The MYO9B Gene Is a Strong Risk Factor for Developing Refractory Celiac Disease
2007; Elsevier BV; Volume: 5; Issue: 12 Linguagem: Inglês
10.1016/j.cgh.2007.08.018
ISSN1542-7714
AutoresVictorien M. Wolters, Wieke H.M. Verbeek, Alexandra Zhernakova, N. Charlotte Onland‐Moret, Marco W.J. Schreurs, Alienke J. Monsuur, Willem Verduijn, Cisca Wijmenga, Chris J. Mulder,
Tópico(s)Galectins and Cancer Biology
ResumoBackground & Aims: Celiac disease (CD) is associated with HLA-DQ2 and HLA-DQ8 and has been linked to genetic variants in the MYO9B gene on chromosome 19. HLA-DQ2 homozygosity is associated with complications of CD such as refractory celiac disease type II (RCD II) and enteropathy-associated T-cell lymphoma (EATL). We investigated whether MYO9B also predisposes to RCD II and EATL. Methods: Genotyping of MYO9B and molecular HLA-DQ2 typing were performed on 62 RCD II and EATL patients, 421 uncomplicated CD patients, and 1624 controls. Results: One single nucleotide polymorphism in MYO9B showed a significantly different allele distribution in RCD II and EATL patients compared with controls (P = .00002). The rs7259292 T allele was significantly more frequent in RCD II and EATL patients compared with CD patients (P = .0003; odds ratio [OR], 3.61; 95% confidence interval [CI], 1.78–7.31). The frequency of the haplotype carrying the T allele of this single nucleotide polymorphism was significantly increased in RCD II and EATL patients (11%), compared with controls (2%) and CD patients (3%) (OR, 6.76; 95% CI, 3.40–13.46; P = 2.27E-09 and OR, 4.22; 95% CI, 1.95–9.11; P = .0001, respectively). Both MYO9B rs7259292 and HLA-DQ2 homozygosity increase the risk for RCD II and EATL to a similar extent when compared with uncomplicated CD patients (OR, 4.3; 95% CI, 1.9–9.8 and OR, 5.4; 95% CI, 3.0–9.6, respectively), but there was no evidence for any interaction between these 2 risk factors. Conclusions: We show that both MYO9B and HLA-DQ2 homozygosity might be involved in the prognosis of CD and the chance of developing RCD II and EATL. Background & Aims: Celiac disease (CD) is associated with HLA-DQ2 and HLA-DQ8 and has been linked to genetic variants in the MYO9B gene on chromosome 19. HLA-DQ2 homozygosity is associated with complications of CD such as refractory celiac disease type II (RCD II) and enteropathy-associated T-cell lymphoma (EATL). We investigated whether MYO9B also predisposes to RCD II and EATL. Methods: Genotyping of MYO9B and molecular HLA-DQ2 typing were performed on 62 RCD II and EATL patients, 421 uncomplicated CD patients, and 1624 controls. Results: One single nucleotide polymorphism in MYO9B showed a significantly different allele distribution in RCD II and EATL patients compared with controls (P = .00002). The rs7259292 T allele was significantly more frequent in RCD II and EATL patients compared with CD patients (P = .0003; odds ratio [OR], 3.61; 95% confidence interval [CI], 1.78–7.31). The frequency of the haplotype carrying the T allele of this single nucleotide polymorphism was significantly increased in RCD II and EATL patients (11%), compared with controls (2%) and CD patients (3%) (OR, 6.76; 95% CI, 3.40–13.46; P = 2.27E-09 and OR, 4.22; 95% CI, 1.95–9.11; P = .0001, respectively). Both MYO9B rs7259292 and HLA-DQ2 homozygosity increase the risk for RCD II and EATL to a similar extent when compared with uncomplicated CD patients (OR, 4.3; 95% CI, 1.9–9.8 and OR, 5.4; 95% CI, 3.0–9.6, respectively), but there was no evidence for any interaction between these 2 risk factors. Conclusions: We show that both MYO9B and HLA-DQ2 homozygosity might be involved in the prognosis of CD and the chance of developing RCD II and EATL. Celiac disease (CD) is an immune-mediated enteropathy following the ingestion of gluten. It is characterized by a permanent intolerance for the gluten proteins present in dietary wheat, rye, and barley. The pathogenesis of disease involves interactions between environmental, genetic, and immunologic factors.1Kagnoff M.F. Celiac disease: a gastrointestinal disease with environmental, genetic, and immunologic components.Gastroenterol Clin North Am. 1992; 21: 405-425Abstract Full Text PDF PubMed Google Scholar, 2Sollid L.M. Markussen G. Ek J. et al.Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer.J Exp Med. 1989; 169: 345-350Crossref PubMed Scopus (807) Google Scholar Genes play a key role in the pathogenesis of CD. The class II HLA-DQ2 and HLA-DQ8 loci are the most important genetic contributors identified so far. The HLA DQ2 heterodimer is encoded by the DQA1*0501 and DQB1*02 alleles and is present in 95% of all individuals diagnosed correctly with CD. Almost all the remaining patients express HLA-DQ8.2Sollid L.M. Markussen G. 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HLA susceptibility genes in celiac disease: genetic mapping and role in pathogenesis.Gastroenterology. 1993; 105: 910-922Abstract PubMed Google Scholar Several studies found evidence for linkage to regions on different chromosomes, including chromosomes 5, 6, and 19.10Greco L. Corazza G. Babron M.C. et al.Genome search in celiac disease.Am J Hum Genet. 1998; 62: 669-675Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 11Greco L. Babron M.C. Corazza G.R. et al.Existence of a genetic risk factor on chromosome 5q in Italian coeliac disease families.Ann Hum Genet. 2001; 65: 35-41Crossref PubMed Scopus (81) Google Scholar, 12Percopo S. Babron M.C. Whalen M. et al.Saturation of the 5q31-q33 candidate region for coeliac disease.Ann Hum Genet. 2003; 67: 265-268Crossref PubMed Scopus (23) Google Scholar, 13van Belzen M.J. Meijer J.W. Sandkuijl L.A. et al.A major non-HLA locus in celiac disease maps to chromosome 19.Gastroenterology. 2003; 125: 1032-1041Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 14van Belzen M.J. Vrolijk M.M. Meijer J.W. et al.A genomewide screen in a four-generation Dutch family with celiac disease: evidence for linkage to chromosomes 6 and 9.Am J Gastroenterol. 2004; 99: 466-471Crossref PubMed Scopus (22) Google Scholar Our group found strong evidence for linkage to genetic variants on chromosome 19, in the myosin IXB gene (MYO9B), which might lead to an impaired intestinal barrier and thereby play a role in the pathogenesis of CD.15Monsuur A.J. de Bakker P.I. Alizadeh B.Z. et al.Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect.Nat Genet. 2005; 37: 1341-1344Crossref PubMed Scopus (205) Google Scholar Individuals homozygous for the at-risk MYO9B allele had a 2.3 times higher risk of CD. So far, the only treatment for CD is a life-long gluten-free diet (GFD). However, a small subgroup (2%–5%) of CD patients diagnosed at adult age fails to improve on a GFD.16Wahab P.J. Meijer J.W. Mulder C.J. Histologic follow-up of people with celiac disease on a gluten-free diet: slow and incomplete recovery.Am J Clin Pathol. 2002; 118: 459-463Crossref PubMed Scopus (257) Google Scholar In this group of patients, the enteropathy persists despite adherence to the diet or recurs after an initially good response to the diet. These patients are regarded as suffering from refractory celiac disease (RCD), defined as persisting villous atrophy with crypt hyperplasia and increased IELs, despite maintaining a strict GFD for more than 12 months, or when severe symptoms necessitate intervention independent of the duration of the GFD.17Abdulkarim A.S. Burgart L.J. See J. et al.Etiology of nonresponsive celiac disease: results of a systematic approach.Am J Gastroenterol. 2002; 97: 2016-2021Crossref PubMed Google Scholar, 18Biagi F. Corazza G.R. Defining gluten refractory enteropathy.Eur J Gastroenterol Hepatol. 2001; 13: 561-565Crossref PubMed Scopus (71) Google Scholar, 19Daum S. Cellier C. Mulder C.J. Refractory coeliac disease.Best Pract Res Clin Gastroenterol. 2005; 19: 413-424Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 20Mulder C.J. Wahab P.J. Moshaver B. et al.Refractory coeliac disease: a window between coeliac disease and enteropathy associated T cell lymphoma.Scand J Gastroenterol Suppl. 