAGTGACAGGGACAC-ph) or TaqMan SNP genotyping assays (assay identification number C__16070505_10; Applied Biosystems, Nieuwerkerk a/d Ijssel, the Netherlands) on an Applied Biosystems 7500 real-time polymerase chain reaction system.

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Statistical Analysis

The wild-type genotype (357SS) was used as reference genotype. Frequencies of the p.S357N variant were compared between cases and controls, and odds ratios were calculated (SS vs SN and SS vs NN). Mantel-Haenszel statistics (PEPI program version 4.0, available at http://sagebrushpress.com/PEPI.html) were used to determine P values for the pooled European analysis and to test heterogeneity between the Dutch, German, and Italian populations. Values were considered significant if P < 0.05. Verification of Hardy-Weinberg equilibrium of genotypes was performed using the Pearson χ2 test.

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RESULTS

Patients

The population consisted of 682 European patients with CF, of which 294, 212, and 176 were, respectively, Dutch, German, and Italian nationals. Of all European patients with CF, 99 (15%) suffered from meconium ileus, of which 42, 21, and 36 were respectively Dutch, German, and Italian nationals.

Mantel-Haenszel statistics showed no heterogeneity between the Dutch, German, and Italian populations for all of the variables, including the CFTR genotype and the CLCA1 p.S357N variant, and therefore data were pooled.

Characteristics of the European patients with CF with and without meconium ileus are described in Table 1. In the European population, age was significantly lower in patients with than in patients without meconium ileus (P < 0.001). No differences were found between meconium ileus and sex.

Table 1
Table 1
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After excluding patients with an unknown CFTR genotype or genotype class, the European population consisted of 551 patients of which 79 (14%) experienced meconium ileus at birth. In the European cohort, we found an association between meconium ileus and severe CFTR genotype (P = 0.003) and between meconium ileus and F508del homozygosity (P = 0.002) (Table 1).

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The p.S357N CLCA1 Variant and Meconium Ileus

The genetic distribution for the p.S357N variants was in Hardy-Weinberg equilibrium. Frequencies of p.S357N in patients with CF were comparable with the CEU-HapMap samples (www.ensembl.org). In all of the patients with CF, without stratifying for CFTR genotype, a trend towards significance was found between meconium ileus and the p.S357N variant (SS vs SN, P = 0.076) (Table 2). Because meconium ileus was associated with CFTR genotype, further analysis was done after stratification for genotype (severe genotype and F508del homozygotes). Stratification for mild CFTR genotype was not possible because of low numbers.

Table 2
Table 2
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In the European population, the 357SS genotype was found to be significantly overrepresented in patients with meconium ileus, both when analyzing patients with severe genotype (SS vs SN, P = 0.009) and when analyzing patients with F508del homozygotes (SS vs SN, P = 0.002) (Table 2).

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DISCUSSION

We found an association between meconium ileus in European patients with CF and the c.1070G>A (p.S357N) CLCA1 variant. The wild-type genotype, 357SS, was found to be significantly overrepresented in patients with meconium ileus and also with a severe genotype or F508del homozygosity. So far, this study represents the first report of an association between a CLCA1 variant and meconium ileus in CF.

In European patients with CF, meconium ileus was strongly associated with a severe CFTR genotype and F508del homozygosity that is in concordance with a large twin and sibling study (1). Because of the strong effect of the CFTR genotype on the development of meconium ileus, we stratified the patients according to the CFTR genotype. The association between the CLCA1 p.S357N variant and meconium ileus was significant in the subgroup with a severe genotype and in the p.F508del homozygote patients, whereas in the total group of patients with CF only a trend towards significance was observed, indicating that meconium ileus is a consequence of interaction between the CFTR genotype and other genes such as CLCA1. This is consistent with the evidence that CFTR and modifier genes interact in influencing the risk of developing meconium ileus (1).

Recently, the CF twin and sibling study in the United States identified regions of suggestive linkage for modifier genes that cause meconium ileus at chromosome 4, 8, and 11 or protect from meconium ileus at chromosomes 20 and 21 by genomewide analysis (1). However, CLCA1 that is located at chromosome 1 is not positioned in one of these regions, suggesting the involvement of additional loci in the development of meconium ileus.

