Background
Subarachnoid hemorrhage (SAH) caused by rupture of a cerebral aneurysm is the reason for approximately 85% of cases with spontaneous SAH [
1]. It accounts for 5% of all stroke cases and is associated with high rate of mortality and morbidity [
2]. Rebleeding and delayed cerebral ischemia are the two major complications that are associated with poor prognosis and high mortality rate in SAH [
3]. The first-degree relatives of patients with SAH have a three-fold increased risk for the rupture of an aneurysm when compared with general population [
4]. But the role of genetic factors which contribute to the risk of SAH is poorly defined. Most candidate gene studies have considered proteins associated with connective tissue organization [
5‐
7]. The reason for SAH occurrence was not only due to weakened vessel wall structure but also due to rupture of vessel wall [
8]. A few studies have investigated the role of fibrinolytic system and coagulation factors association with the risk of aSAH [
9‐
11].
Coagulation factor XIII belongs to transglutaminase family which circulates as a heterotetramer, composed of two A subunits and two B subunits [
12]. During coagulation, thrombin activates the catalytic factor XIII A subunit and crosslinks the fibrin molecules to increase the clot stability [
13]. During fibrinolysis, factor XIII A activates anti plasmin which inhibits the plasmin from degrading the crosslinked fibrin structure [
14]. Thus, factor XIII A subunit plays a significant role both in coagulation and fibrinolysis. Also, it plays a key role in extracellular remodelling, angiogenesis, atherosclerosis, wound healing and tissue repair [
15].
In humans, the Coagulation factor XIII A chain (
F13A) gene is located on chromosome 6p 24–25 [
16]. The factor XIII A is 83 kDa protein, which consists of 732 amino acids [
17].
F13A gene consists of 15 exons and 14 introns [
18]. The nine polymorphisms in
F13A genes are c.103G > T(p.Val35Leu), c.614A > T(p.Tyr204Phe), c.996A > C (p.Pro332Pro), c.1652C > T(p.Thr550Ile), c.1694C > T (p.Pro564Leu), c.1704A > G (p.Glu567Glu), c.1696 T > A (p.Leu588Gl), c.1951G > A (p.Val650Ile) and c.1954G > C (p.Glu652Gln) [
19]. Among them, the common
F13A polymorphisms are c.103G > T (p.Val35Leu)and c.1694C > T (p.Pro564Leu).
In the Asian and Caucasian population, the allele frequency of 34Leu allele is 0.13 and 0.25 [
20]. In Han Chinese population, the c.103G > T (p.Val35Leu) polymorphism was associated with the risk of ischemic cardiovascular and cerebrovascular diseases [
21].In Caucasian population, c.103G > T (p.Val35Leu) polymorphism was associated with the risk of intracerebral hemorrhage and brain infarction [
22,
23].In the Asian and Caucasian population, the allele frequency of 564Leu allele is 0.29 and 0.21 [
17]. The c.1694C > T(p.Pro564Leu) polymorphism was associated with decreased factor XIII plasma levels with increased factor XIII activity [
24]. When stratified by gender c.1694C > T (p.Pro564Leu) polymorphism was associated with risk of haemorrhagic stroke in women aged < 45 years in Caucasian population [
10]. The aim of the present study is to investigate the association of c.103G > T (p.Val35Leu) and c.1694C > T (p.Pro564Leu) polymorphisms with the risk of aSAH in a South Indian population.
Discussion
Spontaneous subarachnoid hemorrhage (non-traumatic) remains as one of the considerable neurosurgical problems that affect 25,000 to 28,000 people yearly [
28]. In cerebrovascular disorders, the role of multifactorial and multigene have been studied progressively. The difference in phenotype in persons carrying same genetic mutation suggests the role of multiple factors in the pathogenesis of the disease [
29]. This study was carried out to analyse whether
F13A polymorphism was associated with the risk of aSAH.
Extracellular matrix remodelling dysfunction, atherosclerosis and fibrinolytic dysfunction were considered as important pathogenic mechanisms in the formation and rupture of a cerebral aneurysm [
30‐
32]. Coagulation factor XIII A chain plays a significant role in extracellular matrix (ECM) remodelling and tissue repair [
33]. Crosslinking of collagen and fibronectin to each other by F13A during extracellular matrix formation and wound healing was an important physiological event in stabilizing the ECM [
34]. F13A in the cellular form plays a significant role in triggering atherosclerosis [
18]. F13A helps in angiotensin I receptor dimerization which activates the monocyte adhesion to endothelium cells and this was considered as one of the pathogenic mechanism in the progression of atherosclerosis [
33]. In the fibrinolytic system, the primary mechanism to prolong fibrinolysis is crosslinking of α
2 -anti plasmin and fibrin by F13A [
35]. It has been shown that properties of F13A were affected by its gene variants [
19] and it was suggested that
F13A variants play a key role in the pathogenesis of a cerebral aneurysm by affecting the vessel wall stability, triggering atherosclerosis and decreasing clot stability [
20].
