Association of the tumor necrosis factor -308 A/G promoter polymorphism with Tourette syndrome.

Several lines of evidence suggest that certain subtypes of obsessive-compulsive and tic disorders might be paediatric manifestations of post-streptococcal autoimmunity caused by cross-reactive autoantibodies. As tumor necrosis factor (TNF) is known to play a seminal role in coordinating the humoral immune response, TNF gene polymorphisms have been proposed as genetic risk factors both in obsessive-compulsive disorder (OCD) and Tourette syndrome (TS). The aim of this study was to investigate two TNF promoter polymorphisms (-238 A/G: rs361525 and -308 A/G: rs1800629) on the genetic susceptibility to OCD and TS in a child psychiatric sample (102 patients with OCD and 117 patients with TS). In the case-control set-up, the genotype and allele frequencies were compared to a control group from the general population (n = 405). As a control child psychiatric sample, 194 children with attention-deficit hyperactivity disorder were also genotyped. Our results revealed that the TNF -308 G-allele was more frequent in children with TS compared to controls (90.2% vs 84.8%, P = 0.037). For confirmation of this genetic association, a family-based analysis, the transmission disequilibrium test was used, which showed preferential transmission of the G-allele to patients with TS (nominal P-value 0.011). Moreover, this allele was also transmitted more frequently to children with tic symptoms (nominal P-value 0.039). No association was found between OCD or obsessive-compulsive symptoms and the studied TNF polymorphisms. Based on these findings, the TNF -308 G-allele can be associated with Tourette syndrome, highlighting the potential pathophysiological role of TNF dysregulation.

Dopamine D4 receptor and serotonin transporter gene effects on the longitudinal development of infant temperament.

Existing studies of the effect on infant temperament of the 48 base pair variable number of tandem repeats polymorphism in exon 3 of the dopamine D4 receptor gene, DRD4 VNTR, and the serotonin transporter-linked polymorphic region, 5-HTTLPR, have provided contradictory results, and age seems to be an important factor. The present study investigated the effect of these two polymorphisms on the stability of infant temperament between 4 and 9 months of age. Furthermore, the effect of a recently discovered single nucleotide polymorphism which modulates the 5-HTTLPR (rs25531) was investigated in relation to infant temperament. The study sample consisted of 90 infants, who were assessed by parental report at the two ages under consideration using the Revised Infant Behavior Questionnaire. It was found that infants carrying the 7-repeat allele of the DRD4 VNTR had higher levels of Negative Affect. Furthermore, there was an interaction between DRD4 VNTR and 5-HTTLPR genotype such that infants with the DRD4 VNTR 7-repeat allele and the highest expressing 5-HTTLPR genotype (L(A) L(A) ) had the highest level of Negative Affect. These effects were largely driven by scores on the Falling Reactivity scale. Genetic effects were stable across age. The results emphasize the need for developmental studies of genetic effects on temperament.

Genetic factors of reaction time performance: DRD4 7-repeat allele associated with slower responses.

Twin studies indicate substantial inherited components in cognitive abilities. One of the most extensively studied candidate genes of cognitive functioning is the dopamine D4 receptor gene (DRD4), which has been suggested to be related to attentional disorders. Based on reaction time data of 245 Caucasians participating in different cognitive tasks, slower responses characterized the group with the 7-repeat allele. This effect was present in both sexes and was not because of fatigue. To our knowledge, this is the first report on significant association (P = 0.0001) between the DRD4 variable number of tandem repeat (VNTR) polymorphism and response latencies in a non-clinical adult sample. Other studied dopaminergic polymorphisms did not show an association with reaction time. These results illustrate that speed-of-performance measures derived from multiple reaction time tasks using standardization procedures could be promising tools to detect unique genetic effects in the background of cognitive abilities.

Polymorphisms of beta defensins are associated with the risk of severe acute pancreatitis.

