Background

Stroke is one of the leading causes of death over the all world and cause major health problem [1]. However, it is the first cause of morality in China [2]. Ischemic stroke (IS) frequently caused dysfunction of the brain and comprises 80% of cases [3]. The reasons for the patho-physiological cause of ischemic stroke are unclear. The concept was widely accepted that stroke is caused by combination of genetic and environment factors. Among genetic factors, it well known that a few specific gene variants of lipid metabolism are modifiable risk factors for the occurrence of IS [1, 4]. The extensive study indicated that atherosclerotic plaques form is the major cause of IS [5, 6], which the occurrence of atherosclerotic plaques are strongly associated with abnormal lipid metabolism [7, 8]. So far, it was known that Apolipoprotein A5 (ApoA5), lipoprotein lipase (LPL), Cholesteryl ester transfer protein (CETP) and low-density lipoprotein receptor (LDL-R) gene mutations had increased the concentration of lipids [9,10,11].

The association of human candidate genes with IS patients may be the confounding effects of ethnic, environment factor and individual life style [12]. ** of the ApoA5, LPL, CETP and LDLR gene SNPs

DNA sample were analyzed by SNaPshot SNP ty** machine (SnaPshot multiplex, ABI, USA). Detail PCR and single-base extension primer sequences were recorded as our previous study [18].

Statistical analysis

The statistical analysis was performed using SPSS 20.0(IBM, USA). Clinical parameter such as serum TC, TGL, HDL-C, LDL-C, ApoA1, ApoB, Lpa glucose, and serumwere measured with automatic machine in IS patients and controls. The allelic frequencies were calculated by Hardy-weinberg equipment method. Multivariate logistic regression analyses were performed for genotype, allele frequencies and lipid profile factor associated with ischemic stroke. Through counting DNA sequencing data, the genotype and allele frequencies can be estimated. The distinction between studied groups were analyzed by Pearson’s X2 test. Then, logistic regression analysis was measured the strength.

Results

Clinical character of subjects

The basic clinical characteristics of the 408 IS patients and 347 control were shown in Table 1. There were no significant difference in age, sex and body mass index (BMI), However, it was greatly differences in hypertension, diabetes mellitus (DM); Total Cholesterol (TC); Triglycerides (TG); low Density Lipoprotein-C (LDL-C); High Density Lipoprotein-C (HDL-C); Apolipoprotein–A1 (ApoA1); Apolipoprotein B (ApoB); lipoprotein a (Lpa) (P < 0.001). Therefore, these clinical parameters provide an evidence that IS patients often have abnormal serum lipoprotein level. It also is closed association with hypertension and DM history.

Table 1 Candidate genes and SNP information
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Lipidsmetabolism relative genes SNP information

After we accessed to http://www.ncbi.nlm.nih.gov/SNP database a, it was found that possible APOA5, LPL, CETP and LDLR genes candidate SNP site were shown in Table 2. These mutations were verified by PCR and DNA product sequencing. These data confirmed that these candidate genes were indeed mutated in IS patients.

Table 2 Candidate Genes and SNP information
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The distribution difference of ApoA5 rs662799, LPL rs320, rs328, CETP rs708277, LDLR rs688 mutation with IS in Han and Uighur population by Hardy-Weinberg equilibrium test

We compared the distribution difference of APOA5 rs662799, LPL rs320, LPL rs328, CETP rs708277 and LDLR rs688 gene loci in IS and control Han and Uighur population of **njiang. Then we calculated the difference in overall case and control group by Hardy-Weinberg equilibrium test. Data were shown in Tables 3 and 4. The frequencies of LDLR rs688 mutation were dramatic increased in IS patients than the controls (P = 0.003). This result strongly supports the association LDLR mutation with IS. However, there was no difference that the frequencies of ApoA5 rs662799, LPL rs320, LPL rs328, CETP rs708272 in Hardy-Weinberg equilibrium test.

Table 3 Hardy-Weinberg equilibrium test of Overall sample case group
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Table 4 Hardy-Weinberg equilibrium test of Overall sample CONTROL group
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The distribution difference of ApoA5 rs662799, LPL rs320, rs328, CETP rs708277, LDLR rs688 mutation with IS and control according to their dominant, recessive and codominant genetic style by pearson χ2 and CHISP test

The next, we compared the difference of ApoA5, LPL, CETP and LDLR SNP mutation between Han and Uighur IS patients and control by pearson χ2 test. The result were shown in Tables 5 and 6. By contrast, either Uighur (Table 5) or Han population (data not shown) was highly significant when compared to respective controls in recessive genetic model. However, associations were not significant in dominant genetic model. Interest, this result also is consistent by CHISP test (Tables 6 and 7). Further, no significant association was found in dominant and codominant genetic model in stroke cases (data not shown).

