Zsuzsanna Elek

DRD4 and TH gene polymorphisms are associated with activity, impulsivity and inattention in Siberian Husky dogs

Both dopamine receptor D4 (DRD4) exon 3 and tyrosine hydroxylase (TH) intron 4 repeat polymorphisms have been linked to activity and impulsivity in German Shepherd dogs (GSDs). However, the results in GSDs may not be generalisable to other breeds, as allelic frequencies vary markedly among breeds. We selected the Siberian Husky for further study, because it is highly divergent from most dog breeds, including the GSD. The study sample consisted of 145 racing Siberian Huskies from Europe and North America. We found that this breed possesses seven DRD4 length variants, two to five more variants than found in other breeds. Among them was the longest known allele, previously described only in wolves. Short alleles of the DRD4 and TH repeat polymorphisms were associated with higher levels of activity, impulsivity and inattention. Siberian Huskies possessing at least one short allele of the DRD4 polymorphism displayed greater activity in a behavioural test battery than did those with two long alleles. However, the behavioural test was brief and may not have registered variation in behaviour across time and situations. Owners also completed the Dog-Attention Deficit Hyperactivity Disorder Rating Scale (Dog-ADHD RS), a more general measure of activity and attention. Siberian Huskies from Europe with two short alleles of the TH polymorphism received higher ratings of inattention on the Dog-ADHD RS than did those with the long allele. Investigation of the joint effect of DRD4 and TH showed that dogs possessing long alleles at both sites were scored as less active-impulsive than were others. Our results are aligned with previous studies showing that DRD4 and TH polymorphisms are associated with activity-impulsivity related traits in dogs. However, the prevalence of variants of these genes differs across breeds, and the functional role of specific variants is unclear.

Glycogen synthase kinase 3 beta gene structural variants as possible risk factors of bipolar depression

The glycogen synthase kinase 3B (GSK3B) is an important target protein of several antidepressants, such as lithium, a mood stabilizer. Recent studies associated structural variations of the GSK3B gene to bipolar disorder (BP), although replications were not conclusive. Here we present data on copy number variations (CNVs) of the GSK3B gene probing the 9th exon region in 846 individuals (414 controls, 172 patients with major depressive disorder (MDD) and 260 with BP). A significant accumulation (odds ratio: 5.5, P = 0.00051) of the amplified exon 9 region was found in patients (22 out of 432) compared to controls (4 of 414). Analyzing patient subgroups, GSK3B structural variants were found to be risk factors of BP particularly (P = 0.00001) with an odds ratio of 8.1 while no such effect was shown in the MDD group. The highest odds (19.7 ratio) for bipolar disorder was observed in females with the amplified exon 9 region. A more detailed analysis of the identified GSK3B CNV by a set of probes covering the GSK3B gene and the adjacent NR1I2 and C3orf15 genes showed that the amplified sequences contained 3′ (downstream) segments of the GSK3B and NR1I2 genes but none of them involved the C3orf15 gene. Therefore, the copy number variation of the GSK3B gene could be described as a complex set of structural variants involving partial duplications and deletions, simultaneously. In summary, here we confirmed significant association of the GSK3B CNV and bipolar disorder pointing out that the copy number and extension of the CNV varies among individuals.

RNA interference links oxidative stress to the inhibition of heat stress adaptation.

UNLABELLED Increased oxidative stress is associated with various diseases and aging, while adaptation to heat stress is an important determinant of survival and contributes to longevity. However, the impact of oxidative stress on heat resistance remains largely unclear. AIM In this study we investigated how oxidative stress impinges on heat stress responses. RESULTS We report that hydrogen-peroxide (H(2)O(2)) pretreatment inhibits both acquired thermotolerance and heat-induced Hsp70 expression in mammalian cells, as well as acquired thermotolerance in the nematode Caenorhabditis elegans, via RNA interference. Moreover, we demonstrate that elimination of RNA interference by silencing key enzymes in microRNA biogenesis, dcr-1 or pash-1, restores the diminished intrinsic thermotolerance of aged and H(2)O(2)-elimination compromised (catalase-2 and peroxiredoxin-2 deficient) worms. INNOVATION AND CONCLUSION These results uncover a novel post-transcriptional element in the regulation of heat stress adaptation under oxidative conditions that may have implications in disease susceptibility and aging.

Association of aggression with a novel microRNA binding site polymorphism in the wolframin gene.

Rare mutations in the WFS1 gene lead to Wolfram syndrome, a severe multisystem disorder with progressive neurodegeneration and diabetes mellitus causing life‐threatening complications and premature death. Only a few association studies using small clinical samples tested the possible effects of common WFS1 gene variants on mood disorders and suicide, the non‐clinical spectrum has not been studied yet. Self‐report data on Aggression, Impulsiveness, Anxiety, and Depression were collected from a large (N = 801) non‐psychiatric sample. Single nucleotide polymorphisms (SNPs) were selected to provide an adequate coverage of the entire WFS1 gene, as well as to include putative microRNA binding site polymorphisms. Molecular analysis of the assumed microRNA binding site variant was performed by an in vitro reporter‐gene assay of the cloned 3′ untranslated region with coexpression of miR‐668. Among the 17 WFS1 SNPs, only the rs1046322, a putative microRNA (miR‐668) binding site polymorphism showed significant association with psychological dimensions after correction for multiple testing: those with the homozygous form of the minor allele reported higher aggression on the Buss–Perry Aggression Questionnaire (P = 0.0005). Functional effect of the same SNP was also demonstrated in a luciferase reporter system: the minor A allele showed lower repression compared to the major G allele, if co‐expressed with miR‐668. To our knowledge, this is the first report describing a microRNA binding site polymorphism of the WFS1 gene and its association with human aggression based on a large, non‐clinical sample.