AIMS: Bacterial translocation from the intestinal tract plays an important role in severe acute pancreatitis (AP). Human ß-defensins are a family of antimicrobial peptides present at the mucosal surface. The aim of this study was to investigate the relevance of single nucleotide polymorphisms (SNPs) in the DEFB1 gene and copy number polymorphisms of the DEFB4 genes in AP. METHODS: 124 AP patients (30 with mild and 94 with severe disease) and 100 healthy controls were enrolled in the study. Three SNPs of the DEFB1 gene [G-20A (c.-20G→A), C-44G (c.-44C→G) and G-52A (c.-52G→A)] were genotyped by Custom TaqMan assay. The DEFB4 gene copy number was determined by means of a TaqMan real-time PCR assay. RESULTS: Significantly higher frequencies of the AA genotype of G-20A (c.-20G→A) and the AA genotype of G-52A (c.-52G→A) were observed among the patients with severe AP (SAP) compared with the healthy controls (38 vs. 20 and 41 vs. 18%, respectively). The GG protective genotype of C-44G (c.-44C→G) SNP was much less frequent (1%) among the patients than among the controls (9%). A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with SAP compared with the healthy controls (62 vs. 24%, respectively). CONCLUSIONS: The variations in the genes encoding human ß-defensin-1 and -2 may be associated with the risk of SAP. and IAP.

Molecular and behavioral analysis of the intron 2 repeat polymorphism in the canine dopamine D4 receptor gene.

Genetic polymorphisms in the human dopamine D4 receptor (DRD4) gene, especially the exon 3 variable number of tandem repeats (VNTR), have been related to several psychiatric disorders and personality traits. A homologous exon 3 VNTR has been described in dogs, and we previously showed an association between the DRD4 exon 3 polymorphism and activity/impulsivity trait in German shepherds. In this study, we present a detailed analysis of the intron 2 VNTR of the DRD4 gene. A short and a long form of the intronic variation were identified in 678 unrelated dogs from five breeds and in 22 wolves. For molecular analysis, the intron 2 region was cloned into a promoterless luciferase reporter vector that led to an elevation in transcriptional activity. Moreover, an allelic difference in promoter activity was detected, and a repressive effect of the long allele was observed. Behavioral analysis of 96 unrelated German shepherds showed a significant association between the social impulsivity endophenotype of the Greeting Test and both the exonic (P = 0.002) and the intronic (P = 0.003) VNTRs of the DRD4 gene. Moreover, an additive effect of the two polymorphisms was also shown (Spearman's rho = 0.356, P = 0.0004). In conclusion, these results give further support to our previous findings that the DRD4 gene is associated with dog behavior. We also present molecular evidence for the functional role of the intron 2 VNTR in the canine DRD4 gene.

Adenoviral vector transduction of the human deoxycytidine kinase gene enhances the cytotoxic and radiosensitizing effect of gemcitabine on experimental gliomas.

The aim of this work was to improve the cytotoxic and radiosensitizing effects of gemcitabine using a gene-directed enzyme prodrug therapy approach. Murine Gl261, rat C6 and human U373 glioma cell lines were transduced with an adenoviral vector encoding the human deoxycytidine kinase gene (Ad-HudCK). Intracranial tumors were established in C57BL/6 mice and Wistar rats using either wild-type or Ad-HudCK-transduced Gl261 and C6 glioma cells. In vitro growing cells and established tumors were treated with gemcitabine and irradiation either alone or in combination. Deoxycytidine kinase overexpression substantially increased both the toxic and radiosensitizing effects of gemcitabine in each cell line, but the enhancement rate varied: it was mild in the Gl261 cells and much stronger in the C6 and U373 cells. In vivo experiments showed a mild radiosensitizing effect of dCK overexpression both in the Gl261 and C6 models. The combination of dCK overexpression, gemcitabine treatment and irradiation improved the survival rate of C6 bearing rats significantly. In conclusion, overexpression of the dCK gene can improve the cytotoxic and radiosensitizing effect of gemcitabine both in vitro and in vivo in a tumor-specific manner.

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs.

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene (DRD4) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a) were detected in this breed, and genotype frequencies were in Hardy-Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele (P = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.

Smoking and a complement gene polymorphism interact in promoting cardiovascular disease morbidity and mortality.

