Introduction

Atherosclerotic disease includes coronary artery diseases, cerebrovascular disease, and peripheral arterial diseases. It is still the leading cause of morbidity and mortality worldwide1,2,3. Vascular calcification occurs as a part of the atherosclerotic process and it is an active process regulated similarly to the process of bone formation4. According to the current theoretical knowledge, the formation of microcalcifications causes plaque instability and is correlated with cardiovascular risk5. Therefore, vascular calcification is supposed to be a strong predictor of cardiovascular events independent of the traditional risk factors6, 7.

Matrix γ-carboxyglutamic acid Gla protein (MGP), a 10-kDa vitamin K-dependent extracellular matrix protein, has been shown to be an inhibitor of vascular calcification8, 9. The mechanism by which MGP inhibits vascular calcification is still unknown. According to the current understanding, MGP regulates vascular calcification by binding and inactivating bone morphogenic protein 2 and preventing the deposition of calcium phosphate in the vascular matrix10,11,12.

Over the past decade, increasing evidence has indicated that several single nucleotide polymorphisms (SNPs) of the MGP gene may play a crucial role in the susceptibility of vascular calcification and atherosclerotic disease9. Genes rs1800801 (G7-A), rs1800802 (T138-C), rs4236 (Thr83-Ala) were most often reported. The association between MGP gene rs1800801, rs1800802, rs4236 polymorphisms and vascular calcification and atherosclerotic disease has been discussed in several studies, but the results have been controversial13,14,15,16,17. These results were inconclusive and did not reach a consensus. Therefore, we conducted this meta-analysis in order to precisely elucidate the genetic roles for the MGP gene rs1800801, rs1800802, rs4236 polymorphisms in the process of vascular calcification and atherosclerotic disease.

Materials and Methods

Literature search and criteria of inclusion

This meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria18. Relevant articles were identified by a systematic search of Medline, Embase and China Knowledge Resource Integrated (CNKI) Database from their inception to December 2016. The following search terms were used: “Matrix Gla protein”, “MGP”, “NTI”, “GIG36”, “MGLAP”, “polymorphism”, “polymorphisms”, “calcification”, “atherosclerosis”, “acute coronary syndrome”, “myocardial infarction”, “stenosis”, “ischemic stroke”, and “cerebral infarction”. Two authors independently confirmed the eligibility of articles and collated the data from the qualifying articles. The reference lists of retrieved articles were also reviewed for eligible studies. There were no language restrictions.

Inclusion criteria

Eligible articles should meet the following criteria: (1) evaluated the association of the MGP gene (rs1800801, rs1800802, rs4236) polymorphism with vascular calcification and atherosclerotic disease, (2) studied on human beings, (3) in a case-control or nested case-control study design.

Exclusion criteria

We excluded studies according to the following criteria: (1) unrelated to the association of MGP gene polymorphism with vascular calcification and atherosclerotic disease; (2) review articles; (3) have no control group; (4) animal studies; (5) data is missing or incomplete, and the authors could not be contacted; (6) data is duplicated.

Data extraction and quality assessment

Relevant information was carefully extracted from all eligible articles. The following data were extracted: first author, year of publication, country of origin, ethnicity, source of controls, frequency of genotypes in cases and controls, and evidence of Hardy-Weinberg equilibrium (HWE) in controls. Two authors independently extracted the data and assessed the study quality based on the Newcastle Ottawa Scale (NOS)19. Any study with a score greater than 7 was considered as “high quality”. Disagreements were resolved by consensus or arbitration by a third reviewer.

Statistical analysis

All analyses were computed in Stata software version 12 (StataCorp, College Station, TX). HWE was assessed for each SNP among controls using a χ2 test20. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of associations of the MGP gene (rs1800801, rs1800802, rs4236) polymorphism with vascular calcification and atherosclerotic disease. The Z test was used to assess the significance of the ORs, and a P value < 0.05 was considered statistically significant. Heterogeneity between studies was tested through chi-square and I-square (I2) tests. A fixed-effects model was used if the I2 value was less than 50% and the p-value was greater than 0.1; otherwise a random-effects model was used. Subgroup analyses were conducted based on ethnicity and source of control. Sensitivity analyses were performed to display possible variability. Begg’s and Egger’s linear regression tests were applied to assess the potential publication bias21, 22.

Results

Characteristics of studies included in this meta-analysis

The study selection process is shown in Fig. 1. From 66 potential articles, 10 articles13,14,15,16,17, 23,24,25,26,27 met the inclusion criteria, including 23 studies consisting of 5280 cases and 5773 controls. Among these included studies, 18 were performed in the Caucasian population and 5 were performed in Asians. All were case-control studies and had been published between 2000 and 2016. The characteristics of eligible studies are shown in Table 1.

