Elevation of collagen type I in fibroblast-keratinocyte cocultures emphasizes the decisive role of fibroblasts in the manifestation of the phenotype of cyclosporin A-induced gingival overgrowth.

BACKGROUND AND OBJECTIVE: Collagen type I elevation in cyclosporin A-induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo-like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth-derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. MATERIAL AND METHODS: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. RESULTS: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. CONCLUSION: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth-phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth-derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A-induced effects under more in vivo-like conditions.

Structural variation of the monoamine oxidase A gene promoter repeat polymorphism in nonhuman primates.

By conferring allele-specific transcriptional activity on the monoamine oxidase A (MAOA) gene in humans, length variation of a repetitive sequence [(variable number of tandem repeat (VNTR)] in the MAOA promoter influences a constellation of personality traits related to aggressive and antisocial behavior and increases the risk of neurodevelopmental and psychiatric disorders. Here, we have analyzed the presence and variability of this MAOA promoter repeat in several species of nonhuman primates. Sequence analysis of MAOA's transcriptional control region revealed the presence of the VNTR in chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), rhesus macaque (Macaca mulatta) and Gelada baboon (Theropithecus gelada). The majority of P. troglodytes and P. paniscus showed a single repeat with a sequence identical to the VNTR sequence in humans. In contrast, analyses of the remaining species revealed shorter sequences similar to the first 18 bp of human VNTR. Compared with other nonhuman primates, the VNTR sequence of M. mulatta showed the highest length variability with allele frequencies of 35, 25 and 40% for the five, six and seven repeat variants, respectively. The extent of variability of the MAOA promoter repeat in both rhesus monkeys and humans supports the notion that there may be a relationship between functional MAOA expression and aggression-related traits in humans and rhesus macaque populations.

Functional PAX-6 gene-linked polymorphic region: potential association with paranoid schizophrenia.

BACKGROUND: Early differentiation of the nervous system and adult CNS neuroplasticity is modulated by PAX-6. We have shown previously that a highly polymorphic, functional AC/AG repeat in the 5' regulatory region of the gene showed significantly increased promoter activity, if containing > or = 29 repeats, and that the heterozygous genotype (< or = 28/> or = 29) revealed increased mRNA PAX-6 levels in human brain tissue compared to the homozygous short variant. METHODS: In a case-control study of 655 unrelated individuals, allele frequencies and genotype distributions of the functional PAX-6 promoter polymorphism were investigated comprising patients with DSM-IV schizophrenia, patients with affective disorders, and population controls. RESULTS: No allelic or genotypic association of the PAX-6 promoter polymorphism to affective disorder or to schizophrenia as one disease entity was observed. After subtyping schizophrenia into paranoid and nonparanoid forms, potential evidence was found for a genotypic association of the high-activity variant with the paranoid subtype of schizophrenia (p = .02). The estimated odds ratio was 1.7 (95% CI .98 to 2.95) for those heterozygous and 1.4 (95% CI .82 to 2.42) for those heterozygous or homozygous for the high-activity variant compared to the homozygous low-activity variant. CONCLUSIONS: Our finding indicates that early developmental genes may be involved in the etiopathogenesis of schizophrenia subtypes via variable transcriptional regulation in the developing and adult human brain.

Association analysis of the functional monoamine oxidase A gene promoter polymorphism in psychiatric disorders.

Functional characterization studies revealed that transcriptional activity of the human monoamine oxidase A (MAOA) gene is modulated by a polymorphic repetitive sequence located approximately 1.2 kb upstream of the ATG codon. To investigate the possible influence of the allelic variants of the MAOA gene-linked polymorphic region (MAOA-LPR) on the genetic predisposition to psychiatric disorders, we have performed a case-control association study. 174 patients with affective disorders and 258 patients with schizophrenia according to DSM-IV, as well as 229 population controls were tested. Statistical analysis showed no significant differences in allele or genotype frequencies between control and patient groups. Our results suggest that there is no association between MAOA-LPR genotype and susceptibility to recurrent major depression, bipolar disorder, and schizophrenia in our population.

Association between a functional polymorphism in the monoamine oxidase A gene promoter and major depressive disorder.

