Genetic association of PROC variants with pulmonary embolism in Northern Chinese Han population

To evaluate SNPs (single nucleotide polymorphism) in PROC (protein C gene) associated with pulmonary embolism (PE) susceptibility in North Chinese Han population. A case-control study design was used, and patients with PE and healthy participants were enrolled from the Emerging Department of the several hospitals in Weifang, Shandong, China. SNPs in PROC were genotyped using Mass ARRAY system. The allele frequency of rs199469469 was significantly different between PE patients and the control [OR (95 % CI) = 5.00 (1.66–15.12), P = 0.004], and the difference remained significantly after controlling for age and gender [OR (95 % CI) = 5.34 (1.47–19.39), P = 0.011). The G(del)G in the haplotype includes rs1799809|rs199469469|rs2069928 was of a significantly difference (P = 0.016) among PE patients and the controls, and remained significant (P = 0.015) after adjustment for age and sex. Our study reports that PROC SNPs (rs199469469) might be associated with PE susceptibility, with the G allele of rs199469469 serving as the protective factors for incidence of PE. These findings may contribute to the understanding and primary prevention of PE.


Background
The major thrombotic medical disorders include venous thromboembolism (VTE), a multifactorial disorder consisting of deep venous thrombosis (DVT) and pulmonary embolism (PE). PE is one manifestation of venous thromboembolism (VTE) and is a frequent, recurrent and potentially fatal disease (Goldhaber and Bounameaux 2012;Goldhaber 2012). PE contributes to 5-10 % of deaths in hospitalized patients and VTE is a leading preventable cause of in-hospitalized death (Alikhan et al. 2004;Cohen et al. 2008). In USA, the incidence of all DVT/PE events is 300,000-600,000 cases per year (approximately 1-2 per 1000 persons per year) and the mortality rates of all DVT/PE events is 60,000-100,000 cases per year (Beckman et al. 2010). However, it is difficult to estimate accurate mortality rates of the PE, because of the presence of related diseases and the large proportion of undiagnosed PE (Laack and Goyal 2004). With the improvement of its diagnosis and the development of access to healthcare, VT in Asian populations is now thought to be rising (Roberts et al. 2009;Zakai and McClure 2011).
PE is caused by both genetic and environmental factors, among which genetic factors account for up to 60 % of risk (Souto et al. 2000). Studies have shown that single nucleotide polymorphisms (SNPs) in genes (protein C gene, protein S gene and antithrombin gene) may contribute to the susceptibility to PE (Roberts et al. 2009;Suehisa et al. 2001). Protein C (PROC, or PC) is a vitamin K-dependent serine protease zymogen, which is an inactive zymogen and can be stimulated by the thrombinthrombomodulin (TM)-endothelial protein C receptor complex on endothelial cell surfaces. TM-bound thrombin cleaves the 158-169 activation peptide of PC and generates activated PC (APC) (Wildhagen et al 2011).
APC is a key component of the anticoagulation system. APC inhibits the coagulation pathway by proteolysis of coagulation factor Va (the activated form of coagulation factor V) and coagulation factor VIIIa (Cramer and Gale 2011). The decreasing of APC level is an independent risk factor for both venous and arterial thrombosis (Soare and Popa 2010).
The human PROC is located on 2q13-q14 and comprises 9 exons. Up to date more than 200 mutations were identified in the PROC gene (D'Ursi et al 2007). Most studies were conducted in Western populations, and few in the Asian population (Gandrille et al. 1995;Miyata et al. 1998;Reitsma et al. 1991;Shen et al. 1997). These studies show that the mutation pattern of PROC is of significant ethnic differences, thus, some mutations (Arg230Cys, Arg178Trp, Gln132X, Val297Met and Pro168Leu) are common in the Caucasian population, while the others (Phe139Val/ rs199469470, Arg169Trp/ rs759316085, Val297Met, Met364Ile, and G8857del) are observed in the Japanese population. So far, few studies have been published to investigate the association between PROC variants with incidence of PE in mainland China.
In this study, we investigated the association between PROC variants and incidence of PE in a case-control study of North China Han population.
All the six polymorphisms in cases and controls were both confirmed to Hardy-Weinberg equilibrium (Table 1). Table 1 showed that the "del" allele of rs199469469 (AAG/del) was of allele difference between the case and the control (OR = 5.00, 95 % CI 1.66-15.12, P = 0.004), and the association remained significant after adjustment for age and sex (OR = 5.34, 95 % CI 1.47-19.39, P = 0.011). For the other 5 SNPs, no statistical significances were observed (Table 1). rs199469469 was associated with PE in genotype and dominant model, but not in recessive model ( Table 2).
The frequencies of Haplotype were evaluated using the Chi-square test, and Logistic regression analysis was performed to adjust for age and gender. The results showed that Haplotype G(del)G frequency (rs1799809|r s199469469|rs2069928) between the case and the control (P = 0.016) was of significant difference, and remained significant (P = 0.015) after adjustment for age and sex (Table 3).
We also described the allele frequency data in the present study as well as 1000 genome project. Except for rs199469469, the allele frequency of other 5 SNPs are comparable among the present study and these in 1000 Genome of different ethnic groups (Table 4). This paper confirms data reported by Tang et al. (2012b) who identified that the rs199469469 in PROC was associated with both decreased protein C anticoagulation activity and an increased risk of thrombosis in Chinese subjects of South China, whereas no allele frequency of rs199469469 were reported in 1000 Genome or other populations. Therefore, the "del" allele of rs199469469 could be considered typical of Chinese people, and should be validated in large cohort of different ethnic groups.