2000; 232: 32-37PubMed Google Scholar, 21Wahab P.J. Meijer J.W. Goerres M.S. et al.Coeliac disease: changing views on gluten-sensitive enteropathy.Scand J Gastroenterol Suppl. 2002; 236: 60-65Crossref PubMed Scopus (43) Google Scholar With immunophenotypic analysis, 2 types of RCD can be recognized, RCD I and RCD II. RCD II patients are characterized by the presence of aberrant (CD7+CD3-CD4-CD8-cytoplasmic CD3+) IELs in the small bowel mucosa, whereas these lymphocytes are not detected in RCD I patients.18Biagi F. Corazza G.R. Defining gluten refractory enteropathy.Eur J Gastroenterol Hepatol. 2001; 13: 561-565Crossref PubMed Scopus (71) Google Scholar, 21Wahab P.J. Meijer J.W. Goerres M.S. et al.Coeliac disease: changing views on gluten-sensitive enteropathy.Scand J Gastroenterol Suppl. 2002; 236: 60-65Crossref PubMed Scopus (43) Google Scholar, 22Cellier C. Delabesse E. Helmer C. et al.Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma: French Coeliac Disease Study Group.Lancet. 2000; 356: 203-208Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar Approximately half of the RCD II patients develop an enteropathy-associated T-cell lymphoma (EATL) within 5 years, whereas RCD I patients seldom develop an EATL.23Al Toma A. Verbeek W.H.M. Mulder C.J. Survival in patients with refractory coeliac disease and enteropathy associated T cell lymphoma.Gut. 2007; 56: 1373-1378Crossref PubMed Scopus (242) Google Scholar, 24Meijer J.W. Mulder C.J. Goerres M.G. et al.Coeliac disease and (extra)intestinal T-cell lymphomas: definition, diagnosis and treatment.Scand J Gastroenterol Suppl. 2004; 241: 78-84Crossref PubMed Scopus (53) Google Scholar In the RCD II and EATL group, significantly more patients were seen to be HLA-DQ2 homozygous compared with uncomplicated CD patients. This difference was even more pronounced when compared with healthy controls.25Al Toma A. Goerres M.S. Meijer J.W. et al.Human leukocyte antigen-DQ2 homozygosity and the development of refractory celiac disease and enteropathy-associated T-cell lymphoma.Clin Gastroenterol Hepatol. 2006; 4: 315-319Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Our aim was to investigate whether the MYO9B gene is an additional risk factor for the development of RCD II and EATL, similar to HLA-DQ2 homozygosity. We also investigated a possible interaction between MYO9B and HLA. A total of 62 Dutch white RCD II and EATL patients were included in the study (50 RCD II patients and 12 EATL patients). All patients were ill and had severe malabsorption; RCD II is difficult to treat. More than half of the RCD II patients have since died. Patients were treated with cladribine therapy,23Al Toma A. Verbeek W.H.M. Mulder C.J. Survival in patients with refractory coeliac disease and enteropathy associated T cell lymphoma.Gut. 2007; 56: 1373-1378Crossref PubMed Scopus (242) Google Scholar and 8 patients underwent autologous stem cell transplantation with good results.26Al Toma A. Verbeek W.H.M. Visser O.J. Disappointing outcome of autologous stem cell transplantation for enteropathy-associated T-cell lymphoma.Dig Liver Dis. 2007; 39: 634-641Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar However, 25 (52%) of the RCD II patients went on to develop EATL. The 2-year survival rate for EATL patients is 15%–20%, despite aggressive treatment with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and alemtuzumab, and 4 patients even had autologous stem cell transplantation, but with disappointing results in contrast to the 8 RCD II patients. All except one of our patients were also included in a former study by Al Toma et al.25Al Toma A. Goerres M.S. Meijer J.W. et al.Human leukocyte antigen-DQ2 homozygosity and the development of refractory celiac disease and enteropathy-associated T-cell lymphoma.Clin Gastroenterol Hepatol. 