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Additive Interaction Between Potentially Modiable Risk Factors And Ethnicity In The Han, Tujia and Miao Populations With First-Ever Ischemic Stroke

Background: As a country with one-fth of the global population, China has had explosive growth in ischemic stroke burden with signicant ethnic and geographic disparities. The aim of this study was to examine the relative risk of potentially modiable risk factors for ischemic stroke among the Han population and two ethnic minorities (Tujia and Miao). Methods: A case-control study was conducted: 324 cases of rst-ever IS from the hospitals of the Xiangxi Tujia and Miao Autonomous Prefecture and 394 controls were surveyed using structured questionnaires from communities covering the same area. Univariate and multivariate logistic regression analyses with adjusted odds ratios (ORs) and 95% condence intervals (CIs) were used to examine the association between risk factors and ischemic stroke. The additive model was used to study the interaction between the modiable risk factor and ethnicity in the R software. Results: Higher high-sensitivity C-reactive protein level(OR 50.54, 95%CI 29.76-85.85), higher monthly family income(4.18, 2.40-7.28), increased frequency of hot pot consumption (2.90, 1.21-6.93), diabetes mellitus (2.62, 1.48-4.62), higher apolipoprotein(Apo)B/ApoA1 ratio(2.60, 1.39-4.85), hypertension(2.52, 1.45-4.40) and moderate-intensity physical activity(0.50, 0.28-0.89) were associated with ischemic stroke. There is an additive interaction between ApoB/ApoA1 ratio and ethnicity in Tujia and Miao populations with rst-ever ischemic stroke (the relative excess risk due to interaction was 5.75, 95%CI 0.58~10.92; the attributable proportion due to interaction was 0.65, 95%CI 0.38~0.91; the synergy was 3.66, 95%CI 1.35~9.93). Conclusions: It is the rst case-control study examining modiable risk factors for ischemic stroke among the Han population and two ethnic minorities (Tujia and Miao) in China, some differences were observed in the impact of risk factors among these ethnic groups. Our results may help interpret health-related data, including surveillance and research, when developing strategies for stroke prevention.


Case Selection
The case group included all IS patients who were admitted to the First A liated Hospital of Jishou University and all 8 County People's Hospitals of the Xiangxi Tujia and Miao Autonomous Prefecture within 5 days of symptom onset and 72 h of hospital admission and with a discharge diagnosis of rst-ever IS. IS was diagnosed according to the World Health Organization (WHO) clinical criteria [12]. Computed tomography(CT) or magnetic resonance imaging(MRI) of the brain was completed within 1 week of presentation. All patients also underwent a range of blood tests, chest radiography, and electrocardiography at admission. According to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria [13], IS was divided into 1) large-artery atherosclerosis, 2) cardioembolism, 3) small-vessel occlusion, 4) stroke of other determined etiology, and 5) stroke of undetermined etiology. Disease severity was estimated by the National Institutes of Health Stroke Scale (NIHSS) score on admission by the physician. For patients unable to communicate adequately, proxy respondents were used. We de ned a valid proxy respondent as a spouse or rst-degree relative who was living in the same home or was aware of the participant's previous medical history and current treatments. The exclusion criteria included those who: (1) were unable to communicate and without a valid surrogate respondent; (2) had subdural hemorrhage, tumor, or brain abscess (i.e., non-vascular causes); (3) were under current hospitalization for acute coronary syndrome or myocardial infarction; and (4) an inability to obtain consent from the patient or surrogate.

Control Selection
Controls were community-based from all 8 counties and had no history of stroke. Each control was matched for age (± 5 years) with the case. Exclusion criteria for controls were identical to those described for cases.