We have demonstrated previously that carriers of a genotype called C4B*Q0 (silent allele of the C4B gene) have a substantially increased risk to suffer from myocardial infarction or stroke, and are selected out from the healthy elderly population. Because smoking carries a major risk for cardiovascular disease (CVD), it seemed worthwhile to study if these two factors interact. Study 1 involved 74 patients with angina pectoris (AP), 85 patients with recent acute myocardial infarction (AMI) and 112 survivors of a previous AMI and 382 controls from Iceland. Study 2 involved 233 patients with severe CVD and 274 controls from Hungary. Smoking habits were registered for each subject. The number of C4A and C4B genes was determined by phenotyping or genotyping. Compared to controls, C4B*Q0 carrier frequency was significantly higher at diagnosis in Icelandic smokers with AP (P = 0.005) and AMI (P = 0.0003) and Hungarian smokers with severe coronary artery disease (P = 0.023), while no such difference was observed in non-smoking subjects. Age-associated decrease in C4B*Q0 observed previously in two remote Caucasian populations was found, in the present study, to be associated strongly with smoking, and to already occur in smokers after age 50 years both in Iceland and Hungary. Our findings indicate that the C4B*Q0 genotype can be considered as a major covariate of smoking in precipitating the risk for AMI and associated deaths.

Deoxycytidine kinase is reversibly phosphorylated in normal human lymphocytes.

The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions.

Effects of intracellular calcium chelation and pifithrin-alpha on deoxynucleotide metabolism in human lymphocytes.

Previously, we have found that activation of deoxycytidine kinase elicited by various DNA-damaging chemical agents could be prevented by BAPTA-AM, a cell-permeable calcium chelator or by pifithrin-alpha, a pharmacological inhibitor of p53. Here, we show that stimulation of deoxycytidine kinase by UV-light also is calcium-dependent and pifithrin-alpha-sensitive in tonsillar lymphocytes, while thymidine kinase 1 activity is stabilised in the presence of BAPTA-AM. Importantly, both UV-irradiation and calcium chelation decreased the incorporation of labelled deoxycytidine and thymidine into DNA. Pifithrin-alpha dramatically reduced the labelling of both the nucleotide and DNA fractions, possibly due to inhibition of transmembrane nucleoside transport.

The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region.

BACKGROUND: The human dopamine D4 receptor (DRD4) is a candidate gene of great interest in molecular studies of human personality and psychiatric disorders. This gene is unique in having an exceptionally high amount of polymorphic sites both in the coding and in the promoter region. RESULTS: We report the identification of a new 27 bp deletion starting 524 bp upstream of the initiation codon (27 bp del) of the dopamine D4 receptor (DRD4) gene, in the close vicinity of the -521C>T SNP. The presence of the 27 bp deletion leads to the misgenotyping of the -616C>G SNP by the Sau96 I RFLP method, thus the genotype determination of the mutation is of additional importance. The frequency of this novel sequence variation is considerably low (allele frequency is = 0.16%), as no homozygotes, and only 3 heterozygote carriers were found in a healthy, unrelated Caucasian sample (N = 955). CONCLUSION: Remarkably, the deleted region contains consensus sequences of binding sites for several known transcription factors, suggesting that the different alleles may affect the transcriptional regulation of the gene. A comparison of methods and results for the allelic variations of the DRD4 gene in various ethnic groups is also discussed, which has a high impact in psychiatric genetic studies.

Stimulation of deoxycytidine kinase results in prolonged maintenance of the enzyme activity.

A number of genotoxic and antiproliferative agents such as 2-chlorodeoxyadenosine (Cladribine; CdA) and aphidicolin (APC) have been shown to stimulate the activity of deoxycytidine kinase, the main deoxynucleoside salvage enzyme in lymphocytes. Here we show that enzyme activation could be prevented by treating cells with the membrane-permeant calcium chelator BAPTA-AM. Long-term incubations demonstrated that CdA and APC not only stimulated but also sustained deoxycytidine kinase activity in the cellular context, as compared to the control and BAPTA-AM treated enzyme activities.

Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene.

DNA-protein interaction in the 5' upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2 x 120 bp) or single copy (1 x 120 bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5' upstream region and suggest enhanced binding capacity of the duplicated form.

A novel A/G SNP in the -615th position of the dopamine D4 receptor promoter region as a source of misgenotyping of the -616 C/G SNP.

The polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene containing several single nucleotide polymorphisms (SNPs) has recently become a focus of association studies in psychiatric genetics. Most SNP genotyping methods are based on the two-step procedure of restriction fragment length polymorphism (RFLP). An alternative technique is a single-step method of allele-specific amplification (ASA), previously introduced for genotyping the -521 C/T SNP of the DRD4 promoter region and applied here for the -616 C/G SNP. Parallel genotyping of individuals with the novel ASA method and the conventionally used Ava II RFLP showed a potential underestimation of the -616 GG genotype frequency by the conventional method. Sequencing the dubious samples clearly demonstrated a novel A/G SNP at the -615th position influencing the Ava II digestion and thus resulting in misgenotyping. To avoid this problem, we introduced the Sau96 I RFLP for the -616 C/G genotyping as this restriction enzyme is not sensitive for the -615 A/G sequence variation. Allele (-616 G = 0.48; -616 C = 0.52) and genotype (-616 GG = 0.25; -616 GC = 0.46; -616 CC = 0.29) frequencies were determined by both the novel ASA and the Sau96 I methods. The obtained genotype frequencies corresponded to the Hardy-Weinberg equilibrium in our healthy Caucasian sample (N = 534, P = 0.168). Using these methods, no association was found between the -616 C/G SNP and personality factors of Cloninger's temperament and character inventory (N = 153) in our population.

Human personality dimensions of persistence and harm avoidance associated with DRD4 and 5-HTTLPR polymorphisms.

The associations of human personality traits as measured by the Temperament and Character Inventory (TCI) with two genetic polymorphisms, the dopamine D4 receptor (DRD4) gene exon III repeat polymorphism (VNTR) and the serotonin transporter-linked functional polymorphism (5-HTTLPR) are presented in a population of 157 ethnically homogeneous Caucasians. No association was found between Novelty Seeking and the DRD4 VNTR, but male individuals with a 7-repeat allele exhibited significantly lower Persistence scores. The 5-HTTLPR polymorphism itself had no significant effect on any of the temperament dimensions, but a significant DRD4 VNTR x 5-HTTLPR interaction was observed for Harm Avoidance, the subgroup with a s/s 5-HTTLPR, 7-repeat DRD4 genotype showed a higher mean Harm Avoidance score than the other groups. These results are discussed in relation to the recent findings on infant temperament. Association between the DRD4 7-repeat allele and Persistence can be theoretically linked to the 7-repeat allele as a risk factor for attention deficit hyperactivity disorder.

Association of D4 dopamine receptor gene and serotonin transporter promoter polymorphisms with infants' response to novelty.

Effects of DRD4 and 5-HTTLPR length polymorphisms have been reported on neonatal and infant temperament as well as adult personality traits. The 7-repeat form of the DRD4 III exon VNTR polymorphism has been associated with childhood ADHD, and recently we have reported its link with attachment disorganization in a nonclinical population of infants. Here, we report associations of these polymorphisms with infant temperament at 12 months of age. Maternal accounts of temperament and observed response to novelty were investigated for 90 infants, who were independently genotyped for the DRD4 III exon, and for 5-HTT-linked promoter region length polymorphisms. Maternal rating of temperament was not affected by these polymorphisms, but we found combined genotype effects for infants' observed responses to a novel, anxiety-provoking stimulus: the appearance of, and approach by, a stranger. Infants with at least one copy of both the 7-repeat DRD4 allele and the long variant of 5-HTTLPR (7(+), l/l&l/s) responded with significantly less anxiety than infants with other genotypes. However, infants with the 7-repeat DRD4 allele and homozygous for the short form of 5-HTTLPR (7(+), s/s) showed more anxiety and resistance to the stranger's initiation of interaction. These genotype effects were not redundant with the previously reported association between the 7-repeat DRD4 allele and disorganized attachment behavior. Although both temperament and attachment behavior were affected by the DRD4 repeat polymorphism, the effect on temperament measures was modified by the infants' 5-HTTLPR genotype.

