Is There an Association Between GNβ3–C825T Genotype and Lower Functional Gastrointestinal Disorders?

GASTROENTEROLOGY 2006;130:1985–1994

Is There an Association Between GN␤3–C825T Genotype and Lower Functional Gastrointestinal Disorders? VIOLA ANDRESEN,* MICHAEL CAMILLERI,*,‡ H. JAE KIM,* DEBRA A. STEPHENS,* PAULA J. CARLSON,* NICHOLAS J. TALLEY,* YURI A. SAITO,* RAUL URRUTIA,‡ and ALAN R. ZINSMEISTER§ *Clinical Enteric Neuroscience Translational and Epidemiological Research (C.E.N.T.E.R.) Program,‡Gastroenterology Research Unit, and the § Department of Health Sciences Research, Division of Biostatistics, Mayo Clinic College of Medicine, Rochester, Minnesota

Background & Aims: GN␤3 influences G-protein translation of a majority of ligand-receptor activations. It has been reported that functional dyspepsia (FD) is associated with homozygous genotypes of the C825T polymorphism in the GN␤3 gene. It is unknown whether the GN␤3 genotype is associated with lower functional gastrointestinal disorders (FGID). We aimed to compare the prevalence of the different GN␤3–C825T genotypes in patients with lower FGID and healthy controls and to test the associations of these genetic variations with subgroups of irritable bowel syndrome (IBS), functional abdominal pain (FAP), lower FGID–FD overlap, and high somatic symptom scores. Methods: GN␤3–C825T polymorphism was analyzed in DNA from blood samples of 233 patients with lower FGID and 152 healthy controls. A validated bowel questionnaire characterized the FGID phenotype: 82 with IBS constipation, 94 with IBS diarrhea, 38 with IBS alternating bowel function, and 19 with FAP. There were 159 patients with lower FGID and overlap FD using Rome II criteria. Regression analyses assessed associations of the GN␤3 genotypes with lower FGID as a group, and subgroups of FGID and somatic symptom scores. Results: GN␤3–C825T genotype distributions were similar between healthy controls (50.7% CC, 40.8% TC) and patients with lower FGID (8.6% TT, 51.5% CC, 40.8% TC, and 7.7% TT). There were no significant associations of GN␤3–C825T polymorphism with lower FGID overall or with the separate symptom subgroups including IBS, FAP, lower FGID–FD overlap, or high somatic symptom scores. Conclusions: In contrast to the reported association with FD, GN␤3– C825T polymorphism is not associated significantly with lower FGID, with different IBS or FAP phenotypes, or lower FGID–FD overlap.

n most communities in Western countries, there is a high prevalence of lower functional gastrointestinal disorders (FGID) such as irritable bowel syndrome (IBS) and functional abdominal pain (FAP).1 These disorders are associated with several pathophysiologic abnormalities including abnormal gastrointestinal motor function,

I

visceral hypersensitivity, psychosocial changes (eg, depression, anxiety, or abuse), autonomic dysfunction, and inflammation.2 Approximately 80% of all known membrane receptors that are linked to intracellular effector systems are coupled to heterotrimeric G proteins. G proteins are expressed in all cells of the human body and play a pivotal role in the signal translation from the cell surface (eg, ligand-receptor interaction) to subsequent cellular responses. Thus, qualitative or quantitative changes in G proteins could result in disease by blocking or enhancing intracellular signal transduction. G proteins are composed of 3 different ␣, ␤, and ␥ subunit isoforms; the ␤␥ subunits form a functional monomer. On receptor activation, the ␣ and ␤␥ subunits dissociate from their respective receptor and, in turn, modulate a large variety of intracellular effector systems. The ␤ subunit protein G␤3 belongs to a superfamily of propeller proteins that are composed of 7 tryptophan– aspartate repeat domains (each consisting of repeats of tryptophan–aspartate residues). These domains are involved in the interaction between the G-protein ␤ and ␥ subunits and in the effect of the ␤␥ dimer.3 The G␤3 protein is encoded by the GN␤3 gene. In the GN␤3 gene, there is a common C825T single nucleotide polymorphism (SNP) with an exchange from cytosine to thymidine, which gives rise to 3 possible genotypes (ie, CC, TC, and TT). The C825T polymorphism is located Abbreviations used in this paper: DI, dyspepsia using definition I (Rome II criteria); DII, dyspepsia using definition II (meal-related symptoms); FD, functional dyspepsia; FAP, functional abdominal pain; FGID, functional gastrointestinal disorders; HWE, Hardy–Weinberg equilibrium principle; IBS, irritable bowel syndrome; IBS-A, IBS with alternating bowel habits; IBS-C, irritable bowel syndrome with predominant constipation; IBS-D, irritable bowel syndrome with predominant diarrhea; OR, odds ratio; SNP, single nucleotide polymorphism; SoSC, somatic symptom scores. © 2006 by the American Gastroenterological Association Institute 0016-5085/06/$32.00 doi:10.1053/j.gastro.2006.03.017

