Association of Matrix Gla protein gene (rs1800801, rs1800802, rs4236) polymorphism with vascular calcification and atherosclerotic disease: a meta-analysis

Association between the MGP gene rs1800801, rs1800802, rs4236 polymorphisms and vascular calcification and atherosclerotic disease was inconsistent. To clarify precise association, we performed this meta-analysis. Medline, Embase and China Knowledge Resource Integrated Database were systematically searched through December 2016. A total of 23 case-control studies, consisting of 5280 cases and 5773 controls, were included. The overall results suggested that the -7A polymorphism was associated with an increased risk for vascular calcification and atherosclerotic disease in the recessive model (OR = 1.50, 95% CI 1.01–2.24, P = 0.045). Subgroup analyses of Caucasians showed significant associations in the allelic model, recessive model, and homozygote model: allelic model (OR = 1.19, 95% CI 1.06–1.34, P = 0.004), 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). Subgroup analysis of the Asian population did not demonstrate any significant associations in any of the genetic models. No significant association was found in any genetic model amongst the rs1800802 and rs4236 polymorphisms. The findings of this meta-analysis indicate that the MGP gene rs1800801 polymorphism is significantly associated with vascular calcification and atherosclerotic disease, especially in the Caucasian population.

. A flow diagram of selection process. Continued between MGP gene rs1800801, rs1800802, rs4236 polymorphisms and vascular calcification and atherosclerotic disease has been discussed in several studies, but the results have been controversial [13][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.

Literature search and criteria of inclusion. This meta-analysis was performed according to the Preferred
Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria 18 . 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,    Table 2. Genotype distribution and allele frequency of the three MGP gene (rs1800801, rs1800802, rs4236) polymorphisms in cases and controls. OR, odds ratio; CI, confidence intervals; I 2 = I-square; PB, population based; HB -hospital based.
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 test 20 . 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 (I 2 ) tests. A fixed-effects model was used if the I 2 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 bias 21,22 .  Table 1.

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.
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) (  Fig. 3 (Table 2). 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).

Sensitivity analyses. As shown in
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).

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 calcification 28 . MGP-deficient mice rapidly developed extensive vascular calcification and died due to blood vessel rupture 8 . In humans, nonsense mutations in MGP cause Keutel syndrome, a rare autosomal recessive disorder characterized by abnormal cartilage calcification 29 .
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 activity 13,26,30 . 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 stroke 13,24,25 . While other studies have found no significant association between these three SNPs with vascular calcification and atherosclerotic disease 14,15,27 . 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 VSMCs 30 . The -7A allele occurred more frequently in patients with vascular calcification, myocardial infarction, and ischemic atherothrombotic stroke 13,25 . 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 men 13,16,31 . While another study found that the rs1800801 polymorphism was associated with an increased risk of ischemic atherothrombotic stroke only in women 25 . 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.