Promoter Specific Methylation of SSTR4 is Associated With Alcohol Dependence in Han Chinese Males

Abstract

Alcohol dependence (AD), a disease can be affected by environmental factors with epigenetic modification like DNA methylation changes, is one of the most serious and complex public health problems in China and worldwide. Previous findings from our laboratory using the Illumina Infinium Human Methylation450 BeadChip suggested that methylation at the promoter of SSTR4 was one of the major form of DNA modification in alcohol-dependent populations. To investigate whether DNA methylation levels of the SSTR4 promoter influence alcohol-dependent behaviors, genomic DNA was extracted from the peripheral blood sample of 63 subjects with AD and 65 healthy controls, and pyrosequencing was used to verify the results of BeadChip array. Linear regression was used to analyze the correlation between the methylation levels of SSTR4 promoter and the scores of alcohol dependence scales. Gene expression of SSTR4 in brain tissue was obtained from the Genotype-Tissue Expression (GTEx) project and Human Brain Transcriptome database (HBT). We found the methylation levels of SSTR4 in AD group were significantly lower than healthy controls (two-tailed t-test, t = 14.723, p < 0.001). In addition, only weak to moderate correlations between the methylation levels of the SSTR4 promoter region and scale scores of Alcohol Use Disorders Identification Test (AUDIT), Life Events Scale (LES) and Wheatley Stress Profile (WSS) based on linear regression analyses (AUDIT: R ² = 0.35, p < 0.001; LES: R ² = 0.27, p < 0.001; WSS: R ² = 0.49, p < 0.001). The hypomethylated status of SSTR4 may involve in the development of AD and increase the risk of AD persistence in Han Chinese males.

Keywords: Han Chinese; SSTR4; alcohol dependence; gene expression; hypomethylation.

FIGURE 1

The flowchart of research. We used the sequence in the box by pyrosequencing to examine the methylation level of cg01471923 (chr20:23015091), a CpG site located at the promoter region of gene SSTR4.

FIGURE 2

The correlation between the methylation value of the SSTR4 promoter region and AUDIT, LES and WSS scores. (A) The methylation value of the SSTR4 promoter region and the AUDIT score were negatively correlated. (B) The methylation value of the SSTR4 promoter region and the LES score were negatively correlated. (C) The methylation value of the SSTR4 promoter region and the WSS score were negatively correlated. (D) Bubble plot for the visualization of association of SSTR4, AUDIT4, LES and WSS.

FIGURE 3

The methylation difference of the SSTR4 promoter region in 10 paired siblings with microarray and case-controls with pyrosequencing. (A) Box plot of the methylation value of the SSTR4 promoter region with microarray and 10 paired siblings. (B) Box plot of the methylation value of the SSTR4 promoter region with pyrosequencing and case-controls. ^∗ p < 0.05,^∗∗ p < 0.001.

FIGURE 4

Gene expression of SSTR4 by GTEx and HBT. (A) Spatial expression pattern of the SSTR4 gene in human brain regions from GTEx. TPM = transcripts per kilobase million. Expression threshold: >0.1 TPM and ≥6 reads in 20% or more of samples. Box plots are shown as median and 25th and 75th percentiles; points are displayed as outliers if they are above or below 1.5 times the interquartile ranges. Data Source: GTEx Analysis Release V8 (dbGaP Accession phs000424. v8. p2). (B) Dynamic expression pattern of the SSTR4 gene in 6 human brain regions across lifespan from HBT. NCX, neocortex; CBC, cerebellar cortex; MD, mediodorsal nucleus of the thalamus; STR, striatum; AMY, amygdal; HIP, hippocampus. Period 1, Embryonic development; Period 2, Early fetal development; Period 3, Early fetal development; Period 4, Early mid-fetal development; Period 5, Early mid-fetal development; Period 6, Late mid-fetal development; Period 7, Late fetal development; Period 8, Neonatal and early infancy; Period 9, Late infancy; Period 10, Early childhood; Period 11, Middle and late childhood; Period 12, Adolescence; Period 13, Young adulthood; Period 14, Middle adulthood; Period 15, Late adulthood.