Discussion
In this study, we investigated the association between PROC variants and the development of PE, and revealed that the rs199469469 (also designated as c.574_576del or p.Lys150del, located in exon 7 of the PROC gene) predispose people to PE. The case-control study showed that variant confers an approximately 5.00-fold increased risk of PE in the north Chinese population, and the results persist after adjustment for age and sex (OR = 5.34, P = 0.011).
PROC, with a single chain and synthesized by hepatocytes, is composed of a short activation peptide, a serine protease domain, carboxy-glutamic acid residue (Gla) domain, two epidermal growth factor (EGF)-like domains (Fisher et al. 1994;Perera et al. 2000). The deletion of lysine in PROC (rs19469469) can caused conformational changes in the protease domain (Chen et al. 2008), resulting in the elimination of a positive charge (Wildhagen et al. 2011). The most important is that the deletion is located at the 'linking peptide (residues 137-157)' , which precedes the activation peptide and excised upon protein C activation. Residues Gly142-Leu155 are well conserved among species, and previously researches showed the region is important for anticoagulant activity (Mesters et al. 1993;Lu et al. 2013). Factor-V-Leiden mutation in the coding sequence of F5 (Dahlbäck et al. 1993;Koster et al. 1993;Bertina et al. 1994) , prothrombin-G20210A mutation in the 3′ UTR of F2 and antithrombin-Cambridge-II mutation in SERPINC1 are common genetic risk factors for VT in whites (Poort et al. 1996;Corral et al. 2007). However, these polymorphisms are rare in Asians, including Chinese populations. Little is known about the genetic background of VT, and no common genetic risk factors have been identified in the Chinese population until recently. Tang et al (2012a) carried out the first study on the genetic background of PC deficiency in the Chinese population, and identified a common mutation in PROC (rs146922325, c.565C>T) in a family study (first-degree relatives bearing this variant had an 8.8-fold increased risk of venous thrombosis), which has been further verified by our casecontrol study (the mutant allele conferred a high predisposition to venous thrombosis (adjusted OR = 7.34, 95 % CI 3.61-14.94). Tang et al (2012bTang et al ( , 2013 further identified that rs199469469 in PROC was associated with both decreased protein C anticoagulant activity and an increased risk of VTE in Chinese (Hubei Province), with an odds ratio of 2.93, and 2.84 (Tang et al. 2012b. The studies by Tang et al (2012aTang et al ( , b, 2013 were performed in Southern China, and our study performed in Northern China confirmed that rs199469469 in PROC was associated with an increased risk of VTE in Chinese.

Conclusion
We firstly showed that the "del" allele of rs199469469 in PROC is associated with the increased risk of PE in a Northern Chinese Han population and the finding was consistent with that in Southern Chinese. Further epidemiologic studies are required to determine this association in larger populations, and functional studies are needed to determine the effect of this mutation contributing to susceptibility to PE. These studies will be a benefit to early predication or primary prevention of PE.  (Torbicki et al. 2008), and the patients with PE for patent foramen ovale were not screened. The inclusion criteria were: (1) Chinese Han population; (2) > or = 18 year-old. While the exclusion criteria were: (1) patients with acute liver disease or nephrotic syndrome; (2) recurrent venous thrombosis or pulmonary embolism; (3) patients with personal or family history of venous thromboembolism or other blood disease; (4) patients with the special history of drug use; (5) patients with chronic diseases such as hypertension; (6) patients with insertion of pacemaker.    The controls were 279 ethnic matched healthy individuals recruited in physical examination. All the patients and the control have given their written consents to participate in the study.

Selection of SNPs and genotyping
For the selection of candidate SNPs, we firstly downloaded all SNPs information in PROC region (including 5000 bp in the upstream and 5000 bp in the downstream) from Hapmap (Version 3, Release 2, Analysis Panel CHB), then we screened the SNPs with Minor Allele Frequency ≥0.10, and P ≥ 0.10 in Hardy-Weinberg Equilibrium test, finally we selected the tag SNPs (r 2 ≥ 0.80) using Tagger function of Haploview (Barrett et al. 2005). Five SNPs near PROC (rs1799809, rs199469469, rs2069928, rs7580688, rs2240817) meet the criteria. rs199469469 was also added because it was associated with both decreased protein C anticoagulation activity and an increased risk of thrombosis in Chinese subjects of South China (Tang et al. 2012b).
Genomic DNA was extracted from whole-blood samples using QIAamp DNA Blood Mini Kit (Qiagen, German) according to the manual instructions. Genomic DNA samples were subsequently diluted to 25 ng/ μl. SNPs were genotyped using Mass ARRAY system (Sequenom, Inc., SanDiego, CA). Sequenom Mass-ARRAY ® Assay Design 3.0 software (Sequenom, Inc., San Diego, CA, USA) was used to design Multiplexed SNP Mass-EXTEND assays (Gabriel et al. 2009). SNP genotyping was performed using the standard protocol recommended by the manufacturer with a Sequenom Mass-ARRAY ® RS1000 (Sequenom, Inc.). Sequenom Typer 4.0 software was used for data management and analyses (Thomas et al. 2007).

Statistical analysis
The Hardy-Weinberg equilibrium (HWE) of each SNP was evaluated using the Chi-square test. Differences between groups were analyzed by a Student's t test or Mann-Whitney U test depending on the distribution of the laboratory data for continuous variables. The associations between PE and specific PROC genotypes were estimated by computing the odds ratios (OR) and 95 % confidence intervals (95 % CI) from the Chi-square test or Fisher's exact test. Logistic regression analysis was performed to adjust for conventional risk factors including age and gender. Two-sided significance level was 0.05. The statistical analyses were performed by SPSS 13.0 (SPSS Inc., Chicago, IL, USA).