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on exon 10, and the 825T allele (TC or TT genotype) is associated with alternative splicing of the gene in exon 9 and the formation of a truncated splice variant G␤3s, a protein product with 41 amino acids less than in the G␤3 wild-type protein.4,5 This corresponds to the lack of the equivalent of one entire tryptophan–aspartate repeat domain.6 The G␤3s protein variant is associated with enhanced G-protein activation7 and the 825T allele corresponding to the formation of G␤3s has been associated with cardiovascular, affective, and metabolic disorders.6,8 –10 The homozygous C allele (CC genotype) results predominantly in the synthesis of the G␤3 wild-type protein and only minute amounts of the ␤3 splice variant. The homozygous CC genotype is associated with diminished G-protein activity, decreased signal transduction responses,4 and was shown to be associated with unexplained or community dyspepsia.11,12 Interestingly, a subgroup of 20 patients with dyspepsia, who also had IBS symptoms, also showed a trend to a significant association with the CC genotype.11 Holtmann et al13 proposed that genetic factors interact with environmental factors in the etiology of FGID. In this model, it is biologically plausible for either the 825C or T allele to be associated with the development of lower FGID, which may correspond to different physiologic effects of either enhanced or decreased G-protein activity (eg, resulting in either rapid or slow gastrointestinal transit). Thus, the aims of this study were to compare the prevalence of the different genotypes of the GN␤3 C825T polymorphism in patients with lower FGID and healthy controls. Furthermore, because genetic associations have been shown for IBS subtypes,14 and because the GN␤3–C825T genotype has been associated with functional dyspepsia (FD)11,12 and affective disorders,15 we also aimed to test associations of these genetic variations with subgroups of lower FGID, lower FGID–FD overlap, and high somatic symptom scores (SoSCs).

Participants and Methods Participants IBS participants (ages, 18 –75 y) were selected from an administrative database of 752 patients with IBS who had been evaluated previously at the Mayo Clinic and were residing within a 150-mile radius of Rochester, Minnesota. All were invited to participate by mailed invitation. The 233 patients who agreed to participate completed a questionnaire and agreed to provide a peripheral blood sample for DNA analysis. All IBS patients had been evaluated previously by a staff gastroenterologist and undergone clinically indicated tests including a colonoscopy with mucosal biopsy examinations as indicated and clinical tests for rectal evacuation disorders. All

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IBS patients had to fulfill Rome II criteria16 by their questionnaire responses. A total of 152 healthy volunteers (ages, 18 –75 y) were recruited by public advertisement in Rochester, Minnesota. All participants signed informed consent for the study, which was approved by the Mayo Foundation Institutional Review Board. The majority of participants in the study came from a cohort evaluated for a genetic association between candidate adrenergic and serotonergic genotypes and lower FGID.14