It was initially suggested that CLCA1 and Clca3 were Ca2+-activated Cl channels (13). Subsequent studies indicated that CLCA1 and Clca3 are, rather, soluble secreted globular proteins (14), likely involved in protein–protein interactions and extracellular signaling (14,15). Although the exact function of Clca3 is unknown, it is obvious that Clca3 plays a major role in intestinal obstruction in Cftr knockout mice. In congenic C57BL/6 Cftr−/− mice that usually die of severe intestinal obstruction (6,7), the intestinal expression of Clca3 is decreased (8,9), whereas correction for this Clca3 deficiency results in amelioration of intestinal pathology and survival (9).

The results of the Cftr−/− mice studies show that Clca3 has an important function in the development of intestinal obstruction in mice (8,9). Furthermore, through genetic and expression studies it was concluded that CLCA1, the human orthologue of murine Clca3, could be involved in the human rectal CFTR independent residual chloride secretion (5). Our study showed that the p.S357N variant in CLCA1 is associated with meconium ileus in humans. This change in amino acid residue at position 357 may affect physicochemical properties of the CLCA1 protein and consequently its function and/or expression, suggesting that CLCA1 may contribute in a similar manner to intestinal obstruction in patients with CF with meconium ileus as in Cftr knockout mice.

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Acknowledgments

The authors wish to thank S. van de Graaf of the Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, the Netherlands, for helping with the discussion. The authors further thank Markus Braun of the Department of Hepatology and Gastroenterology, Charité Universitätsmedizin, Berlin, Germany, for technical assistance.

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REFERENCES

1. Blackman SM, Deering-Brose R, McWilliams R, et al. Relative contribution of genetic and nongenetic modifiers to intestinal obstruction in cystic fibrosis. Gastroenterology 2006; 131:1030–1039.

2. Lai HJ, Cheng Y, Cho H, et al. Association between initial disease presentation, lung disease outcomes, and survival in patients with cystic fibrosis. Am J Epidemiol 2004; 159:537–546.

3. Rozmahel R, Wilschanski M, Matin A, et al. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor. Nat Genet 1996; 12:280–287.

4. Zielenski J, Corey M, Rozmahel R, et al. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Nat Genet 1999; 22:128–129.

5. Ritzka M, Stanke F, Jansen S, et al. The CLCA gene locus as a modulator of the gastrointestinal basic defect in cystic fibrosis. Hum Genet 2004; 115:483–491.

6. Ratcliff R, Evans MJ, Cuthbert AW, et al. Production of a severe cystic fibrosis mutation in mice by gene targeting. Nat Genet 1993; 4:35–41.

7. Snouwaert JN, Brigman KK, Latour AM, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992; 257:1083–1088.

8. Brouillard F, Bensalem N, Hinzpeter A, et al. Blue native/SDS-PAGE analysis reveals reduced expression of the mClCA3 protein in cystic fibrosis knock-out mice. Mol Cell Proteomics 2005; 4:1762–1775.

9. Young FD, Newbigging S, Choi C, et al. Amelioration of cystic fibrosis intestinal mucous disease in mice by restoration of mCLCA3. Gastroenterology 2007; 133:1928–1937.

10. Rosenstein BJ, Zeitlin PL. Cystic fibrosis. Lancet 1998; 351:277–282.

11. Stern RC. The diagnosis of cystic fibrosis. N Engl J Med 1997; 336:487–491.

12. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993; 73:1251–1254.

13. Loewen ME, Forsyth GW. Structure and function of CLCA proteins. Physiol Rev 2005; 85:1061–1092.

14. Gibson A, Lewis AP, Affleck K, et al. hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore are not ion channels. J Biol Chem 2005; 280:27205–27212.

15. Mundhenk L, Alfalah M, Elble RC, et al. Both cleavage products of the mCLCA3 protein are secreted soluble proteins. J Biol Chem 2006; 281:30072–30080.

Cited By:

This article has been cited 1 time(s).

Journal of Biological Chemistry
Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation
Yurtsever, Z; Sala-Rabanal, M; Randolph, DT; Scheaffer, SM; Roswit, WT; Alevy, YG; Patel, AC; Heier, RF; Romero, AG; Nichols, CG; Holtzman, MJ; Brett, TJ
Journal of Biological Chemistry, 287(): 42138-42149.
10.1074/jbc.M112.410282
CrossRef
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Keywords:

CLCA1; cystic fibrosis; meconium ileus; polymorphism

© 2010 Lippincott Williams & Wilkins, Inc.

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