- Email: [email protected]
Association of MYO9B haplotype with type 1 diabetes
Human Immunology (2008) 69, 112–115
Association of MYO9B haplotype with type 1 diabetes Jose Luis Santiagoa, Alfonso Martíneza, Concepción Núñeza, Hermenegildo de la Calleb, Miguel Fernández-Arqueroa, Emilio G. de la Conchaa, E. Urcelaya,* a b
Immunology Department, Hospital Universitario San Carlos, Madrid, Spain Endocrinology Department, Hospital Ramón y Cajal, Madrid, Spain
Received 29 October 2007; received in revised form 20 December 2007; accepted 10 January 2008
KEYWORDS MYO9B polymorphisms; Type 1 diabetes; Genetic susceptibility
Summary MYO9B (myosin IXB) polymorphisms were associated with celiac disease and ulcerative colitis susceptibility, presumably through alteration of the intestinal permeability. Recently this gene was also associated with several diseases with an autoimmune component, such as rheumatoid arthritis and systemic lupus erythematosus. We aimed to test, for the first time, the potential role of MYO9B polymorphisms in type 1 diabetes (T1D), an autoimmune condition preceded by changes in intestinal barrier integrity. Three previously associated MYO9B polymorphisms (rs962917, rs2279003, and rs2305764) were studied in 316 T1D patients and 706 ethnically matched controls. Minor alleles of those polymorphisms were more frequent in diabetic patients than in controls and the haplotype carrying major alleles in those positions, rs962917*G/rs2279003*C/rs2305764*G, significantly reduced the risk of T1D in the Spanish population (p ⫽ 0.004; OR [95% confidence interval] ⫽ 0.68 [0.52– 0.90]). Our data suggest an involvement of this MYO9B chromosomal region in T1D predisposition, indicating extensive influence on autoimmune diseases. © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Introduction Type 1 diabetes (T1D) is an immune-mediated disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets, culminating in the destruction of insulin-producing  cells. The strongest T1D genetic determinant lies in the major histocompatibility complex class II * Corresponding author. Fax: 91 3303344. E-mail address: [email protected] (E. Urcelay).
region on 6p21 and more than 10 T1D susceptibility loci have been found [1,2]. Efforts to define the genetics of this polygenic disorder were hampered by the low penetrance of each individual locus. The T1D complex etiology reflects gene– gene and gene– environment interactions. Although the exact environmental trigger has not been described, the rapid rise in incidence of the disease worldwide [3], together with the concordance rate for monozygotic twins, estimated between 30 and 50% [4], supports the idea that environmental factors influence T1D risk. Infectious agents as well as
0198-8859/$ -see front matter © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.01.003
113
MYO9B association with T1D
ABBREVIATIONS CI MYO9B OR RR T1D
confidence interval myosin IXB gene odds ratio relative risk type 1 diabetes
dietary factors have been hypothesized to play a role in diabetes susceptibility, but the ascertainment of the specific antigen leading to the aberrant immune response remains elusive. Nonetheless, considering that the gastrointestinal tract is the largest interface with the environment, the contribution of an increased intestinal permeability to T1D pathogenesis has been the focus of attention. Loss of barrier integrity was observed in both diabetic patients [5– 8] and animal models [9 –11]. Moreover, zonulin, a protein that modulates intestinal permeability by acting on tight junctions, was recently determined to be involved in the pathogenesis of autoimmune diabetes [12]. High serum zonulin levels were correlated with increased intestinal permeability and upregulation of the expression of integral membrane tight junction protein genes such as claudin-1 and myosin IXB in diabetic patients. Polymorphisms in the myosin IXB (MYO9B) gene were recently associated with celiac disease and ulcerative colitis in certain populations [13–16], and a role of the gene in increasing susceptibility to rheumatoid arthritis and systemic lupus erythematosus was also reported [17], suggesting that an influence of this chromosomal locus might underlie different autoimmune diseases. MYO9B encodes a protein with a Rho-GTPase-activating domain [18] and Rho family GTPases participate in remodeling of the cytoskeleton and tight junction assembly; therefore, they are involved in the establishment of epithelial barriers [19]. It has been speculated that variants within the 3’ end of the MYO9B gene lead to a mutated Rho-GTPase-activating domain and a subsequent impaired barrier function. The MYO9B gene maps to human chromosome 19p13 and wholegenome scans in T1D patients revealed linkage to this locus [20,21]. Therefore, we aimed to test, for the first time, whether the MYO9B gene previously associated with predisposition to some autoimmune diseases is also involved on T1D risk.