Questionnaires All participants completed a validated bowel disease questionnaire17 including questions that corresponded to Rome II criteria16 and a somatic symptom checklist. The latter questionnaire has been used in the literature to identify people with a propensity to report somatic symptoms.18 The symptoms surveyed were as follows: headache, backache, wheezing, trouble breathing, difficulty sleeping, fatigue (tiredness), depression (feeling sad or blue), general stiffness, palpitations, joint pains, eye pain associated with reading, dizziness, weakness, nervousness (or shakiness), hot or cold spells, and high blood pressure. Data on the SoSCs were available for more than 90% of all participants. The SoSC was summarized as a mean of the frequency and severity scores over the 16 items each participant recorded on a scale of 0 – 4. Participants were classified as having a high somatic score when their mean score across the 16 domains was higher than .75, which was the 90th percentile of mean scores for the healthy participants in this study. We and others have used these 2 questionnaires extensively in epidemiologic studies (eg, in people with FGID).18,19 All patients were symptomatic with their functional bowel disorder at the time of the questionnaire assessment and were categorized in disease phenotypes using the validated bowel disease questionnaire17 in accordance with Rome II criteria. There is evidence of a significant likelihood of category transitions between constipation-predominant IBS (IBS-C) and functional constipation, and similar transitions in the categories of diarrhea-predominant IBS (IBS-D) and functional diarrhea.20 Hence, patients whose symptoms at the time of the questionnaire suggested IBS-C and functional constipation were grouped into 1 group (designated IBS-C), or those with IBS-D and functional diarrhea into a second group (designated IBS-D). Other independent categories were IBS with alternating bowel habits (IBS-A) and FAP. The bowel disease questionnaire also was used to determine the co-existence of dyspepsia, using 2 different definitions for dyspepsia. Definition 1 (DI) was based on the Rome II definition of dyspepsia and used the following criteria: frequent upper-abdominal pain or frequent early satiety within the past year. Definition 2 (DII) also included frequent early satiety but was stricter in requiring that the upper-abdominal pain be associated either with frequent nausea (at least once a week) or occurred after meals. The latter definition was added given the observation that approximately 60% of dyspeptic people in a community study experienced meal-related symptoms.18

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Table 1. Patient Characteristics and Mean SoSCs in 233 Patients With Lower FGID and 152 Healthy Controls

N Age, mean (range) Sex, n (% female) SoSC,a mean (⫾SD) [N] Caucasians, n (%) Hispanics, n (%) African Americans, n (%) Asians, n (%) Other, n (%) aThe

Controls

All lower FGID

IBS-C

IBS-D

IBS-A

FAP

152 42 (18–81) 112 (74) .35 (.36) [134] 139 (92) 4 (3) 1 (.7) 7 (5) 1 (.7)

233 50 (18–82) 197 (85) .9 (.59) [215] 228 (98) 1 (.4) 0 (0) 2 (.9) 2 (.9)

82 52 (23–82) 76 (93) .90 (.57) [79] 79 (96) 1 (1.2) 0 (0) 1 (1.2) 1 (1.2)

94 49 (19–77) 77 (82) .89 (.58) [80] 93 (99) 0 (0) 0 (0) 1 (1.1) 0 (0)

38 47 (18–69) 30 (79) .92 (.65) [38] 37 (97) 0 (0) 0 (0) 0 (0) 1 (2.6)

19 47 (26–78) 14 (74) .88 (.62) [18] 19 (100) 0 (0) 0 (0) 0 (0) 0 (0)

SoSC was not available for all participants.

GN␤3 Genotyping Venous blood drawn from a forearm vein was stored as de-identified samples. Genomic DNA was isolated from whole blood in 385 participants by the alkaline lysis method using the QIAamp DNA Blood Maxi Kit (Qiagen Inc., Valencia, CA). GN␤3 genotypes were determined by methods described in the literature.6 Real-time polymerase chain reaction using TaqMan chemistries (Applied Biosystems, Foster City, CA) was used to determine alleles present in each sample. Real-time polymerase chain reactions were performed in an Applied Biosystems 7500 machine (Applied Biosystems). TaqMan primer-probe assays for GN␤3 SNPs C825T (rs6489738) were purchased from Applied Biosystems. Each reaction volume was 25 ␮L and consisted of 13.75 ␮L of a master mix containing 12.5 ␮L 2⫻ TaqMan Universal reaction mix (Applied Biosystems), 1.25 ␮L of a 20⫻ primer-probe assay mix (Applied Biosystems), and 11.25 ␮L (20 ng) genomic DNA. Amplification conditions consisted of 10 minutes at 95°C followed by 40 cycles at 92°C for 15 seconds and 60°C for 60 seconds. Fluorescence intensity was measured before and after amplification and then analyzed using automated software (SDS 2.1; Applied Biosystems) to determine the genotype of each sample. Genotypes were confirmed or assessed selectively by direct sequencing as previously described.12

