A hypervariable segment in the human dopamine receptor D4 (DRD4) gene

The human dopamine D₄ receptor contains a novel polymorphismwithin the putative third cytoplasmic loop of the protein. Thepolymorphism is characterized by a varying number of directimperfect 48-bp repeats in the gene. Pharmacological characterizationhas suggested that this receptor is the site through which theatypical neuroleptic clozapine exerts its antipsychotic actionand that some polymorphic variants display different pharmacologicalproperties. Further analysis of the repeat region using innovativetechnologies indicates that the alleles vary not only in thenumber of repeats (2–8 or 10 repeat units) but also inthe sequence of the repeats and the order in which they appear.In 178 unrelated chromosomes we have identified 19 differentrepeats in 25 different haplotypes coding for 18 different predictedamino acid sequences, making this one of the most variable functionalproteins currently described.     

Dopamine D4 receptor variants in chinese sporadic and familial schizophrenics

Variation in the number of tandem repeats of a 48 base pair (bp) unit was found in the gene of the dopamine D4 receptor (DRD4). The number of repetitions of the 48bp unit was shown to influence the binding of clozapine, which suggests that different alleles may function differently in vivo and affect the pathogenesis of schizophrenia. Genotypes of DRD4 polymorphism were analyzed for 47 schizophrenic probands who had at least one living sibling with a diagnosis of schizophrenia, 35 unaffected siblings of the schizophrenic proband, 42 sporadic schizophrenic patients, and 43 healthy controls without a family history of psychosis. There was no significant difference in genotypic or allelic distributions among the four groups. Significant differences in the frequencies of two- and seven-repeats alleles between the Chinese and Caucasians controls were noted. The present study did not support that a particular allele or genotype of the 48bp-repeat of DRD4 was associated with schizophrenia. Am. J. Med. Genet. 74:412–415, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystropothhy

The sequence of the tandem repeat sequence (D4Z4) associatedwith facioscapulohumeral muscular dystrophy (FSHD) has beendetermined: each copy of the 3.3 kb repeat contains two homeoboxesand two previously described repetitive sequences, LSau anda GC-rich low copy repeat designated hhspm3. By Southern blotting,FISH and isolation of cDNA and genomic clones we show that thereare repeat sequences similar to D4Z4 at other locations in thehuman genome. Southern blot analysis of primate genomic DNAindicates that the copy number of D4Z4-like repeats has increasedmarkedly within the last 25 million years. Two cDNA clones wereisolated and found to contain stop codons and frameshifts withinthe homeodomains. An STS was produced to the cDNAs and analysisof a somatic cell hybrid panel suggests they map to chromosome14. No cDNA clones mapping to the chromosome 4q35 D4Z4 repeatshave been Identified, although the possiblilty that they encodea protein cannot be ruled out. Although D4Z4 may not encodea protein, there is an association between deletions withinthis locus and FSHD. The D4Z4 repeats contain LSau repeats andare adjacent to 68 bp Sau3A repeats. Both of these sequencesare associated with heterochromatic regions of DNA, regionsknown to be involved in the phenomenon of position effect variegation.We postulate that deletion of D4Z4 sequences could produce aposition effect.     

Association analysis of the DRD4 and COMT genes in methamphetamine abuse.

We analyzed two polymorphisms in genes encoding proteins of the dopamine system, the Val158Met polymorphism in the catechol-O-methyltransferase gene and the 120-bp VNTR polymorphism in the promoter of the dopamine D4 receptor gene for association with methamphetamine abuse. We used a case/control design with 416 methamphetamine abusing subjects and 435 normal controls. All subjects were Han Chinese from Taiwan. We found an excess of the high activity Val158 allele in the methamphetamine abuser group, consistent with several previous reports of association of this allele with drug abuse. The 120-bp VNTR polymorphism in the promoter of the dopamine D4 receptor gene itself did not show significant association with methamphetamine abuse. However, analysis of the 120-bp VNTR polymorphism and the exon 3 VNTR in the dopamine D4 receptor as a haplotype showed significant association with methamphetamine abuse, which gave an empirical P value 0.0034 for a heterogeneity model. Moreover, there were significant interactive effects between polymorphisms in the catechol-O-methyltransferase and dopamine D4 genes. The evidence of interaction between COMT 158 Val/Met and DRD4 48-bp VNTR polymorphisms (P = 0.0003, OR = 1.45, 95% CI: 1.148-1.77), and between COMT 158 Val/Met and DRD4 120 bp promoter polymorphisms (P = 0.01, OR = 1.10, 95% CI: 1.10-1.18) were significant but the latter was weak. We conclude that genetic variation in the dopamine system may encode an additive effect on risk of becoming a methamphetamine abuser.     

