MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

The involvement of the canonical Wnt-signaling receptor LRP5 and LRP6 gene variants with ADHD and sexual dimorphism: Association study and meta-analysis.

Wnt-signaling is one of the most abundant pathways involved in processes such as cell-proliferation, -polarity, and -differentiation. Altered Wnt-signaling has been linked with several neurodevelopmental disorders including attention-deficit/hyperactivity disorder (ADHD) as well as with cognitive functions, learning and memory. Particularly, lipoprotein receptor-related protein 5 (LRP5) or LRP6 coreceptors, responsible in the activation of the canonical Wnt-pathway, were associated with cognitive alterations in psychiatric disorders. Following the hypothesis of Wnt involvement in ADHD, we investigated the association of genetic variations in LRP5 and LRP6 genes with three independent child and adolescent ADHD (cADHD) samples (total 2,917 participants), followed by a meta-analysis including previously published data. As ADHD is more prevalent in males, we stratified the analysis according to sex and compared the results with the recent ADHD Psychiatric Genomic Consortium (PGC) GWAS. Meta-analyzing our data including previously published cADHD studies, association of LRP5 intronic rs4988319 and rs3736228 (Ala1330Val) with cADHD was observed among girls (OR = 1.80 with 95% CI = 1.07-3.02, p = .0259; and OR = 2.08 with 95% CI = 1.01-4.46, p = .0026, respectively), whereas in boys association between LRP6 rs2302685 (Val1062Ile) and cADHD was present (OR = 1.66, CI = 1.20-2.31, p = .0024). In the PGC-ADHD dataset (using pooled data of cADHD and adults) tendency of associations were observed only among females with OR = 1.09 (1.02-1.17) for LRP5 rs3736228 and OR = 1.18 (1.09-1.25) for LRP6 rs2302685. Together, our findings suggest a potential sex-specific link of cADHD with LRP5 and LRP6 gene variants, which could contribute to the differences in brain maturation alterations in ADHD affected boys and girls, and suggest possible therapy targets.

Potential salivary biomarkers and their genetic effects in a pilot study of adolescent boys with externalizing problems.

AIMS: Beside the well-known stress response marker cortisol, salivary alpha-amylase is receiving increasing attention. Numerous studies have investigated the potential biomarker properties of cortisol mirroring abnormal hypothalamic-pituitary-adrenal axis activity in connection to both internalizing and externalizing behavior problems. The other major physiological system involved in stress reactivity, the sympathetic nervous system activity can be also measured by the surrogate marker of salivary alpha-amylase. Most of the studies applied a stressful situation to obtain inter-individual differences in stress-reactivity, although differences in the baseline level of cortisol have been also shown in relation to externalizing problems. To test the relevance of another (easier) biomarker, we selected to study baseline circadian salivary cortisol and alpha-amylase levels among adolescent boys with externalizing problems. METHODS: Saliva samples were collected at 3 time-points (morning, noon, evening) during 3 consecutive days from 37 inpatient boys (mean age 12.4±1.0). Cortisol and alpha-amylase levels were measured by enzyme-linked immunosorbent and kinetic enzyme assays, respectively. Genetic variants in the hypothalamic-pituitary-adrenal axis and the norepinephrine transporter or catecholamine metabolizing enzymes were tested for potential moderating effects at these salivary biomarkers. RESULTS: Saliva cortisol showed the classical diurnal fluctuation in boys with externalizing problems (possibly from a lower morning level), but it was not modified by the presence of either conduct, oppositional defiant or attention-deficit/hyperactivity disorder. The diurnal fluctuation of the salivary alpha-amylase levels was also typical, but the presence of conduct disorder was associated with significantly lower alpha-amylase activity (p=0.024) among boys with externalizing problems. The catechol-O-methyltransferase Val158Met (rs4680) polymorphism had an additional effect on salivary alpha-amylase: boys with homozygote genotypes had lower alpha-amylase activity at all 3 time-points compared to Val/Met heterozygotes (p=0.045). CONCLUSIONS: Our preliminary data suggest that salivary alpha-amylase might be used to further characterize subgroups within externalizing problems, however, this biomarker might be modified by certain genetic polymorphisms.

Association analysis of norepinephrine transporter polymorphisms and methylphenidate response in ADHD patients.

AIMS: Methylphenidate (MPH) is the most frequently prescribed drug in Attention Deficit Hyperactivity Disorder (ADHD). Hitherto mostly the dopamine transporter gene has been studied in MPH-response and only a few studies analyzed the norepinephrine transporter (NET, SLC6A2) gene, although MPH is a potent inhibitor of both dopamine and norepinephrine transporters. We aimed to analyze this monoamine transporter gene in relation to ADHD per se and MPH-response in particular to gain further knowledge in ADHD pharmacogenetics using a Caucasian sample. METHODS: Six single nucleotide polymorphisms (rs28386840, rs2242446, rs3785143, rs3785157, rs5569, rs7194256 SNP) were studied across the NET gene in 163 ADHD children (age: 9.3±2.6; 86.5% male) using ADHD-RS hyperactivity-impulsivity and inattention scales. For case-control analysis 486 control subjects were also genotyped. At the MPH-response analysis responders had minimum 25% decrease of ADHD-RS total score after 2months of treatment, and chi-square test compared 90 responders and 32 non-responders, whereas ANOVA was used to assess symptom improvement after the first month among the 122 ADHD patients. RESULTS: The classical case-control analysis did not yield any association with ADHD diagnosis, which was supported by meta-analysis conducted on the available genetic data (combining previously published and the present studies). On the other hand, the intronic rs3785143 showed nominal association with inattention symptoms (p=0.01). The haplotype analysis supported this association, and indicated the importance of the first haploblock encompassing the intronic and 2 promoter SNPs. With MPH-response only the promoter rs28386840 showed nominal association: Those with at least one T-allele were overrepresented in the responder group (42% vs 19%, p=0.08), and they had better improvement on the hyperactivity-impulsivity scale compared to the AA genotype (p=0.04). CONCLUSION: Although none of our single SNP findings remained significant after correcting for multiple testing, our results from the MPH-response analysis indicate the potential importance of promoter variants in the NET gene.