Assessment Of Risk Factors
Data on occupation, education, fertility, monthly family income, smoking status, frequency of fast food consumption, frequency of hot pot consumption, moderate-intensity physical activity(MIPA), and history of HT, DM and hyperlipidemia were collected by the study physicians using structured questionnaires.
Occupation was classi ed as either mental or manual work. Education was classi ed as either 9 years of school education or ≥ 9 years (senior high school or above). The history of fertility was classi ed as having given birth to ≤ 2 children or to ≥ 3 children. Smoking status was classi ed as never smoked and current smoking (smoking ≥ 1 cigarette per day within the year prior to the interview and included those who had quit smoking less than a year). MIPA was de ned as 4 h or more per week, including brisk walking, dancing, gardening, housework and domestic chores, traditional hunting and gathering, general building tasks (e.g., roo ng, thatching, painting), carrying/moving moderate loads (< 20 kg), and so on [14]. HT was de ned as under treatment with antihypertensive medication, a previous HT diagnosis, or current HT according to the 2003 WHO criteria (blood pressure of 140/90 mmHg or higher). For those with a history of DM or treated diabetes preceding IS, diabetes was de ned according to the 1999 WHO criteria as fasting plasma glucose (FPG) ≥ 7.0 mmol/L (126 mg/dL), a 2-h oral glucose tolerance test of ≥ 11.1 mmol/L (200 mg/dL), or glycated hemoglobin (HbA1c) ≥ 6.5%. We de ned dichotomous components of hyperlipidemia as total cholesterol (TC) ≥ 6.2 mmol/L (240 mg/dL), triglyceride (TG) ≥ 2.3 mmol/L (200 mg/dL), low-density lipoprotein cholesterol (LDL-C) ≥ 4.1 mmol/L (160 mg/dL), or high-density lipoprotein cholesterol (HDL-C) < 1.0 mmol/L (40 mg/dL) [15].
Waist and hip circumferences were measured in the standing and supine positions. If patients were unable to stand because of disability, these measurements were completed only in the supine position [16]. Fasting blood samples (10 mL) were taken from cases and controls within 72 h of recruitment, separated by centrifugation, and frozen at -70 °C immediately after processing. Samples were shipped in packaging incorporating dry ice cooling agents by courier from every site to a blood storage site, where they were stored at -70 °C. FPG, HbA1c, TP, TC, TG, LDL-C, HDL-C, ApoA1, ApoB and high-sensitivity Creactive protein (hs-CRP) concentrations were measured at Dian Diagnostics Group with a Beckman Coulter (Brea, CA) AU680 Clinical Chemistry Analyzer and Beckman Coulter reagents. Detailed cutoffs for WHR [17], HDL-C [15], ApoB/ApoA1 ratio [18] and hs-CRP [19] appear in Additional le 1: Table S1.

Statistical analysis
Statistical analyses were produced with IBM SPSS Statistics for Windows (version 23.0; Armonk, NY) and R for Windows(Version 4.0.3). All statistical tests were two-sided.
First, we performed a comparison between case and control groups using the Chi-squared tests and calculated univariate odds ratios (ORs) and 95% con dence intervals (CIs) for all dichotomized or multinomial risk factors. Second, binary multivariable logistic regression was applied, with backward stepwise (likelihood ratio) variable removal at the level of P < 0.10, and adjusted ORs and 95% con dence intervals (CIs) were calculated. The Breslow-Day test was used for the homogeneity of the association between modi able risk factors and IS by ethnicity [20], if P < 0.05, we will detect the biological interaction between the modi able risk factor and ethnicity. Three measures of biological interaction: RERI, the relative excess risk due to interaction; AP, the attributable proportion due to interaction; and S, the synergy [21]; index with corresponding 95% con dence intervals were calculated in R [22]. RERI and AP equal to 0 and S equal to 1 were de ned as no interaction [21]. The names and values of variables in univariate and multivariate logistic regression analyses also appear in Additional le 1: Table S1.

Basic information
The study included 324 patients and 394 controls, with ages ranging from 22 to 80 years. Descriptive characteristics of patients, including age, sex, TOAST classi cation, and NIHSS score, are reported in Table 1. One hundred and seventy (50.1%) patients had minor stroke and 115 (33.9%) had moderate stroke.  Table 2 provides the univariate analysis results for the entire population, we found all 15 risk factors to be statistically associated with rst-ever IS. Higher hs-CRP level, higher ApoB/ApoA1 ratio, DM, higher monthly family income, HT, increased frequency of fast food consumption, lower HDL-C level, increased frequency of hot pot consumption, hyperlipidemia, higher education level, higher WHR, current smoking, higher fertility number of women were all associated with an increased risk of IS, and manual work and moderate-intensity physical activity were both associated with a reduced risk of IS. We analyzed the association between all the modi able risk factors and IS and tested the homogeneity in different ethnic group with the Breslow-Day test, the results showed that only the difference of the association between higher ApoB/ApoA1 ratio and rst-ever IS in Tujia and Miao population had a statistical signi cance(P < 0.05)( Table 3).

Multivariate Logistic Regression Analysis of Risk Factors for First-Ever Ischemic Stroke
Sex, age, ethnicity and all 15 risk factors showed statistical association with IS in the univariate comparisons were included in binary multivariate logistic regression analysis. In the entire population, six signi cant risk factors for the rst-ever IS included(in decreasing order of risk) higher hs-CRP level, higher monthly family income, increased frequency of hot pot consumption, DM, higher ApoB/ApoA1 ratio, HT. Moderate-intensity physical activity was signi cantly associated with a reduced risk of IS (Table 4).  Abbreviation: Apo B, apolipoprotein B; Apo A1, apolipoprotein A1; RERI, the relative excess risk due to interaction; AP, the attributable proportion due to interaction; and S, the synergy.