Subjects and methods Patients We studied 316 White unrelated Spanish T1D patients (50% women) diagnosed according to the criteria of the American Diabetes Association [22] and 706 healthy controls recruited among blood donors from the Madrid area (51% women). All subjects were insulin-dependent at the time of the study. Informed consent was obtained from all subjects included in the study, which was approved by the Ethics Committee of the Hospital Clínico San Carlos.
Genotyping The MYO9B polymorphisms (rs2305767, rs962917, rs1457092, rs2279003, rs2305765, and rs2305764) were analyzed in a 7900HT
following the manufacturer’s suggestions by predeveloped TaqMan allelic discrimination assays (C__1654927_20, C__7493866_1_, C__1654895_10, C__11698814_10, C__1654877_1_, and C__ 1654873_1_, respectively) from Applied Biosystems (Foster City, CA, USA). The genotyping success rate is over 98%.
Statistical analysis Case– control analysis were performed by applying 2 statistics or Fisher’s exact test when necessary. The genotype association with T1D was estimated by the odds ratio (OR) with 95% confidence interval (CI). Statistical analysis was performed using Epi Info v. 6.02 software (Centers for Disease Control, Atlanta, GA, USA). Haplotypic frequencies were estimated using the expectation– maximization algorithm implemented in Arlequin v2.000 software [23], with the number of iterations set at 5,000 and initial conditions at 50, with an epsilon value of 10⫺7.
Results This study was performed by analyzing three tagging polymorphisms and the results obtained through comparison between diabetic patients and healthy controls are summarized in Table 1. No significant difference was observed in allele or genotype distributions of any tested variant between T1D and control cohorts, although minor alleles were consistently more frequent in cases than in healthy controls. Four major haplotypes were estimated by applying the expectation–maximization algorithm [23] and their overall comparison evidenced a significant difference between patients and controls (2 ⫽ 8.7; p ⫽ 0.033). The haplotype carrying major alleles of each polymorphism (rs962917*G/ rs2279003*C/rs2305764*G) significantly reduces the risk of T1D in the Spanish population (Table 2). The statistical significance of this haplotype withstands Bonferroni’s correction.
Table 1 Allele and genotype frequencies of the MYO9B polymorphisms.
rs962917 GG GA AA G A rs2279003 CC CT TT C T rs2305764 GG GA AA G A
T1D patients (%)
Controls (%)
(n ⫽ 316) 114 (36.1) 153 (48.4) 49 (15.5) 381 (60.3) 251 (39.7) (n ⫽ 316) 96 (30.4) 159 (50.3) 61 (19.3) 351 (55.5) 281 (44.5) (n ⫽ 316) 99 (31.3) 165 (52.2) 52 (16.5) 363 (57.4) 269 (42.6)
(n ⫽ 706) 280 (39.6) 319 (45.2) 107 (15.2) 879 (62.2) 533 (37.8) (n ⫽ 695) 221 (31.8) 366 (52.7) 108 (15.5) 808 (58.1) 582 (41.9) (n ⫽ 693) 249 (35.9) 326 (47.0) 118 (17.0) 824 (59.4) 562 (40.6)
114 Table 2
J.L. Santiago et al. Frequency of haplotypes conformed by MYO9B polymorphisms in T1D patients and controls. T1D patients (2n ⫽ 632)
Controls (2n ⫽ 1362)
rs962917
rs2279003
rs2305764
Frequency
n
Frequency
n
OR (95% CI)
p
G A G G
T C C C
G A G A
0.440 0.397 0.134 0.024
278 251 85 15
0.407 0.370 0.186 0.021
554 504 253 29
1.15 1.12 0.68 1.12
0.16 0.25 0.004 0.73
Discussion The association of the MYO9B polymorphisms was originally described with celiac disease in the Dutch population [13]. Three pairs of MYO9B polymorphisms (rs962917 and rs1457092, rs2279003 and rs2305767, rs2305764 and rs2305765; complete linkage disequilibrium within each pair) were associated with two celiac Dutch cohorts and were used to tag haplotypes. Each pair of polymorphisms was also in complete linkage disequilibrium in our Spanish control cohort and every polymorphism conformed to Hardy– Weinberg equilibrium [24]. Their association with ulcerative colitis in several independent cohorts [14 –16] and also with other autoimmune diseases [17] was recently reported. Unexpectedly, the haplotype that increased susceptibility to those conditions (the second by frequency) is not the one associated with T1D (Table 2). When diabetic patients and healthy controls were compared, no difference was observed between the