تعامل محیط زیست ژن با یک متغیر مهارکننده های مونوآمینواکسیداز؛ یک تقویت کننده رونویسی با اختلال شخصیت ضد اجتماعی در ارتباط است

Abstract Monoamine Oxidase A (MAOA) is a critical enzyme in the catabolism of monoaminergic neurotransmitters. MAOA transcriptional activity is thought to be regulated by a well characterized 30 base pair (bp) variable nucleotide repeat (VNTR) that lies approximately ∼1000 bp upstream of the transcriptional start site (TSS). However, clinical associations between this VNTR genotype and behavioral states have been inconsistent. Herein, we describe a second, 10 bp VNTR that lies ∼1500 bp upstream of the TSS. We provide in vitro and in silico evidence that this new VNTR region may be more influential in regulating MAOA transcription than the more proximal VNTR and that methylation of this CpG-rich VNTR is genotype dependent in females. Finally, we demonstrate that genotype at this new VNTR interacts significantly with history of child abuse to predict antisocial personality disorder (ASPD) in women and accounts for variance in addition to that explained by the prior VNTR.

Introduction Monoamine Oxidase A (MAOA) is perhaps one of the best characterized genes in behavioral sciences. The gene consists of 15 exons that give rise to two splice variants of 2.1 and 5 kb that both code for a 527 amino acid protein ( Billett, 2004 and Chen et al., 1991). Transcription of MAOA is thought to be moderated by two regulatory motifs. The first is a 30 base pair (bp) variable nucleotide repeats (VNTRs) whose biological activity has been extensively examined with the majority of studies concluding that 4 repeat (4R) allele is associated with greater transcriptional activation than the 3 repeat (3R) allele ( Beach et al., 2010, Cirulli and Goldstein, 2007, Guo et al., 2008 and Hotamisligil and Breakefield, 1991). The second regulatory motif is a set of two CpG islands flanking this VNTR ( Philibert et al., 2008a). Despite this understanding of transcriptional regulation at MAOA and extensive evidence that alterations in MAOA protein activity are associated with behavioral illness including smoking, depression and aggression ( Berlin and Anthenelli, 2001, Brunner et al., 1993, Fowler et al., 1996 and Shih et al., 1999), the association between genotype at this VNTR and any behavioral illness remains ambiguous ( Craig and Halton, 2009, Fan et al., 2010 and Li and He, 2008). The potential reasons for this ambiguity are numerous and include the possibility that difficulties in quantitating gene–environment interactions at this locus may be confounding attempts to directly link VNTR genotype to phenotype ( Caspi et al., 2002 and Kim-Cohen et al., 2006). However, another possibility is that previously unappreciated genetic variation may also be confounding our efforts to link VNTR genotype to phenotype. This is particularly important for our studies of antisocial personality disorder (ASPD) in the Iowa Adoption Studies (IAS), the largest case and control adoption study of substance use and ASPD in the United States. In previous studies of this cohort, Cadoret and colleagues have shown strong gene–environment interactions (G × E) effects for ASPD and ASPD spectrum behavior (Cadoret et al., 1995, Cadoret et al., 2003 and Riggins-Caspers et al., 2003). Spurred by the seminal findings of Caspi and colleagues who demonstrated significant G × E effects for ASPD at the previously described MAOA VNTR ( Caspi et al., 2002), we recently examined our cohorts and found evidence supporting the original findings ( Beach et al., 2010). These confirmatory findings using the IAS are particularly invigorating because the randomized adoption paradigm implemented by Dr. Cadoret ensures independence of genetic and environmental variables ( Yates et al., 1998). However, the effect was weaker than expected given the richness of the IAS for the expected outcomes. In addition, although we were also able to confirm prior in vitro findings showing an effect of the VNTR variation on gene activation ( Beach et al., 2010), the effects were rather modest and the association of methylation with genotype was not entirely consistent with our understanding of the role of methylation in the regulation of this gene ( Philibert et al., 2010). Therefore, we began to look for alternative genetic variation. Specifically, we hypothesized that there may be other genetic variation near the transcription start site of MAOA besides the previously described VNTR that could account for some of the discrepancies observed in the literature. During the course of this examination, we noted a CpG rich region near the previously described VNTR that had the hallmarks of a repetitive DNA element. In this communication, we report the discovery of this second VNTR, which we designate MAOA P2, approximately 1500 bp upstream of the previously described VNTR, which we designate MAOA P1. We present evidence that it is functional and describe its genotypic distribution and relationship to DNA methylation. Then using the IAS, we present evidence that G × E interplay at this locus may help improve our prediction of antisocial personality disorder (ASPD) in women.

