Human catechol-O-methyltransferase pharmacogenetics: description of a functional polymorphism and its potential application to neuropsychiatric disorders.

Catechol-O-methyltransferase (COMT) inactivates catecholamines and catechol drugs such as L-DOPA. A common genetic polymorphism in humans is associated with a three-to-four-fold variation in COMT enzyme activity and is also associated with individual variation in COMT thermal instability. We now show that this is due to G-->A transition at codon 158 of the COMT gene that results in a valine to methionine substitution. The two alleles can be identified with a PCR-based restriction fragment length polymorphism analysis using the restriction enzyme Nla III. The identification of a gentic marker associated with significant alterations in enzyme activity will facilitate the analysis of a possible role for the COMT gene in neuropsychiatric conditions in which abnormalities in catecholamine neurotransmission are believed to occur, including mood disorders, schizophrenia, obsessive compulsive disorder, alcohol and substance abuse, and attention deficit hyperactivity disorder. In addition, this polymorphism may have pharmacogenetic significance in that it will help make it possible to identify patients who display altered metabolism of catechol drugs.

Bipolar spectrum disorders in patients diagnosed with velo-cardio-facial syndrome: does a hemizygous deletion of chromosome 22q11 result in bipolar affective disorder?

OBJECTIVE: The purpose of this study was to conduct a systematic assessment of psychiatric illness in patients diagnosed with velo-cardio-facial syndrome, a genetic syndrome that involves over 40 somatic anomalies, learning disabilities, and behavioral disorders and is associated with a microdeletion on chromosome 22q11. METHOD: Subjects were referred for psychiatric diagnostic evaluation without regard to age or previous psychiatric history. In order to establish DSM-III-R consensus clinical diagnoses for patients who ranged in age from 5 to 34 years, the Diagnostic Interview for Children and Adolescents--Revised or the Structured Clinical Interview for DSM-III-R (SCID) was used. A review of available medical and psychiatric records and a clinical interview performed by two research psychiatrists to validate specific symptoms and syndromes reported in the Diagnostic Interview for Children and Adolescents--Revised and the SCID were used to elucidate the chronological appearance and duration of symptoms. RESULTS: Sixty-four percent (N = 16 of 25) of this unselected series of patients with velo-cardio-facial syndrome met DSM-III-R criteria for a spectrum of bipolar disorders with full syndromal onset in late childhood or early adolescence (mean age at onset = 12 years, SD = 3). In addition, 20% (N = 5) met DSM-III-R criteria for attention deficit hyperactivity disorder (ADHD), while 16% (N = 4) met criteria for attention deficit disorder without hyperactivity. In contrast to previous reports of a high prevalence of schizophrenia, none of the patients was diagnosed with schizophrenia, and only four had psychotic symptoms during a phase of their illness, all in their 20s or 30s. CONCLUSIONS: Given that the prevalence of bipolar disorder in the general population is estimated to be 1.5% and that the average age at onset is 24, these findings support an unusually strong association between velo-cardio-facial syndrome and early-onset bipolar disorder and suggest that a gene deleted at the 22q11 chromosomal locus may be involved in its pathogenesis. If confirmed, these findings may provide a new and fruitful line of investigation into the molecular basis of bipolar spectrum disorders.

Analysis of the pseudoautosomal X-linked gene SYBL1in bipolar affective disorder: description of a new candidate allele for psychiatric disorders.

The absence of father-to-son transmission has been observed in a subset of families with bipolar disorder (BPD), suggestive of a susceptibility gene on the sex-linked portion of the X chromosome. This is supported by some genetic linkage studies that have provided evidence for a susceptibility locus near Xq28. We have analyzed one candidate gene on Xq28, SYBL1, which maps to the Xq pseudoautosomal region (PAR). SYBL1 encodes a member of the synaptobrevin family of proteins that is involved in synaptic vesicle docking and membrane transport. Genes in the PAR generally escape X-chromosome inactivation and have an active homolog on the Y chromosome, which would result in an increase in same-sex concordance in paternal transmitted traits. However, SYBL1 is neither expressed on the Y chromosome nor the inactive X chromosome and would therefore be expected to show typical sex-linked transmission. We have screened SYBL1 for mutations that could be tested as candidate alleles in the development of BPD. Following single-strand conformation polymorphism (SSCP) analysis and DNA sequencing, four single nucleotide polymorphisms were detected: a silent mutation at codon 108, two intron mutations without any obvious biological significance, and a G-->C transversion in the polypyrimidine tract at the 3' splice acceptor site preceding exon 8. This polymorphism, which creates a perfect 16/16 stretch of pyrimidines, was analyzed in 110 patients with BPD not selected for sex-linked transmission and 119 control subjects. The results show a statistical trend toward an increase in the frequency of the C allele in males with BPD but not females. Males: chi(2) = 3.46, 1 df, p =.06; Females: chi(2) =.20, 1 df, p =.66.