Various polymorphisms of the X-chromosomal monoamine oxidase A (MAO-A) gene were investigated for association with affective disorders. However, none of the studied variants could consistently be associated with either major depressive or bipolar affective disorder. Recently, a positive association between panic disorder and a novel functional repeat polymorphism in the MAO-A gene promoter, with the longer alleles being more active, was reported. Since monoaminergic neurotransmission is supposed to play an important role in affective disorders, we investigated a potential association of this polymorphism with major depressive illness in a sample of 146 unrelated patients of German descent and a control group of 101 individuals with a negative life history for affective disorders. Similarly to the recent findings in panic disorder, we observed a significantly increased frequency of genotypes containing only long alleles in female patients with recurrent major depression in comparison with age- and sex-matched controls. Thus, our data suggest that an excess of high-activity MAO-A gene promoter alleles resulting in an elevated MAO-A activity is a risk factor for major depressive disorder in females. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:801-803, 2000.

Regulation of PAX-6 gene transcription: alternate promoter usage in human brain.

We have isolated and characterized the 5'-flanking regulatory region of the human PAX-6 gene. Mapping of transcription initiation sites revealed the existence of an additional non-coding 5' exon, exon 1A. Functional analyses indicated that PAX-6 transcription is regulated by two distinct promoters, A and B, resulting in alternative transcription of exon 1A or 1B and joint transcription of exons 2 to 13. While a single initiation site was identified for exon 1A, transcription of exon 1B appears to be initiated from more than one site downstream of the promoter B-associated TATA motif. Multiple potential binding sites for transcription factors were found in the regions of promoter A and B. Although a 1.1-kb fragment of promoter A and a 1.5 kb fragment of promoter B, which had been fused to a reporter gene and transiently expressed in cell lines, displayed constitutive promoter activity, transcription of PAX-6 driven by promoter B was considerably higher than by promoter A in various regions of human postmortem brain. Transcript PAX-6B was primarily expressed in cerebellar cortex, whereas relatively low concentrations were detected in other brain areas. Functional dissection by serial deletions revealed several clusters of both activating elements and cell-selective silencers within the regulatory regions upstream of exon 1A and 1B. Coexpression of the promoter B constructs with a vector expressing PAX-6 modulated promoter B activity, thus indicating autoregulation by PAX-6 transcription. In conclusion, our findings suggest that PAX-6 transcription is regulated by alternate usage of promoter A and B, and that in adult human brain expression of PAX-6 is primarily controlled by promoter B. Alternate promoter usage and differential PAX-6 transcription are likely to play a critical role in brain development and neuroplasticity.

Association analysis of a regulatory promoter polymorphism of the PAX-6 gene with idiopathic generalized epilepsy.

The PAX-6 gene is a member of the paired-box-containing (PAX) gene family, encoding a transcriptional activator, that plays an important role in the development of the central nervous system. The present association study tested the hypothesis that length variation of a novel regulatory dinucleotide repeat polymorphism in the promoter region of the PAX-6 gene (PAX-6 gene-linked polymorphic region, PAX-6LPR) confers susceptibility to the epileptogenesis of common subtypes of idiopathic generalized epilepsy (IGE). The repeat length of the regulatory dinucleotide repeat polymorphism was assessed in 354 German control subjects and 125 German IGE patients, comprising 70 patients with juvenile myoclonic epilepsy (JME) and 55 patients with an idiopathic absence epilepsy (IAE). The allelic distribution of the PAX-6LPR did not deviate significantly between the controls and the IGE patients (Wilcoxon Rank-Sum test: P > 0.76), or both subgroups of either JME patients (P > 0.78) or IAE patients (P > 0.87). Our results do not provide evidence that length variation of the polymorphic dinucleotide sequence in the PAX-6LPR contributes a frequent and relevant effect to the pathogenesis of common subtypes of IGE.

The serotonin transporter in Alzheimer's and Parkinson's disease.

