Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos

Genetic Risk Factors in Lupus Nephritis and IgA Nephropathy - No Support of an Overlap

Background: IgA nephropathy (IgAN) and nephritis in Systemic Lupus Erythematosus (SLE) are two common forms of glomerulonephritis in which genetic findings are of importance for disease development. We have recently reported an association of IgAN with variants of TGFB1. In several autoimmune diseases, particularly in SLE, IRF5, STAT4 genes and TRAF1-C5 locus have been shown to be important candidate genes. The aim of this study was to compare genetic variants from the TGFB1, IRF5, STAT4 genes and TRAF1-C5 locus with susceptibility to IgAN and lupus nephritis in two Swedish cohorts. Patients and Methods: We genotyped 13 single nucleotide polymorphisms (SNPs) in four genetic loci in 1252 DNA samples from patients with biopsy proven IgAN or with SLE (with and without nephritis) and healthy age-and sex-matched controls from the same population in Sweden. Results: Genotype and allelic frequencies for SNPs from selected genes did not differ significantly between lupus nephritis patients and SLE patients without nephritis. In addition, haplotype analysis for seven selected SNPs did not reveal a difference for the SLE patient groups with and without nephritis. Moreover, none of these SPNs showed a significant difference between IgAN patients and healthy controls. IRF5 and STAT4 variants remained significantly different between SLE cases and healthy controls. In addition, the data did not show an association of TRAF1-C5 polymorphism with susceptibility to SLE in this Swedish population. Conclusion: Our data do not support an overlap in genetic susceptibility between patients with IgAN or SLE and reveal no specific importance of SLE associated SNPs for the presence of lupus nephritis.Original Publication: Mai Tuyet Vuong, Iva Gunnarsson, Sigrid Lundberg, Elisabet Svenungsson, Lars Wramner, Anders Fernström, Ann-Christine Syvanen, Lieu Thi Do, Stefan H. Jacobson and Leonid Padyukov, Genetic Risk Factors in Lupus Nephritis and IgA Nephropathy - No Support of an Overlap, 2010, PLOS ONE, (5), 5. http://dx.doi.org/10.1371/journal.pone.0010559 Licensee: Public Library of Science (PLoS) http://www.plos.org/</p

CO carbonylation and first evaluation as a P-gp tracer in rats

BACKGROUND: At present, several positron emission tomography (PET) tracers are in use for imaging P-glycoprotein (P-gp) function in man. At baseline, substrate tracers such as R-[(11)C]verapamil display low brain concentrations with a distribution volume of around 1. [(11)C]phenytoin is supposed to be a weaker P-gp substrate, which may lead to higher brain concentrations at baseline. This could facilitate assessment of P-gp function when P-gp is upregulated. The purpose of this study was to synthesize [(11)C]phenytoin and to characterize its properties as a P-gp tracer. METHODS: [(11)C]CO was used to synthesize [(11)C]phenytoin by rhodium-mediated carbonylation. Metabolism and, using PET, brain pharmacokinetics of [(11)C]phenytoin were studied in rats. Effects of P-gp function on [(11)C]phenytoin uptake were assessed using predosing with tariquidar. RESULTS: [(11)C]phenytoin was synthesized via [(11)C]CO in an overall decay-corrected yield of 22 ± 4%. At 45 min after administration, 19% and 83% of radioactivity represented intact [(11)C]phenytoin in the plasma and brain, respectively. Compared with baseline, tariquidar predosing resulted in a 45% increase in the cerebral distribution volume of [(11)C]phenytoin. CONCLUSIONS: Using [(11)C]CO, the radiosynthesis of [(11)C]phenytoin could be improved. [(11)C]phenytoin appeared to be a rather weak P-gp substrate

Immunoseq: the identification of functionally relevant variants through targeted capture and sequencing of active regulatory regions in human immune cells