Analysis of GNAZ gene polymorphism in bipolar affective disorder.

Evidence for a bipolar disorder (BPD) susceptibility locus on chromosome 22q11 has been provided in several studies. One candidate gene that maps to this region is the G-protein alpha subunit gene Galphaz (GNAZ). We have identified a common silent polymorphism in GNAZ exon 2 by single strand conformation polymorphism analysis. The frequency of this polymorphism was determined in a control population (n=84) and in patients with BPD (n=88). The data showed a statistical trend toward a difference in the distribution of alleles in patients with BPD compared with control subjects (chi square=3.2, 1 df, P=0.073, two-tailed). No significant difference was detected when the GNAZ polymorphism was analyzed in control subjects and schizophrenia patients (n=63, P=0.92). These data continue to provide some support for a BPD susceptibility gene on 22q11, possibly in linkage disequilibrium with the GNAZ 309 polymorphism.

Association of codon 108/158 catechol-O-methyltransferase gene polymorphism with the psychiatric manifestations of velo-cardio-facial syndrome.

Velo-cardio-facial-syndrome (VCFS) is a common congenital disorder associated with typical facial appearance, cleft palate, cardiac defects, and learning disabilities. The majority of patients have an interstitial deletion on chromosome 22q11. In addition to physical abnormalities, a variety of psychiatric illnesses have been reported in patients with VCFS, including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. The psychiatric manifestations of VCFS could be due to haploin-sufficiency of a gene(s) within 22q11. One candidate that has been mapped to this region is catechol-O-methyltransferase (COMT). We recently identified a polymorphism in the COMT gene that leads to a valine-->methionine substitution at amino acid 158 of the membrane-bound form of the enzyme. Homozygosity for COMT158met leads to a 3-4-fold reduction in enzymatic activity, compared with homozygotes for COMT158val. We now report that in a population of patients with VCFS, there is an apparent association between the low-activity allele, COMT158met, and the development of bipolar spectrum disorder, and in particular, a rapid-cycling form.

Linkage studies suggest a possible locus for bipolar disorder near the velo-cardio-facial syndrome region on chromosome 22.

Velo-cardio-facial syndrome (VCFS) is a congenital anomaly characterized by multiple dysmorphisms, cleft palate, cardiac anomalies, and learning disabilities, that results from a microdeletion of chromosome 22q11. An increased prevalence of psychiatric illness has been observed, with both schizophrenia and bipolar disorder commonly being diagnosed. For these reasons, the VCFS region is an interesting candidate region for bipolar disorder. We examined this region in 17 bipolar families from three populations: 13 families from the general North American population (University of California, San Diego/University of British Columbia, UCSD/UBC), three larger families from New York, and a portion of Old Order Amish pedigree 110. Three microsatellite markers spanning 13 cM around the VCFS region were genotyped in all the families. A maximum lod score of 2.51 was obtained in the UCSD/UBC families under a dominant model at D22S303. In the combined family set, maximum lod scores of 1.68 and 1.28 were obtained at this marker under dominant and recessive models, respectively. Four additional markers were subsequently typed in selected positive families, and yielded positive lods at 6 of 7 markers spanning 18 cM in this region. Nonparametric, multipoint analyses using the affected pedigree member (APM) method also yielded suggestive evidence for linkage in both the UCSD/UBC family set (P = 0.0024) and in the combined families (P = 0.017). Affected sibpair analyses were similarly positive in the UCSD/UBC families (P = 0.017), and in the combined families (P = 0.004). These results are suggestive of a possible locus for bipolar disorder near the VCFS region on chromosome 22.

