Lack of Association Between a 3 ` UTR VNTR Polymorphism of Dopamine Transporter Gene (SLC6A3) and ADHD in a Brazilian Sample of Adult Patients

Objective: To investigate a possible association between a 3`UTR VNTR polymorphism of the dopamine transporter gene (SLC6A3) and ADHD in a Brazilian sample of adult patients. Method: Study Case-control with 102 ADHD adult outpatients (DSM-IV criteria) and 479 healthy controls. The primers` sequence used were: 3`UTR-Forward: 5`TGT GGT GAT GGG AAC GGC CTG AG 3` and 3`UTR-Reverse: 5`CTT CCT GGA GGT CAC GGC TCA AGG 3`. Alleles of the 3`UTR were coded according to their number of repeats: 6- repeat 320 bp (allele 6), 8- repeat 400 bp (allele 8), 9-repeat 480 bp (allele 9), 10- repeat 480 bp (allele 10), and 11- repeat 520 bp (allele 11). Results: There were no allelic (chi(2) = 2.67, 5df, p = .75) and genotypic (chi(2) = 7.20, 1df, p = .61) association between adult ADHD and VNTR 3`UTR polymorphism of SLC6A3. Conclusion: Our findings do not support SLC6A3 as marker genetic susceptibility factor in adult ADHD. More comprehensive polymorphism coverage within the SLC6A3 region should be conducted in larger samples, including comparisons in clinical subgroups, and in samples with different ethnic backgrounds. (J. of Att. Dis. 2011; 15(4) 305-309)

Cytogenetic molecular delineation of a terminal 18q deletion suggesting neo-telomere formation

Deletion of the long arm of chromosome 18 is one of the most common segmental aneusomies compatible with life and usually involves a deletion of the terminal chromosomal region. However, the mechanisms implicated in the stabilization of terminal deletions are not well understood. In this study, we analyzed a girl with moderate mental retardation who had a cytogenetically visible terminal 18q deletion. In order to characterize the breakpoint in the terminal 18q region, we used fluorescence In situ hybridization (FISH) with bacterial artificial chromosomes (BACs) and pan-telomeric probes and also the array technique based on comparative genomic hybridization (array-CGH). FISH with pan-telomeric probes revealed no signal in the terminal region of the deleted chromosome, indicating the absence of normal telomere repeat (TTAGGG)n sequences in 18q. We suggest that neo-telomere formation by chromosome healing was involved in the repair and stabilization of this terminal deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.

Candidate-Gene Approach in Posttraumatic Stress Disorder After Urban Violence: Association Analysis of the Genes Encoding Serotonin Transporter, Dopamine Transporter, and BDNF

Posttraumatic stress disorder (PTSD) is a prevalent, disabling anxiety disorder marked by behavioral and physiologic alterations which commonly follows a chronic course. Exposure to a traumatic event constitutes a necessary, but not sufficient, factor. There is evidence from twin studies supporting a significant genetic predisposition to PTSD. However, the precise genetic loci still remain unclear. The objective of the present study was to identify, in a case-control study, whether the brain-derived neurotrophic factor (BDNF) val66met polymorphism (rs6265), the dopamine transporter (DAT1) three prime untranslated region (3`UTR) variable number of tandem repeats (VNTR), and the serotonin transporter (5-HTTPRL) short/long variants are associated with the development of PTSD in a group of victims of urban violence. All polymorphisms were genotyped in 65 PTSD patients as well as in 34 victims of violence without PTSD and in a community control group (n = 335). We did not find a statistical significant difference between the BDNF val66met and 5-HTTPRL polymorphism and the traumatic phenotype. However, a statistical association was found between DAT1 3`UTR VNTR nine repeats and PTSD (OR = 1.82; 95% CI, 1.20-2.76). This preliminary result confirms previous reports supporting a susceptibility role for allele 9 and PTSD.

Chronic Acetonemia Alters Liver Oxidative Balance and Lipid Content in Rats. A Model of Nash?

