Patients
Medical records of all newly diagnosed proven patients with diabetes and hyperlipidemia admitted to Shanghai Tongji Hospital from October 2016 to November 2018. This study was approved by the ethics committee of Shanghai Tongji Hospital and was conducted according to guidelines of the Declaration of Helsinki. All participants provided informed consent. If the subjects were unable to communicate, written consent was obtained from their legal relatives.
Patients who met the following criteria were included: (1) fasting blood glucose ≥7.0 mmol/L or OGTT 2 h≥11.1 mmol/L, (2) TG≥1.7 mmol/L or LDL-C≥3.37 mmol/L or CHOL≥5.18 mmol/L, (3) diagnosis of IS meets the standards of Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2014, and (4) all the patients were Shanghai han population and unrelated.
Patients who met the following criteria were excluded: (1) diagnosis of other types of cerebrovascular disease (e.g., intracranial hemorrhage, subarachnoid hemorrhage, cerebrovascular malformation, or cerebral aneurysm) and (2) severe systemic diseases such as cancer, severe inflammatory diseases, and serious chronic diseases (e.g., hepatic failure or renal failure).
In the control group a total of 336 individuals without history of cerebrovascular disease were included. Age, sex, use of oral contraceptives and history of thrombotic events or drug abuse were recorded. There were no statistical difference in age and sex compared with IS group. The baseline characteristics of patients and controls were shown in S-table 1.
Serum Lipid and Glucose Measurement
Approximately 3 mL blood samples of fasting blood were collected from each study participant then centrifuged at 2500 rpm for 10 min (Backman Coulter, SPINCHRONTM DLX Centrifuge, USA). Serum levels of total cholesterol (CHOL), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) were analyzed by automatic biochemical analyzer (Backman Coulter, DXC800, USA).
DNA extraction and genetic analysis
Genomic DNA was extracted from peripheral blood leukocytes using the TIANamp Genomic DNA Kit (TIANGEN Biotech, Beijing). Polymorphisms were genotyped using the restriction fragments length polymorphism (RFLP) or allele specific Polymerase chain reaction (PCR) method. PCR using the sequence of primers are shown in S-table 2 (Sangon Biotech Co., Ltd., Shanghai). The composition of PCR mixes includes Ex Taq enzyme, dNTP and 10x buffer (TaKaRa). Amplified PCR products of intron 16 of the polymorphic ACE gene were separated on 2% agarose gel (BIO-RAD Laboratories, Hercules, California, USA) (Fig. 1). The presence of 191 bp fragments represented the D allele the presence of 480 bp fragments represented I allele, and the presence of 191 bp and 480 bp fragments represented D/I allele. PCR was performed by denaturation at 95℃ for 30 s, annealing segment at 55℃ for 30 s, and extension at 72℃ for 30 s, repeated for 35 cycles. Amplification products of polymorphic b-Fg-455 gene were digested by Hae III (Thermo, FD0154) and separated on 2% agarose gel (Fig. 1). The presence of 343 bp, 383 bp, 575 bp fragments represented the GG allele, the presence of 343 bp and 958 bp fragments represented AA allele, the the presence of 343 bp, 383 bp, 575 bp, 958 bp fragments represented G/A allele. PCR was performed by denaturation at 94℃ for 30 s, annealing segment at 55℃ for 30 s, and extension at 72℃ for 80 s, repeated for 35 cycles. Amplification products of polymorphic b-Fg-148 gene were digested by Hind III (Thermo, FD0505) and separated on 2% agarose gel (Fig. 1). The presence of 100 bp and 200 bp fragments represented the CC allele, the presence of 300 bp fragments represented TT allele, the presence of 100 bp, 200 bp, 300 bp fragments represented T/C allele. PCR was performed by denaturation at 94℃ for 30 s, annealing segment at 58.5℃ for 30 s, and extension at 72℃ for 30 s, repeated for 35 cycles. Amplification products of polymorphic MTHFR gene were digested by Hinf (Thermo, FD0804) and separated on 2% agarose gel (Fig. 1). The presence of 100 bp and 200 bp fragments represented the TT allele, the presence of 300 bp fragments represented CC allele, the presence of 100 bp, 200 bp, 300 bp fragments represented T/C allele. PCR was performed by denaturation at 94℃ for 30 s, annealing segment at 58℃ for 30 s, and extension at 72℃ for 30 s, repeated for 35 cycles. Amplification products of polymorphic PAI-1 gene were separated on 2% agarose gel. The PCR products were recovered and submitted to sequencing. PCR was performed by denaturation at 94℃ for 30 s, annealing segment at 58.5℃ for 45 s, and extension at 72℃ for 30 s, repeated for 35 cycles. Amplification products of polymorphic ApoE e2,3,4 gene were separated on 2% agarose gel. The PCR products were recovered and submitted to sequencing. PCR was performed by denaturation at 95℃ for 30 s, annealing segment at 60℃ for 45 s, and extension at 72℃ for 55 s, repeated for 35 cycles. First-generation sequencing technique was used in this study ( Jinweizhi Biotech co. LTD, Suzhou, China).
Statistical analysis
We used SPSS statistical software version 21.0 for the statistical analysis. Pearson chi-square test or Fisher's exact test were used for counting data. Continuous variables were displayed as mean ± SD. Single factor of variance was used to analysis patient’s age, GLU, CHOL, TG, LDL-C. We used odds ratio (OR) and 95% confidence intervals (95% CI) to assess the correlation between polymorphism sites and abnormal risk of laboratory parameters. Anova was used to calculate the best control value of laboratory parameters. P<0.05 was considered to be obviously statistical significance.