first and the second more frequent haplotypes (OR [95% CI] ⫽ 0.99 [0.80 –1.23]; p ⫽ 0.94). These data would indicate either that the statistical power to detect the previously described effect is compromised in our diabetic cohort or that different causative polymorphisms in linkage disequilibrium with the two associated haplotypes are responsible for the detected effects in distinct diseases. The existence of more than one haplotype within a chromosomal region modifying susceptibility is not a novel situation; the three polymorphisms within the nucleotide-binding oligomerization domain containing 2 gene, NOD/CARD15, the main genetic susceptibility factor for Crohn’s disease, are not arranged in a unique haplotype and the greatest relative risk was seen in patients possessing a variant allele at more than one of these loci (simple heterozygote relative risk [RR] ⫽ 2.4; homozygote RR ⫽ 9.8; compound heterozygote RR ⫽ 29.3 [25]). Moreover, this is not the first example of opposing susceptibility effects for a specific locus in different diseases, i.e., the DRB1*1501-DQB1*0602 haplotype, the strongest genetic susceptibility determinant to multiple sclerosis, confers a protective effect for T1D [26]. Several studies pointed to different putative etiologic variants in the MYO9B gene, although firm experimental support is lacking. The variant originally suggested by Monsuur and collaborators [13], rs2305764, located in intron 28 of MYO9B and supposedly able to explain the association with celiac disease by itself, was not replicated in many of the independent populations studied. The nonsynonymous change in exon 20, Ala1011Ser (rs1545620), was proposed by Van Bodegraven and collaborators [15] as the etiological polymorphism in inflammatory bowel diseases. They indi-
(0.94–1.39) (0.92–1.37) (0.52–0.90) (0.57–2.18)
cated it might affect the processive motor activity of the protein. However, the authors observed a genetic association of similar magnitude, as measured by OR values, for the variant rs1457092. Therefore, from a genetic standpoint it is difficult to distinguish between the putative causal polymorphism rs1545620 and both rs1457092 and rs962917, provided that all of them are found in very strong linkage disequilibrium in Caucasian populations, as detailed in http://www. hapmap.org. Certainly, although the polymorphism leading to an amino acidic change is the a priori best candidate, replication in populations with different ethnicity is warranted to confirm this hypothesis. Intronic polymorphisms affected alternative splicing and, as recently reported, abnormal kinetics of mRNA translation may arise from a silent single nucleotide polymorphism, thus yielding a protein with different function [27]. Therefore, no single nucleotide polymorphism can be disregarded as etiological and the identification of the real causative variant must be supported by information derived from genetic analyses in independent populations. The entheropathy associated with alterations of this chromosomal region might allow unregulated passage of environmental antigens that could potentially initiate the autoimmune response leading to disease in genetically susceptible individuals. In fact, increased intestinal permeability precedes the clinical onset of T1D and is altered during the whole course of the disease, as recently demonstrated by Bosi et al. [28]. Moreover, downstream of the MYO9B gene, a conserved region is found with approximately 100 residues in common with the eukaryotic occludin proteins, OCEL1. The barrier protein occludin is decreased in experimental diabetes and vascular permeability is associated with reduced endothelial occludin content [29]. Diabetes increases blood– brain barrier permeability via a loss of tight junction proteins [9] and the cerebral occludin content in diabetic rats is significantly reduced compared with insulin-treated diabetic rats or control rats [30]. Therefore, MYO9B might not be the only susceptibility gene within the locus. Whatever the case, our data suggest the association of the MYO9B chromosomal region with pathogenic implications in T1D and confirmation of this effect in independent populations should be sought.