Figure 1
figure 1

A flow diagram of selection process.

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Table 1 Characteristics of studies included in this meta-analysis.
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Meta-analysis results

Distribution and allele frequency of the three MGP gene polymorphisms in the cases and controls are shown in Table 1. The main results of this meta-analysis are presented in Table 2.

Table 2 Genotype distribution and allele frequency of the three MGP gene (rs1800801, rs1800802, rs4236) polymorphisms in cases and controls.
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A meta-analysis of the rs1800801 polymorphism with the risk of vascular calcification and atherosclerotic disease

Nine studies with a total of 2015 cases and 2423 controls evaluated the association of the rs1800801 polymorphism with vascular calcification and atherosclerotic disease. There was a significant association of rs1800801 gene polymorphism with vascular calcification and atherosclerotic disease in the recessive model (OR = 1.50, 95% CI 1.01–2.24, P = 0.045) (Table 2, Fig. 2). No significant association was found in the other genetic models: allelic model (OR = 1.13, 95% CI 0.96–1.32, P = 0.141), dominant model (OR = 1.06, 95% CI 0.92–1.22, P = 0.402), homozygote model (OR = 1.50, 95% CI 0.95–2.38, P = 0.082), and heterozygote model (OR = 1.00, 95% CI 0.87–1.15, P = 0.996) (Table 2).

Figure 2
figure 2

Forest plots of association of rs1800801 polymorphism with vascular calcification and atherosclerotic disease in recessive model.

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A subgroup analysis stratified by ethnicity showed a significant association amongst Caucasians in the allelic model, recessive model and homozygote model: allelic model (OR = 1.19, 95% CI 1.06–1.34, P = 0.004), dominant model (OR = 1.12, 95% CI 0.95–1.33, P = 0.170), recessive model (OR = 1.60, 95% CI 1.26–2.03, P < 0.001), homozygote model (OR = 1.83, 95% CI 1.18–2.81, P = 0.006), and heterozygote model (OR = 1.02, 95% CI 0.86–1.22, P = 0.822) (Table 2, Fig. 3). A subgroup analysis of the Asian population found no association in any genetic models: allelic model (OR = 0.85, 95% CI 0.56–1.28, P = 0.427), dominant model (OR = 0.94, 95% CI 0.73–1.20, P = 0.596), recessive model (OR = 0.51, 95% CI 0.21–1.27, P = 0.150), homozygote model (OR = 0.48, 95% CI 0.19–1.20, P = 0.118), and heterozygote model (OR = 0.96, 95% CI 0.75–1.23, P = 0.755) (Table 2).

Figure 3
figure 3

Forest plots of subgroup analyses in Caucasians, (A) allelic model; (B) recessive model; (C) homozygote model.

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A meta-analysis of the association between the rs1800802 polymorphism and the risk of vascular calcification and atherosclerotic disease

Nine studies, consisting of 2073 cases and 2177 controls, evaluated the association between the rs1800802 polymorphism and vascular calcification and atherosclerotic disease. Overall, no significant association was found in any of the genetic models: allelic model (OR = 0.98, 95% CI = 0.83–1.16, P = 0.842), dominant model (OR = 0.93, 95% CI = 0.75–1.14, P = 0.457), recessive model (OR = 1.13, 95% CI = 0.92–1.39, P = 0.232), homozygote model (OR = 1.10, 95% CI = 0.89–1.36, P = 0.395), and heterozygote model (OR = 0.88, 95% CI = 0.72–1.08, P = 0.231). Similar findings were seen in the subgroup analyses in different ethnicities and source of controls (Table 2).

A meta-analysis of rs4236 polymorphism with the risk of vascular calcification and atherosclerotic disease

Five studies, consisting of 1192 cases and 1173 controls, evaluated the association of the rs4236 polymorphism with vascular calcification and atherosclerotic disease. Overall, no significant association was found in any of the genetic models: allelic model (OR = 0.94, 95% CI = 0.81–1.09, P = 0.428), dominant model (OR = 0.93, 95% CI = 0.77–1.11, P = 0.408), recessive model (OR = 0.94, 95% CI = 0.65–1.37, P = 0.764), homozygote model (OR = 0.91, 95% CI = 0.61–1.35, P = 0.637), and heterozygote model (OR = 0.93, 95% CI = 0.77–1.12, P = 0.465). The subgroup analyses in different ethnicities and source of controls showed similar results (Table 2).

