We found a clearly significant difference between males (mean; 10.7) and females (mean; 12.1) (t = 4.69; p ≤ 0,0001), as well as between smokers and non-smokers in mean trbc-MAO activity (t = 5,86; p =< 0,0001). Smokers showed a 23% lower trbc-MAO activity compared to non-smokers. Females with a depressed state showed a significantly higher mean trbc-MAO activity than unaffected females (t = 2,02; p = 0,04).

Approximately 4.5 kb of both the MAOA and MAOB gene promotor, including the first exons, totaling 9 kb, were sequenced from a total of 148 X chromosomes. Power to discover SNPs with frequencies greater than 1% and 3% for this sample size was 77% and 100%, respectively. No variants were found in the MAOB gene. In contrast, one previously reported variation was confirmed (rs3788863) for the MAOA gene, lying within the first intron, as well as two additional variants further down stream with a minor allele frequency greater than 1%. Both the recorded and most distal variants showed complete LD with each other, therefore only the recorded variant was chosen for further analysis.

The genotyping error rate was calculated at 0,4% through males scoring as heterozygotes and from MZ twins where both twins in a pair were genotyped. These errors could not be scored differently from the sequence and therefore most likely reside in the handling of samples, e.g. contamination or labelling error.

In addition to resequencing the upstream regions, we genotyped reported SNPs in the remainder of the gene clusters by Pyrosequencing. Six of the previously reported SNPs could not be confirmed as polymorphic (rs1014876, rs3027464, rs6324, rs1040398 and two SNPs reported by Balciuniene et al.) The remaining nine polymorphic variants; one in the Norrie gene (rs766117), four SNPs in MAOB (rs1181252, rs2283729, rs3027452 and rs1799836) and four SNPs in MAOA (rs1801291, rs979605, rs6323, rs388863) were sequenced in the total sample.

The LD map (Figure 2) displays a clear structure of the MAO locus with strong LD across the MAOA gene in a distinct block spanning approximately 65 kb. The MAOB gene also displays a similar block-like structure, although the pattern of LD is not as robust as for MAOA. This is perhaps due to the inconsistencies in allele frequencies across MAOB. Interestingly, weak LD is observed at the tail ends between the two MAO loci.

Furthermore, because there was no LD between the Norrie gene variant, located >66 kb upstream of MAOB, and any other variant in the MAO region, we decided not use this variant further in the haplotype assessment. Modest deviations from Hardy-Weinberg equilibrium were noted in rs766117 in the Norrie gene (p = 0,022) and rs979605 in intron 10 of the MAOA gene (p = 0,028). This could reflect the underlying LD structure 29, as demographic influences would act over larger regions 30. However to clarify this, a denser set of SNPs would need to be genotyped.

In the male population we could identify five distinct haplotypes in the MAOB gene and four in the MAOA gene with frequencies ≥1% (Figure 1.). When analyzing the MAO locus as one large block using eight SNPs, we found ten distinct locus haplotypes with a frequencies ≥1% (data not shown). In the female population, "PHASE" assembled identical higher frequency haplotypes as were identified in the male sample, with minor discrepancies in lower frequency haplotypes due to unknown phase (Figure 1).

In the total sample, no single variant of any of the individual SNPs was associated with trbc-MAO activity. However, in females the C/C and C/T genotypes of rs979605 in the MAOA gene were associated with a significant decrease in trbc-MAO activity, (-2,9; CI 95%: -5,2 – -0,6) and (-2,4; CI 95%: -4,7 – -0,1) respectively.

Analyzed by gender, depressed state was associated with the A-allele of MAOB SNP rs1181252 in males (OR = 4,5; CI 95%: 1,0 – 21,7) and both GG and GA of rs766117 (OR = 2,2; CI 95%: 1,1 – 4,3) in females. It should be noted that the "A" allele of rs1181252 only had a population frequency of 6%.

There was no association between any of the MAOB haplotypes and trbc-MAO activity. Two MAOA haplotypes, A1 and A3, both sharing identical alleles at the three first haplotype positions (CCA-) (Figure 1), were associated with a significant decrease in trbc-MAO activity (Table 1). Analyses of the entire MAO locus and trbc-MAO activity did not reveal any significant findings (data not shown). We could not find any significant associations between depressed state and any specific haplotype in men or women (Table 2).

The participants were taken from a longitudinal twin study of aging, the Swedish Adoption/Twin Study of Aging (SATSA) with up to five occasions of measurement 47. SATSA is a sub-sample of the population based Swedish Twin Registry 48. All participants are Caucasian and born in Sweden. For the present analyses we selected all individuals who participated in an in-person testing session during which questionnaires were administered and a blood sample was drawn. The mean age of the sample was 61,3 years at the time of testing. Twenty two percent of the participants were current smokers; 35% of the males and 15% of the females.

Zygosity was initially based on self-reports of similarity and confirmed by serological analyses and comparisons of up to 10 DNA markers.