Acknowledgments We thank Carmen Martínez for expert technical assistance. Alfonso Martínez and Jose Luis Santiago are recipients of FIS
MYO9B association with T1D contracts (CP04/00175 and CM05/00216, respectively). Elena Urcelay works for the “Fundación para la Investigación Biomédica–Hospital Clínico San Carlos.” This work was supported by grants from Fundación Areces and Fundación Mutua Madrileña.
References [1] Hirschhorn JN. Genetic epidemiology of type 1 diabetes. Pediatr Diabetes 2003;4:87. [2] Todd JA, Walker NM, Cooper JD, Smyth DJ, Downes K, Plagnol V, et al. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat Genet 2007;39:857-864. [3] Green A, Patterson CC. Trends in the incidence of childhoodonset diabetes in Europe 1989 –1998. Diabetologia 2001;44 (Suppl 3):B3. [4] Redondo MJ, Yu L, Hawa M, Mackenzie T, Pyke DA, Eisenbarth GS, et al. Heterogeneity of type I diabetes: analysis of monozygotic twins in Great Britain and the United States. Diabetologia 2001;44:354. [5] Carratu R, Secondulfo M, de Magistris L, Iafusco D, Urio A, Carbone MG, et al. Altered intestinal permeability to mannitol in diabetes mellitus type I. J Pediatr Gastroenterol Nutr 1999; 28:264. [6] Damci T, Nuhoglu I, Devranoglu G, Osar Z, Demir M, Ilkova H. Increased intestinal permeability as a cause of fluctuating postprandial blood glucose levels in type 1 diabetic patients. Eur J Clin Invest 2003;33:397. [7] Kuitunen M, Saukkonen T, Ilonen J, Akerblom HK, Savilahti E. Intestinal permeability to mannitol and lactulose in children with type 1 diabetes with the HLA-DQB1*02 allele. Autoimmunity 2002;35:365. [8] Secondulfo M, Iafusco D, Carratu R, deMagistris L, Sapone A, Generoso M, et al. Ultrastructural mucosal alterations and increased intestinal permeability in non-celiac, type I diabetic patients. Dig Liver Dis 2004;36:35. [9] Hawkins BT, Lundeen TF, Norwood KM, Brooks HL, Egleton RD. Increased blood-brain barrier permeability and altered tight junctions in experimental diabetes in the rat: contribution of hyperglycaemia and matrix metalloproteinases. Diabetologia 2007;50:202. [10] Meddings JB, Jarand J, Urbanski SJ, Hardin J, Gall DG. Increased gastrointestinal permeability is an early lesion in the spontaneously diabetic BB rat. Am J Physiol 1999;276:G951. [11] Neu J, Reverte CM, Mackey AD, Liboni K, Tuhacek-Tenace LM, Hatch M, et al. Changes in intestinal morphology and permeability in the biobreeding rat before the onset of type 1 diabetes. J Pediatr Gastroenterol Nutr 2005;40:589. [12] Sapone A, de Magistris L, Pietzak M, Clemente MG, Tripathi A, Cucca F, et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 2006;55:1443. [13] Monsuur AJ, de Bakker PI, Alizadeh BZ, Zhernakova A, Bevova MR, Strengman E, et al. Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect. Nat Genet 2005;37:1341.