Differential Genetic Effect of the Norepinephrine Transporter Promoter Polymorphisms on Attention Problems in Clinical and Non-clinical Samples.

Among the monoaminergic modulatory neurotransmitters, norepinephrine is involved in task orienting, hence noradrenergic genetic variants have been studied in connection to attentional processes. The role of this catecholamine system is also highlighted by the selective norepinephrine transporter blocking atomoxetine, which has proved to be effective in the pharmacological treatment of Attention Deficit Hyperactivity Disorder (ADHD). In the present genetic association study three single nucleotide polymorphisms (rs28386840, rs2242446, rs3785143 SNPs) were analyzed from the 5' region of the norepinephrine transporter (NET, SLC6A2) gene, which have been linked to ADHD previously. Attention problems scores of the mother-rated Child Behavior Checklist (CBCL) were used in separate analyses of 88 preschoolers (59.1% male, 6 years of age) recruited from the general population and 120 child psychiatry patients with ADHD diagnosis (85.8% male, age: 9.8 ± 2.9). The NET SNPs showed associations with attention problems, but the direction was different in the two groups. Regarding the promoter variant rs28386840, which showed the most consistent association, the T-allele-carrier patients with ADHD had lower CBCL attention problems scores compared to patients with AA genotype (p = 0.023), whereas T-allele-carriers in the community sample had more attention problems (p = 0.042). Based on previous reports of lower NE levels in ADHD children and the inverted-U shape effect of NE on cognitive functions, we propose that rs28386840 (-3081) T-allele, which is associated with lower NET expression (and potentially higher synaptic NE level) would support attention processes among ADHD patients (similarly as atomoxetine increases NE levels), whereas it would hinder cortical functions in healthy children.

[Formulation of a capsule preparation with high active ingredient content using re-crystallized drug compound]. / Nagy hatóanyagtartalmú kapszulázott készítmény formulálása attkristályosított hatóanyag alkalmazásával.

Richter Gedeon Company aimed to manufacture and launch a generic product, that's original is already on market in Hungary. The goal of a generic development is to produce an essentially similar product to the original one. It seems to be simple and routine work, but during the formulation a couple of problems have to be solved. Our active agent was voluminous therefore we could not fill the whole quantity of the active, even without any excipients. In addition the flow property of the powder mixture were very bad, and it may cause inaccurate capsule filling and significant differences in the weight of the filled capsules. During the formulation we managed to assure the appropriate physical properties of the agent by re-crystallisation. We changed the excipients used in the first trials. Chemically these were the same, but with different physical features.

[The generic Research and Development possibilities after Hungary accessed th European Patent Convention]. / A magyar generikus készítményfejlesztés lehetósége az iparjogvédelem tükrében.

January 1, 2003 Hungary became a member of the Munich Convention on the Grant of European Patents (European Patent Convention, EPC). As from January 1, 2003 European patents may be claimed and obtained also for Hungary and European applications may be filed with the Hungarian Patent Office as well. This paper focuses on the exceptional generic R&D possibilities which can harmonize in harmony with all the claims of the Union in spite of the fact that it does not follow general European limitations and practises. Based on the amended Hungarian Patent Law (No. XXXIII of 1995 on the Protection of Inventions by Patents; Article 19 (6)b)) the exclusive right of exploitation shall not extend to acts done for experimental purposes relating to the subject matter of the invention, including experiments and tests necessary for the registration of medicines as well. The national and international legal history and background of the operative Hungarian Patent Law are also presented roughly. The paper deals with the divergence of the originator-generic industry and the EU-USA industry and with the incoherence within the EU itself on the early testing permissions and limitations.

Carboxylesterase 1 gene polymorphism and methylphenidate response in ADHD.

Methylphenidate (MPH) is the most frequently prescribed drug in the treatment of attention deficit hyperactivity disorder (ADHD). Several pharmacogenetic studies suggested that catecholamine candidate genes influence individual MPH-responses, but these results are mostly contradictory. Genetic analyses of MPH metabolizing carboxylesterase 1 enzyme (CES1) have not been carried out, whereas, meta-analysis of CYP2D6 genetic variants has been already indicated significant pharmacogenetic differences in atomoxetine treatment. Here we present an association analysis of the CES1 Gly143Glu functional polymorphism in a Hungarian ADHD group (n = 173). The genotype frequencies were similar to that of the general population (5.8% vs 4.1% of Gly/Glu heterozygote). Pharmacogenetic analysis was conducted among 122 ADHD children treated with MPH. Neither the categorical analysis comparing 90 responders vs 32 non-responders, nor the dimensional analysis of Inattention and Hyperactivity-Impulsivity score reduction showed a significant main genotype effect. However, analyzing the daily dose, we observed an association with the rare 143Glu-variant: 5 patients in the responder group carrying the Glu-allele required lower doses of MPH for symptom reduction (0.410 +/- 0.127 vs 0.572 +/- 0.153 mg/kg, t(1,88) = 2.33, p = 0.022). This result warrants for further investigations of the CES1 gene in larger ADHD samples.