Sensitivity analyses

As shown in Table 1, two studies were not consistent with the HWE in controls (P < 0.05). Hence we conducted a sensitivity analyses and observed no statistically significant changes in the pooled ORs when omitting any of the studies, which demonstrated that our results are stable and reliable (Fig. 4).

Figure 4
figure 4

Sensitivity analysis of the rs1800801 polymorphism in recessive model.

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Detection for heterogeneity

There was no significant publication bias based on the visual inspection of the funnel plots (Fig. 5). Similarly, no significant publication bias was found in the Begg’s test or Egger’s test (P > 0.05).

Figure 5
figure 5

Begg’s funnel plots for assessing publication bias for (A) rs1800801; (B) rs11614913; (C) rs4236.

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Discussion

MGP is a mineral-binding extracellular matrix protein secreted by chondrocytes and vascular smooth muscle cells. It is thought to be a key regulator of vascular calcification28. MGP-deficient mice rapidly developed extensive vascular calcification and died due to blood vessel rupture8. In humans, nonsense mutations in MGP cause Keutel syndrome, a rare autosomal recessive disorder characterized by abnormal cartilage calcification29.

The MGP gene (NCBI-Gene ID: 4256) is located on the short arm of chromosome 12 (12p12.3). There is increasing evidence that genetic variation at the MGP locus could modulate the development of vascular calcification and atherosclerotic disease. Previous studies have shown that MGP genes rs1800801, rs1800802 and rs4236 polymorphisms have an important impact on the promoter activity13, 26, 30. T-138, A-7 and Ala-83 alleles of the MGP gene may contribute to the risk of vascular calcification and atherosclerotic disease, such as acute coronary syndrome and ischemic atherothrombotic stroke13, 24, 25. While other studies have found no significant association between these three SNPs with vascular calcification and atherosclerotic disease14, 15, 27. The findings have been inconsistent and inconclusive, which may be attributed to clinical heterogeneity, different ethnic populations, inadequate statistical power, and small sample sizes. Therefore, we conducted this meta-analysis and used subgroup analyses to make a more precise and convictive assessment. To our knowledge, this is the first systematic review and meta-analysis published on the association of MGP polymorphisms with vascular calcification and atherosclerotic disease.

In this meta-analysis, we investigated the association between three SNPs in the MGP gene with the risk of vascular calcification and atherosclerotic disease in 23 case-control studies (consisting of 5280 cases and 5773 controls). The overall results revealed that only the rs1800801 polymorphism was associated with the risk of vascular calcification and atherosclerotic disease. Stratification analysis by ethnicity indicated that the association was significant among Caucasians, but not among Asians in rs1800801 polymorphism. The reason why this association varies among different ethnicities is not clear, the small number of studies or the natural selection in different ethnicities may explain it. No significant association was found in the rs1800802 and rs4236 polymorphisms. Stratification analyses by ethnicity and source of control showed similar results in the rs1800802 and rs4236 polymorphisms.

The in vitro study revealed that the rs1800801–7A variant had an approximately 1.5-fold higher activity than -7G variant in VSMCs30. The -7A allele occurred more frequently in patients with vascular calcification, myocardial infarction, and ischemic atherothrombotic stroke13, 25. Therefore, the -7A allele of the MGP gene may confer an increased risk of vascular calcification and atherosclerotic disease, and therefore may be a novel promising target for prevention and treatment.

There are several limitations to this study. First, the association of the MGP gene polymorphism with vascular calcification and atherosclerotic disease may be influenced by gender. In some studies, the association of MGP polymorphisms with vascular calcification and atherosclerotic disease was only observed in men13, 16, 31. While another study found that the rs1800801 polymorphism was associated with an increased risk of ischemic atherothrombotic stroke only in women25. However, since detailed gender specific data could not be obtained for the studies included in this meta-analysis, we were unable to perform a sub-analysis by gender. Second, as the heterogeneity in different ethnicities influenced the results significantly, the stratification analysis of the source of control was not conducted in the SNP rs1800801. Third, only three studies were performed in Asians, therefore the findings from the Asian based studies were not convictive enough; more studies focusing on the Asian population are needed.

In conclusion, findings from this meta-analysis indicate that the MGP rs1800801 polymorphism is associated with an increased risk of vascular calcification and atherosclerotic disease. Furthermore, this association might only exist in Caucasians. The MGP gene rs1800802 and rs4236 polymorphisms are not associated with an increased risk of calcification and atherosclerotic disease. A larger number of epidemiological studies are required to confirm our findings.