A family-based study does not confirm the association of MYO9B with celiac disease in the Italian population

Abstract

Association between Myosin IXB (MYO9B) gene polymorphisms and celiac disease (CD) was recently detected by a case–control association study in the Dutch, but not confirmed in the British and Swedish/Norwegian populations. We tested the association between CD and the three most associated single nucleotide polymorphisms (SNPs) in the Dutch study by the transmission disequilibrium test in the Italian population. A total of 252 pediatric patients and 504 parents were genotyped. No transmission distortion was detected either for the single SNPs or for their haplotypic combinations. Control allele frequencies, calculated from untransmitted alleles, were significantly different from those of the Dutch control population. Conversely, allele frequencies were very similar in Italian, British, Swedish/Norwegian and Dutch patients. In conclusion, MYO9B is not involved in CD susceptibility in the Italian population. The difference with the Dutch result might be explained by an imperfect selection of the Dutch controls.

Introduction

Gluten-sensitive enteropathy, also known as celiac disease (CD), is a common disorder characterized by malabsorption and small intestinal injury owing to intolerance to certain storage proteins found in wheat (gluten), barley and rye.

CD is a typical multifactorial disease with both environmental and genetic susceptibility factors. The major environmental factor is well known: ingested gluten. A strong genetic component is suggested by the very high concordance rate among monozygotic twins (71.4%, the highest concordance so far detected for a complex autoimmune disease) compared to that of dizygotic twins (9.1%).1, 2 The principal genetic factor (CELIAC1) lies in the human lymphocyte antigen (HLA) region. More than 90% of CD patients carry the HLA-DQ2 alpha/beta heterodimer encoded by the DQA1*05 and DQB1*02 alleles. Most of the remaining patients carry the DQ8 molecules encoded by the DQA1*03 and DQB1*0302 alleles. However, although necessary, HLA alone is not sufficient for disease manifestation. Only a small fraction of DQ2-positive individuals in the population ever develop CD despite eating gluten. Further, it does not explain the whole genetic susceptibility. In fact, concordance rate between monozygotic twins (70%) is higher than that between HLA-identical sibs (30%). The HLA locus was estimated to account for about 30% of the familial clustering of CD in Italy.3 Other genetic components are therefore likely to be involved.

Three non-HLA CD susceptibility loci are reported as CELIAC2, CELIAC3 and CELIAC4 in the OMIM catalog (http://www.ncbi.nlm.nih.gov/omim/).

Evidence for a genetic risk factor located in the 5q31–q33 region (CELIAC2) comes from the meta- and mega-analyses performed on whole-genome linkage data from a total of 442 Italian, Scandinavian, British and Finnish families with at least two sibs affected by CD.4 The data were analyzed using the Zlr statistics.5 Apart from the HLA region, only 5q31–33, with a maximum Zlr=4.39 (P=6 × 10−6), showed genome-wide significant linkage according to standard thresholds.6 Linkage of CD to this region was originally identified by Greco et al.7 and was also found by Naluai et al.8 and Liu et al.9

Evidence for a risk factor in 2q33 (CELIAC3), likely corresponding to the CTLA4/ICOS genes, comes from association studies mainly in Northern Europe populations.10

The CELIAC4 locus was mapped to 19p13.1 with a maximum logarithm of odds score=4.43 in a cohort of 101 affected sib pairs belonging to 82 Dutch families.11 However, this chromosomal location does not show any evidence for linkage (Zlr=1.65 for the peak marker D19S221) in the pooled families of the European Cluster study,4 which does not include Dutch families.

Recently, Monsuur et al.12 identified the myosin IXB gene (MYO9B) as the likely susceptibility gene CELIAC4 in the Dutch population. A highly significant association was detected by a case–control study with common variants in the 3′end of MYO9B. The most significantly associated single nucleotide polymorphism (SNP) was rs2305764 (A/G) in intron 28, for which the A allele was enriched in patients when compared to controls (P=2.1 × 10−6) with an odds ratio (OR)=1.51 (confidence interval: 1.28–1.80). According to the authors, this SNP alone can completely explain the association observed at MYO9 and could be considered a marker for CD. However, this association was not confirmed in a case–control study in a large British cohort13 and in a family-based and case–control association study in the Swedish/Norwegian population.14 The lack of confirmation could be explained either by a false positive in the Dutch study or by genetic heterogeneity between the Dutch and the other two populations. Further association studies in different populations are necessary to resolve whether MYO9B variants truly predispose one to CD.