genotype frequencies for the controls participating in the study from that expected based on the Hardy–Weinberg equilibrium principle (HWE).21 The univariate association between the GN␤3 genotypes and symptom groups (overall FGID group and, separately, different symptom phenotype subgroups including the lower FGID–FD overlap group vs the control group) were assessed using a ␹2 test or Fisher exact test as appropriate. Logistic regression models were used to estimate the odds ratios for specific phenotypes of IBS in participants with different GN␤3 genotypes relative to the wild type. Specifically, the odds ratios (and the 95% confidence intervals) for a specific phenotype were computed from the estimated logistic regression model coefficients (and their standard errors) examining the CC genotype relative to the combined TC and TT genotypes. Race and sex were included as covariates in each of the logistic regression models. The association of the somatic symptom checklist score with the genotype subgroup was assessed using a linear regression model with genotype (coded as dummy regression variables with TC subtype as the reference level) as the primary predictor variable, adjusting for age and sex. The statistical software used for all analyses was SAS (SAS Institute Inc., Cary, NC). After the study was completed, we estimated the effect size detectable with 80% power to identify significant associations given the number of participants in different subgroups included in this study.

Data and Statistical Analysis Statistical analyses assessed the associations of the 3 different genotypes (CC, TC, and TT) and of CC vs combined TC and TT with lower FGID. The latter analysis was of specific interest because the CC genotype relative to non-CC (or combined TC and TT) has been associated with an increased risk for unexplained dyspepsia and dyspepsia in a community-based sample.11,12 A ␹2 test was used to assess the deviation of the observed

Results Lower FGID Symptoms, Lower FGID–FD Overlap, and High SoSCs The symptom phenotypes of patients with lower FGID, for whom DNA was available for analysis, were as follows: 82 IBS-C, 94 IBS-D, 38 IBS-A, and 19 FAP.

Table 2. Distribution of the GN␤3–C825T Genotypes in 233 Patients With Lower FGID and 152 Healthy Controls Across Overall Groups and Subtypes GN␤3–C825T genotype CC, % TC, % TT, %

Controls (N ⫽ 152)

All lower FGID (N ⫽ 233)

IBS-C (N ⫽ 82)

IBS-D (N ⫽ 94)

IBS-A (N ⫽ 38)

FAP (N ⫽ 19)

50.7 40.8 8.6

51.5 40.8 7.7

51.2 39 9.8

57.5 35.1 7.4

39.5 57.9 2.6

47.4 42.1 10.5

NOTE. Values are column percentages. ␹2 analysis for controls and FGID subgroups: P ⫽ .51.

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Table 3. The Distribution of Controls and Different IBS Symptom Phenotypes in Categories of the GN␤3–C825T Genotype IBS phenotype in each genotype

CC (N ⫽ 197)

TC (N ⫽ 157)

TT (N ⫽ 31)

39.1 21.3 27.4 7.6 4.6 .65 (.52)

39.5 20.4 21.0 14.0 5.1 .72 (.62)

41.9 25.8 22.6 3.2 6.5 .76 (.63)

Controls IBS-C IBS-D IBS-A FAP SoSC (mean ⫾ SD) NOTE. Values are column percentages, except as noted.

groups are shown in Table 2. The proportions of GN␤3 C825T genotypes in patients with lower FGID or subgroups were not significantly different from controls. When the analysis used 6 FGID phenotypes by separating IBS-C from functional constipation and IBS-D from functional diarrhea, there were no significant associations with the GN␤3–C825T genotype (data not shown).

There were 159 patients with lower FGID–FD overlap according to DI and 143 patients with lower FGID–FD overlap according to DII. With both FD definitions, there were patients in all 4 lower FGID phenotypes who had evidence of overlap with FD. Thus, almost all patients with IBS-A and FAP and about 50 patients in each of the IBS-D and IBS-C groups had FD. In 14 patients with lower FGID, the FD status could not be assessed because of incomplete questionnaire data. The demographics of patients in the different lower FGID subgroups are shown in Table 1. Female participants predominated in the patient (85%) and control (74%) groups; 92% of controls and 98% of patients were Caucasians; there were 5% Asian and 3% Hispanic healthy participants. Other racial groups constituted less than 1% in the 2 groups. FGID subgroups were associated significantly with high SoSCs; patients in the different symptom subgroups had higher scores than the healthy controls (P ⬍ .01).

