br Materials and methods br

Materials and methods
Results
Discussion In this study, we uncovered a female-specific haplotype-MDD vulnerability association, by which a two-marker haplotype TG of rs4633-rs4680 may link to enhanced MDD risk in females. Female haplotype TG carriers were estimated to have a 9.17-fold increased risk to develop MDD compared to their counterparts. Homozygous TG is associated with elevated total-COMT mRNA in MDD and controls, and high MB-COMT mRNA in MDD subjects. The TG female control group has relatively higher MB-COMT protein triapine than non-TG male MDD subjects and controls, and the TG male and female MDD groups. In contrast, non-TG female controls have relatively higher S-COMT protein expression. Of controls, all groups have relatively higher COMT specific activity; also, homozygous TG, CG group and non-TG female controls have relatively lower COMT activity than the other groups. Our findings indicate that in females harboring haplotype TG of rs4633-rs4680 may be linked to relatively higher MB-COMT protein and COMT specific activity, which serves as a predisposing factor leading to enhanced MDD risk. rs4633, a synonymous SNP in the COMT coding region, was reported as one of 4 central SNPs (rs6269, rs4633, rs4818 and rs4680) in the COMT locus combining into three common haplotypes (high: GCGG, intermediate: ATCA, and low: ACCG). These haplotypes were also characterized as affecting the local stem-loop structure of mRNA molecules, where the low COMT activity haplotype retains relatively stable mRNA structure and consequently, less-transcribed low protein production and enzyme activity (Nackley et al., 2006). Another group studying the same 4 central COMT SNPs pointed out that haplotype GT, GTG, and GTGG were more frequently detected in MDD subjects than in controls (Kocabas et al., 2010), in which rs4633 and rs4680 are carrying the T and G allele, respectively. In this study, rs4633 was estimated to share high LD with rs4680, forming haplotype TG and modulating MDD risk, probably through interacting with female gender and mRNA regulation, MB-COMT protein content, and COMT activity in controls. The previous study pointed out that MB-COMT is predominantly expressed in neurons, and its C-terminus catalytic domain appears responsible for the degradation of synaptic and postsynaptic dopamine (Chen et al., 2011). In contrast, S-COMT is suggested to be expressed more abundantly in lymphocytes, and S-COMT is characterized with higher catalytic activity but lower substrate affinity than MB-COMT (Lotta et al., 1995). At the mRNA level, we only observed that the homozygous TG group had a significantly higher total-COMT mRNA expression than other groups. Also, the homozygous TG MDD group also highly expressed MB-COMT mRNA. In general, the MB-COMT mRNA levels were significantly higher in different haplotype MDD groups than in controls. Nevertheless, this finding is not consistent with the data for COMT protein expression and enzyme activity. Homozygous TG MDD had the highest expression of MB-COMT mRNA in our analysis, but we thought that the MB-COMT mRNA data should be interpreted with great caution. At the protein level, unlike the findings at the mRNA level, the homozygous TG and CG MDD groups had lower MB-COMT expression compared to controls; also, the homozygous TG MDD expressed less S-COMT protein than other groups. Further analysis revealed that female TG controls had higher MB-COMT and S-COMT protein expression though the S-COMT protein level was not the highest. Compared with the non-TG female control group, female TG controls had relatively higher MB-COMT protein and COMT specific activity but not higher S-COMT protein levels.