We tested an Italian population for association between CD and the three most associated SNPs in the Dutch study, namely rs962917 (A/G), rs1457092 (A/C) and rs2305764 (A/G).

We performed on the Italian data a family-based association study, the transmission disequilibrium test (TDT), thus avoiding the danger of a spurious association derived from an imperfect match between cases and controls.

Results and discussion

The three selected SNPs, rs962917, rs1457092 and rs2305764, were genotyped in 252 trio families (Table 1). We did not type rs2305765, which is also very strongly associated with CD in the Dutch population, because both the Dutch and the British data show that it is in almost complete linkage disequilibrium with rs2305764.

Table 1 Characteristics of the patients

TDT results at the single SNP level are shown in Table 2. No transmission distortion was detected for any of the tested SNPs, not even for rs2305764 reported to be the strongest CD-associated MYO9B variant in the Dutch population.

Table 2 MYO9B single-marker TDT

For the AAA haplotype, which was significantly (P=0.000018) enriched in the Dutch CD patients, we did not observe any excess of transmission from 238 heterozygous parents to their affected offspring (117 transmitted vs 121 untransmitted; Table 3).

Table 3 MYO9B haplotype TDT

Similarly to the other tested populations, we observed a strong positive LD between the A alleles of the three SNPs (rs962917 vs rs1457092: D′=0.94, r2=0.87; rs962917 vs rs2305764: D′=0.92, r2=0.80; rs962917 vs rs2305764: D′=0.95, r2=0.94, as determined by Haploview version 3.11; www.hapmap.org/).

In order to compare directly our results with those obtained in the Dutch, British and Norwegian/Swedish case–control association studies,12, 13, 14 we calculated the frequencies of the transmitted and untransmitted alleles, which represent the allele frequencies in Italian cases and controls, respectively. Allele frequencies from the CEU (CEPH-UTAH) population typed in the HapMap (www.hapmap.org/) were also considered for comparison to an additional European population. As shown in Table 4, the frequencies of rs962917, rs1457092 and rs2305764 were very similar in all the examined control populations with the exception of the Dutch, where they were very significantly different (P=3 × 10−5, P=8 × 10−7 and P=10−3, respectively).

Table 4 Allele frequencies in controls and in CD patients in different Caucasoid populations

Notably, gene frequencies were similar for all the patient samples even if they were not homogeneous for age at diagnosis (pediatric for Italian and Swedish/Norwergian and adult for Dutch and British samples).

Thus in our sample of 252 Italian CD patients, we did not replicate the association observed in the Dutch population. An effect similar to that of the SNP most strongly associated with CD in the Dutch study (rs2305764, OR=1.51) would have been detected in our sample data with a power of 90%. Thus, heterogeneity in this gene's involvement in CD in the two populations could be suspected as also suggested by the different linkage results.11, 4 However, the lack of replication in the British and Swedish/Norwegian studies,13 along with the observation that that marker distributions are more similar across the four patient samples than across the control samples, makes the possibility of a spurious result in the Dutch population more likely. If this is true, our efforts for unraveling the non-HLA CD genetic component and for understanding the pathogenesis of CD must again be redirected. In order to definitively confirm or reject the possibility of a MYOB9 gene effect specific to the Dutch population, it would be necessary to type the available parents of the Dutch patients and to perform a family-based test. This strategy has the huge advantage of ensuring a perfect match between patient and control gametes for the association test and also to simultaneously test for linkage and association.

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Acknowledgements

This work was supported by ELFID (European Laboratory for Food Induced Disease), the Italian Ministero della Salute (Project 2000, no. 0AB/F), the Italian Ministero dell'Istruzione, Università e Ricerca (MIUR-PRIN no. MM06 187812) and by CARIPLO and ‘Compagnia S Paolo’ foundations. MM was supported by a fellowship from Regione Piemonte. We are grateful to the patients and their parents.

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Giordano, M., Marano, C., Mellai, M. et al. A family-based study does not confirm the association of MYO9B with celiac disease in the Italian population. Genes Immun 7, 606–608 (2006). https://doi.org/10.1038/sj.gene.6364331

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Keywords

  • celiac disease
  • myosin IXB
  • transmission disequilibrium test

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