Table 5 The distribution of different genotype of recessive between two Uighur groups
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Table 6 The distribution of different genotype of recessive between two Han groups
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Table 7 The distribution of different genotype of recessive between two Uighur groups
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Association of ApoA5 rs662799, LPL rs320, rs328, CETP rs708277, LDLR rs688 mutation with IS in Han and Uighur population by binary logistic regression analysis

We further compared the difference of IS patients and control by binary logistic regression analysis. It was observed significantly associated with rs688 both addictive model (TT/CC, adjusted OR = 1.47, 95% CI = 1.04–2.07, P < 0.01) and recessive model (TT/CT + CC, adjusted OR = 2.66, 95% CI = 1.37–5.14, p = 0.004) (Tables 8 and 9). In Han population, ischemic stroke was observed significantly associated with rs688 both in addictive model (TT/CC, adjusted OR = 3.27, 95% CI = 1.06–10.05, P < 0.05). In Uighur population, no significant association was found between gene polymorphisms and the risk of ischemic stroke (data not shown). Combined analysis of multiple gene and loci, interaction effects of LDLR rs688 C/T, ApoA5 rs662799 A/G and CETP rs708272 C/T denoted a significant influence on IS susceptibility (p < 0.05).

Table 8 Additive model analysis of different genes with stroke
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Table 9 Recessive model analysis of different genes with stroke
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Association of LPL rs328 and CETP rs708277 with serum lipid protein levels and clinical profile in total population

To explore the association of lipid metabolism gene SNPs with serum lipid protein levels and clinical profile, we detected major serum lipid levels, glucose, height, weight, BMI and blood Pressure with LPL rs328 and CETP.rs70877 in total population. Data were shown in Tables 10 and 11. We found that there are significant lower TG in rs328 GG than in rs328 CC and GC population (Table 10). Low SBP also was measured in CETP.rs70877 GA and AA (Table 11).

Table 10 Association comparison of rs328 genotype with stroke in study population
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Table 11 Association comparison of rs708272 genotype with stroke in study population
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These results indicated that different lipid protein metabolism relative gene SNPs is associated with special clinical profile.

Discussion

The risk factors of stroke were extensively studied [10, 20, 21]. Recent data revealed that a few protein genetic polymorphisms were significantly association with the risk for stroke [22,23,24]. Here, we investigated that ApoA5, LPL, CETP and LDLR gene polymorphisms in IS patients from **njiang region. The results provided linkage of different genetic background, life style and the occurrence of ischemic stroke.

Our study showed the presence of ApoA5 rs662799, LPL rs320, LPL rs328(Ser474 Ter), CETP rs708277 and LDLR rs688 in IS patients from **njiang region. Surprisingly, it was observed that rs688 genetic polymorphism had significantly more difference in IS patients than in control in recessive model analysis. This phenomenon also was seen by binary logistic regression analysis of total studied population. IS patients was observed significantly associated with rs688 both addictive model (TT/CC, adjusted OR = 1.47, 95% CI = 1.04–2.07, P < 0.01) and recessive model (TT/CT + CC, adjusted OR = 2.66, 95% CI = 1.37–5.14, p < 0.01). In Han population, ischemic stroke was observed significantly associated with rs688 both in addictive model (TT/CC, adjusted OR = 3.27, 95% CI = 1.06–10.05, P < 0.05). However, This phenomenon weren’t observed in Uighur population. It was well known that a single gene may have different genetic polymorphism with different kind of disease [25,26,27,28]. The same gene in one kind of disease may have diverse SNPs owing to different race and place. It was widely reported that LDLR genetic polymorphisms are associated with many disease such as essential hypertension [28], coronary artery disease (CAD) [29] and high cholesterol [30]. These SNP site were C1773T, rs2228671, rs1122608 and so on. A similar study were reported that rs688 is associated with IS patients in Taiwanese population [24]. Gao et al. revealed that rs688 increase exon12 alternative splicing and affected LDL receptor function [27]. Zhu et al. showed that rs688 promote a high serum cholesterol by modulating LDLR exon12 splicing efficiency [30]. Therefore, we speculated that rs688 firstly increase serum cholesterol and then cause atherosclerotic plaques form.

ApoA5 had similar phenomenon like LDLR. It was reported that ApoA5 T-1131C, T1259C, and IVS3 + G476A are associated with IS, DM and CAD [11, 25]. These SNPs result in high triglycerides. It also was widely observed that some gene SNPs are closely associated with special clinical profile in **njiang. Our study indicated that SNPs of lipid metabolism relative genes is associated with response of drug [18] Zhang et al. reported that there are significant difference in β3-adrenergic receptors (ADRβ3) gene polymorphisms rs6986132 of Han and Uighur populations in **njiang. They also observed the ADRβ3 rs2298423 G allele carriers increase risk for TC and LDL-C level in the Uighur populations of **njiang [31]. Abulizi et al. found that ApoA5 gene c553G-T polymorphism is associated with high TG levels in Han and Uighur population [32]. Our present data showed that LPL rs328 CC and GC allele people have high TG than rs328 GG allele population. People of CETP rs708277 GG allele had high SBP than it in rs708277 GA or AA allele people.

Conclusion

Our results indicated that SNPs of lipid metabolism relative gene is closely associated with different population of **njiang. In Han population, ischemic stroke was significantly associated with LPLR rs688 polymorphisms. However, this phenomenon didn’t detect in Uighur population. We also found that different special SNPs allele have different serum lipid and blood pressure levels. These results demonstrated that we need take care of some specific SNPs people because they is easy to develop ischemic strokes.