Chronic inflammatory conditions and autoimmune disorders are typically characterized by a deregulated adaptive and innate immune system. The innate defense consists of multiple components that include physical barriers, antimicrobial molecules, pattern recognition receptors, circulating phagocytes, and the complement system (Hoebe et al. 2004
). A breach of the epithelial barrier and loss of microbial containment is often the first of a series of events that trigger or sustain chronic inflammatory diseases (Tlaskalova-Hogenova et al. 2004
) as described, e.g., in Crohn’s disease, atopic eczema, asthma, and psoriasis (Schreiber et al. 2005
). In CD, the gut–lumen separation is undermined by dietary gluten that evokes a combined innate and adaptive immune response (Londei et al. 2005
). It is the joined action of gluten peptides, environmental factors, and genetic determinants that precipitates this enteropathy. The human leukocyte antigen locus is the major genetic contribution to the adaptive Th1 reaction (Koning et al. 2005
). Recently, we identified MYO9B
as a susceptibility gene in the Dutch population that possibly has an effect on epithelial barrier integrity (Monsuur et al. 2005
). Several other studies have underscored the involvement of innate immunity in CD, however, without identification of underlying causative gene variants (Londei et al. 2005
). Interestingly, it was also reported that the epithelial glycocalyx and the bacterial composition in the CD gut is distinct (Forsberg et al. 2004
; Tjellstrom et al. 2005
In search of genes that may have a primary contribution to CD pathogenesis, we focused our attention to the SPINK family of serine protease inhibitors that play an important role in tissue preservation through the containment of uncontrolled proteolysis and bacterial growth. In this study, we demonstrated differential gene expression of mucosal SPINK4 in CD. Crypt hyperplasia is a feature of the Marsh III and Marsh II stages of CD, and the concomitant increase in the number of goblet cells may contribute to the increased SPINK4 expression. However, the observed sharp decrease in gene expression sets in during the MIII/MII transition, whereas crypt normalization is observed only later at the MII/MI recovery phase. This suggests that SPINK4 downregulation sets in soon after commencement of the gluten-free diet. This SPINK4 differential expression probably reflects altered goblet cell activity, but its functional significance and regulatory mechanism in CD pathology remains to be established.
The combination of functional relevance and mapping to CD linkage intervals pointed to the SPINK family members as attractive functional and positional candidate genes. We have chosen a robust strategy for genetic association testing based on haplotype tagging SNPs and linkage-disequilibrium structure of the SPINK loci applied to a considerably sized Dutch case-control cohort. With our study design, we had 75% power to confirm association with SPINK1, -2, and -5 (relative risk 2.0; allele frequency 0.1–0.45; 95% confidence interval), whereas this was even 95% (RR 2.0) and 80% (RR 1.6) for SPINK4. These power estimates reflect a Type I error rate of 0.05, which is appropriate for testing a previously reported result. Initial detection of a new genetic association would require much more stringent criteria to assure reproducibility, and power would be correspondingly less.
In parallel, we examined the extended Dutch CD family for variants and deletions in SPINK4. We hypothesized that a specific SPINK4 mutation, although rare in the general population, could have a dramatic impact on mucus composition, bacterial containment, and gluten sensitivity, thereby explaining the apparent dominant and high penetration inheritance pattern in our extended CD family. With both approaches, we were not able to establish a genetic involvement of the SPINK genes tested. However, we cannot completely rule out the possibility of a rare noncoding mutation in SPINK4 (outside the splice donor and acceptor regions) that might specifically segregate in this atypical CD family, characterized by an exceptional high prevalence of affected members.
Despite this negative result in the Dutch CD population, we cannot formally rule out the possibility of genetic contribution of SPINK
genes to CD in other European populations like the Italian in whom, unlike the Dutch (van Belzen et al. 2003
), chromosome 5q linkage was established (Greco et al. 1998
; Percopo et al. 2003
). Genuine population heterogeneity has been reported before, e.g., between CARD15
and Crohn’s disease (Lesage et al. 2002
; Croucher et al. 2003
) and between SPINK5
and asthma (Blumenthal 2005
; Jongepier et al. 2005
). The new SPINK
members on chromosome 5q (SPINK5L2
, and SPINK9
) were not part of this study. Currently, no functional annotation is available for these genes that are located near SPINK1
in a chromosomal region that appears to have been subjected to gene duplication during evolution. Therefore, we cannot exclude their possible involvement in CD or any other inflammatory disorder.