Acute lithium treatment enhances neuropeptide Y gene expression in rat hippocampus.

It has been suggested that the therapeutic action of lithium in affective disorders may be due to its inhibition of signal transduction and second messenger synthesis, in particular of the phosphoinositide (PI) pathway. Yet, previous work in neuronal cell lines indicates that lithium has an enhancing effect on gene expression mediated by protein kinase C, which is activated by the PI pathway. In this report, we have analyzed the effect of lithium on two neuropeptide encoding genes that are regulated by second messenger systems; neuropeptide Y (NPY) and proenkephalin (Enk). We find that acute treatment with lithium, resulting in serum levels that are within the therapeutic range effective in patients with mood disorders, significantly enhances basal expression of the NPY gene in rat hippocampus. In contrast, no effect on Enk expression was detected. This selective effect in a limbic structure supports the hypothesis that gene expression may be an important target of lithium's therapeutic action.

Hippocampal neuropeptide Y mRNA is reduced in a strain of learned helpless resistant rats.

The learned helpless rat is considered to be one of the better animal models of depression. A genetically inbred strain with a high vulnerability to develop helplessness (LH), as well as a highly resistant strain (NLH) have both been developed. Since the brain peptide neuropeptide Y (NPY) is involved in the regulation of a number of behaviors known to be altered in clinical depression as well as in learned helplessness, we measured the relative level of NPY mRNA in the hippocampus and cortex of control Sprague Dawley (SD), LH and NLH rats. We find that NLH rats have approximately a 30-35% decrease in basal hippocampal NPY mRNA compared with SD and LH rats. By contrast, cortical NPY mRNA and hippocampal pre-proenkephalin and somatostatin mRNA levels were not significantly different in the 3 strains. The data suggest that the regulation of NPY gene expression may be involved in the reduced vulnerability of NLH rats to develop learned helplessness.

Lack of association of catechol-O-methyltransferase (COMT) functional polymorphism in bipolar affective disorder.

Abnormal catecholamine transmission has been implicated in the pathogenesis of mood disorders. Consequently, alterations in genes that are involved in catecholamine metabolism could be potential candidates for bipolar affective disorder (BPD) vulnerability. One such candidate is catechol-O-methyltransferase (COMT). A functional polymorphism has recently been characterized that is responsible for substantial variability in COMT enzymatic activity. A relatively low activity allele is associated with a methionine residue at amino acid 158 of membrane bound COMT whereas a high activity variant has a valine at this site. We have now screened 63 unrelated patients with BPD for this functional polymorphism. However, no significant association was detected. This suggests that the codon 158 COMT polymorphism is not a susceptibility gene in BPD.

Lithium augments fos protoonocogene expression in PC12 pheochromocytoma cells: implications for therapeutic action of lithium.

It has been proposed that lithium's antimanic action is due to an effect on phosphoinositide metabolism. Second messengers generated by this pathway regulate calcium mobilization and the activity of the serine and threonine kinase, protein kinase C (PKC). Included among the targets of PKC is activation of fos protooncogene expression, a well-established component of the AP-1 transcription factor. Because of these interactions, we investigated the effect of lithium on fos gene expression in PC12 pheochromocytoma cells. We find that lithium increases the level of fos mRNA that occurs in response to receptor and postreceptor activation of PKC. Treatment with lithium also leads to an augmentation of muscarinic cholinergic-mediated fos gene expression in cells that are down-regulated as a result of excessive cholinergic stimulation. The ability of lithium to enhance the response of a down-regulated cholinergic system suggests a model for its therapeutic efficacy in affective disorders.

Lithium augments pilocarpine-induced fos gene expression in rat brain.