Association of dopamine D4 receptor (DRD4) polymorphisms with attention deficit hyperactivity disorder in Indian population.

Attention deficit hyperactivity disorder (ADHD) is a childhood onset neurobehavioral disorder. Several studies worldwide have implicated a possible association between ADHD and transmission of different polymorphisms of the dopamine D4 receptor gene (DRD4) in different ethnic groups. However, this is the first report on the transmission of different polymorphisms of DRD4 in Indian subjects. Association of 5' flanking 120-bp duplication, exon 1 12-bp duplication, and exon 3 48-bp variable numbers of tandem repeats (VNTR) were analyzed in 50 ADHD cases. Haplotype-based haplotype relative risk (HHRR) analysis and transmission disequilibrium test (TDT) were carried out to ascertain the association of these polymorphisms with the disorder. Linkage disequilibria (LD) between the polymorphisms were calculated using EH+ and 2LD programs. Our preliminary data showed lack of association between ADHD and transmission of the 5' flanking 120-bp duplication and exon 1 12-bp duplication. But, the transmissions of 6 and 7 repeat alleles of exon 3 48-bp VNTR showed significant association with ADHD. We have also examined the haplotype frequencies and biased transmission of one haplotype was observed in ADHD probands. LD analysis showed very strong disequilibrium between exon 1 12-bp duplication and exon 3 48-bp VNTR. Strong LD was also observed between the 5' flanking 120-bp duplication and exon 1 12-bp duplication. The observed association between higher repeat alleles of exon 3 48-bp VNTR and Indian ADHD children is consistent with some of the earlier reports.     

Autosomal location of a new subtype of 1.688 satellite DNA ofDrosophila melanogaster

During the screening of aDrosophila melanogaster YAC library with DNA from the minichromosomeDp(1;f)1187 we isolated a clone, yw20D5, which contains a new subtype of 1.688 satellite DNA. Although the sequences of several monomers subcloned from the YAC show a considerable variation in length, the derived consensus sequence is 356-bp long. This new subtype and the one constituted by the 353-bp repeats are both located on the left arm heterochromatin of chromosome 3, arranged in separate arrays. Despite their autosomal location, phylogenetic relationships among 1.688 satellite sequences suggest that they may have originated from the 359-bp repeats of the X chromosome heterochromatin. We have used the new 356-bp repeats to investigate whether sequences related to the 1.688 satellite are dispersed along the euchromatic arms of the autosomes in a similar way to that in which they are found along the X chromosome euchromatin.accepted for publication by D. Ward     

Alleles at the dopamine D4 receptor locus do not contribute to the genetic susceptibility to schizophrenia in a large Swedish kindred

The discovery of a functional polymrphism within the dopamine D₄ receptor gene (DRD4) has not only strengthened the hypotheses implicating DRD4 in the etiology of neuropyschiatic disorders, but also provided a genetic marker for testing these hypotheses. The possibility of the dopamine D₄ receptor as a candidate gene for schizophrenia was investigated in a large Swedish kindred segregating for schizophrenia. Linkage to schizophrenia was tested by linkage analyses of 6 polymorphic markers (at 4 loci) in chromosome 11p15.5 including the dopamine D₄ receptor (DRD4) and the tyrosine hydroxylase (TH) loci. Schizophrenia was excluded from close linkage to the DRD4 locus using two of the polymorphisms located within the dopamine D₄ receptor gene. The first DRD4 polymorphism consists of variation in the number of a 48 bp imperfect direct repeat in the third exon; the second consists of a variable number of repeated G nucleotides in the first intron. In addition, some of the individuals homozygous for four or seven copies of 48 bp repeat alleles were tested for previously reported sequence variation among repeats. No single haplotype of the DRD4 alleles or haplotype of other markers in chromosome 11p15.5 was found to be common to the schizophrenic individuals in this family. Therefore, we find no evidence for linkage of the D₄ receptor, or this region of 11p15.5, with genetic susceptibility to schizophrenia in this kindred. © 1993 Wiley-Liss, Inc.     