Relation of GN␤3 Genotypes With Lower FGID Subgroups and SoSCs Table 3 summarizes the distribution of controls and of lower FGID subgroups within the 3 GN␤3 genotypes (CC, TC, and TT). No association between GN␤3 genotype and phenotype (overall lower FGID vs controls, or phenotypic subgroups vs controls) was detected (P ⬎ .05, Table 3). Tables 4 and 5 summarize the distribution of the 3 different GN␤3 genotypes in controls and patients with lower FGID–FD overlap, presented separately for the overall lower FGID group (Table 4), or for the different lower FGID subgroups (Table 5). The proportions of GN␤3 C825T genotypes were not significantly different in controls and in patients with lower FGID without or with FD overlap (Table 4, Fisher exact test: P ⫽ .18 for DI and P ⫽ .29 for DII). Similarly, the GN␤3–C825T genotype was not associated significantly with any lower FGID subgroup with or without overlapping FD when compared with controls (Table 5, data are presented only for DI).

Distribution of Genotypes in Healthy Controls The observed genotype frequencies of the GN␤3 polymorphism in the control participants are shown in Table 2. The genotype frequencies in the control group were in accordance with the HWE. Distribution of Genotypes in Patients With Lower FGID The observed genotype frequencies of the GN␤3 polymorphism in patients with lower FGID and sub-

Table 4. Distribution of the GN␤3–C825T Genotypes in Patients With Overall Lower FGID With (⫹D) and Without (⫺D) Overlap FD by Two Different Dyspepsia Definitions

GN␤3–C825T genotype CC, % TC, % TT, %

Using DI Controls (N ⫽ 152)

Any FGID ⫺DI (N ⫽ 60)

⫹DI (N ⫽ 159)

50.7 40.8 8.6

58.3 30.0 11.7

49.7 45.3 5.0 P ⫽ .18a

Using DII Controls (N ⫽ 152)

Any FGID ⫺DII (N ⫽ 76)

⫹DII (N ⫽ 143)

50.7 40.8 8.6

56.6 32.9 10.5

49.6 45.4 4.9 P ⫽ .29a

NOTE. A total of 14 IBS-D participants could not be classified as to dyspepsia status. DI, Rome II; DII, early satiety or frequent abdominal pain in relation to meal or frequent abdominal pain with frequent nausea. aTest for association of genotype with phenotype (including controls) and dyspepsia status using the Fisher exact test; P values are unadjusted for multiple comparisons.

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Table 5. Distribution of the GN␤3–C825T Genotypes in Patients With Lower FGID Subgroups With (⫹D) and Without (⫺D) Overlap FD by DI FGID IBS-C GN␤3–C825T genotype CC, % TC, % TT, %

FGID IBS-D

FGID IBS-A

FGID FAP

Controls (N ⫽ 152)

⫺DI (N ⫽ 32)

⫹DI (N ⫽ 50)

⫺DI (N ⫽ 28)

⫹DI (N ⫽ 52)

⫺DI (N ⫽ 0)

⫹DI (N ⫽ 38)

⫺DI (N ⫽ 0)

⫹DI (N ⫽ 19)

50.7 40.8 8.5

62.5 21.9 15.6

44.0 50.0 6.0 P ⫽ .11a

53.6 39.3 7.1

63.5 32.7 3.8 P ⫽ .59a

0 0 0

39.5 57.9 2.6 P ⫽ .16a

0 0 0

47.4 42.1 10.5 P ⫽ .94a

NOTE. A total of 14 IBS-D participants could not be classified as to dyspepsia status. DI, Rome II. aTest for association of genotype with phenotype (including controls) and dyspepsia status using the Fisher exact test; P values are unadjusted for multiple comparisons.

Figure 1 shows the odds ratios of the GN␤3–C825T CC genotype relative to the combined TC and TT genotypes for the different lower FGID subgroups. There were no significant associations detected, implying that the GN␤3 genotypes were unable to predict the symptom phenotypes. Although symptom subgroup (essentially overall lower FGID) was a significant predictor of high SoSCs, there was no significant association between the GN␤3 C825T polymorphism and the SoSC (Table 3). Statistical Power to Detect Associations Given that a statistically significant association was not observed, we addressed the study’s power to detect such an association. Although ideally a random sample from the general population would provide the best theoretic basis for such an assessment, data from the current study was used to mimic obtaining samples from