Lithium salts are considered the most effective agents used in treating manic-depression. Previous studies in PC12 pheochromocytoma cells indicate that lithium has a dramatic augmenting effect on expression of the fos proto-oncogene, a component of the AP-1 transcription factor. Although fos expression is activated by agonists that function through different signal transduction pathways, the lithium augmenting effect appears to be specific for receptor and post-receptor stimulators of protein kinase C (PKC). In particular, fos induction mediated by the m1 muscarinic receptor linked to PKC activation was found to be exquisitely sensitive to lithium enhancement. We now show that a similar augmenting effect can be demonstrated in rat brain. Following treatment with the muscarinic agonist pilocarpine, fos mRNA accumulates in the cortex, an effect that is blocked by the m1 antagonist pirenzepine. Rats treated with a single intraperitoneal injection of lithium chloride exhibited a substantial increase in pilocarpine-mediated fos expression. In contrast, fos expression induced in several brain regions by a single electroconvulsive shock is not augmented by lithium. The finding that short-term treatment with lithium enhances fos expression in the brain suggests a mechanism for its therapeutic action.

Effects of the antiglucocorticoid RU 38486 on the induction of learned helpless behavior in Sprague-Dawley rats.

Learned helplessness (LH) is induced by exposure to an inescapable or uncontrollable stressor which results in an inability to escape or avoid the same stressor when subsequently presented in a different context. In order to understand which central mechanisms may influence the expression of the learned helpless phenotype, we have pursued an experimental approach that seeks to elucidate the behavioral effects of glucocorticoid (GC) hormones in this animal model of depression. We have previously shown that the induction of LH behavior is enhanced by adrenalectomy, an effect that is reversed by corticosterone. In this study, our aim was to attempt to locate CNS sites responsible for the observed effects of glucocorticoids on learned helpless behavior by introducing the type II GC receptor antagonist, RU 38486 to discrete brain regions. We did not observe a significant effect in LH with acute systemic, acute dentate gyrus or intracerebroventricular injection of RU 38486 in contrast to previous studies using the Porsolt swim test, another animal model of depression. However, we were able to observe a significant change upon chronic administration to the dentate gyrus. These findings suggest that glucocorticoids exert a long-term influence on stress-induced behavior, presumably by affecting glucocorticoid responsive genes in the dentate gyrus.

Alterations in glucocorticoid inducible RNAs in the limbic system of learned helpless rats.

Glucocorticoids (GC) have an important effect on mood in humans and influence learned helplessness, an escape avoidance paradigm that is considered one of the best animal models of depression. A strong genetic component underlies the development of learned helplessness as shown by the emergence of a line of highly vulnerable rats (LH strain) through selective inbreeding. In addition, hormonal factors play a role. Adrenalectomy (adx) for example is known to increase the vulnerability to acquire learned helplessness, an effect that is reversed by glucocorticoids (GC). Since GC function primarily by modulating gene expression, hormone mediated alterations in mRNAs expressed in the brain may be important in the development of an adequate escape avoidance response. Conversely, we postulate that the deficit in escape avoidance behavior exhibited by the LH strain may be associated with an alteration in GC-mediated gene expression in the brain. To test this hypothesis, we analyzed GC-responsive mRNAs that are expressed in the hippocampus. Control Sprague-Dawley (SD) rats showed consistent alterations in mRNAs that are modulated by GC, such as type II GC receptor (GR) and metallothionein-1 (MT-1). Under our experimental conditions, both GR and MT-1 mRNA are significantly increased in the hippocampus of hormone-treated SD rats. An increase in hypothalamic GR mRNA was also observed. However, under the same experimental conditions, LH rats showed more selective hormone induced changes since GC had no effect on hypothalamic and hippocampal GR mRNA whereas a significant increase in MT-1 mRNA was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal signal transduction: a hypothetical model for bipolar affective disorder.

The molecular basis of bipolar affective disorder is poorly understood at this time. The episodic nature of the condition in which relatively euthymic periods of variable duration separate periods of mania and depression, and the specificity of lithium therapy suggests that a molecular target of the illness may be a system that bidirectionally influences neurotransmission and is affected by lithium. Signal transduction pathways, which are important mediators of neurotransmitter generated signals, may represent such a system because they: 1) generate second messenger molecules that stimulate neurotransmission and also mediate negative feedback mechanisms, and 2) appear to be a direct target of lithium's action on cells. In this paper, we present a model in which abnormal regulation of signal transduction could lead to the episodic accumulation of biologically active transducers or second messengers. These alterations may result in prolonged effector stimulation which may underlie mania, followed by excessive receptor desensitization, which may result in depression. Using our model we suggest a plausible hypothesis that can explain the clinical spectrum of the disorder and the therapeutic action of lithium.