115 [14] Nuñez C, Oliver J, Mendoza JL, Gómez-García M, Piñero A, Taxonera C, et al. MYO9B polymorphisms in patients with inflammatory bowel disease. Gut 2007;56:1321-1322. [15] van Bodegraven AA, Curley CR, Hunt KA, Monsuur AJ, Linskens RK, Onnie CM, et al. Genetic variation in myosin IXB is associated with ulcerative colitis. Gastroenterology 2006;131:1768. [16] Latiano A, Palmieri O, Valvano MR, D’Inca R, Caprilli R, Cucchiara S, et al. Association of myo9b gene in Italian patients with inflammatory bowel diseases. Aliment Pharmacol Ther. 2007 17 Oct [Epub ahead of print]. [17] Sanchez E, Alizadeh BZ, Valdigem G, Ortego-Centeno N, Jimenez-Alonso J, et al. MYO9B gene polymorphisms are associated with autoimmune diseases in Spanish population. Hum Immunol 2007;68:610. [18] Wirth JA, Jensen KA, Post PL, Bement WM, Mooseker MS. Human myosin-IXb, an unconventional myosin with a chimerinlike rho/rac GTPase-activating protein domain in its tail. J Cell Sci 1996;109:653. [19] Matter K, Aijaz S, Tsapara A, Balda MS. Mammalian tight junctions in the regulation of epithelial differentiation and proliferation. Curr Opin Cell Biol 2005;17:453. [20] Concannon P, Gogolin-Ewens KJ, Hinds DA, Wapelhorst B, Morrison VA, Stirling B, et al. A second-generation screen of the human genome for susceptibility to insulin-dependent diabetes mellitus. Nat Genet 1998;19:292. [21] Mein CA, Esposito L, Dunn MG, Johnson GC, Timms AE, Goy JV, et al. A search for type 1 diabetes susceptibility genes in families from the United Kingdom. Nat Genet 1998;19:297. [22] Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 1997;20:1183. [23] Schneider S, Excoffier L. Arlequin: A software for population genetics data analysis. Geneva, University of Geneva, 2000. [24] Nunez C, Marquez A, Varade J, Martinez A, Polanco I, Maluenda C, et al. No evidence of association of the MYO9B polymorphisms with celiac disease in the Spanish population. Tissue Antigens 2006;68:489. [25] Ahmad T, Armuzzi A, Bunce M, Mulcahy-Hawes K, Marshall SE, Orchard TR, et al. The molecular classification of the clinical manifestations of Crohn’s disease. Gastroenterology 2002;122: 854. [26] Noble JA, Valdes AM, Cook M, Klitz W, Thomson G, Erlich HA. The role of HLA class II genes in insulin-dependent diabetes mellitus: molecular analysis of 180 Caucasian, multiplex families. Am J Hum Genet 1996;59:1134. [27] Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, et al. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science 2007;315:525. [28] Bosi E, Molteni L, Radaelli MG, Folini L, Fermo I, Bazzigaluppi E, et al. Increased intestinal permeability precedes clinical onset of type 1 diabetes. Diabetologia 2006;49:2824. [29] Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW. Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group. Diabetes 1998;47: 1953. [30] Chehade JM, Haas MJ, Mooradian AD. Diabetes-related changes in rat cerebral occludin and zonula occludens-1 (ZO-1) expression. Neurochem Res 2002;27:249.