2006; 4: 315-319Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Because our main interest was to look for genetic association in RCD II patients, we refer to our cohort of RCD II and EATL patients as RCD II. The diagnosis of CD was confirmed by histologic examination, with a documented histologic response to gluten withdrawal.21Wahab P.J. Meijer J.W. Goerres M.S. et al.Coeliac disease: changing views on gluten-sensitive enteropathy.Scand J Gastroenterol Suppl. 2002; 236: 60-65Crossref PubMed Scopus (43) Google Scholar, 27When is a coeliac a coeliac? report of a working group of the United European Gastroenterology Week in Amsterdam, 2001.Eur J Gastroenterol Hepatol. 2001; 13: 1123-1128Crossref PubMed Scopus (202) Google Scholar All patients and their adherence to GFD were regularly checked in the outpatient clinic by a dietitian. Patients with CD were considered to be refractory if their symptoms of malabsorption caused by villous atrophy persisted despite strict adherence to a GFD or recurred after an initially good response to the diet. Their histopathology showed at least partial villous atrophy (Marsh IIIA) according to the modified Marsh criteria and after excluding other possible causes of villous atrophy.18Biagi F. Corazza G.R. Defining gluten refractory enteropathy.Eur J Gastroenterol Hepatol. 2001; 13: 561-565Crossref PubMed Scopus (71) Google Scholar, 21Wahab P.J. Meijer J.W. Goerres M.S. et al.Coeliac disease: changing views on gluten-sensitive enteropathy.Scand J Gastroenterol Suppl. 2002; 236: 60-65Crossref PubMed Scopus (43) Google Scholar Additional information about the diagnosis, evaluation, HLA typing, and IEL phenotyping is given in Table 1 and in the supplementary information on RCD II patients (see supplementary information online at www.cghjournal.org).Table 1Baseline Demographic Characteristics of RCD II GroupsPatient characteristicTotal groupRCD II totalEATL after RCD IIEATL onlyTotal (male:female)62 (29:33)50 (19:31)25 (10:15)12 (10:2)Age at diagnosis of CD, y (± SD) (range)56 (± 10.2) (33–74)54 (± 10.6) (33–74)55 (± 9.4) (34–71)63 (± 3.8) (56–70)Age at diagnosis of RCD II/EATL, y (± SD) (range)58 (± 8.5) RCDII, 62 (± 7.4) EATL (39–79)58 (± 8.5) (39–74)61 (± 8.6) (52–79)64 (± 4.0) (56–70)DQ2 total60 (96%)49 (98%)24 (96%)11 (92%) Heterozygosity22 (35%)19 (38%)5 (20%)3 (25%) Homozygosity38 (61%)30 (60%)19 (76%)8 (67%)Myosin IXB Heterozygosity12 (19%)10 (20%)4 (16%)2 (16%)SD, standard deviation. Open table in a new tab SD, standard deviation. A total of 421 Dutch white subjects with uncomplicated CD were included in the study. They were a subgroup of CD cases described previously,15Monsuur A.J. de Bakker P.I. Alizadeh B.Z. et al.Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect.Nat Genet. 2005; 37: 1341-1344Crossref PubMed Scopus (205) Google Scholar but from which we excluded all individuals with RCD I (n = 22), RCD II (n = 9), RCD II and EATL (n = 10), RCD II and ulcerative jejunitis (n = 1), and EATL (n = 1). HLA data were available for 407 individuals. A total of 1624 Dutch white controls were included in the study. The control group comprised the controls used in the article by Monsuur et al15Monsuur A.J. de Bakker P.I. Alizadeh B.Z. et al.Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect.Nat Genet. 2005; 37: 1341-1344Crossref PubMed Scopus (205) Google Scholar expanded with 938 extra blood bank controls.28van Bodegraven A.A. Curley C.R. Hunt K.A. et al.Genetic variation in myosin IXB is associated with ulcerative colitis.Gastroenterology. 