Discussion
In this case-control study, we found marked differences in IS risk pro les among the Han, Tujia, and Miao populations, which may help clinicians to establish population-speci c IS prevention programs and aid in future research.
Ischemic stroke is mainly caused by atherosclerosis and thrombotic obstruction of cerebral blood ow [23][24][25][26]; therefore, the identi cation of biomarkers for atherosclerosis-artery stenosis or occlusion is essential for the early prevention of stroke. Considerable clinical observational studies of different populations have demonstrated that serum hs-CRP is a useful and powerful in ammatory marker in predicting future cardiovascular and cerebrovascular events [19,[27][28][29][30][31]. Our ndings are in accordance with those of previous studies. Higher hs-CRP level was rst demonstrated as an independent predictor for IS in all ethnic groups of the Xiangxi Tujia and Miao Autonomous Prefecture. Although CRP should be considered only as a surrogate biomarker of upstream cytokines (IL-6 and IL-1β) [32], the classical acute-phase reactant that can be measured with high-sensitivity assays seems to be relevant for risk prediction. Speci cally, higher hs-CRP levels appeared to increase the risk of recurrent stroke and vascular events in a Hs-CRP sub-study in J-STARTS [33], and hs-CRP level may be useful in individuals classi ed as "intermediate risk for IS by traditional risk factors" in the Atherosclerosis Risk in Communities (ARIC) study [34]. The ARIC study suggested that determination of plasma hs-CRP concentration could be used as an adjunct for risk assessment in primary and secondary prevention of IS, especially in different ethnic populations. However, in our study we didn't nd higher Hs-CRP concentration and ethnicity had a signi cant additive interaction effect on rst-ever IS in the Han, Tujia and Miao populations.
Consistent with previous studies, our ndings showed that HT and DM were signi cant risk factors for IS [5,35]. HT and DM are global epidemic and have been recognized as common modi able risk factors for both cardiovascular disease and stroke. Some studies have found marked ethnic differences in association between BP parameters and stroke [36][37][38]. A 10-mm Hg difference in SBP was associated with an 8%(95CI, 0%-16%) increase in stroke risk for Caucasians, but a 24%(95%CI, 14%-35%) increase for African-Americans (P interaction =0.02) [38]. Limited data exists on understanding ethnic differences in association between HT and stroke in China, and in our study there had not any additive interaction effect on rst-ever IS between HT and ethnicity in the Han, Tujia and Miao populations.
In multivariate analyses of our study, the ApoB/ApoA1 ratio was a stronger risk predictor of IS than hyperlipidemia, WHR and HDL-C level. An elevated ApoB/ApoA1 ratio mirrors an adverse imbalance between proatherogenic and antiatherogenic lipoprotein particles, which may result in enhanced atherosclerotic burden [39][40]. There are still controversies regarding lipid pro le results as risk markers of IS events [41][42][43], and recent evidence from various large studies suggested that the ApoB/ApoA1 ratio was better than the lipid pro le and in detecting IS risk [44][45], which is in agreement with our results. A study demonstrated that increased ApoB/ApoA1 ratio was independently associated with occurrence of IS in young patients [46]. Furthermore, in our study we found that ApoB/ApoA1 ratio and ethnicity have an additive interaction effect on rst-ever ischemic stroke(RERI 5.75, 95%CI 0.58 ~ 10.92). The ethnic difference in the impact of the ApoB/ApoA1 ratio on stroke risk may be related to genetics or metabolism. Further studies are needed to clarify this mechanism. Overall, there is a clear need to focus on the ApoB/ApoA1 ratio to improve the prevention and treatment of IS in patients of different ethnicities.
In our study, we also found that higher monthly family income and increased frequency of hot pot consumption were signi cantly associated with IS. Hot pot is a local cuisine in the Tujia and Miao ethnic populations, and it has been appearing on tables all year round in the Xiangxi Tujia and Miao Autonomous Prefecture. There are always chock-full of Sichuan peppercorns, chili pepper, and sour pickles in broth of the Tujia and Miao-style hot pot. And most of the ingredient are salted or pickled food, such as pickled sh, pork and chicken, in the Tujia and Miao-style hot pot. Hot pot was rst identi ed as the important risk factor for IS, and further research is needed for clari cation of the mechanism. Recent study demonstrated that moderate-to-vigorous physical activity doses equivalent to meeting the current recommendations attenuate or effectively eliminate the association between sitting and all-cause and CVD mortality risk among the least physically active adults [47], similar to the ndings reported by the current study. This study suggested that targeted interventions that promote physical activity and a healthy diet for different ethnic populations could substantially reduce the burden of IS.

Limitations And Strengths
Our study has several potential limitations. First, there may be some potential confounding factors that may not have been taken into account. Second, our sample size may be inadequate to provide reliable information about the importance of each risk factor in different ethnic groups.
This study also has some strengths. Our study included the unique ethnic customs of Tujia and Miao populations as risk factors, such as local cuisine, which may add substantial information to other commonly modi able risk factors.

Conclusions
In

Consent of publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.