Association of MYO9B haplotype with type 1 diabetes Jose Luis Santiagoa, Alfonso Martíneza, Concepción Núñeza, Hermenegildo de la Calleb, Miguel Fernández-Arqueroa, Emilio G. de la Conchaa, E. Urcelaya,* a b
Immunology Department, Hospital Universitario San Carlos, Madrid, Spain Endocrinology Department, Hospital Ramón y Cajal, Madrid, Spain
Received 29 October 2007; received in revised form 20 December 2007; accepted 10 January 2008
KEYWORDS MYO9B polymorphisms; Type 1 diabetes; Genetic susceptibility
Summary MYO9B (myosin IXB) polymorphisms were associated with celiac disease and ulcerative colitis susceptibility, presumably through alteration of the intestinal permeability. Recently this gene was also associated with several diseases with an autoimmune component, such as rheumatoid arthritis and systemic lupus erythematosus. We aimed to test, for the first time, the potential role of MYO9B polymorphisms in type 1 diabetes (T1D), an autoimmune condition preceded by changes in intestinal barrier integrity. Three previously associated MYO9B polymorphisms (rs962917, rs2279003, and rs2305764) were studied in 316 T1D patients and 706 ethnically matched controls. Minor alleles of those polymorphisms were more frequent in diabetic patients than in controls and the haplotype carrying major alleles in those positions, rs962917*G/rs2279003*C/rs2305764*G, significantly reduced the risk of T1D in the Spanish population (p ⫽ 0.004; OR [95% confidence interval] ⫽ 0.68 [0.52– 0.90]). Our data suggest an involvement of this MYO9B chromosomal region in T1D predisposition, indicating extensive influence on autoimmune diseases. © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Introduction Type 1 diabetes (T1D) is an immune-mediated disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets, culminating in the destruction of insulin-producing  cells. The strongest T1D genetic determinant lies in the major histocompatibility complex class II * Corresponding author. Fax: 91 3303344. E-mail address: [email protected] (E. Urcelay).
region on 6p21 and more than 10 T1D susceptibility loci have been found [1,2]. Efforts to define the genetics of this polygenic disorder were hampered by the low penetrance of each individual locus. The T1D complex etiology reflects gene– gene and gene– environment interactions. Although the exact environmental trigger has not been described, the rapid rise in incidence of the disease worldwide [3], together with the concordance rate for monozygotic twins, estimated between 30 and 50% [4], supports the idea that environmental factors influence T1D risk. Infectious agents as well as
0198-8859/$ -see front matter © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.01.003
113
MYO9B association with T1D
ABBREVIATIONS CI MYO9B OR RR T1D
confidence interval myosin IXB gene odds ratio relative risk type 1 diabetes
dietary factors have been hypothesized to play a role in diabetes susceptibility, but the ascertainment of the specific antigen leading to the aberrant immune response remains elusive. Nonetheless, considering that the gastrointestinal tract is the largest interface with the environment, the contribution of an increased intestinal permeability to T1D pathogenesis has been the focus of attention. Loss of barrier integrity was observed in both diabetic patients [5– 8] and animal models [9 –11]. Moreover, zonulin, a protein that modulates intestinal permeability by acting on tight junctions, was recently determined to be involved in the pathogenesis of autoimmune diabetes [12]. High serum zonulin levels were correlated with increased intestinal permeability and upregulation of the expression of integral membrane tight junction protein genes such as claudin-1 and myosin IXB in diabetic patients. Polymorphisms in the myosin IXB (MYO9B) gene were recently associated with celiac disease and ulcerative colitis in certain populations [13–16], and a role of the gene in increasing susceptibility to rheumatoid arthritis and systemic lupus erythematosus was also reported [17], suggesting that an influence of this chromosomal locus might underlie different autoimmune diseases. MYO9B encodes a protein with a Rho-GTPase-activating domain [18] and Rho family GTPases participate in remodeling of the cytoskeleton and tight junction assembly; therefore, they are involved in the establishment of epithelial barriers [19]. It has been speculated that variants within the 3’ end of the MYO9B gene lead to a mutated Rho-GTPase-activating domain and a subsequent impaired barrier function. The MYO9B gene maps to human chromosome 19p13 and wholegenome scans in T1D patients revealed linkage to this locus [20,21]. Therefore, we aimed to test, for the first time, whether the MYO9B gene previously associated with predisposition to some autoimmune diseases is also involved on T1D risk.
Subjects and methods Patients We studied 316 White unrelated Spanish T1D patients (50% women) diagnosed according to the criteria of the American Diabetes Association [22] and 706 healthy controls recruited among blood donors from the Madrid area (51% women). All subjects were insulin-dependent at the time of the study. Informed consent was obtained from all subjects included in the study, which was approved by the Ethics Committee of the Hospital Clínico San Carlos.
