Methylation Analysis in Uygur and Kazak Patients with Hypertension Complicated with Left Ventricular Hypertrophy: Ethnic Difference Based On High-density Genomic DNA Methylation Chips

Background:This study aimed to analyze the methylation level in Uygur and Kazak patientswith hypertension complicated with left ventricular hypertrophy using high-density DNA methylation chips. Methods: Three Uygur and three Kazak patients with hypertension complicated with left ventricular hypertrophy were selected for DNA extraction and bisulfite conversion. Left ventricular hypertrophy was defined by echo Penn convention as left ventricular mass index≥115 g/m2 in men and ≥95 g/m2 in women. Illumina HD450K Infmium Methylation BeadChip was applied to detect 450,000 methylation sites of the whole human genome.The cluster analysis and significance test were carried out to analyze the different methylation levelsin Uygur and Kazak patients with left ventricular hypertrophy. The Delta Data were screened for statistics.Results: A total of 236genes (including 84 hypermethylated and 152 hypomethylated) showed significant differences in the degree of methylation between Uygur and Kazak patients.The average methylation level of significantly different genes was higher in the experimental group compared with the control group.The chromosome distribution of aberrant methylationgenes was quite even. The cluster analysis of the candidate genes showed thataberrant methylation genes in Uygur and Kazak patients with hypertension complicated with left ventricular hypertrophy mainly participated in cell adhesion,signal transduction, and cell cycle.Conclusions:Different methylation genes were found in Uygur and Kazak patients with hypertension complicated with myocardial hypertrophy, providing the basis for further studies on the molecular mechanism and prevention and treatment ofhypertension complicated with target organ damage among different nationalities in Xinjiang.


Background
Kazak nationals in Xinjiang is a high-risk group of hypertension. The prevalence of 3 hypertension and blood pressure among adolescents is higheramong Kazak nationals than among Uygur nationals, and the prevalence increases more obviously after the age of 25 years [1] . Data show that the increase in the relative risk of hypertension and the degree of blood pressure is higher in Kazak people than in Uygur people [2,3] , but the prevalence of myocardial hypertrophy, heart failure, and atrial fibrillation caused by hypertension is lower than thatin Uygur people.
The cases of 12,449 patients with hypertension at the First Affiliated Hospital of Xinjiang Medical University in May 2015 were reviewed. Themerged disease prevalence of Kazak revealed ethnicity-related differences in patients with left ventricular hypertrophy [4] . The blood pressure level could not fully explain the differences in hypertension complicated by myocardial hypertrophy [5] .
The role played by DNA methylation in the occurrence and development of hypertension and target organ damage in different ethnic groups and its pathophysiological mechanism need further exploration and research.

Participants
Uygur and Kazak patients with hypertension complicated with left ventricular hypertrophy admitted to the First Affiliated Hospital of Xinjiang Medical University in 2016 were selected for diagnosing primary hypertension according to the diagnostic criteria of hypertension in the guidelines for the prevention and treatment of hypertension in China 2018 [6] . Left ventricular hypertrophy was defined as left ventricular mass index 110g/m 2 in males and 95g/m 2 in females measured by color Doppler. All patients diagnosed with secondary hypertension based on medical history inquiry, physical examination, and related auxiliary examination and those not associated with severe coronary heart disease, cardiac valvular disease, cardiomyopathy, pulmonary heart disease, hyperthyroidism, diabetes, hepatic and renal insufficiency, malignant tumors, and connective tissue disease were excluded from the study. Three Uygur and three Kazak patients with matching age, gender, hypertension course, blood pressure level, and left ventricular quality index were selected, with a male-to-female ratio of 2:1. The average age of Uygur and Kazak patients was 52.7 and 52.1 years, respectively.

Screening of differential methylation sites and genes
The beta score was determined using the log (Beta)/(1 -Beta) formula.Benjaminiand Hochberg method was used for calculating FDR. The P value was less than 0.05, and the average beta score difference between the two groups was greater than 0.2. The differentially methylated regions (DMRs) were screened.The results showed that DMRs had 236 genes (84 with increased methylation degree and 152 with decreased methylation degree ( Figures. 1 and 2).   (Table 1)

Pathway analysis
The database was used to analyze the genes corresponding to different methylation loci, and the significance of gene enrichment in each item was calculated using statistical tests. The analysis of the differentially expressed genes revealed entries enriched with 7 differentially expressed genes. The differentially expressed genes in different samples that might be related to the changes in cell pathways were searched. It was found that these gene pathways mainly focused on endocytosis, cell adhesion, immune system, carbohydrate metabolism, and other aspects ( Figure. 6)

Discussion
The etiology of primary hypertension is closely related to genetic and environmental factors. Of these, genetic factors, namely susceptibility genes, may play a decisive role in the occurrence of hypertension [7,8] . Relevant studies suggested ethnic and national differences in left ventricular remodeling in patients with essential hypertension. The left ventricular remodeling was higher in Uygur patients than in the Han and Kazak patients.
Also, the tolerance to high blood pressure load of Kazak patients was high, but left ventricular hypertrophy was lower than that inUygur patients [9] .With the development of molecular genetics and molecular biology technology in recent years, the etiology of hypertension has been analyzed from the perspective of genes.The incidence of hypertension may be related to some susceptible genes [10] . The pathogenesis and pathological process of hypertension are more complex and difficult to explain.
Consequently, the research on DNA methylation modification and hypertension has not achieved much progress. However, some studiessuggested that abnormal DNA methylation modification might affect the expression of some candidate genes related to hypertension and ultimately play a role in the disease process [11,12] .

Conclusions
In this study, the genome-wide methylation levels were compared among Uygur and Kazak Competing interests: The authors declare that they have no competing interests.

Funding:
Natural science foundation of xinjiang autonomous region. Financial support was provided for the design of research, the collection, analysis and interpretation of data.  Figure 1 Different methylation sites of Uygur and Kazak groups.

Authors' contributions:Xi
13 Figure 2 Different methylation genes of Uygur and Kazak groups.
14 Figure 3 Volcanic map of the degree of difference between the two sample groups.
15 Figure 4 Cluster diagram of different methylation sites and samples.