Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder

Abstract

The variability in phenotypic presentations and the lack of consistency of genetic associations in mental illnesses remain a major challenge in molecular psychiatry. Recently, it has become increasingly clear that altered promoter DNA methylation could play a critical role in mediating differential regulation of genes and in facilitating short-term adaptation in response to the environment. Here, we report the investigation of the differential activity of membrane-bound catechol-O-methyltransferase (MB-COMT) due to altered promoter methylation and the nature of the contribution of COMT Val158Met polymorphism as risk factors for schizophrenia and bipolar disorder by analyzing 115 post-mortem brain samples from the frontal lobe. These studies are the first to reveal that the MB-COMT promoter DNA is frequently hypomethylated in schizophrenia and bipolar disorder patients, compared with the controls (methylation rate: 26 and 29 versus 60%; P=0.004 and 0.008, respectively), particularly in the left frontal lobes (methylation rate: 29 and 30 versus 81%; P=0.003 and 0.002, respectively). Quantitative gene-expression analyses showed a corresponding increase in transcript levels of MB-COMT in schizophrenia and bipolar disorder patients compared with the controls (P=0.02) with an accompanying inverse correlation between MB-COMT and DRD1 expression. Furthermore, there was a tendency for the enrichment of the Val allele of the COMT Val158Met polymorphism with MB-COMT hypomethylation in the patients. These findings suggest that MB-COMT over-expression due to promoter hypomethylation and/or hyperactive allele of COMT may increase dopamine degradation in the frontal lobe providing a molecular basis for the shared symptoms of schizophrenia and bipolar disorder.

Figure 1

Representative examples of the MSP and sequence analyses of MB-COMT promoter. Brain samples from schizophrenia and bipolar disorder patients and control subjects were analyzed using MSP as outlined under Materials and Methods. (A) MSP: lanes U and M indicate the presence of unmethylated and methylated templates, respectively. Placental DNA (PD) and in vitro methylated DNA (ID) served as negative and positive controls, respectively. A no-template sample (H₂O) was used to detect DNA contamination in the PCR reactions. Samples 1–3 indicate the presence of a methylated promoter DNA and samples 4–7 represent an unmethylated promoter. (B) Bisulfite sequencing of the MB-COMT promoter of an un-methylated template (sample 4) correlates with conversion of cytosine (C) to thymine (T) in the DNA sequence traces in the promoter region corresponding to CpGs (U). However, the samples exhibiting methylation (sample 1) contained sequence traces for Cs in addition to Ts at several CpGs (which include two SP1 binding sites), indicating that the template is a mixture of unmethylated and methylated DNA (M). The color codes for the nucleotides are as follows: red, thymine; green, adenine; black, guanine and blue, cytosine. The original DNA sequence is indicated at the top of the trace; red Cs are within CpG sequences and are targets for methylation.

Figure 2

The effect of laterality of brain in MB-COMT promoter DNA methylation. The frequency of methylated MB-COMT promoter in the left and right hemispheres of the brains of patients with schizophrenia (SCZ) or bipolar disorder (BPD) and normal controls.

Figure 3

MB-COMT expression profiles of schizophrenia and bipolar disorder and the relationship between methylation status and MB-COMT expression. (A) Semi-quantitative RT-PCR of schizophrenia and control samples was performed as described under Materials and Methods. Control samples were loaded in lanes 1–5 and samples from schizophrenia patients were loaded in lanes 6–10. βActin was used as an internal standard to normalize the abundance of the RT-PCR product derived from the MB-COMT gene. (B) qRT-PCR analysis of the patients and control subjects was performed as described under Materials and Methods. For each sample (1–35, X-axis), the relative quantitation of the MB-COMT expression compared with the mean of the controls is sorted from minimum to maximum level (Y-axis). As indicated earlier, most of the schizophrenic (SCZ) and bipolar disorder patients (BPD) show higher levels of expression of the MB-COMT in the frontal lobe compared with the controls. (C) MSP analysis showing levels of methylation in representative samples from (A).

Figure 4

Relationship between the expression of MB-COMTand DRD1. The upper (A) and lower (B) panels represent the gene expression profiles of high and low expressive MB-COMT groups, respectively, in total samples, control subjects, schizophrenia (SCZ) and bipolar disorder patients (BPD). As it is shown, when MB-COMT expression is high, the expression of DRD1is low and vice versa, both in control subjects and in the patient group implying that, in general, hyper-expression of MB-COMT is associated with hypo-expression of DRD1.

Figure 5

COMTVal158Met polymorphism in schizophrenia and bipolar disorder. The COMT Val158Met polymorphism analysis was performed as described under Materials and Methods. Briefly, COMT gene-specific PCR products encompassing the polymorphic region were cleaved by the NlaIII restriction enzyme to an 88 bp fragment and two variable fragments 96 and 114 bp in length, which indicated the Met and Val alleles, respectively. The first lane contains a 50 bp DNA ladder used as the marker. Representative examples are shown for heterozygosity (lanes 2, 4, 5 and 7), Met homozygosity (lanes 3 and 9) and Val homozygosity (lanes 6, 8 and 10).