Genotyping The MYO9B polymorphisms (rs2305767, rs962917, rs1457092, rs2279003, rs2305765, and rs2305764) were analyzed in a 7900HT
following the manufacturer’s suggestions by predeveloped TaqMan allelic discrimination assays (C__1654927_20, C__7493866_1_, C__1654895_10, C__11698814_10, C__1654877_1_, and C__ 1654873_1_, respectively) from Applied Biosystems (Foster City, CA, USA). The genotyping success rate is over 98%.
Statistical analysis Case– control analysis were performed by applying 2 statistics or Fisher’s exact test when necessary. The genotype association with T1D was estimated by the odds ratio (OR) with 95% confidence interval (CI). Statistical analysis was performed using Epi Info v. 6.02 software (Centers for Disease Control, Atlanta, GA, USA). Haplotypic frequencies were estimated using the expectation– maximization algorithm implemented in Arlequin v2.000 software [23], with the number of iterations set at 5,000 and initial conditions at 50, with an epsilon value of 10⫺7.
Results This study was performed by analyzing three tagging polymorphisms and the results obtained through comparison between diabetic patients and healthy controls are summarized in Table 1. No significant difference was observed in allele or genotype distributions of any tested variant between T1D and control cohorts, although minor alleles were consistently more frequent in cases than in healthy controls. Four major haplotypes were estimated by applying the expectation–maximization algorithm [23] and their overall comparison evidenced a significant difference between patients and controls (2 ⫽ 8.7; p ⫽ 0.033). The haplotype carrying major alleles of each polymorphism (rs962917*G/ rs2279003*C/rs2305764*G) significantly reduces the risk of T1D in the Spanish population (Table 2). The statistical significance of this haplotype withstands Bonferroni’s correction.
Table 1 Allele and genotype frequencies of the MYO9B polymorphisms.
rs962917 GG GA AA G A rs2279003 CC CT TT C T rs2305764 GG GA AA G A
T1D patients (%)
Controls (%)
(n ⫽ 316) 114 (36.1) 153 (48.4) 49 (15.5) 381 (60.3) 251 (39.7) (n ⫽ 316) 96 (30.4) 159 (50.3) 61 (19.3) 351 (55.5) 281 (44.5) (n ⫽ 316) 99 (31.3) 165 (52.2) 52 (16.5) 363 (57.4) 269 (42.6)
(n ⫽ 706) 280 (39.6) 319 (45.2) 107 (15.2) 879 (62.2) 533 (37.8) (n ⫽ 695) 221 (31.8) 366 (52.7) 108 (15.5) 808 (58.1) 582 (41.9) (n ⫽ 693) 249 (35.9) 326 (47.0) 118 (17.0) 824 (59.4) 562 (40.6)
114 Table 2
J.L. Santiago et al. Frequency of haplotypes conformed by MYO9B polymorphisms in T1D patients and controls. T1D patients (2n ⫽ 632)
Controls (2n ⫽ 1362)
rs962917
rs2279003
rs2305764
Frequency
n
Frequency
n
OR (95% CI)
p
G A G G
T C C C
G A G A
0.440 0.397 0.134 0.024
278 251 85 15
0.407 0.370 0.186 0.021
554 504 253 29
1.15 1.12 0.68 1.12
0.16 0.25 0.004 0.73
Discussion The association of the MYO9B polymorphisms was originally described with celiac disease in the Dutch population [13]. Three pairs of MYO9B polymorphisms (rs962917 and rs1457092, rs2279003 and rs2305767, rs2305764 and rs2305765; complete linkage disequilibrium within each pair) were associated with two celiac Dutch cohorts and were used to tag haplotypes. Each pair of polymorphisms was also in complete linkage disequilibrium in our Spanish control cohort and every polymorphism conformed to Hardy– Weinberg equilibrium [24]. Their association with ulcerative colitis in several independent cohorts [14 –16] and also with other autoimmune diseases [17] was recently reported. Unexpectedly, the haplotype that increased susceptibility to those conditions (the second by frequency) is not the one associated with T1D (Table 2). When diabetic patients and healthy controls were compared, no difference was observed between the first and the second more frequent haplotypes (OR [95% CI] ⫽ 0.99 [0.80 –1.23]; p ⫽ 0.94). These data would indicate either that the statistical power to detect the previously described effect is compromised in our diabetic cohort or that different causative polymorphisms in linkage disequilibrium with the two associated haplotypes are responsible for the detected effects in distinct diseases. The existence of more than one haplotype within a chromosomal region