Aggression,Digit Ratio,and Variation in the Androgen Receptor,Serotonin Transporter,and Dopamine D4 Receptor Genes in African Foragers: The Hadza

The role of genes in the expression of aggression and masculinity traits in humans has been a focus of recent behavioral genetic studies. This is the first study on the variation in aggression, the digit ratio (the ratio between the second and the fourth digits, 2D:4D), the directional asymmetry in 2D:4D (D_R-L) and polymorphisms of the AR, DRD4, and 5-HTTL genes in simple hunter-gatherers, namely the Hadza of Tanzania (142 adult men). The distribution of AR, DRD4E3, and 5-HTTLPR genotypes and allele frequencies in Hadza was compared to other African populations on which the data were available. Hadza and Ariaal differed significantly in the distributions of frequencies of AR alleles with different numbers of CAG repeats. Hadza population was similar to other African populations in the distribution of allelic frequencies of the DRD4E3 locus, and to Afro-Americans in the distribution of allelic types of the 5-HTTLPR locus. We found no influence of AR gene on the right hand 2D:4D ratio, D_R-L, and any of aggression subscales of the Buss-Perry Aggression Questionnaire (AQ). Although, a weak positive correlation between CAG repeats and the left hand 2D:4D was found. The multiple regression analysis with digit ratios, D_R-L and aggression subscales of AQ as dependent variables and the three gene candidates (AR, DRD4E3, and 5-HTTLPR) as independent variables revealed the following: men with lower number of CAG repeats had significantly lower left hand 2D:4D ratio; men with higher numbers of 48-bp unit copies in exon 3 of a VNTR polymorphism in the DRD4 gene had significantly lower digit ratios on both hands; no effect of the 5-HTTLPR gene on either the digit ratio or aggressive behavior. These findings demonstrate the complexity of gene effects on digit ratios and aggression and call for simultaneous analysis of more candidate genes. It is noteworthy that these results were obtained for a human population that is still practicing foraging and has been subjected to a high selective pressure due to harsh environments and practically has no access to modern medical care. Hadza are highly egalitarian, and their culture does not favor persons with a dominant or aggressive behavior. It is still to be found to what extent the relationships observed in this study are similar to those in other human populations.     

Family-based and case-control association studies of DRD4 and DAT1 polymorphisms in Chinese attention deficit hyperactivity disorder patients suggest long repeats contribute to genetic risk for the disorder.

Molecular genetic studies of attention deficit hyperactivity disorder (ADHD) have implicated the variable number of tandem repeat (VNTR) polymorphisms of two candidate genes, the dopamine D4 receptor (DRD4) and the dopamine transporter (DAT1). We sought to determine if these genes were relevant to the etiology of ADHD in China by using both family-based (N = 202 nuclear ADHD families) and case-control (N = 340 ADHD cases, and 226 controls) association study designs. Diagnoses and subtypes were ascertained according to Clinical Diagnostic Interview Scales (CDIS) using DSM-IV criteria. The repeat numbers at the DRD4 VNTR ranged from 2 to 6 repeats in the Han Chinese controls, with the most common being the 4-repeat (77%) and 2-repeat (19.4%) alleles. Neither the 7-repeat allele nor longer repeats were found. For the DAT1 VNTR, the repeat numbers ranged from 6 to 7 repeats and 9 to 11 repeats. The 10-repeat allele was the most frequent (90.7%). The long-repeat alleles of DRD4 (ranging from 4 to 6 repeats) and DAT1 (ranging from 11 to 12 repeats), were present more frequently in ADHD probands than controls (P < 0.05), although there was no significant allelic association when the alleles were analyzed separately from each other and there findings were not supported by within family tests of association. An exploratory stratification by gender suggests that long-repeat alleles of DRD4 and DAT1 may increase the risk for ADHD in Han Chinese children.     

Stability of Marek’s disease virus 132-bp repeats during serial in vitro passages

Summary. The Marek’s disease virus (MDV) genome contains 2 sets of 132-bp tandem repeat sequences. An increase in 132-bp repeat units has been associated with attenuation of oncogenicity during in vitro passage. By cloning entire genomes, we demonstrated that the copy number of 132-bp repeats can differ within an individual MDV genome. The stability of the 132-bp repeats during cell passage depended on the initial copy number. When both sets of repeats contained 2 copies, the copy number remained stable, while if even 1 set of repeats contained 6 copies, repeat expansion occurred relatively quickly. This expansion did not affect the in vitro growth curve. However, when MDV clones with low and high copy numbers were passed together, genomes with expanded repeats rapidly predominated, mimicking the behavior of naturally-occurring MDV. These results suggest that the preponderance of high-copy repeats after passage reflects intracellular copy number within individual infected cells rather than an influence on the spread of the virus.     

Compound microsatellite repeats: practical and theoretical features.