Polymorphism in the serotonin receptor 2a (HTR2A) gene as possible predisposal factor for aggressive traits.

Aggressive manifestations and their consequences are a major issue of mankind, highlighting the need for understanding the contributory factors. Still, aggression-related genetic analyses have so far mainly been conducted on small population subsets such as individuals suffering from a certain psychiatric disorder or a narrow-range age cohort, but no data on the general population is yet available. In the present study, our aim was to identify polymorphisms in genes affecting neurobiological processes that might explain some of the inter-individual variation between aggression levels in the non-clinical Caucasian adult population. 55 single nucleotide polymorphisms (SNP) were simultaneously determined in 887 subjects who also filled out the self-report Buss-Perry Aggression Questionnaire (BPAQ). Single marker association analyses between genotypes and aggression scores indicated a significant role of rs7322347 located in the HTR2A gene encoding serotonin receptor 2a following Bonferroni correction for multiple testing (p = 0.0007) both for males and females. Taking the four BPAQ subscales individually, scores for Hostility, Anger and Physical Aggression showed significant association with rs7322347 T allele in themselves, while no association was found with Verbal Aggression. Of the subscales, relationship with rs7322347 was strongest in the case of Hostility, where statistical significance virtually equaled that observed with the whole BPAQ. In conclusion, this is the first study to our knowledge analyzing SNPs in a wide variety of genes in terms of aggression in a large sample-size non-clinical adult population, also describing a novel candidate polymorphism as predisposal to aggressive traits.

Candidate gene copy number analysis by PCR and multicapillary electrophoresis

Genetic polymorphisms are often considered as risk factors of complex diseases serving as valuable and easily detectable biomarkers, also stable during the whole lifespan. A novel type of genetic polymorphism has been identified just recently, referred to as gene copy number variation (CNV) or copy number polymorphism. CNV of glycogen synthase kinase 3 beta and its adjacent gene, Nr1i2 (pregnane X receptor isoform), has been reported to associate with bipolar depression. In our study we introduced multicapillary electrophoresis for gene copy number analysis as an affordable alternative to real‐time PCR quantification with TaqMan gene probes. Our results show the reliability of the developed method based on conventional PCR followed by separation of products by multicapillary electrophoresis with quantitative evaluation. This method can be readily implemented for the analysis of candidate gene CNVs in high throughput clinical laboratories and also in personalized medicine care of depression‐related risk factors.

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus.

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ 2-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene.

The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high‐throughput SNP and VNTR analysis. Association studies were carried out in a case–control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele‐ and genotype‐wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

Komplex jellegek genetikai hátterének elemzése

Although the Human Genome Project discovered the sequence of the human genetic information 15 years ago, genetic background of the diseases - primarily that of complex disorders - is still not known. The sum of the not yet discovered inherited risk factors is termed the missing heritability; the identification of these genetic components is, however, essential, as it is the base of the understanding of the molecular pathomechanism of diseases. It is not only of theoretical importance: this knowledge can be used in the clinical practice, as it offers the possibility of improvement of diagnostics, prevention as well as targeted and individualized therapy. Application of novel and more efficient molecular biological tools contribute to the discovery of unknown genetic factors, the complete goal can only be achieved, however, by re-conceptualization of several clinical and genetic points. Our knowledge was established by genome-wide studies, however, further knowledge must be acquired according to the following points: (1) genotype and association analysis of repeat variations (VNTRs and CNVs) besides SNPs, (2) investigation of gene-gene and gene-environment interactions, (3) epigenetic studies, (4) assessing the biological function of polymorphisms, (5) application of biologically relevant diagnostic categories and endophenotypes. Although it is only 1.2% of the whole genome that codes for proteins, however, as much as 90% is transcribed to RNA, consequently it can be hypothesized that gene expression analyses might offer promising starting points for further studies, as they can shed light on the molecular processes that contribute to the development of diseases. Orv Hetil. 2018; 159(31): 1254-1261.

ABCA1 Polymorphism, a Genetic Risk Factor of Harm Avoidance

Even though cholesterol homeostasis and self-harm behaviors have shown to be associated, gene polymorphisms of the cholesterol system have not been studied yet in the context of self-harm related personality traits. Here we present an association study between six ABCA1 polymorphisms and temperament scales measured by Cloninger’s Temperament and Character Inventory on 253 young adults. An association between ABCA1 rs4149264 and harm avoidance has been observed. This association remained significant after Bonferroni correction. Haplotype analysis confirmed an independent association between rs4149264 and harm avoidance. ABCA1, a cholesterol homeostasis gene, is a candidate gene for harm related personality traits.