$\textbf{BACKGROUND}$: The observation that the genetic variants identified in genome-wide association studies (GWAS) frequently lie in non-coding regions of the genome that contain cis-regulatory elements suggests that altered gene expression underlies the development of many complex traits. In order to efficiently make a comprehensive assessment of the impact of non-coding genetic variation in immune related diseases we emulated the whole-exome sequencing paradigm and developed a custom capture panel for the known DNase I hypersensitive site (DHS) in immune cells - "Immunoseq". $\textbf{RESULTS}$: We performed Immunoseq in 30 healthy individuals where we had existing transcriptome data from T cells. We identified a large number of novel non-coding variants in these samples. Relying on allele specific expression measurements, we also showed that our selected capture regions are enriched for functional variants that have an impact on differential allelic gene expression. The results from a replication set with 180 samples confirmed our observations. $\textbf{CONCLUSIONS}$: We show that Immunoseq is a powerful approach to detect novel rare variants in regulatory regions. We also demonstrate that these novel variants have a potential functional role in immune cells.This work was supported by grants from the Canadian Institute of Health Research (CIHR), the UK Medical Research Council (G1100125), the Swedish Research Council (DO283001) and Knut and Alice Wallenberg Foundation (KAW). We also acknowledge the use of subjects from the Cambridge BioResource and the support of the Cambridge NIHR Biomedical Research Centre. AM was supported by the Fond de Recherche Santé Québec Doctoral training award. TP and CL holds a Canada Research Chair

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia

Peer reviewe

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection

Sequence-selective detection of double-stranded DNA sequences using pyrrole-imidazole polyamide microarrays

We describe a microarray format that can detect double-stranded DNA sequences with a high degree of sequence selectivity. Cyclooctyne-derivatized pyrrole-imidazole polyamides were immobilized on azide-modified glass substrates using microcontact printing and a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. These polyamide-immobilized substrates selectively detected a seven-base-pair binding site incorporated within a double-stranded oligodeoxyribonucleotide sequence even in the presence of an excess of a sequence with a single-base-pair mismatc

Genomics meets HIV-1

Genomics is now a core element in the effort to develop a vaccine against HIV-1. Thanks to unprecedented progress in high-throughput genotyping and sequencing, in knowledge about genetic variation in humans, and in evolutionary genomics, it is finally possible to systematically search the genome for common genetic variants that influence the human response to HIV-1. The identification of such variants would help to determine which aspects of the response to the virus are the most promising targets for intervention. However, a key obstacle to progress remains the scarcity of appropriate human cohorts available for genomic research

Ancient horizontal gene transfer and the last common ancestors

Background The genomic history of prokaryotic organismal lineages is marked by extensive horizontal gene transfer (HGT) between groups of organisms at all taxonomic levels. These HGT events have played an essential role in the origin and distribution of biological innovations. Analyses of ancient gene families show that HGT existed in the distant past, even at the time of the organismal last universal common ancestor (LUCA). Most gene transfers originated in lineages that have since gone extinct. Therefore, one cannot assume that the last common ancestors of each gene were all present in the same cell representing the cellular ancestor of all extant life. Results Organisms existing as part of a diverse ecosystem at the time of LUCA likely shared genetic material between lineages. If these other lineages persisted for some time, HGT with the descendants of LUCA could have continued into the bacterial and archaeal lineages. Phylogenetic analyses of aminoacyl-tRNA synthetase protein families support the hypothesis that the molecular common ancestors of the most ancient gene families did not all coincide in space and time. This is most apparent in the evolutionary histories of seryl-tRNA synthetase and threonyl-tRNA synthetase protein families, each containing highly divergent “rare” forms, as well as the sparse phylogenetic distributions of pyrrolysyl-tRNA synthetase, and the bacterial heterodimeric form of glycyl-tRNA synthetase. These topologies and phyletic distributions are consistent with horizontal transfers from ancient, likely extinct branches of the tree of life. Conclusions Of all the organisms that may have existed at the time of LUCA, by definition only one lineage is survived by known progeny; however, this lineage retains a genomic record of heterogeneous genetic origins. The evolutionary histories of aminoacyl-tRNA synthetases (aaRS) are especially informative in detecting this signal, as they perform primordial biological functions, have undergone several ancient HGT events, and contain many sites with low substitution rates allowing deep phylogenetic reconstruction. We conclude that some aaRS families contain groups that diverge before LUCA. We propose that these ancient gene variants be described by the term “hypnologs”, reflecting their ancient, reticulate origin from a time in life history that has been all but erased”.National Science Foundation (U.S.) (Grant DEB 0830024)Exobiology Program (U.S.) (Grant NNX10AR85G)United States. National Aeronautics and Space Administration (Postdoctoral Program