the general population with these genotypes by dividing all participants in this study (patients and controls) into 2 groups: 1 group with the non-CC (ie, combined TT and TC) genotype, and 1 group with the CC genotype. Table 6 summarizes the degree of association (difference in corresponding proportions of participants with specific symptom complexes) between the GN␤3– 825C genotype (TT/TC vs CC) and the presence/absence of specific symptom subgroups (including overall lower FGID) that could have been detected with approximately 80% power. The first column lists the observed proportions of study participants with a specific phenotype status among all study participants (ie, patients with lower FGID and healthy controls) with the TT or TC genotype. By using these observed proportions of symptomatic participants in the non-CC genotype group, the size difference in the proportions of participants with the CC genotype and a specific phenotype that could have been detected with 80% or greater power was calculated based on a 2-sample test for proportions using a 2-sided ␣ value of .05. The data indicate that the study could have detected (with at least 80% power) a difference in the prevalence of the specific symptom phenotype in the CC genotype vs the TC/TT genotype corresponding to 13%– 14% for all lower FGID, and 10%–13% for IBS-C and IBS-D. Thus, the study sample size had sufficient power to detect differences in CC vs TC/TT genotypes that were observed in functional11 or uninvestigated12 dyspepsia, and the study had sufficient power to detect a clinically meaningful association of lower FGID or IBS-C or IBS-D with the CC genotype.

Discussion

Figure 1. Odds ratios with 95% confidence intervals for IBS-D, IBS-C, IBS-A, or FAP of the GN␤3–C825T CC genotype relative to the combined TC and TT genotypes.

This study evaluated the association of the GN␤3 C825T gene polymorphism with lower FGID. This polymorphism results in differences in G-protein activation and potentially predisposes to several diseases.22 The distributions of the GN␤3 genotypes in 233 patients

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Table 6. Statistical Power of this Study to Detect Phenotype Differences Based on Genotype

Phenotype Healthy Any FGID IBS-C IBS-D

Observed % participants with specific phenotypes among all participants with TC or TT genotype (N ⫽ 188)

Significantly different proportions (%) of participants with specific phenotypes in the CC genotype vs the TC/TT genotype group that could have been detected with ⱖ80%

39% 61% 21% 21%

⬎53% ⬎74% ⬎34% ⬎34%

or or or or

⬍26% ⬍47% ⬍11% ⬍11%

Actual observed % participants with specific phenotypes among all participants with the CC genotype (N ⫽ 197) 40% 60% 21% 27%

NOTE. This analysis includes all participants (n ⫽ 385, of which 188 had TC/TT genotypes and 197 had the CC genotype) in this study sample.

with lower FGID, 159 patients with lower FGID–FD overlap, and 152 controls were not significantly different. These results contrast with recent studies that have identified a significant association of the homozygous GN␤3 CC11,12 or TT12 polymorphism with FD. FGIDs are multifactorial disorders that generally are considered to develop by the interaction of genetic predisposition and environmental factors.13 Studies showing familial aggregation of these disorders provide further evidence for an involvement of genetic factors.19,23,24 Several candidate genetic markers have been evaluated in genotype–phenotype association studies. The markers were chosen based on their potential role in the mechanism leading to the disorder, such as inflammation, or in the pathophysiologic manifestations of the disorder including effects of neurohormonal substances. To date, the studies evaluating the associations of gene polymorphisms with IBS, including SNPs in the genes controlling the anti-inflammatory cytokine interleukin-1025,26 or the promoter of the gene controlling synthesis of the serotonin transporter protein,14,27,28 have provided inconclusive or conflicting results. One report documents the association of ␣2A-1291 (C¡G) and ␣2C Del 322– 325 adrenoceptor polymorphisms with IBS-C.14 Moreover, the ␣2C polymorphism was associated with a high SoSC.14 FGIDs are characterized by a variety of symptoms that often are transient and may change over time within the same patient. FGIDs also are associated functionally with very different pathophysiology (eg, diarrhea or constipation, accelerated or slow transit29). In such complex genetic disorders, the contribution of a SNP to the phenotype is likely to be relatively small, and the detected odds ratios for a variant genotype relative to the wild type rarely exceed 2.0. The potential for 1 SNP to be associated with the diverse manifestations or the overall syndrome might be based on the biological effects of the different GN␤3 genotypes, which may contribute to different or even diametrically opposite symptoms. Thus, although the C allele codes for a less-active G