F13A polymorphism was associated with the severity of outcome in atherothrombotic ischemic stroke [
36], primary intracerebral hemorrhage [
37] brain infarction [
38] and deep vein thrombosis [
39]. Many case-control studies reported the association of
F13A polymorphism and risk of aSAH. Ladenvall et al. reported that 34Leu and 564Leu carriers had an increased risk of aSAH in the Swedish population [
9], but there was no association between c.103G > T (p.Val35Leu) variant and nonfatal haemorrhagic stroke in young white women in U.S population [
10]. Another study done by Rugriok et al. reported that c.103G > T (p.Val35Leu) and c.1694C > T (p.Pro564Leu) polymorphisms did not have any association with the risk for aSAH in Caucasian population [
11]. In Spanish population, the prevalence of 34 Leu allele was higher in aSAH than in primary intracerebral hemorrhage group [
40]. The meta-analysis of four studies including the present study suggested that there was no significant association with c.103G > T (p.Val35Leu) polymorphism and risk of aSAH, whereas the c.1694C > T (p.Pro564Leu) polymorphism showed significant association with risk of aSAH.
The c.103G > T (p.Val35Leu) polymorphism present at exon 2 of
F13A gene increases the activation rate of coagulation and affects the fibrin structure [
41]. The fibrin clot which is crosslinked by 34Leu variants has thinner fibres, smaller pore and altered permeation characteristics when compared with fibrin clot crosslinked by Val34 variant [
19]. Also, the clot formation time was shorter for 34Leu variant samples [
42]. The c.1694C > T (p.Pro564Leu) polymorphism present at exon 12 affects the specific activity of the enzyme. Also, c.1694C > T (p.Pro564Leu) variant causes lower plasma F13A levels and increases F13A activity [
43]. In the present study, 34Leu allele was associated with lower risk and 564Leu allele was associated with the higher risk for aSAH.
The protective effect of the
F13A c.103G > T (p.Val35Leu) polymorphism is not well understood and needs to be elucidated. The protective effect c.103G > T (p.Val35Leu) polymorphism was reported in few studies on myocardial infraction and venous thrombosis [
44‐
47]. .An increased F13A activity was reported in 34Leu carriers, higher activity in Leu homozygotes and intermediate activity in Leu heterozygote [
43]. This was because of proximity of polymorphism to the thrombin activation site. Kohler et al. reported that the higher F13A activation results in ineffective cross linking [
48]. Van Wersch et al. reported that in pregnant women, F13A levels were higher in smokers than in non -smokers [
49]. In our study number of smokers in patients were higher than that in controls. Elbaz et al. reported that the ORs associated with smoking were lower in 34Leu carriers than in noncarriers. This suggests that the protective effect of polymorphism was more significant than effect of smoking in 34Leu carriers [
22]. The investigation of F13A activity in healthy controls while taking the effect of smoking and c.103G > T (p.Val35Leu) polymorphism in to account will be helpful for better understanding.
Basilar top aneurysm is the most common aneurysm seen in the posterior fossa circulation. It was characterised with higher bleeding tendency and worst clinical outcome after rupture [
50]. In this study, 42.8% of patients with basilar top aneurysm had WFNS grade 1 and 71.4% of patients had WFNS grade 2 and 3. Therefore, most of the patients with basilar top aneurysm had the worst clinical outcome in this study.34Leu variant affects clot stability and thereby associated with the bleeding tendency [
51]. Basilar top aneurysm was characterised by bleeding tendency and this explains the reason for the association between Leu/Leu genotype and basilar top aneurysm in this study.
The SIFT algorithm predicts the ‘damaging’ and ‘non-damaging’ (tolerated) SNPs based on the sequence homology and physical properties of sequence submitted [
52]. The PolyPhen-2 algorithm predicts the nsSNPs in three distinct categories: ‘probably damaging’, ‘possibly damaging’ and ‘benign’ SNPs based on the structural homology-based approach using functional point of view [
53]. The SNPs predicted as damaging /deleterious in both sequence and structural homology-based approach are considered as ‘high-confidence’ nsSNP, since they have higher impact on the function of protein [
52,
54]. The rs5985 and rs5982 SNPs do not have any direct structural-functional effect on factor XIII A protein according to SIFT and PolyPhen-2 annotation programs. But the studied SNPs might have effect on factor XIII A protein through other indirect pathway.
There are previous reports of linkage disequilibrium (LD) between the variants of
F13A gene [
9,
10]. LD is the non-random association of alleles in two or more loci [
55]. LD block (haplotype) is clinically important for the identification of disease causing genes and the origin of mutations [
56]. Haplotypes occurs when SNPs are situated near to each other in the chromosome and are inherited in blocks [
57]. In both the haplotypes, we found a significant association with the risk of aSAH. Haplotypes are more powerful than individual polymorphism for detecting susceptibility alleles associated with diseases [
56,
57].