992 research outputs found
Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer
No full textAncient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position –3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222–228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17
Combination of 768-well microplate array diagonal gel electrophoresis with duplex PCR of X and Y chromosome markers for quality control of epidemiological DNA banks
No full textLarge DNA banks for human epidemiological studies have become an increasingly important research tool. The power of genotype-phenotype studies is dependent both on the quality of phenotyping and of genotyping and of correct linking of phenotypes to genotypes. Samples must be tracked through numerous steps between subject or patient and post-genotypic data. Only one phenotype, sex, has a perfect and binary correlation with genotype. In mixed sex studies, it may be advantageous for purposes of quality control to keep sexes mixed during the steps from acquisition to DNA bank, in order to be able to check later for sample swaps. We have designed a duplex PCR combining an amplicon from MAOA marking the X chromosome and an amplicon from DDX3Y marking the Y chromosome. We combined this with a simple economical palmtop sized 768-well microplate compatible electrophoresis system developed in-house for examination of duplex PCR products. We applied this quality control test in the validation of two DNA banks
Molecular pathogenesis of subarachnoid haemorrhage
No full textSubarachnoid haemorrhage (SAH) results from leakage of blood into the subarachnoid space and carries high morbidity and mortality. However, there is limited understanding to date, of the risk factors, cellular, intermediate biochemical and genetic traits predisposing to SAH. Nevertheless, in conjunction with improved methods of diagnostic imaging and less invasive approaches to preventing aneurysmal rupture, there may be utility in gaining a better understanding of the pathogenesis and in identifying pre-disease markers. Additionally, it is not impossible that drugs of value (e.g. matrix or endothelial modifiers) could become available. Several different clinical subtypes can be recognised, distinguished by arterial or venous involvement, presence of unruptured arterial aneurysms, and apparently ‘sporadic’ and ‘familial’ occurrences. Epidemiological risk factors include alcohol consumption and smoking: hypertension is a risk factor for rupture. About 10% seem to reflect strong family history and this subset may be particularly illuminating with respect to the molecular pathogenesis. Haemodynamic stress and poor vascular structure may be the main mechanisms of pathogenesis. The epidemiological and statistical evidence for familial megaphenic genes and modifier genes is reviewed. This review focuses on the pathogenesis, as opposed to inflammatory response to SAH. It sets in context the roles of specific genes and their protein products, such as polycystin (PKD1), fibrillin (FBN1), collagen III (COL3A1), elastin (ELN), collagen IV, protease inhibitor or ?1-antitrypsin (PI) and proteases. These considerations illustrate the shortfalls in current knowledge, the needs of future biochemical and cellular research and their potential implications for future prevention of this often fatal condition
Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping
No full textElectrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid–protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient ‘dry’ gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5–10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated. <br/
Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle
No full textPolymorphic dinucleotide repeat loci (microsatellite markers) are found in varying abundance throughout the genomes of most organisms. They have been extensively used for genetic studies, but conventional techniques used for their genotyping require sophisticated equipment. Microplate array diagonal gel electrophoresis (MADGE) has previously been extended to economical high-throughput genotyping of trinucleotide and tetranucleotide microsatellite amplicons. However, the capability of this technique to resolve the alleles of dinucleotide repeat loci has not been explored previously. Here we show that a modified microsatellite-MADGE approach can provide sufficient resolution for dinucleotide repeat typing. This enables economical and convenient set up for analysis of single markers in many samples in parallel, suitable, for example, for population association studies
Non-recombining chromosome Y haplogroups and centromeric HindIII RFLP in relation to blood pressure in 2,743 middle-aged Caucasian men from the UK
No full textEvidence from rodents and association analyses in humans suggest the presence on chromosome Y of one or more genes affecting blood pressure (BP). The HindIII centromeric alphoid polymorphism has been reported to be associated with BP in three independent human populations, although other studies reported null associations with this trait. Our objective was to test for association between BP and genetic variation of the Y chromosome. To this end, 2,743 unrelated Caucasian men recruited from nine UK practices were analysed for five SNPs (including the HindIII site) and two microsatellites spanning the non-recombining region of the Y chromosome. Systolic and diastolic BP were analysed both as quantitative traits and as categorical variables. Differences between locations were tested. Haplotypic and linkage disequilibrium (LD) analyses were also performed. Overall, no significant association was found between any of the loci analysed and BP, although post hoc analyses suggest a possible relation of specific Y haplogroups to BP. The HindIII polymorphism marks major structural differences in the Y centromere which could infuence mitotic loss during ageing, or other somatic events. However, this study does not support a causal effect on BP, although association of one or more Y haplogroups cannot be excluded
Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.
No full textThe QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD
MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers
No full textBackground: various software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.Results: we have developed a software package (MIDAS – Multiallelic Interallelic Disequilibrium Analysis Software) for the estimation and graphical display of interallelic linkage disequilibrium. Linkage disequilibrium is analysed for each allelic combination (of one allele from each of two loci), between all pairwise combinations of any type of multiallelic loci in a contig (or any set) of many loci (including single nucleotide polymorphisms, microsatellites, minisatellites and haplotypes). Data are presented graphically in a novel and informative way, and can also be exported in tabular form for other analyses. This approach facilitates visualisation of patterns of linkage disequilibrium across genomic regions, analysis of the relationships between different alleles of multiallelic markers and inferences about patterns of evolution and selection.Conclusion: MIDAS is a linkage disequilibrium analysis program with a comprehensive graphical user interface providing novel views of patterns of linkage disequilibrium between all types of multiallelic and biallelic markers
Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies
No full textRecuento de la inmigración mexicana. Historias. Revista de la Dirección de Estudios Históricos Num. 83 (2012) septiembre-diciembre
No full textDelia Salazar, Las cuentas de los sueños. La presencia extranjera en México a través de las estadísticas nacionales, 1880-1914, México, INM/DGE/INAH, 2010
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