protein and a decreased signal transduction (eg, slow transit or constipation), the T allele is associated with a highly active G protein and enhanced signal transduction22 that leads to increased function (eg, diarrhea). The literature confirms associations of the different GN␤3 genotypes and disease. For example, the T-allele has been associated with hypertension,6,30 depression,31 IgA nephritis,32 atherosclerosis,33 and insulin resistance.34 On the other hand, the homozygous CC genotype was increased significantly in patients with FD and with dysmotility-like symptoms.11 A recent study of community uninvestigated dyspepsia in Olmsted County, Minnesota, confirmed this observation and also detected an association of the homozygous TT genotype with meal-unrelated dyspepsia.12 In contrast to the reports on functional dyspepsia, the results of the present study do not indicate any association of the GN␤3–C825T polymorphism with lower FGID. Given the substantial overlap in symptomatology, the frequent co-existence of these disorders, and the common pathophysiologic models of altered gut motility and visceral hypersensitivity in the 2 disorders, this observation appears surprising. Indeed, 68% of participants with lower FGID had Rome II criteria for functional dyspepsia and 61% fulfilled more restrictive DII criteria that explored meal-aggravated dyspepsia. Lower FGID with FD overlap also was not associated significantly with the GN␤3–C825T polymorphism, whether as a single lower FGID group, or as the different lower FGID phenotypes. Possible explanations for the discrepancies with the earlier studies should be considered. First, it is conceivable that we failed to show a true association owing to a type 2 error. As indicated in Table 6, our study sample sizes allowed for detecting meaningful differences (⌬ prevalence 10%–14%) between genotype distributions in lower FGID, IBS-C, and IBS-D, with a power of greater than 80%. The sample of participants (patients and controls) in our study had a genotype distribution that is very similar to that of more than 10,000 Caucasians reported in the literature (Table 7). Hence, the

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Table 7. Compilation of the Distribution of GN␤3–C825T Genotypes in Controls in the Published Literature Country Caucasians with CC ⬎ TC Rosskopf et al40 Exton et al31 Dzida et al41 von Beckerath et al33 Wascher et al34 Krippl et al42 Meirhaeghe et al43 Siffert et al44 Rankinen et al45 Hengstenberg et al46 Sarrazin et al47 Zagradisnik et al48 Overall Caucasians with CC > TC Caucasians with TC ⬎ CC Thibaudin et al32 Martin et al49 Hanon et al50 Holtmann et al (study A, asymptomatic BD)11 Holtmann et al (study B, normal BD)11 Camilleri et al12 Overall Caucasians with TC > CC

Overall n

CC, %

TC, %

TT, %

Germany Germany Poland Germany Austria Austria France Germany United States Germany Germany Slovenia

1855 190 172 340 932 500 842 277 473 2052 1781 220 9634

48.8 48.8 54 51.5 46.8 50.1 48.2 46 52.2 46.2 48.6 51.8 48.3

42.9 41.6 37 40.6 44.9 42.4 42.9 44 40.8 43.5 42.7 40 42.8

8.7 10 9 7.9 8.3 7.5 8.9 10 7 10.3 8.8 8.2 8.9

France Spain France Germany Germany United States

303 78 306 161 112 39 999

45.2 37.2 37 41.6 41.1 43 40.9

47.2 41.1 49 47.8 55.5 54 49

7.6 16.7 14 10.6 3.6 3 10.1

10,633

47.6

43.4

9.0

152 139

50.7 53.2

40.8 40.3

8.6 6.5

Overall Caucasians Controls current study Controls current study (Caucasians only) Africans/African Americans Siffert et al44 Rankinen et al45 Rosskopf et al40 Asians Siffert et al44 Rosskopf et al40 Aboriginal Canadians Pollex et al51 Arabs Mahmood et al52

Zimbabwe South Africa United States Zimbabwe

713

3

29

68

248 99

5.2 3

38.3 29

56.5 68

China China

960 222

27 33.3

51 48.6

22 18.1

Canada

515

19.4

58.4

22.2

United Arab Emirates

211

20.4

55.9

23.7

NOTE. Data are classified by different ethnicities. Note that among Caucasians, 6 of 18 studies showed a greater proportion of TC vs CC (TC ⬎ CC). BD, blood donor.