The etiology of late-onset Alzheimer's disease (AD) and idiopathic Parkinson's disease (PD) is not known. In both disorders there is an extensive degeneration of serotonergic neurons, with corresponding losses of the serotonin (5HT) transporter (5HTT), which is responsible for the reuptake of 5HT from the synaptic cleft. An increasing body of evidence indicates that allelic variation of the 5HTT gene promoter (5HTT gene-linked polymorphic region, 5HTTLPR) determines high or low 5HT uptake in normal human brain. Association studies show that the low-activity allele of the 5HTTLPR is a risk factor for late-onset AD. In PD, the 5HTTLPR influences the risk of developing depression, a common symptom in PD patients. A compromised serotonergic system thus plays an important role in the pathophysiology of both AD and PD.

Association of a regulatory polymorphism in the promoter region of the monoamine oxidase A gene with antisocial alcoholism.

We analyzed a novel functional 30-bp repeat polymorphism in the promoter region of the X-chromosomal monoamine oxidase A gene (MAOA) to test whether length variation of the repeat polymorphism contributes to variation in the individual vulnerability to antisocial behavior and liability to alcohol dependence. The repeat number (3-5) of the MAOA polymorphism was assessed in 488 male subjects of German descent, a sample comprising 185 psychiatrically screened control subjects and 303 alcohol-dependent subjects including 59 alcoholics with antisocial personality disorder. The frequency of the low-activity 3-repeat allele was significantly increased in 59 antisocial alcoholics compared to 185 control subjects (51 vs. 35%; P = 0.031) and to 244 alcoholics without antisocial personality disorder (51 vs. 32%; P = 0.008), respectively. We found no significant difference in the frequency of the 3-repeat allele between 244 alcoholics without an antisocial personality disorder and the control subjects. Our findings suggest that the low-activity 3-repeat allele of the MAOA promoter polymorphism confers increased susceptibility to antisocial behavior rather than alcohol dependence per se in alcohol-dependent males.

Polymorphic MAO-A and 5-HT-transporter genes: analysis of interactions in panic disorder.

Recurrent panic attacks, anticipatory anxiety and phobic avoidance characterise panic disorder. The influence of genetic factors on liability to the disease has been the object of several linkage and association studies and appears to relate to an oligo- or polygenic rather than a monogenic mode of inheritance. Recently, an excess of high activity monoamine oxidase A (MAO-A) gene promoter alleles was found in female patients with panic disorder. An analysis of possible synergistic effects of the MAO-A gene promoter variant and the short serotonin transporter (5-HTT) gene promoter variant in panic disorder was performed in a German and an Italian sample (combined panic disorder n = 144, combined controls n = 175). There was no significant difference in odds ratios, suggesting that the observed increase of genetic liability by the long MAO-A gene promoter allele is not modified by the 5-HTT gene promoter polymorphism.

Association analysis of the functional monoamine oxidase A gene promotor polymorphism in migraine.

Migraine affects about 15% of the adult population. Serotonergic and dopaminergic systems are believed to be involved in its pathophysiology. One of the key enzymes in the degradation of serotonin and to a lesser extent of dopamine is monoamine oxidase A (MAO-A). In this study we investigated a functionally relevant gene-linked polymorphic repetitive sequence (LPR) located approximately 1.2 kb upstream of the ATG codon in the MAO-A-promotor gene. 119 patients with migraine and 229 controls were tested. The allelic distribution of the controls and the migraine patients did not show significant differences with respect to the low- and high-activity alleles. Moreover, effectiveness of the potent serotonergic antimigraine agents, triptans, which are metabolized by MAO-A, was clinically not affected by the MAO-A-LPR in our patients. These findings thus indicate that there is no association between the functional MAO-A-LPR and susceptibility to migraine.

Beta-thalassemia in the German population: mediterranean, Asian and novel mutations. Mutations in brief no.228. Online.

The beta-thalassemia mutations of 13 unrelated heterozygous Germans who remained unidentified in a previous study of 40 subjects were investigated at the DNA level. Two Mediterranean, one Asian and three novel mutations (CD6 -G, CDs 108 /112-12nt, CDs 130/131 + GCCT) were identified. Altogether, in 30 of the 35 subjects (86%) in which a mutation in the beta-globin gene was identified, the mutation was of Mediterranean origin. The geographical distribution suggests recent migration from the Mediterranean region as cause of the high proportion of frequent Mediterranean beta-thalassemia mutations in the German population. Our results support the notion that the majority of beta-thalassemia genes in the western and central European population are of Mediterranean origin.

Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.

PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.

