The Study On The Effect Of SLC6A4 Gene Methylation And Family Environment On Anorexia Nervosa

[Objectives]1.To clarify the characteristics of DNA methylation of the SLC6A4 gene promoter region among untreated AN patients and its association with the clinical symptoms;2.To explore the predictive effect of the change of SLC6A4 gene methylation rates on the change of clinical symptoms in AN patients after 12 weeks follow-up;3.To explore the effect of the family environment on SLC6A4 gene methylation and clinical symptoms in patients with AN.[Methods]Ninety-one untreated outpatients meeting DSM-IV-TR criteria of AN and 87 matched normal controls(NC)were recruited.All participants underwent the test of SLC6A4 gene methylation and clinical symptoms at baseline;51 AN patients were followed-up with regular treatment in clinc and 28 AN patients were followed after 12 weeks,and underwent the tests of SLC6A4 gene methylation,mRNA expression and platelet 5-HT;41 AN and 46 NC cases underwent the evaluation of family environment.The analysis of SLC6A4 gene methylation rates,the mRNA expression of SLC6A4 gene and the 5-HT concentration in platelet was measured using MassARRAY compact system,Taqman real-time PCR and high performance liquid chromatography,respectively.The related clinical symptoms of AN were evaluated using scales including general state examination,EAT-26,EDE-Q6.0.The related family environment of AN were evaluated using FES-CV,EMBU.[Results]1.The characteristics of SLC6A4 gene methylation in untreated AN patients and its relationship with clinical symptoms in the cross-sectional study(AN=91,NC=87):(1)the AN patients showed hypermethylation of promoter region of SLC6A4 gene,the methylation rates of CpG3,CpG4,CpG6 sites and mean methylation in AN group were significantly higher than those in NC group(all p values<0.05);(2)Compared with the NC group,the AN patients had lower mRNA expression level in PBMC and lower 5-HT concentration in platelet(all p values<0.05);there was a trend for significant negative association between the SLC6A4 methylation and mRNA expression level in AN group(r=-0.277,p=0.065);(3)the mathylation rate of CpG3 site was positive associated with EDEQ-6.0 total score in AN group(r=0.226,p values<0.05).2.The predictive effect of the change of SLC6A4 gene methylation after 12 weeks treatment on the change of clinical symptoms in AN in the longitudinal study(AN=28):(1)the mRNA expression level,platelet 5-HT concentration,BMI,the total scores of EAT-26 and EDE-Q6.0 differed significantly after 12 weeks treatment(all p values<0.05),except the methylation rates of SLC6A4 gene;(2)the change of methylation rates of CpG1 site was positively associated with the change of EDE-Q6.0 total score after 12 weeks treatment(r=0.556,p=0.028);the other associations between the methylation rates of CpG sits and mRNA expression level,5-HT concentration were not statistically significant(all p values>0.05);(3)The comprehensive analysis using ANCOVA showed that the effect of CpG1 on the change of EDEQ-6.0 were not significant(p>0.05).3.The effect of family environment on SLC6A4 methylation and clinical symptoms in the retrospective study(AN=40,NC=41):(1)the scores of fathers' rejection denial and over-protection factors in EMBU were significantly associated with the methylation rate of CpG3 site in the AN group,respectively(r=0.425,p=0.038;r=0.362,p=0.048);(2)the score of cohesion factor in the FES-CV was positively associated with the total score of EDE-Q6.0 in the AN group(r=-0.291,p=0.050);all of the scores of fathers' rejection and denial factor,fathers' over protection factor in the EMBU were positively associated with the total score of EDE-Q6.0 in the AN group(r=0.330~0.437,all p values<0.05);(3)The comprehensive analysis of the effects of parent rearing styles and CpG3 on the AN clinical severity showed that only the faters' s over protection factor was significant(p=0.030).[Conclusions]1.The DNA methylation of the SLC6A4 gene promoter region plays a crucial role in the pathogenesis of the AN,and the characteristic of the SLC6A4 gene among untreated young women AN patients was hypermethylation and low mRNA expression level;2.Our preliminary follow-up study in AN patients demonstrated that the SLC6A4 gene methylation may not change during 12 weeks regular clinical treatments,and have no predictive effect on the change of clincal symptoms;3.Family environment,especially parental rearing styles,might not influence the methylation rate of SLC6A4 gene,but influence the severity of the clinical symptoms of AN patients.