br Materials and methods br Results br Discussion
Materials and methods
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 new relationships 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. Haplotype CG of rs4633-rs4680 was reported to be significantly higher in controls (p < 0.0001) than in patients with endometrial cancer (Hirata et al., 2008), where endometrial cancer tissues with genotype CC of rs4633 and genotype GG of rs4680 had relatively higher COMT expression than samples with genotype TT of rs4633 and genotype AA of rs4680. In this study, we also observed that haplotype CG is more prevalent in controls (71.4% vs. 62.6%) and homozygous CG is more frequently detected in female controls than in female MDD patients (51.2% vs. 36.6%). Nevertheless, haplotype CG is not correlated to MDD vulnerability after stratification of gender effects in logistic regression analysis. Haplotype CG was only associated with higher MB-COMT mRNA expression in MDD compared to different control groups. The homozygous CG MDD group had relatively lower MB-COMT protein expression, but the homozygous CG control group had relatively higher S-COMT protein expression. Of the different control groups, only the homozygous CG control group had higher COMT activity than the homozygous TG group. No difference was found between different haplotype groups in MDD subjects since the activities were all low and very close to each other. In agreement with previous findings, harboring haplotype CG of rs4633-rs4680 involves higher S-COMT expression and enzyme activity in controls but not in MDD. In this study, we defined haplotype CA of rs4633-rs4680 as a minor allele in our study population since it was only prevalent at 1.8% and not different between MDD (1.7%) and controls (1.8%) (P = 0.653). However, in our qPCR assay, due to sample restriction we were unable to determine whether this haplotype carrier has relatively low COMT mRNA expression. Even in the western blot and enzyme activity measurements, only one haplotype CA heterozygote (1 CG/1 CA allele carriage) in MDD subjects was collected and analyzed. Therefore, quantitative western blot data of this sample remains unrevealed and its COMT activity did not look very different from that of other MDD haplotype groups. Alternatively, we chose non-CG and TG allele carriers as the counterpart of haplotype CG (as a protective allele due to its relatively lower MDD risk) and TG (a risk allele) for the purpose of enlarging case numbers. As expected, homozygous TG allele carriers had higher total-COMT mRNA expression than other groups including the non-TG, CG allele group in both MDD and control subjects (P < 0.01).