Most linkage and population genetic studies that use microsatellites assume that the polymorphism observed at these loci is due simply to variation in the number of units of a single repeat. Variation is far more complex, however, for the numerous microsatellites that contain interruptions within the repeat or contain more than one type of repeat. We observed that for D18S58, a compound microsatellite containing (CG)(m), as well as (CA)(n) repeats, the apparent length of certain alleles varied between genotyping experiments. Similar results were obtained with other (CG)(m)-(CA)(n) repeats. Sequencing demonstrated that the D18S58 alleles demonstrating variable mobility contained longer (CG)(m) stretches than those alleles whose length did not appear to vary between experiments. These results suggest that (CG)(m) repeats, which are frequently present in compound human microsatellites, are prone to form an unusually stable secondary structure. We discuss the relative frequency of different classes of compound microsatellites identified through database searches, as well as their patterns of sequence and variation. Further characterization of such variation is important for elucidating the origin, mutational processes, and structure of these widely used, but incompletely understood, sequences.     

Unique sequence requirements for the P1 plasmid replication origin

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although five boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.     

Lack of association between dopamine receptor D4 variable numbers of tandem repeats gene polymorphism and smoking

Nicotine addiction, related to cigarette smoking, develops as a product of the complex interactions between social, environmental and genetic factors. Genes encoding the components of the dopaminergic system are thought to be associated with smoking. Literature data showed not only an association, but also a lack of association between variable number of tandem repeats (VNTR) polymorphism located in the third exon of dopamine D4 receptor (DRD4) gene and smoking. Repetitive sequence of DRD4 VNTR is 48 bp long and maximum 11 tandem copies were reported in humans. Presence of alleles with 6 and more repeats (i.e. long alleles) was associated with greater tendency to novelty seeking and addictive behaviors than the presence of 5 and less alleles (short alleles). The aim of this study was to determine the association between VNTR in DRD4 gene and present smoking status in ethnically homogenous Caucasian population from the Eastern European (Croatian) origin. Genotyping was done in 565 healthy subjects, 511 men and 54 women, respectively, who were subdivided into 176 smokers and 389 nonsmokers. Logistic regression analyses, adjusted for age and sex, revealed the lack of significant (p > 0.05) effect of the 4/4, 4/7 and 7/7 genotypes, or carriers of the long and short allele, or all genotypes of the DRD4 VNTR on smoking status. The results of this study failed to confirm the hypothesis that long allele of the DRD4 VNTR is associated with smoking status in Caucasian subjects.     

Chicks from a high and low feather pecking line of laying hens differ in apomorphine sensitivity

Proactive rodents show a larger behavioral response to apomorphine (APO) than reactive copers, suggesting a more sensitive DA system in proactive individuals. Previously, chicks from a high feather pecking (HFP) and low feather pecking line (LFP) have been suggested to display a proactive and reactive coping strategy, respectively. Therefore, at approximately 4 weeks of age, the behavior of 48 LFP and 48 HFP chicks in response to an APO injection was studied using an open field. Another objective of the present study was to determine whether behavioral variation (in an open field) between HFP and LFP birds, after APO injection, is also reflected by variation of D(1) and D(2) receptor densities in the brain. Receptor binding capacities were assessed by measuring specific binding of tritiated D(1) and D(2) receptor ligands in different regions of the brain of control HFP and LFP chicks. In the present study, it is shown that indeed HFP chicks display a more enhanced behavioral response to acute APO treatment (0.5 mg/kg BW) than LFP birds in an open field. This difference was not reflected by variation of D(1) and D(2) receptor densities in the brain between both lines.     

Variable numbers of simple tandem repeats make birds of the order Ciconiiformes heteroplasmic in their mitochondrial genomes

We have analyzed a variable domain of the mitochondrial DNA control region of 18 avian species. intra-individual length variation was identified and characterized in 15 species. The occurrence of heteroplasmy among species is phylogenetically consistent with a current classification of birds. Polymerase chain reaction amplifications, direct sequencing, and Southern analysis of mitochondrial DNA showed that the heteroplasmy is due to variable numbers of direct repeats in a tandem organization, located in the control region close to the tRNA^Phe gene. The tandem repeats consist of short sequence motifs that vary in size from 4 to 32 base pairs between species. Sequence complexity of the repeat motifs was low, with almost exclusively Ts and Gs in the heavy-strand. Extensive variation in the copy number of the repeats was seen both intra-specifically and within individuals. This is the first report of mitochondrial heteroplasmy characterized at the sequence level in birds.