Association between Polymorphism of the G-protein Β3 Subunit C825t and Essential Hypertension: an Updated Meta-analysis Involving 36,802 Subjects

Purpose: The G-protein β3-subunit gene C825T polymorphism (GNB3-C825T) has been reported to be associated with essential hypertension (EH), but results from previous studies are conflicting. The present study aimed at investigating the association between this polymorphism and risk of EH using a meta-analysis on the published studies. Materials and Methods: PubMed, Embase, CBM (China Biological Medicine Database), Wanfang and VIP databases were searched to identify eligible studies published in English and Chinese before March 2013. Data were extracted using standardized methods. The association was assessed by the odds ratio (OR) with 95% confidence intervals (CI). Begg's test was used to measure publication bias. Results: A total of 40 case-control studies containing 16,518 EH patients and 20,284 controls were involved in this meta-analysis. Overall, a significant association was found between GNB3 C825T polymorphism and risk of EH when all studies were pooled with a random-eff ects model for T versus C No associations were detected between GNB3-C825T and the risk of EH overall in Asian and Japanese people. Conclusions: Meta-analysis results suggest that the GNB3-C825T polymorphism is associated with risk of EH in the overall population, the Caucasians and the Chinese. The eff ect of the variants on the expression levels and the possible functional role of the variants in EH should be addressed in further studies.


INTRODUCTION
Essential hypertension (EH), a complex disease which accounts for ~95% of hypertensive cases, is an increasingly serious worldwide public-health challenge and is generally considered as a paradigmatic multi-factorial disease that is determined by a combination of genetic factors, environmental stimuli and their interaction (O'Shaughnessy, 2001) .It was estimated that 20-60% of the inter-individual variation in blood pressure (BP) is genetically controlled (Kurtz and Spence, 1993).Accordingly, the discovery of many potential hypertension-susceptibility genes has allowed for a better understanding of the disease etiology.Candidate polymorphisms of the genes involved in the pathways of Na + /H + exchange, the renin-angiotensinaldosterone system and the autonomic nervous system such as G-protein β3-subunit (GNB3)-C825T, angiotensin-converting enzyme insertion/deletion, and β2-adrenergic receptor A46G have been investigated in diff erent ethnic populations (Puddu et al., 2007).
in a certain group of patients with EH and revealed that enhanced G protein activation led to this common disorder (Siff ert et al., 1995).The GNB3 gene is one of the five genes coding for a G-protein β-subunit that have been identified in the human genome (Elefsinioti et al., 2004).One widelystudied polymorphism of the GNB3 gene is the C825T polymorphism, which consists of a substitution of C by T at position 825 in exon 10.Siff ert et al. (1998) first found that the GNB3-825T allele was associated with EH.Since then, many researchers have studied the relationship between the GNB3-C825T polymorphism and EH in diff erent populations with controversial results (Snapir et al., 2001;Huang et al., 2003;Potoczna et al., 2004).To estimate fully and comprehensively the association between this gene polymorphism and EH risk across diff erent ethnic populations we conducted an updated meta-analysis, including more studies in the Asian population than the meta-analysis of Bagos et al. carried out in 2007, to derive a more precise estimation of the association.

Literature search
To search for all the studies that examined the association of the GNB3-C825T polymorphism with essential hypertension risk we conducted a computerized literature search from PubMed, Embase, CBM (China Biological Medicine Database), Wanfang and VIP databases, using the following keywords and subject terms: (hypertension or blood pressure or essential hypertension) AND (GNB3-C825T or G-protein beta3) AND (allele or genotype or polymorphism or variant or variation).The full text of the retrieved articles was scrutinized to inspect whether data on the topic of interest were included.We systematically searched eligible studies reported before March 1, 2013.
Eligible publications had to be written in either Chinese or English.The references of all retrieved articles were also screened.To prevent data duplication, when a report overlapped with another study, only the more detailed study was included.If an article reported results on diff erent ethnic sub-populations, each sub-population was treated as a separate study.

Inclusion/exclusion criteria
Studies included in the meta-analysis had to meet all the following criteria: (1) the evaluation of the relationship between the GNB3-C825T polymorphism and essential hypertension; (2) the use of case-control study design or cross-sectional study design; (3) the available genotype/allele frequency of the GNB3-C825T allele between cases and controls; (4) all subjects were well ethnicity-matched; (5) hypertension was defi ned as systolic BP equal to or above 140 mmHg or diastolic BP equal to or above 90 mmHg or treatment with antihypertensive medication; however, studies evaluating secondary hypertension or other types of monogenic hypertension were excluded from the present investigation.If the genotype frequency was not reported, we contacted the original authors by e-mail to obtain the missing data.

Data extraction
To minimize the selection bias, the data were independently gathered in duplicate by two investigators on the basis of a standard protocol.The data extracted from the studies included such details as the first author, the year of publication, study population (region, ethnicity and sex), total number of cases and controls and genotype information.The excluded studies were comprised of studies of poor research quality, providing little or insuffi cient data, violating the inclusion criteria, and repeated publications.If the same research result appeared in diff erent articles, the result was only adopted once in the present meta-analysis.If there was discrepancy between them, it was settled by discussion until a consensus was reached.