. Results The sequence for the MAOA P2 VNTR is given in Fig. 1. The sequence contained within the GenBank reference represents the consensus sequence for the 10 repeat (10R) P2 allele. The repetitive region consists of two decamer repeats, CCCCTCCCCG (A Repeat) and CTCCTCCCCG (B Repeat). The sequence of the first 60 bp (6 repeat units) of the element is invariant in all subjects examined with the exception of a C to T polymorphism present at bp 6365508 (see Fig. 1). Variation in the enhancer region's length results from a variable number of “A” repeats after the first 60 bp. For example, the 7 repeat (7R has a structure of ABABABA, the 8R allele has a structure of ABABABAA, while the 10R allele has a structure of ABABABAAAA. The sequence and structure of the MAOA P2 VNTR region. Sequence numbering is ... Fig. 1. The sequence and structure of the MAOA P2 VNTR region. Sequence numbering is from the GRCh37 reference assembly. Two decamer repeat units are found in the region: CTCCTCCCCG (red) and CCCCTCCCCG (yellow). In areas with consecutive repetitive repeats, the boundary between the repeated domains is illustrated by alternating single and double underlining. The VNTR region is relatively enriched in CpG residues, which are illustrated in blue. The position of the primers used to amplify the repeat is double underlined. The position of a C to T polymorphism (the T allele is present in the 10R allele only) is denoted by the box at bp 6365508. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Figure options The region was genotyped in the Iowa Adoption Studies (IAS) population as described in the methods for both the MAOA P1 and P2 VNTRs. Table 1 gives the clinical characteristics of the cohort while Table 2 gives the distribution of the alleles for both the MAOA P1 and P2 alleles. As Table 1 illustrates, the adoptees are largely a largely White population who at the time of phlebotomy during the 2004–2008 wave, were in their late 40s. Consistent with the intentional loading of the cohort for the biological diathesis for ASPD, the participants tend to have elevated ASPD symptom counts. As shown in Table 2, in this population, the 9R and 10R alleles are far and away the most common at the P2 site. Distribution of genotypes at both the P1 and the P2 VNTR were consistent with Hardy–Weinberg equilibrium. Table 1. Clinical characteristics of the Iowa Adoptions Studies. Age Male 46 ± 8 (N = 259) Female 45 ± 7 (N = 312) Ethnicity African American 14 White 535 Hispanic 14 American Indian 2 Asian 0 Unknown/other 6 Lifetime DSM-IVASPD symptom count Count Males Females 0 37 107 1 75 110 2 55 40 3 29 20 4 25 16 5 24 8 6 11 11 7 3 0 Table options Table 2. MAOA P1 and P2 distribution in the Iowa Adoption Studies. Genotype Female Male P1 VNTR 2, 2 0 2 2, 4 1 3, 3 39 87 3, 3.5 1 3, 4 112 3, 5 2 3.5, 3.5 0 7 3.5, 4 2 4, 4 123 149 4, 5 3 5, 5 0 1 Total 283 246 P2 VNTR 8, 8 0 2 8, 10 3 9, 9 179 175 9, 10 76 9, 11 19 10, 10 15 49 10, 11 4 10, 12 1 11, 11 2 15 Total 299 241 Table options The haplotype distribution of the alleles in White male subjects is given in Table 3. As table illustrates, in these subjects, the 4R repeat at P1 is in complete linkage disequilibrium with the 9R repeat at MAOA P2. However, the situation with respect to the 3R allele at P1 is more complex with the 3R P1 allele being found in association with the 7R, 8R, 9R, 10R and 11R P2 genotypes. Accordingly some individuals who would be characterized as low-activity based on their P1 allele might not be characterized as low-activity based on their P2 allele, raising the potential for characterization at P2 to account for additional variance in outcomes. Table 3. MAOA P1 and P2 haplotype distribution in the Iowa Adoption Studies. P1 genotype P2 genotype Observed 2R 10R 1 3R 8R 2 3R 9R 17 3R 10R 46 3R 11R 13 3.5R 9R 6 4R 9R 136 5R 9R 1 Because haplotypes may differ as a function of ethnicity, the current results are only for the White subjects. Results for the other subjects are available on request. Table options Because of the region's proximity to the transcription start site (TSS) of MAOA and enriched GC content, the sequence of the each of the P2 alleles was analyzed for possible enhancer activity using Proscan Version 1.7. As Table 4 indicates, significant promoter activity (cutoff score for no significant activity is 53) is predicted in both strands with score in the reverse strand remaining constant but with the score in the forward strand varying with the number of repeats. The 9R and 10R alleles are predicted to have higher activity as compared to the 8R and the 11R alleles. Interestingly, according to the ProScan results, the region containing the P1 VNTR does not contain Pol II promoter activity (data not shown). Table 4. Promoter scan analysis of MAOA P2 alleles. Allele DNA segment length Forward strand score Reverse strand score 8R 646 bp 59.61 97.91 9R 656 bp 109.53 97.91 10R 667 bp 109.53 97.91 11R 677 bp 99.88 97.91 Table options In order to further examine and validate these predictions, we constructed luciferase expression vectors to examine the relationship between sequence variation at the P2 VNTR and transcriptional activation. The complete sequence of the insert is given in supplemental Fig. 1. Briefly, at the P1 locus each ∼1.7 kb construct contains the sequence for the 3R P1 allele with constructs varying at the P2 site (i.e. having the 8R, 9R, 10R or 11R sequence). The results of the transfections are given in Fig. 2. As the figure demonstrates, the 9R repeat has the greatest amount of luciferase activity while the 10R repeat, which had the least amount of activity (9R vs. 10R, p < 0.001). The 8R