Acetone is considered to be a substance that can disturb cellular oxidative status, being also associated with the production of glucose during its metabolization. The objective of the present study was to determine the effects of chronic treatment with acetone in oxidative stress and metabolic parameters in rats. Twenty male Wistar rats were divided into two groups: control (CG) and chronic acetone group (CAG). After 28 days of acetone ingestion in a 5% aqueous solution (CAG) or water (CG) the animals were euthanized and urine, plasma and liver were collected for the determination of acetone, glucose, lipemia, hepatic fat, malondialdehyde (MDA), reduced glutathione (GSH), and vitamin E. As expected, urinary and plasma acetone levels were higher in CAG. There was no difference in hepatic MDA values between groups, whereas hepatic GSH was lower in CAG than in CG and hepatic vitamin E was higher in CAG than in CG. There was also an increase in glycemia, cholesterolemia and hepatic fat in CAG compared to CG. Chronic treatment with a 5% acetone solution produced an increase in acetonemia that was able to promote changes in hepatic oxidative metabolism and in lipid content in rats similar to those observed in nonalcoholic steatohepatitis.

Effects of eNOS polymorphisms on nitric oxide formation in healthy pregnancy and in pre-eclampsia

Pre-eclampsia (PE) is associated with decreased nitric oxide (NO) formation. However, no previous study has examined whether genetic variations in the endothelial NO synthase (eNOS) affect this alteration. We hypothesized that PE decreases NO formation depending on eNOS polymorphisms. We examined how three eNOS polymorphisms [T-786C, rs2070744; Glu298Asp, rs1799983; 27 bp variable number of tandem repeats (VNTR) in intron 4] affect plasma nitrite concentrations in 205 pregnant women [107 healthy pregnant (HP) and 98 PE]. Genotypes were determined and eNOS haplotypes were inferred using the PHASE 2.1 program. The plasma nitrite concentrations were determined using an ozone-based chemiluminescence assay. The Glu298Asp polymorphism had no effects on the plasma nitrite concentrations. Higher nitrite levels were found in HP women with the CC versus TT genotype for the T-786C polymorphism (277.9 +/- 19.5 versus 140.6 +/- 8.2 nM; P < 0.05). Lower nitrite levels were found in healthy women with the 4a4a versus 4b4b genotype for the VNTR polymorphism (95.1 +/- 3.3 versus 216.1 +/- 16.8 nM; P < 0.05). No effects of genotypes were found in PE women (all P > 0.05). The `C Glu b` haplotype was more frequent in the HP group than in the PE group (20 versus 5; P = 0.0044). This haplotype was associated with higher nitrite concentrations than the other haplotypes in healthy pregnancies (P < 0.05). No differences in nitrite concentrations were found among PE women with different eNOS haplotypes (P > 0.05). These findings indicate that eNOS polymorphisms affect endogenous NO formation in normal pregnancy, but not in PE, and that the `C Glu b` haplotype may protect against the development of PE by increasing endogenous NO formation.

Huntington`s Disease-Like 2 in Brazil-Report of 4 Patients

Huntington`s disease-like 2 (HDL2) is a neurodegenerative disorder found in people of African ancestry with clinical, radiological, and neuropathological manifestations similar to Huntington`s disease (HD). HDL2 is caused by a pathological expansion of CAG/CTG triplets in exon 2A of the JPH3 gene. We describe four cases of HDL2 from four unrelated families, and discuss their clinical findings. HDL2 should be considered in every patient with an HD-like phenotype who tests negative for the HD mutation, even if African ancestry is not immediately apparent. (C) 2008 Movement Disorder Society

Development and characterization of 15 microsatellite loci for Cariniana estrellensis and transferability to Cariniana legalis, two endangered tropical tree species

From a genomic library enriched for GA/CA repeats, 15 highly polymorphic microsatellite markers were developed for Cariniana estrellensis, a tropical forest tree. The microsatellite loci were screened in 49 mature trees found between Pardo river and Mogi-Gua double dagger u river basins, in the state of So Paulo, Brazil. A total of 140 alleles were detected with an average of 9.33 alleles per locus. The expected heterozygosity ranged from 0.37 to 0.88. These loci showed a high probability of paternity exclusion. Additionally, 12 loci were effectively transferred to Cariniana legalis. High levels of polymorphism make the present SSR markers useful for population genetic studies.

Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Matrix metalloproteinase 9 gene haplotypes affect left ventricular hypertrophy in hypertensive patients

Background: Matrix metalloproteinases (MMPs) are involved in cardiac remodeling and are encoded by genes showing genetic polymorphisms that have functional implications. We examined whether MMP-9 genetic polymorphisms are associated with hypertension and with left ventricular (LV) remodeling in hypertensive patients. Methods: We studied 173 hypertensive patients and 137 age, race and gender matched healthy controls. Heart echocardiography was performed in all patients and the following MMP-9 genetic polymorphisms were analyzed: C-(1562)T (rs3918242). -90 (CA)(14-24) (rs2234681) and Q279R (rs17576). Haplo.stats analysis was used to assess whether MMP-9 haplotypes are associated with hypertension. Linear regression analysis was performed to assess whether MMP-9 haplotypes affect LV mass index (LVMI) and other echocardiography parameters. Results: MMP-9 90 (CA)14-24 ""HH"" genotype (H allele defined by number of CA repeats >= 21) was associated with hypertension (P = 0.0085; OR = 2.321, 95% confidence interval = 1.250 to 4.309). While one MMP-9 haplotype (""C. H, Q"") protects against LVMI and end-diastolic diameter increases due to remodeling (P = 0.0490 and P = 0.0367), another MMP-9 haplotype apparently has detrimental effects over both parameters in hypertensive patients (""T, H. Q"", P = 0.0015 and P = 0.0057. respectively). Conclusion: Genetic polymorphisms in MMP-9 gene may modify the susceptibility of hypertensive patients to LV remodeling. Further studies are necessary to examine whether these polymorphisms affect clinical events in hypertensive patients. (C) 2010 Elsevier B.V. All rights reserved.

Curupira-1 and Curupira-2, two novel Mutator-like DNA transposons from the genomes of human parasites Schistosoma mansoni and Schistosoma japonicum

Transposons of the Mutator superfamily have been widely described in plants, but only recently have metazoan organisms been shown to harbour them. In this work we describe novel Mutator superfamily transposons from the genomes of the human parasites Schistosoma mansoni and S. japonicum, which we name Curupira-1 and Curupira-2. Curupira elements do not have Terminal Inverted Repeats (TIRs) at their extremities and generate Target Site Duplications (TSDs) of 9 base pairs. Curupira-2 transposons code for a conserved transposase and SWIM zinc finger domains, while Curupira-1 elements comprise these same domains plus a WRKY zinc finger. Alignment of transcript sequences from both elements back to the genomes indicates that they are subject to splicing to produce mature transcripts. Phylogenetic analyses indicate that these transposons represent a new lineage of metazoan Mutator-like elements with characteristics that are distinct from the recently described Phantom elements. Description of these novel schistosome transposons provides new insights in the evolution of transposable elements in schistosomes.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

Functional polymorphisms in the promoter of the matrix metalloproteinase-9 (MMP-9) gene are not linked with significant plasma MMP-9 variations in healthy subjects

Background: Matrix metalloproteinase-9 (MMP-9) is involved in the degradation of the extracellular matrix during physiological and pathological processes. Two functional polymorphisms [C(-1562)T and microsatellite (CA)(13-25)] in the promoter region of the MMP-9 gene have been associated with several diseases. The aim of this study was to examine whether these MMP-9 polymorphisms and haplotypes are linked with plasma MMP-9 variations in healthy subjects. Methods: We studied 177 healthy male white volunteers (age range 20-55 years) who were non-smokers and not taking any medication. Genomic DNA was extracted from whole blood and genotypes for the C(-1562)T and the microsatellite (CA)(n) polymorphisms were determined. MMP-9 levels were measured in plasma samples by gelatin zymography. Results: The frequency of the alleles C and T for the C(-1562)T polymorphism were 90% and 10%, respectively. The frequency of the alleles with less than 21 CA repeats Q and with 21 repeats or higher (H) were 47% and 53%, respectively. We found no differences in plasma MMP-9 levels among the genotype groups or among different haplotypes (all p > 0.05). Conclusions: These findings suggest that functional polymorphisms in the promoter of the MMP-9 gene are not linked with significant plasma MMP-9 variations in healthy subjects.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different Solanum species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1-4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613-884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species.

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed ""RaTART"" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.