Noninvasive genotyping of dopamine receptor D4 (DRD4) using nanograms of DNA from substance-dependent patients.

A noninvasive DNA sampling method has been implemented collecting buccal mucosa cells by cotton wool swabs. An amount of 0.2 2 microg DNA per patient was obtained after the phenol-extraction procedure and 0.2 2 ng DNA template was sufficient for PCR amplification of the polymorphic 48 basepair repeat region of dopamine receptor D4 (DRD4) gene. PCR products were visualized during microfabricated electrophoretic separation by laser-induced fluorescent detection and automatic data registration. Initial data of genotyping drug-dependent subjects shows a relatively high ratio of heterozygotes, possessing either longer or shorter variants beside the common 4-repeat DRD4 allele.

Further evidence for the role of the dopamine D4 receptor (DRD4) gene in attachment disorganization: interaction of the exon III 48-bp repeat and the -521 C/T promoter polymorphisms.

In non-clinical low-risk populations 15% of infants show disorganized attachment behavior(1,2) with their caregivers in the Strange Situation,(3) a mildly stressful laboratory procedure testing infants' ability to cope with separation anxiety. Disorganization of early attachment has been primarily ascribed to inadequate parenting,(2,4,5) and has been associated with childhood behavior problems(6,7)and adolescent psychopathological tendencies.(5) We have recently reported an association between the DRD4 exon III 48 basepair repeat polymorphism and disorganization of infants' attachment behavior towards their mother in a low-social-risk group of 1-year-old infants:(8) the risk for disorganized attachment among infants carrying the 7-repeat allele was fourfold. Here we report further evidence for the involvement of the dopamine D4 receptor gene in attachment disorganization. The same group of infants was genotyped for the functional -521 C/T single nucleotide polymorphism (SNP) in the upstream regulatory region of the DRD4 gene(9) in order to test the association with attachment disorganization both alone and in interaction with the DRD4 exon III 7-repeat allele. While the -521 C/T genotype itself had no effect on attachment status (chi(2) = 0.41, df = 2, P = 0.82), there was an interaction between the structural 48-bp repeat polymorphism and the -521 C/T promoter polymorphism: the association between disorganized attachment and the 7-repeat allele was enhanced in the presence of the -521 T allele (chi(2) = 6.61 and 6.67, df = 1, P < 0.025 for CT and TT genotypes, respectively). In the presence of both risk alleles the odds ratio for disorganized attachment increased tenfold. This result supports our previous postulation that the DRD4 gene plays a role in the development of attachment behavior in low-risk, non-clinical populations.

Analysis of mutations in the plasma cholinesterase gene of patients with a history of prolonged neuromuscular block during anesthesia.

Decreased activity of plasma cholinesterase is responsible for prolonged apnea during anesthesia using neuromuscular blockers such as suxamethonium and mivacurium. More than 20 mutations have been identified so far in the BCHE gene resulting in impaired plasma cholinesterase activity. Biochemical tests are not always able to differentiate between pathological and normal sera; hence in some cases unanticipated complications can still occur during anesthesia even after measurements of enzyme activity and dibucaine numbers within the normal range. Therefore, molecular genetic testing is required for the accurate diagnosis of this deficiency. Here we present a study of plasma cholinesterase activity and BCHE genotyping of patients with a history of prolonged neuromuscular block and most of their pedigrees. All four exons of the BCHE gene were directly sequenced from samples and a number of mutations responsible for the reduction of plasma cholinesterase activity were identified. In most cases the atypical mutation in exon 2 (nt 209A --> G, Asp70 --> Gly) was found together with the K-variant mutation in exon 4 (nt 1615G --> A, Ala539 --> Thr), which is in good agreement with previous data suggesting that these mutations along with two others (at nt -116 and nt 1914) are in linkage disequilibrium.

High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.