and 11R had an intermediate level of luciferase activation both of which were significantly less than that of the 9R allele (p < 0.02 and p < 0.03, respectively). The Luciferase transfection assessment of enhancer activity. On the Y-axis, the ... Fig. 2. The Luciferase transfection assessment of enhancer activity. On the Y-axis, the normalized luciferase activity (construct luciferase activity/cotransfected control plasmid activity) is given for each observation (n = 4 for each group). The 9R repeat construct had the greatest activity the 8R (9R vs. 8R; p < 0.03) and 11R (9R vs. 11R; p < 0.02) repeat constructs had intermediate activity and the 10R repeat construct had the lowest activity (9R vs. 10R; p < 0.001). Figure options In prior examinations, we have demonstrated that the amount of methylation of the two promoter associated CpG islands at this locus was dependent on MAOA P1 genotype in female subjects ( Philibert et al., 2008a and Philibert et al., 2009). Using the same methylation data, we examined the relationship of methylation with respect to the P2 VNTR. As Fig. 3 demonstrates, those female subjects homozygous for the 9R allele (n = 97) had significantly less methylation than those either homozygous or heterozygous for the 10R allele (n = 46; p < 0.03). There was no significant effect of P2 genotype on MAOA methylation in the male subjects (p < 0.92). Average MAOA promoter associated CpG island methylation for the 9R and 10R ... Fig. 3. Average MAOA promoter associated CpG island methylation for the 9R and 10R alleles for male and female subjects. On the Y-axis, the average methylation Z score is indicated. Females with at least one 10R repeat (n = 46) had significantly more methylation than those who were homozygous for the 9R allele (n = 97; 9R vs. 10R; p < 0.03). There was no difference in methylation for the male subjects. Figure options Building upon prior work with this sample that reported significant interactions between history of child abuse and MAOA genotype in the prediction of adult symptoms of ASPD (see Beach et al., 2010), we wondered whether variation in the newly described promoter might enhance the prediction of ASPD. As before, we used data from the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA-II) to assess maximum lifetime symptom levels for ASPD. The SSAGA is a polydiagnostic instrument that assesses depression and antisocial personality disorder, among others, in a manner consistent with DSM-III-R, DSM-IV, and Feighner RDC (Research Diagnostic Criteria). Sample alpha coefficient was .71 for ASPD lifetime symptom scales, respectively. The correlation of lifetime ASPD symptoms between the two waves of assessments was .72, providing the average ASPD symptom scale used in the current research with an effective reliability of .84 ( Rosenthal and Rosnow, 1991). In keeping with the luciferase expression results we treated the 10 allele at the P2 promoter as the low activity allele and contrasted those who had a 10 allele with others. We examined the predictive effect of the two promoters considered simultaneously to see whether unique variance in outcomes was predicted by the interaction of child abuse with variation in each promoter region. Follow-up analyses of significant effects were conducted to explicate direction of effects. In all cases analyses were conducted separately by sex to allow characterization of the impact of the genotype variable as a function of sex. We examined the two regions (P1 vs. P2) simultaneously for those participants with complete data (N = 518). Setting direct effects of the VNTRs to zero and freely estimating both interaction terms simultaneously, we found that for males (N = 234), symptoms of ASPD were not significantly uniquely associated with the interaction term for either locus. For females (N = 284) we found that the interaction of P2 with history of child abuse accounted for significant unique variance in symptoms of ASPD (P2: β = −.229, p = 0.007, two-tailed) whereas the interaction of child abuse with variation at P1 did not account for unique variance (P1: β = .073, NS). Fit of the overall model was good (P2: χ2(2) = 2.88, NS). Controlling for age or ethnicity produced no change in the pattern of significant results. We next examined the model dropping the non-significant interaction of P1 with CMI. Symptoms of ASPD were again significantly associated with the interaction term of MAOA P2 × CMI (β = −.177, p = 0.002, two-tailed). Fit of the overall model was good (P2: χ2(1) = 2.28, NS). To explicate direction of effects at P2, we examined the correlation of CMI with ASPD within low MAOA P2 activity level. For the low activity allele females (i.e. those females who had at least one 10R allele at P2) the correlation of CMI to APSP was r(97) = .459, p < 0.001. Conversely among those with no low activity alleles, the correlation of CMI with ASPD symptoms in adulthood was r(187) = .162, p = 0.026. Thus, presence of the low activity allele at P2 was associated with significantly greater vulnerability to the disruptive effects of CMI for females. To explicate the impact of variability at P2 beyond any shared variance with the P1 promoter region, we also examined the subset of females with a low expressing variant of the P1 promoter (2R or 3R). In this subsample of N = 159 females with a 2R or 3R allele at P1, we found that those who also had a low activity allele at P2 had a correlation of CMI to APSP of r(96) = .458, p < 0.001. Conversely those with a 2R or 3R allele at P1 who did not have a low activity allele at P2 were found to have a correlation of CMI with ASPD symptoms of only r(63) = .23, p = 0.06.