Statistical Analysis
As case-control or cross-sectional studies were used, odds ratios (ORs) corresponding to a 95% confidence interval (CI) were implemented to assess the intensity of the association between the GNB3-C825T polymorphism and EH; the ORs were calculated according to the method described by Woolf (WOOLF 1955).We examined the contrast of the T vs. C, TT vs. CC, TC vs. CC, and also examined the dominant genetic model TT+TC vs. CC and the recessive model TT vs. TC+CC.
We applied two models of meta-analysis for dichotomous outcomes in Review-Manager 5.0 software (The Cochrane Collaboration, Oxford, UK): the fixed-eff ects model and the random-eff ects model.When there was inter-study heterogeneity a pooled OR was estimated by the random-effects model (DerSimonian and Laird's method) (DerSimonian and Laird, 1986); otherwise the fixed-effects model (Mantel-Haenszel method) was used (Mantel and Haenszel, 1959).The fixed-eff ects model assumes homogeneity among study estimates and is used when there is no evidence for heterogeneity.Conversely, when heterogeneity exists a random-eff ects model is usually more appropriate because it takes into account the inter-study variability.We used a χ 2 -based Q statistic to assess the interstudy heterogeneity (Lau et al.,1997), which is considered to be significant for P < 0.10 because of the low power of the statistic, as well as the I 2 statistic for estimation of inconsistency in metaanalysis (Trikalinos et al., 2006).Heterogeneity was assessed by the I 2 statistic, which was documented for the percentage of the observed inter-study variability due to heterogeneity rather than chance; it ranges from 0 to 100%, where a value of 0% indicates no observed heterogeneity, and larger values indicate an increasing degree of heterogeneity (roughly suggested cut-off points include: I 2 =0-25%, no heterogeneity; I 2 =25-50%, moderate heterogeneity; I 2 =50-75%, large heterogeneity; I 2 =75-100%, extreme heterogeneity (Higgins et al., 2003).The fixed-eff ects model (if P≥0.10) or the random-eff ects model (if P<0.10) was used to pool the results.The significance of the pooled ORs was determined by the Z-test, and P<0.05 was considered significantly diff erent.
Sensitivity analyses were conducted by deleting a single study each time involved in the meta-analysis to identify the potential influence of the individual dataset on the pooled ORs.
A funnel plot was used to estimate the potential publication bias.The asymmetry of the funnel plot was assessed by Egger's linear regression test (Song and Gilbody, 1998).

Studies included in the meta-analysis
A total of forty relevant studies concerning the association between GNB3-C825T and the risk of EH were identifi ed.These studies involved 16,518 patients and 20,284 controls, containing the studies of fourteen Caucasian populations, twenty-four Asian populations and two African-American populations.Because the sample population in the African-American group was inadequate, ethnicity-specific metaanalysis was conducted on Caucasian and Asian populations.In the subgroup analysis, eight studies in Japanese populations and fi fteen studies in Chinese populations were included in Asian-specifi c groups.Among those forty studies there were four that contained information about distinct independent populations and thus they were considered as different studies that should be counted twice (Brand et al., 1999;Kunugi et al., 2002;Ishikawa et al., 2000;Dai et al., 2002 .Out of all those studies, we found that the control groups were in Hardy-Weinberg equilibrium (HWE) except for four studies (Benjafi eld et al.,1998;Andersen et al., 2006;Yamamoto et al., 2004;Suwazono et al., 2004).The main characteristics of included studies are listed in Table 1.

Results of Meta-analysis
In the meta-analysis of all involved studies, the Q test showed heterogeneity in the 40 studies -we therefore used the random eff ects model to calculate the combined eff ects in the following genetic models.The OR (95% CI) values (Fig. 1 2).Heterogeneity among studies was identifi ed in all the comparisons (P for heterogeneity Q test <0.1).Furthermore, the I 2 statistics suggested the existence of moderate heterogeneity for all comparisons in all studies.Therefore, a sub-group analysis of the studies by ethnicity grouping was suggested.We found a signifi cant association when all Caucasian population studies were pooled with the fi xed-eff ects model for T versus C (OR=1.16, 95% CI 1.08-1.24;P=0.216 for heterogeneity, Fig. 2), TT+TC versus CC (OR=1.33,95% CI 1.13-1.56;P=0.324 for heterogeneity), TT versus TC + CC (OR=1.13,95% CI 1.02-1.20;P=0.152 for heterogeneity).The same association was found in the Chinese population when all studies were pooled with the random-eff ects model for TT vs. CC (OR=1.23,95% CI 1.06-1.57;P<0.001 for heterogeneity, Fig. 3).No associations were detected between GNB3-C825T and the risk of EH in Asians or the Japanese (Table 2).

Sensitivity Analysis
We removed the studies by Benjafield AV et al. (in the Caucasian population) and Yamamoto M et al. (in the Asian population) in the meta-analysis, since the genotype distribution in the control groups of the study deviated slightly from HWE.We found that the corresponding pooled ORs were not substantially altered (Table 3), which indicated the reliability of our results.