The genomic organization of the murine Mlc1 (Wkl1, KIAA0027) gene.

The human MLC1( WKL1, KIAA0027) gene encodes a putative transmembrane protein expressed exclusively in brain. Recessive mutations within this gene cause megalencephalic leukoencephalopathy with subcortical cysts (MLC, MIM 604004, 605908). Furthermore, a missense mutation in this gene is suggestively linked with hereditary catatonic schizophrenia in a large pedigree. The murine gene Mlc1is composed of 12 exons spanning approximately 20 kb, and all exon-intron boundaries conform to the GT/AG consensus. The single copy transcript after splicing is approximately 2.8 kb in length, it contains 496 bp of 5' untranslated region (5'-UTR) and 1143 bp of 3'-UTR, and encodes a protein of 382 amino acids. Potential binding sites for transcription factors including CCAAT-boxes are present in the 5'-flanking region. Fluorescent in situ hybridization localizes the gene to mouse chromosome 15E-F, a region syntenic to human chromosome 22q13. The characterization of the genomic structure of the murine gene will facilitate studies of gene function and physiological properties of the encoded protein in transgenic mouse models.

Functional characterization of the human PAX3 gene regulatory region.

Spatiotemporal expression of the PAX3 gene is tightly regulated during development. We have isolated and sequenced the 5'-flanking regulatory region of human PAX3. Primer extension and ribonuclease protection mapping revealed that transcription is initiated from a single start site downstream of a TATA-like motif in human brain and peripheral tissues. Functional dissection of the gene's 5'-flanking region, which had been fused to a luciferase reporter gene and transiently expressed in rhabdomyosarcoma (RD) and cos-7 cells, indicated that the upstream region of PAX3 contains multiple positive and negative cis-acting regulatory elements. While the basal promoter is likely to be driven by two CCAAT boxes located at nucleotide positions -90 and -135, a cluster of regulatory elements acting as a strong repressor was detected between nucleotides -1200 and -650. Comparison of human and murine sequences revealed more than 90% identity in this segment. A polymorphic (CA)n repeat sequence and a G/C substitution are located 337 bp and 328 bp upstream of the transcription start site, respectively. PCR-based systematic screening for length variations in 225 unrelated individuals of a Caucasian population showed a bimodal distribution of multiple alleles containing between 13 and 30 repeat units. Although the (CA)25 variant of this PAX3 gene-linked polymorphic region (PAX3LPR) conferred lower transcriptional efficiency on the PAX3 promoter, a regulatory impact of the PAX3LPR on PAX3 expression related to brain plasticity and function remains to be demonstrated.

A promoter-associated polymorphic repeat modulates PAX-6 expression in human brain.

The PAX-6 gene plays a critical role in neurodevelopment and brain plasticity. While transcription of human PAX-6 is regulated by alternate usage of two distinct promoters termed A and B, expression in adult human brain is primarily controlled by promoter B. We now report that a novel polymorphic dinucleotide repeat sequence with the structure (AC)m(AG)n is located approximately 1 kb upstream of the transcription initiation site associated with promoter B. PCR-based systematic screening for length variations in a caucasian population showed a skewed distribution of multiple alleles containing between 24 and 36 repeat units. In 217 unrelated individuals, the frequency of alleles in the range between 25 and 29 repeats was 90%, with the 26 repeat allele alone accounting for 50%; the heterozygosity rate was 65%. Variants of this PAX-6 gene-linked polymorphic region (PAX-6LPR) had different transcriptional efficiencies when fused to a luciferase reporter gene and transfected into Cos-7 cells. Promoter activity of variants with >/=29 repeats was 4- to 9-fold higher than that of the 26 repeat allele. The influence of the PAX-6LPR on PAX-6 expression was confirmed in postmortem cerebellum from individuals with different genotypes. mRNA levels were 2-fold higher in genotypes with long alleles compared to those with short alleles. Allelic variation in PAX-6 expression may be a determinant of interindividual differences in brain plasticity and function.

Allelic variation of serotonin transporter expression is associated with depression in Parkinson's disease.