For preliminary screening of the promoter, the first exon and intron regions for novel variants, 94 Swedish male blood donors were randomly selected from a larger sample set collected to study MAOB regulation. All were between the ages of 20 to 40 years and non-smokers.

This study was reviewed and approved by the Ethics Committee of the Karolinska Institute, the Swedish Data Inspection Board, and the IRBs at the University of Southern California and the Pennsylvania State University. All subjects provided informed consent.

DNA samples were available from 573 twins. Trbc-MAO activity measures were available from 565 twins. The trbc-MAO activity is expressed as nmoles of 2-phenylethylamine oxidized per minute and per 10¹⁰ platelets. Trbc-MAO activity measures have previously been described in detail 20.

Depressive symptoms were measured with the Center for Epidemiologic Studies Depression Scale (CES-D), a 20-item self-report instrument developed for use in the community and well established for use with older adults 4950. The scale has been shown to have minimal overlap with physical illness 51 and assesses current symptoms during the past week. Respondents scoring 16 or higher on the CES-D scale are considered to have a clinically relevant depressed state. In this study population of 574 participants, 144 were classified as having a depressed state, 17.9% of the males and 30.2% of females.

Approximately 4.5 kb of each gene was initially sequenced in search of novel SNPs in both MAOA and MAOB, first in 94 Swedish males and later 45 twins with CES-D scores (36 males and 9 females). Power to detect minor allele frequencies (q) between 1 and 5% was determined as by Glatt et al. 52, 1-(1-q)^N where N is the number of chromosomes. Amplification and nested sequencing primers were designed with the CPrimers programme from Genbank entry GI:8671203 containing the promoter, coding exon 1 and flanking intronic sequence of MAOA (~5.0 kb, nucleotides 46490–51454) and Genbank entry GI:2440066 spanning the same characterized sequences of MAOB (~4 kb nucleotides 35033–39021).

Direct sequencing reactions were performed using DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences) and separated using a Megabace 1000. Reads were base called with Phred 53, assembled using Phrap and viewed using Consed Version 13 54. All SNPs were documented and cross validated with dbSNP at NCBI.

Twelve SNPs identified from public databases (dbSNP at NCBI) and two novel SNPs previously reported (introns 3 and 10 of MAOB) a Swedish sample 5 were sequenced in 95 participants (142 chromosomes) by Pyrosequencing to confirm their presence in this population. For Pyrosequencing, either the forward or the reverse primer in each primer pair was biotinylated. Sequencing primers with a length of 14 and 18 bases were placed within one base of the SNP. The PCR reaction was performed in a 50 μl reaction volume, containing 5 ng of genomic DNA, 10 pmoles of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl₂ and 1.5 U of Taq. Thermal cycling was performed in a PTC-225 DNA machine (MJ Research Inc., Cambridge, MA, USA) at 95°C for 5 min followed by 50 cycles of 95°C for 30 s, 45 s of annealing at an optimized temperature, followed by 72°C for 30 s and a final extension of 5 min at 72°C. The biotinylated PCR product was immobilized onto streptavidin-coated sepharose beads and DNA strands were separated by denaturation with 0.2 M NaOH. The pyrosequencing reaction was performed on a PSQ96™ Instrument from Pyrosequencing AB (Uppsala, Sweden) as described by 5556. Detailed primer and assay information are available upon request.

Male haplotypes could be extrapolated directly since the MAO locus is located on the X chromosome and males are thereby hemizygous. Female bi-allelic haplotypes were estimated using an EM algorithm (Sham 1998) and the pair-wise LD measures D' 57 and Δ² 58. We used "PHARE" (by David G Cox, available at http://bioinformatics.org/macroshack/programs/PHARE) to create input files for "PHASE" 5960 to construct female haplotypes.

We used linear regression to estimate the association between trbc-MAO activity and genotypic information using a generalized estimating equation (GEE) approach and alternating logistic regression (ALR) 61 to estimate the association between depressed state and genotypic information. We first modeled the association between single SNPs and each of the two outcomes and then modeled the association between haplotype constructs and the two outcomes. All estimates were adjusted for current smoking status. We estimated both dominance and co-dominance models. Explanatory variables in the dominance models were binary whereas in the co-dominance models they were coded as the number of reference alleles (i.e., 0, 1, or 2 for females and 0 or 1 for males). The parameter estimates for the co-dominance models represent the change in the outcome (trbc-MAO activity or odds of being in a depressed state) per affected allele. Due to the continuous nature of the trbc-MAO measure, only one individual from each complete twin pair and single participating individuals were analyzed (N = 340). Among females we also estimated the effect of being homozygote compared to heterozygote. If the co-dominance model is a good fit to the data then these estimates should be similar to the "per allele" estimates from the co-dominance model. All statistical analyses were performed in SAS 8.01 using GENMOD procedure (SAS Institute Inc. Cary, NC).