Modulation of learned helplessness by 5-hydroxytryptamine2A receptor antisense oligodeoxynucleotides.

Serotonergic systems have been implicated in the pathogenesis of major depression in humans as well as in learned helplessness (LH), an animal model of depression. To understand the significance of neuronal responses in depression and LH that are mediated by serotonin (5-hydroxytryptamine, 5HT) receptors, we used intracerebroventricular injections to introduce a unique antisense oligonucleotide (ASO) to the 5HT2A receptor and determined its effect on LH behavior in Sprague-Dawley rats as determined by an escape-avoidance strategy. Of the rats injected with the 5HT2A receptor ASO, 8/16 rats met criteria for LH. By contrast, only 1/15 of the control group injected with 5HT2A sense oligonucleotide (SO) met criteria for LH. Quantitative receptor autoradiography revealed significant differences in 5HT2A receptor density between ASO and control sense oligonucleotides (SO), in close proximity to the injection site. Significant decreases in 5HT2A receptor density caused by oligonucleotide blockade were found in the CA3 hippocampal region. These data support the view that central 5HT, mediated by the 5HT2A receptor, participates in regulating behaviors that are affected by inescapable stress, and that the induction of behavioral depression may be specifically regulated via serotonergic pathways that terminate in this hippocampal subfield.

A molecular model for bipolar affective disorder.

The biological basis of bipolar disorder is not known. Models for the illness have been proposed that were based on the neurobiological effects of pharmacological agents that affect mood. Although of great interest, these models have not adequately explained the striking clinical pattern of illness in which patients may experience either unipolar episodes or bipolar cycles of mania and depression. We now present a new model suggesting that the unique clinical heterogeneity found in patients with bipolar disorder could be explained by a defect in a 'downstream' portion of a signal transduction pathway that can regulate two or more neurotransmitter systems that have opposite effects on neuronal activity. This model may target specific candidate genes for involvement in bipolar disorder.

Ultra-ultra rapid cycling bipolar disorder is associated with the low activity catecholamine-O-methyltransferase allele.

Bipolar spectrum disorders are recurrent illnesses characterized by episodes of depression, hypomania, mania or the appearance of mixed states. Great variability is evident in the frequency of episode recurrence and duration. In addition to regular circannual episodes, a spectrum of cycle frequencies has been observed, from the classical rapid cycling (RC) pattern of four or more episodes per year, to those with distinct shifts of mood and activity occurring within a 24-48 h period, described as ultra-ultra rapid cycling (UURC) or ultradian cycling. RC has a female preponderance, and occurs with greater frequency premenstrually, at the puerperium and at menopause. Tricyclic antidepressants and MAOIs, both of which increase functional monoamines norepinephrine, dopamine and serotonin, are known to precipitate mania or rapid-cycling in an estimated 20-30% of affectively ill patients. We have recently reported a strong association between velo-cardio-facial syndrome (VCFS) patients diagnosed with rapid-cycling bipolar disorder, and an allele encoding the low enzyme activity catechol-O-methyltransferase variant (COMT L). Between 85-90% of VCFS patients are hemizygous for COMT. Homozygosity for the low activity allele (COMT LL) is associated with a 3-4 fold reduction of COMT enzyme activity compared with homozygotes for the high activity variant (COMT HH). There is nearly an equal distribution of L and H alleles in Caucasians. Individuals with COMT LL would be expected to have higher levels of transynaptic catecholamines due to a reduced COMT degradation of norepinephrine and dopamine. We therefore hypothesized that the frequency of COMT L would be greater in RC BPD ascertained from the general population. Significantly, we found that the frequency of COMT L was higher in the UURC variant of BPD than among all other groups studied (P = 0.002). These findings indicate that COMT L could represent a modifying gene that predisposes to ultra-ultra or ultradian cycling in patients with bipolar disorder.

Differential effect of lithium on fos protooncogene expression mediated by receptor and postreceptor activators of protein kinase C and cyclic adenosine monophosphate: model for its antimanic action.