power calculation approximates what would be expected from a random sample of the same (predominantly) Caucasian cohort. Therefore, the possibility of having missed a true association between GN␤3 and lower FGID in our study appears unlikely. A second consideration is that previous identifications of a significant association between GN␤3 and FD may not be replicable owing to a type 1 error in the prior studies. It is worth noting that the 2 dyspepsia studies had relatively small sample sizes of patients compared with the current study: 4712 and 12311 compared with 233 participants with lower FGID and 159 with FD overlap in the current study. Associations are based on identifying a significant odds ratio for a genotype to be associated with a

phenotype. This calculation is critically dependent on the genotype distributions in controls. Thus, another consideration is that the genotype distributions in controls of both dyspepsia studies11,12 were relatively atypical, compared with the distribution of the GN␤3 genotype in the Caucasian population, as recorded in more than 10,000 healthy controls in the literature (Table 7). Table 7 shows that the controls of both dyspepsia studies had lower frequencies of the CC genotype, which is typically the highest proportion in the Caucasian population. The coincidence of unusually low frequencies of CC in the control groups and the relatively higher frequencies of CC in the dyspepsia groups may have led to the significant odds ratio for genotype CC with dyspepsia.11,12 The association

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may not be replicated if the control group had a distribution of genotypes closer to that of the mean distribution of the Caucasian population. Analysis of the HWE in controls is used to assess whether controls deviate from the expected distribution.35 The HWE21 states that the genotype frequencies at a single gene locus will become fixed at a certain equilibrium value. For the 2 alleles C and T, with the frequencies p (major allele C) and q (minor allele T), the expected genotype frequencies would be p2 for CC, 2pq for TC, and q2 for TT. By using the absolute numbers of alleles and calculated allele frequencies, each population sample can be tested for deviation from the HWE. The controls of the current study are in accordance with the HWE and have the same GN␤3 allele frequencies as the pooled Caucasian controls (Table 7). A few reports in Caucasian people and several studies in other ethnic groups show that the distributions are quite different from the mean genotype distributions in Caucasians (Table 7). When evaluating associations of any disorder with the GN␤3–C825T polymorphism, the great variation of the GN␤3 genotype distributions across different ethnicities should be taken into account (Table 7). Although in Caucasians the most frequent genotype is CC (allele frequencies: C ⫽ 70%, T ⫽ 30%), the most frequent genotypes in Africans is TT (allele frequencies: C ⫽ 20%–30%, T ⫽ 70%– 80%) and in Asians is TC (allele frequencies: C ⫽ 50%, T ⫽ 50%). Therefore, the wild type of the GN␤3 polymorphism differs between ethnic groups. It has been proposed that the increased susceptibility of Africans and African Americans to the development of hypertension might be associated partly with the high frequency of the T allele.22 Accordingly, if FGID was associated with GN␤3 TT, one would expect an increased prevalence of FGID in Africans and African Americans or, conversely, in the case of an association of FGID with GN␤3 CC, an increased prevalence of FGID in Caucasians. However, epidemiology studies do not provide evidence for differences in prevalence across ethnic groups.1,36,37 On the other hand, the results of this study should not be extrapolated to other ethnic groups that were not represented by large patient numbers in this predominantly Caucasian study. Apart from the potential predisposition to disease, the GN␤3–C825T polymorphism has been associated with altered drug responses.38 Interestingly, there are preliminary data to support the hypothesis that the GN␤3 CC 825 genotype influenced the response to a variety of pharmacologic therapies used in chronic dyspepsia in a referral setting.39 Our study does not address whether the GN␤3 status influences the course and the response to

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treatment of lower FGID. Further studies are needed to assess the pharmacogenomics of FGID in relation to variations in GN␤3. In summary, the results of the present study indicate that the GN␤3–C825T polymorphism is not associated with the development of lower FGID in general, with a subgroup of lower FGID, or lower FGID with overlapping FD. Our study highlights the importance of differences in the genotype distributions in controls between studies. This might account for the discrepancies in the conclusions of different studies. This study also shows the importance of reviewing the genotype distribution in large numbers of healthy people of the same ethnic distribution before reaching conclusions regarding such disease associations.

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Received October 14, 2005. Accepted March 9, 2006. Address requests for reprints to: Michael Camilleri, MD, Mayo Clinic, Charlton 8-110, 200 First Street SW, Rochester, Minnesota 55905. e-mail: [email protected]. Supported by grants RO1-DK54681 and K24-DK02638 (to M.C.), and K23-DK066271 (to Y.A.S.) from the National Institutes of Health, and by the Gerhardt Katsch grant from the German Society of Digestive and Metabolic Diseases (to V.A.). The authors thank Cindy Stanislav for excellent secretarial support.