2006; 131: 1768-1774Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The total control cohort (n = 1624) comprised blood donors from Amsterdam (n = 429), Leiden (n = 475), and Utrecht (n = 500) and healthy spouses from different, non-autoimmune projects (n = 220). HLA data were available for 477 controls. Whole blood was obtained from CD and RCD II patients for typing of HLA-DQA1* and DQB1* alleles, performed with a combined single-stranded conformation polymorphism/heteroduplex (HD) method by a semiautomated electrophoresis and gel staining method on the Phastsystem (Amersham Pharmacia Biotech, Uppsala, Sweden). Individuals were designated HLA-DQ2 if alleles DQA1*0501 and DQB1*02 were present and HLA-DQ8 if alleles DQA1*03 and DQB1*0302 were present.29O'Mahony S. Howdle P.D. Losowsky M.S. Review article: management of patients with non-responsive coeliac disease.Aliment Pharmacol Ther. 1996; 10: 671-680Crossref PubMed Scopus (124) Google Scholar, 30Carrington M. Miller T. White M. et al.Typing of HLA-DQA1 and DQB1 using DNA single-strand conformation polymorphism.Hum Immunol. 1992; 33: 208-212Crossref PubMed Scopus (63) Google Scholar, 31Csizmadia C.G. Mearin M.L. Oren A. et al.Accuracy and cost-effectiveness of a new strategy to screen for celiac disease in children with Down syndrome.J Pediatr. 2000; 137: 756-761Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar We did not examine the compound heterozygote of DQA1*05 and DQA1*0201 with DQB1*0201 and DQB1*0202, and we have not classified this combination. In controls, DQA1, DQB1, and DRB1 typing was performed by polymerase chain reaction with sequence-specific biotin-labeled oligonucleotides as described.32Verduyn W. Doxiadis II, Anholts J. et al.Biotinylated DRB sequence-specific oligonucleotides: comparison to serologic HLA-DR typing of organ donors in eurotransplant.Hum Immunol. 1993; 37: 59-67Crossref PubMed Scopus (135) Google Scholar HLA data of a part of the control patients were collected from the ITI two panel (the ITI panel is a DNA panel from the Immunogenetics and Transplantation Immunology Section and consists of 477 unrelated, randomly selected, Dutch blood donors). Eight tag single nucleotide polymorphisms (SNPs) (Table 2, Table 3) completely tagging the 3′ region of MYO9B (from rs7259292–rs388484) were typed.28van Bodegraven A.A. Curley C.R. Hunt K.A. et al.Genetic variation in myosin IXB is associated with ulcerative colitis.Gastroenterology. 2006; 131: 1768-1774Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Three of these SNPs (rs2305767, rs2305764, and rs1457092) were also used by Monsuur et al15Monsuur A.J. de Bakker P.I. Alizadeh B.Z. et al.Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect.Nat Genet. 2005; 37: 1341-1344Crossref PubMed Scopus (205) Google Scholar and van Bodegraven et al.28van Bodegraven A.A. Curley C.R. Hunt K.A. et al.Genetic variation in myosin IXB is associated with ulcerative colitis.Gastroenterology. 2006; 131: 1768-1774Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarTable 2Allele Distribution of MYO9b SNPs in RCD II Patients (n = 62) Compared With Healthy Controls (n = 1624)SNPMajor/minor alleleHealthy controlsRCD II patientsP valueOR95% CIMajor allele (frequency)Minor allele (frequency)Major allele (frequency)Minor allele (frequency)rs7259292C_T3119 (0.97)91 (0.03)112 (0.90)12 (0.10).000023.902.10–7.25rs2305767A_G1764 (0.56)1392 (0.44)79 (0.65)43 (0.35).051.430.99–2.09rs1545620A_C2056 (0.64)1148 (0.36)67 (0.55)55 (0.45).041.471.03–2.12rs1457092C_A2120 (0.67)1042 (0.33)77 (0.63)45 (0.37).371.200.83–1.74rs8107108C_T2959 (0.92)245 (0.08)110 (0.90)12 (0.10).371.410.77–2.57rs2305766G_C2114 (0.67)1050 (0.33)76 (0.62)46 (0.38).301.230.85–1.78rs2305764G_A1958 (0.62)1204 (0.38)71 (0.60)47 (0.40).701.080.75–1.58rs2279002A_G2263 (0.71)933 (0.29)81 (0.66)41 (0.34).301.240.85–1.82 Open table in a new tab Table 3Allele Distribution of MYO9b SNPs in RCD II Patients (n = 62) Compared With Uncomplicated CD Patients (n = 421)SNPMajor/minor alleleCD patientsRCD II patientsP valueOR95% CIMajor allele (frequency)Minor allele (frequency)Major allele (frequency)Minor allele (frequency)rs7259292C_T815 (0.97)25 (0.03)112 (0.90)12 (0.10).00033.611.78–7.31rs2305767A_G508 (0.62)316 (0.38)79 (0.65)43 (0.35).510.880.59–1.31rs1545620A_C485 (0.58)349 (0.42)67 (0.55)55 (0.45).501.140.78–1.67rs1457092C_A510 (0.61)328 (0.39)77 (0.63)45 (0.37).630.920.62–1.35rs8107108C_T774 (0.93)58 (0.07)110 (0.90)12 (0.10).261.540.81–2.92rs2305766G_C508 (0.62)314 (0.38)76 (0.62)46 (0.38).920.990.67–1.46rs2305764G_A445 (0.54)381 (0.46)71 (0.60)47 (0.40).200.780.53–1.15rs2279002A_G546 (0.66)282 (0.34)81 (0.66)41 (0.34).920.990.66–1.48 Open table in a new tab SNPs were genotyped by using Taqman assays (Applied Biosystems, Foster City, CA) and were performed according to the manufacturer's specifications. Genotypes were analyzed by using a TaqMan 7900HT (Applied Biosystems). In the controls, the frequency of all SNPs was in Hardy-Weinberg equilibrium (P > .05). The haplotype structure and linkage disequilibrium in the region were investigated with HAPMAP data (a haplotype map of the human genome, www.hapmap.org)33The International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4880) Google Scholar with the Haploview (Broad Institute of MIT and Harvard, Cambridge, MA) application.34Barrett J.C. Fry B. Maller J. et al.Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11813) Google Scholar Allele and genotype distributions in cases and controls were compared by using the COCAPHASE module of the UNPHASED statistical package.35Dudbridge F. Pedigree disequilibrium tests for multilocus haplotypes.Genet Epidemiol. 2003; 25: 115-121Crossref PubMed Scopus (1047) Google Scholar Haplotype association was estimated with the same package. Hardy-Weinberg equilibrium was tested by comparing the expected and observed genotypes in 2 × 3 χ2 table. Odds ratios (ORs) were calculated, and the confidence intervals (CIs) were approximated by using Woolf's method with Haldane's correction.36Haldane J.B. The estimation and significance of the logarithm of a ratio of frequencies.Ann Hum Genet. 1956; 20: 309-311Crossref PubMed Scopus (798) Google Scholar To investigate the added value of MYO9B to HLA-DQ2 homozygosity, we used a logistic regression model. First, HLA-DQ2 homozygosity (no/yes) was added to the model in which we compared RCD II patients with CD patients. Then we added the MYO9B variant (combining the heterozygous and the homozygous variants) that was most strongly associated to the disease and tested whether the model improved by using a likelihood ratio test. We also evaluated whether the MYO9B variant was independently associated to the risk of RCD II compared with CD patients by evaluating the OR and 95% CI. We tested for possible interaction between HLA-DQ2 homozygosity and the MYO9B variant by including the interaction term in the logistic regression model. We used Stata (Stata/SE 8.2 for Windows; StataCorp LP, College Station, TX) for these analyses. We observed that SNPs rs7259292 showed a different allele distribution in RCD II patients compared with the control group, with minor allele frequencies of 10% in RCD II patients compared with 3% in controls (P = .00002; OR, 3.90; 95% CI, 2.10–7.25) (Table 2). The association of rs1545620 with RCD II compared with controls was borderline statistically significant (OR, 1.47; 95% CI, 1.03–2.12; P = .04) (Table 2). All tested SNPs were located in 1 haploblock and were in strong linkage disequilibrium with each other. We therefore continued by constructing 8 SNP haplotypes. Five haplotypes occurred with a frequency of more than 5% in RCD II cases or controls. Haplotype h5, which carries the associated rs7259292*T allele, occurred significantly more frequently in RCD II patients than in controls (11% vs 2%; P = 2.27E-09; OR, 6.76; 95% CI, 3.40–13.46) (Table 4).Table 4Haplotype Analysis of Myosin IXB SNPs in RCD II Patients (n = 62) Compared With Healthy Controls (n = 1624) and Uncomplicated CD Patients (n = 421)HaplotypeaThe order of SNPs is as follows: rs7259292, rs2305767, rs1545620, rs1457092, rs8107108, rs2305766, rs2305764, rs2279002.RCD II patientsControlsRCD II patients compared with controlsCD patientsRCD II patients compared with CDCount (frequency)Count (frequency)P valueOR95% CICount (frequency)P value, single haplotypeOR95% CIh1C-A-A-C-C-G-G-A10 (0.09)223.9 (0.08).261.580.79–3.1852.56 (0.07).331.530.73–3.22h2C-A-A-C-T-G-G-A9.33 (0.08)228.2 (0.08).411.460.71–2.9850.37 (0.07).381.500.70–3.21h3C-A-C-A-C-C-A-G32.91 (0.29)824.7 (0.29).271.330.83–2.13251.4 (0.33).991.010.61–1.64h4C-G-A-C-C-G-G-A37.65 (0.34)1249 (0.43)Reference1—287.3 (0.37)Reference1—h5T-A-C-C-C-G-G-A12 (0.11)61.2 (0.02)2.27E-096.763.40–13.4621.99 (0.03).00014.221.95–9.11Overall P value.0004.002a The order of SNPs is as follows: rs7259292, rs2305767, rs1545620, rs1457092, rs8107108, rs2305766, rs2305764, rs2279002. Open table in a new tab On comparing the allele frequencies of the 8 tested SNPs in MYO9B between RCD II patients and CD patients, we observed a statistically significant difference in the frequency of rs7259292, as well as in the frequency of haplotype h5 in RCD II patients compared with CD patients (P = .0003; OR, 3.61; 95% CI, 1.78–7.31 and P = .0001; OR, 4.22; 95% CI, 1.95–9.11 on a single SNP and haplotype h5, respectively) (Table 3, Table 4). Strong association of RCD II and EATL with homozygosity for the HLA-DQ2 allele (DQA1*0501 and DQB1*0201) was recently reported by Al-Toma et al.25Al Toma A. Goerres M.S. Meijer J.W. et al.Human leukocyte antigen-DQ2 homozygosity and the development of refractory celiac disease and enteropathy-associated T-cell lymphoma.Clin Gastroenterol Hepatol. 2006; 4: 315-319Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar In the cohort of RCD II patients included in our study, 38 of 61 HLA-typed patients (62%) were DQ2 homozygous (DQA1*0501 and DQB1*0201), whereas only 97 of the 407 HLA-typed CD patients (24%) and 10 of the 477 HLA-typed controls (2%) carried 2 copies of the DQ2 allele. The OR for RCD II patients compared with uncomplicated CD was 5.2 (95% CI, 2.9–9.0; P = .27E-10). Compared with controls, the OR was 69.1 (95% CI, 31.1–153.6; P = 2.16E-54). The frequencies of individuals who carry both HLA-DQ2 homozygosity and rs7259292*T are higher in our patient cohort (Figure 1). Both MYO9B rs7259292 and HLA-DQ2 homozygosity increase the risk for RCD II and EATL to a similar extent when compared with uncomplicated CD patients (OR, 4.3; 95% CI, 1.9–9.8 and OR, 5.4; 95% CI, 3.0–9.6, respectively), and we found no evidence for interaction between these 2 risk factors. We performed a logistic regression model including both HLA-DQ2 homozygosity and the presence of rs7259292*T allele, comparing RCD II patients with CD cases, and observed that both HLA-DQ2 homozygosity and the presence of rs7259292*T allele are independently associated with the development of RCD II (Table 5, Table 6). In the likelihood ratio test, the model fit improved when the rs7259292*T allele was included in the model subsequent to HLA-DQ2 homozygosity (P < .0009).Table 5Distribution of Genotypes and Alleles of rs7259292 SNP in Groups StudiedGroups studiedCC (frequency)CT (frequency)TT (frequency)Allele C (frequency)Allele T (frequency)Controls (n = 1624)1517 (0.95)85 (0.05)3 (0.00)3119 (0.97)91 (0.03)Controls HLA-DQ2 homozygous (n = 10)10 (1.00)0 (0.00)0 (0.00)20 (1.00)0 (0.00)CD (n = 421)396 (0.94)23 (0.05)1 (0.00)815 (0.97)25 (0.03)CD HLA-DQ2 homozygous (n = 97)93 (0.96)4 (0.04)0 (0.00)190 (0.98)4 (0.02)RCD II (n = 62)50 (0.81)12 (0.19)0 (0.00)112 (0.90)12 (0.10)RCD II HLA-DQ2 homozygous (n = 38)30 (0.79)8 (0.21)0 (0.00)68 (0.89)8 (0.11)RCD II non–HLA-DQ2 homozygous (n = 23)19 (0.83)4 (0.17)0 (0.00)42 (0.91)4 (0.09) Open table in a new tab Table 6Logistic Regression Model for HLA-DQ2 Homozygosity and the Presence of rs7259292*T Allele in RCD II Patients Compared With Uncomplicated CD PatientsRisk factorsP > |z|OR95% CIHLA-DQ2 homozygous<.0005.43.0–9.6rs7259292*T allele<.0004.31.9–9.8 Open table in a new tab To test for possible interaction between HLA-DQ2 homozygosity and the rs7259292 genotype, we included an interaction term in the model. It was not statistically significant, and we therefore concluded that there was no significant interact
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