Lithium salts are the most effective agents used in treating manic-depressive illness. It has been suggested that lithium's therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have investigated the effect of lithium on these two signal transduction pathways in PC12 pheochromocytoma cells by studying a common effector target, expression of the fos protooncogene. We find that lithium, at therapeutic doses, has an augmenting effect on phosphatidylinositol (PI)-mediated fos expression induced by activating a muscarinic cholinergic pathway, whereas it has no effect, at tenfold the therapeutic dose, on fos expression induced by receptor or postreceptor activators of cyclic adenosine monophosphate (cAMP). The lithium augmenting effect is also observed when the cells are treated with phorbol esters, which directly activate protein kinase C (PKC), suggesting that the level of lithium's interaction with the IP pathway is at the postreceptor level. We also show that phorbol esters induce extensive down regulation of subsequent cholinergic and phorbol ester responsiveness as well as heterologous down regulation of cAMP responses. Treatment of down-regulated cells with lithium leads to an enhanced responsiveness when cells are rechallenged with agonists that activate PKC but not by agonists that stimulate cAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on cAMP-mediated fos but no effect on the IP pathway. The opposite effects of lithium and carbamazepine on two critical transducing systems suggest a model for the antimanic action of these agents.

Polymorphism in SNAP29 gene promoter region associated with schizophrenia.

Linkage studies indicate that chromosome 22q contains a locus, or loci, for schizophrenia (SZ) and bipolar disorder (BPD). Furthermore, the congenital disorder velo cardio facial syndrome (VCFS), which is usually caused by a 22q11 microdeletion, is associated with an increased prevalence of psychiatric disease, including SZ and BPD. One plausible candidate gene that maps to 22q11, in a region deleted in the most common form of VCFS, is SNAP29, a member of the SNAP-25 family of SNARE proteins. To search for possible functional mutations in SNAP29 that could be analyzed as candidates for 22q11-linked psychiatric problems, exons, intron-exon junctions and the promoter region were screened. No coding variants were found, although a silent mutation at codon 6 and three single nucleotide polymorphisms (SNPs) were identified in the 5' untranslated and promoter regions. One SNP, an A-->G transition 923 [corrected] nucleotides upstream of the transcription start site, showed a moderately significant difference in the distribution of alleles and genotypes in patients with SZ compared with controls (allele frequency: chi(2) = 5.57, 1 df, P = 0.018; genotype: chi(2) = 9.49, 2 df, P = 0.009; odds ratio = 1.59, 95% Cl = 1.08--2.34). No significant difference was found in patients with BPD. Although the functional significance of this mutation is not known, the tetranucleotide core sequence of the ets and IK2 families of transcription factors is altered as a result of the SNP. These data suggest that a mutation in the SNAP29 gene promoter region, or a mutation in linkage disequilibrium with the promoter SNP, may be involved in the pathogenesis of chromosome 22-linked SZ.

Mutation analysis of SYNJ1: a possible candidate gene for chromosome 21q22-linked bipolar disorder.

Genes involved in the regulation of synaptic vesicle function are potential candidates for the development of psychiatric disorders. In addition to experimental and theoretical considerations, a number of genes involved in synaptic vesicle function map to regions of the genome that have been linked to bipolar disorder (BPD) and schizophrenia (SZ). One is synaptojanin 1 (SYNJ1) which maps to 21q22.2, a chromosomal region that has been linked to BPD in a subset of families in several studies. Synaptojanin 1 is an inositol 5-phosphatase that has an important role in synaptic vesicle endocytosis. Mutation screening of 32 exons, intron--exon junctions, and 839 bases of 5'-flanking DNA resulted in the identification of 11 mutations of which four were very common and seven were very rare. Of the 11 mutations identified, several may have functional significance including two coding variants, two that may affect the binding of a transcription factor, and two that involve known splicing regulatory domains. Five bipolar patients out of 149 analyzed were found who have one of the four rare variants that were most likely to have functional significance compared with 0/148 controls. The allele frequencies for three of the four common variants were very similar in bipolar patients and controls. A slight difference in allele frequency was found for an interesting mutation we detected in intron 12 in which two non-adjacent thymidine residues are deleted in a poly-AT tract located near the exon 12 splice donor site (chi(2) = 2.45, P = 0.12, 2-tailed). Although we failed to unequivocally identify a specific SYNJ1 allele that could be responsible for putative chromosome 21q22-linked BPD, several interesting variants were found to be increased in bipolar subjects and should be further investigated.