modifying susceptibility is not a novel situation; the three polymorphisms within the nucleotide-binding oligomerization domain containing 2 gene, NOD/CARD15, the main genetic susceptibility factor for Crohn’s disease, are not arranged in a unique haplotype and the greatest relative risk was seen in patients possessing a variant allele at more than one of these loci (simple heterozygote relative risk [RR] ⫽ 2.4; homozygote RR ⫽ 9.8; compound heterozygote RR ⫽ 29.3 [25]). Moreover, this is not the first example of opposing susceptibility effects for a specific locus in different diseases, i.e., the DRB1*1501-DQB1*0602 haplotype, the strongest genetic susceptibility determinant to multiple sclerosis, confers a protective effect for T1D [26]. Several studies pointed to different putative etiologic variants in the MYO9B gene, although firm experimental support is lacking. The variant originally suggested by Monsuur and collaborators [13], rs2305764, located in intron 28 of MYO9B and supposedly able to explain the association with celiac disease by itself, was not replicated in many of the independent populations studied. The nonsynonymous change in exon 20, Ala1011Ser (rs1545620), was proposed by Van Bodegraven and collaborators [15] as the etiological polymorphism in inflammatory bowel diseases. They indi-
(0.94–1.39) (0.92–1.37) (0.52–0.90) (0.57–2.18)
cated it might affect the processive motor activity of the protein. However, the authors observed a genetic association of similar magnitude, as measured by OR values, for the variant rs1457092. Therefore, from a genetic standpoint it is difficult to distinguish between the putative causal polymorphism rs1545620 and both rs1457092 and rs962917, provided that all of them are found in very strong linkage disequilibrium in Caucasian populations, as detailed in http://www. hapmap.org. Certainly, although the polymorphism leading to an amino acidic change is the a priori best candidate, replication in populations with different ethnicity is warranted to confirm this hypothesis. Intronic polymorphisms affected alternative splicing and, as recently reported, abnormal kinetics of mRNA translation may arise from a silent single nucleotide polymorphism, thus yielding a protein with different function [27]. Therefore, no single nucleotide polymorphism can be disregarded as etiological and the identification of the real causative variant must be supported by information derived from genetic analyses in independent populations. The entheropathy associated with alterations of this chromosomal region might allow unregulated passage of environmental antigens that could potentially initiate the autoimmune response leading to disease in genetically susceptible individuals. In fact, increased intestinal permeability precedes the clinical onset of T1D and is altered during the whole course of the disease, as recently demonstrated by Bosi et al. [28]. Moreover, downstream of the MYO9B gene, a conserved region is found with approximately 100 residues in common with the eukaryotic occludin proteins, OCEL1. The barrier protein occludin is decreased in experimental diabetes and vascular permeability is associated with reduced endothelial occludin content [29]. Diabetes increases blood– brain barrier permeability via a loss of tight junction proteins [9] and the cerebral occludin content in diabetic rats is significantly reduced compared with insulin-treated diabetic rats or control rats [30]. Therefore, MYO9B might not be the only susceptibility gene within the locus. Whatever the case, our data suggest the association of the MYO9B chromosomal region with pathogenic implications in T1D and confirmation of this effect in independent populations should be sought.
Acknowledgments We thank Carmen Martínez for expert technical assistance. Alfonso Martínez and Jose Luis Santiago are recipients of FIS
MYO9B association with T1D contracts (CP04/00175 and CM05/00216, respectively). Elena Urcelay works for the “Fundación para la Investigación Biomédica–Hospital Clínico San Carlos.” This work was supported by grants from Fundación Areces and Fundación Mutua Madrileña.
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