Idiopathic Parkinson's disease (PD) is a common neurodegenerative disorder with prominent motor symptoms. However, depression is common in PD, affecting about 40% of PD patients. Since there is extensive evidence of degeneration of serotonin (5HT) neurons and loss of the 5HT transporter (5HTT) in PD, we assessed whether a functional polymorphism in the promoter of the 5HTT gene (5HTT gene-linked polymorphic region, 5HTTLPR), which determines high or low 5HT uptake, is associated with depressive symptomatology in PD patients. We found that patients with the short allele of the 5HTTLPR had significantly higher scores on the Hamilton Depression Scale. A functional promoter polymorphism of the monoamine oxidase A (MAOA) gene showed no association. Thus, the 5HTTLPR but not the MAOA gene promoter-associated polymorphism may be a risk factor for depression in PD patients, while neither polymorphism increases the risk for development of Parkinson's disease itself.

A missense mutation in a novel gene encoding a putative cation channel is associated with catatonic schizophrenia in a large pedigree.

Schizophrenia is a common and etiologically heterogeneous disorder. Although inheritance of schizophrenic syndromes is complex with genetic and environmental factors contributing to the clinical phenotype, periodic catatonia, a familial subtype of catatonic schizophrenia, appears to be transmitted in an autosomal dominant manner. We report here that a Leu309Met mutation in WKL1, a positional candidate gene on chromosome 22q13.33 encoding a putative non-selective cation channel expressed exclusively in brain, co-segregates with periodic catatonia in an extended pedigree. Structural analyses revealed that this missense mutation results in conformational changes of the mutant protein. Our results not only underscore the importance of genetic mechanisms in the etiology of schizophrenic syndromes, but also provide a better understanding of the pathogenesis and incapacitating course of catatonic schizophrenia and related disorders.

Excess of high activity monoamine oxidase A gene promoter alleles in female patients with panic disorder.

A genetic contribution to the pathogenesis of panic disorder has been demonstrated by clinical genetic studies. Molecular genetic studies have focused on candidate genes suggested by the molecular mechanisms implied in the action of drugs utilized for therapy or in challenge tests. One class of drugs effective in the treatment of panic disorder is represented by monoamine oxidase A inhibitors. Therefore, the monoamine oxidase A gene on chromosome X is a prime candidate gene. In the present study we investigated a novel repeat polymorphism in the promoter of the monoamine oxidase A gene for association with panic disorder in two independent samples (German sample, n = 80; Italian sample, n = 129). Two alleles (3 and 4 repeats) were most common and constituted >97% of the observed alleles. Functional characterization in a luciferase assay demonstrated that the longer alleles (3a, 4 and 5) were more active than allele 3. Among females of both the German and the Italian samples of panic disorder patients (combined, n = 209) the longer alleles (3a, 4 and 5) were significantly more frequent than among females of the corresponding control samples (combined, n = 190, chi2 = 10.27, df = 1, P = 0.001). Together with the observation that inhibition of monoamine oxidase A is clinically effective in the treatment of panic disorder these findings suggest that increased monoamine oxidase A activity is a risk factor for panic disorder in female patients.

Association analysis of a PAX-6 gene promoter-associated polymorphic repeat with alcohol dependence.

The human paired box-containing gene PAX-6 participates in the development and plasticity of the brain including the limbic system, the neural system that plays a crucial role in reward processes. We have reported recently a polymorphic dinucleotide repeat sequence with the structure (AC)m(AG)n, which is located approximately 1 kb upstream of the transcription initiation site associated with promoter B and confers allelic variation of PAX-6 expression in the human brain. In the present association study we tested whether length variation of PAX-6 gene-linked polymorphic region (PAX-6 LPR) influences susceptibility to alcohol dependence.The repeat length of the PAX-6 LPR was assessed in 354 control subjects and 328 alcohol-dependent patients, including four subgroups with a presumed substantial genetic predisposition: (a) with a history of withdrawal complications (n=100); (b) with a history of parental alcoholism (n=115); (c) with early onset (n=67) and (d) with dissocial personality disorders (n=54). Allelic distribution of the PAX-6 LPR did not differ significantly between the controls and the entire group of alcohol-dependent patients χ²=0.015, df 1, p=0.904), or any of the subgroups of patients with severe alcoholism. Our results do not provide evidence that length variation of the PAX-6 LPR contributes to the pathogenesis of alcohol dependence.