Publication Bias
Begg's test and a funnel plot were performed to access the publication bias of the studies.In this study there was no signifi cant publication bias for GNB3-C825T polymorphism (T versus C: overall population, P=0.106; Caucasian population, P=0.472; Asian population, P=0.645).The funnel plot showed a symmetrical distribution of the studies (funnel plot of GNB3- 825T versus C and susceptibility of EH in overall population is shown in Fig. 4).

DISCUSSION
Our results indicated that the prevalence of the GNB3-825T allele varied from 37.75-76.28% in diff erent ethnic groups, the lowest being demonstrated for Caucasians and the highest for African-Americans.Furthermore, meta-analysis results showed that an association existed between the GNB3-C825T allele frequency and risk of EH in the overall population (T versus C, OR=1.09, 95% CI 1.04-1.19),and the Caucasian (T versus C, OR=1.16, 95% CI 1.08-1.24;TT+TC versus CC, OR=1.33, 95% CI 1.13-1.56;TT versus TC + CC, OR=1.13, 95% CI 1.02-1.20)and Chinese populations (TT vs. CC, OR=1.23, 95% CI 1.06-1.57).Conversely, no associations were detected in Asian or Japanese people.
G-proteins comprise a family of ubiquitously distributed signal-transduction proteins.Most membrane receptors rely on heterotrimeric G-proteins to activate or inhibit intracellular signaling cascades.G-proteins are infl uenced by hormones   , 1998).This allele has also been associated with enhanced G-protein signaling (Siff ert et al., 1998;Sun et al., 2005), presumably through abnormal stability or functional interactions of the shortened G-proteins.
In recent years many studies have reported a correlation between GNB3-C825T and EH in Caucasian, African, South American and Asian populations.Our analysis showed that the GNB3-C825T allele was associated with EH in the overall population, which is in agreement with the results of a previous multi-ethnic population meta-analysis (Bagos et al., 2007).These authors reported that TT versus CC + CT: OR=1.08, 95% CI: 1.01-1.15,P<0.001 and TT + CT versus CC: OR=1.17, 95% CI: 1.06-1.29,P<0.001.Further subgroup analysis led to a similar result, i.e., significant associations from studies in the Caucasian population and no associations from studies in Asians.However, we did observe that the GNB3-825T allele (TT vs. CC, OR=1.23, 95% CI 1.06-1.57)was a risk factor for EH in Chinese people.Our studies of the Chinese population involving GNB3-C825T and EH risk include not only the mainland investigations but also Taiwanese ones.
In addition, we calculated the average frequency of the T allele in different ethnic people, which was lowest in Caucasians, intermediate in Asian, and the highest in Africans.Siff ert et al. analyzed the distribution frequencies of GNB3-C825T from a German cohort, and Chinese and African populations, and found that T allele frequencies differed significantly among different ethnic groups and were the lowest in Germans (31.9%), intermediate in Chinese (47.7%) and highest in Africans (81.4-84.1%)(Siff ert et al., 1998).The diff erent genotypic frequencies of GNB3-825T allele might influence the phenotypes related to hypertension such as salt sensitivity in diff erent ethnic populations (Franco et al., 2006), which may contribute to diff erent correlations between Chinese and other populations.It is worth noting that the number of studies from the African population was much lower than for other races.More studies from African people are needed to re-estimate the frequency.Some limitations of this meta-analysis should be discussed.First, this meta-analysis focused only on papers published in the English and Chinese languages and the ones that reported in other languages may bias the present results, even though publication bias was not detected with Begg's test.Additionally, there may be other eligible studies that were not published and indexed by electronic databases.Third, signifi cant between-study heterogeneity was observed.Although we used the random-eff ects model to pool ORs, it may aff ect the precision of results.Finally, lack of individual participants' data restricted the further adjustments by other co-variables such as smoking, body mass index, hyperlipidemia, etc.
Figure1: Pooled OR (T versus C) and 95% CI of individual studies and pooled data for the association between polymorphism of GNB3 C825T and EH in the overall population

Figure 2 :
Figure 2: Pooled OR (T versus C) and 95% CI of individual studies and pooled data for the association between polymorphism of GNB3-C825T and EH in Caucasian population

Figure 4 :
Figure 4: Funnel plot for detecting publication bias among all studies for allele comparison (GNB3-825T versus C) in the overall population.Logor (y axis) the log of OR,s.e. of logor (x axis) the standard error of lor(OR).OR: odds ratio.

TABLE 2
The results of meta-analysis between GNB3-C825T polymorphism and EH TC versus CC: 1.11 (0.72-1.54)P=0.132.However, no signifi cant association was identifi ed in the dominant model or in the recessive model (Table

TABLE 3
Main results of the pooled OR in sensitivity analysis and neurotransmitters and act to regulate blood pressure.Polymorphisms of the GNB3 gene have received considerable attention as candidate genes for EH.The GNB3-C825T allele was demonstrated to lack 41 amino acids in the β3 subunit of trimeric G-proteins (Siff ert et al.