Localization of the gene encoding the GABAA receptor β3 subunit to the Angelman/Prader-Willi region of human chromosome 15

Deletions of the proximal long arm of chromosome 15 (bands 15q11q13) are found in the majority of patients with two distinct genetic disorders, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). The deleted regions in the two syndromes, defined cytogenetically and by using cloned DNA probes, are similar. However, deletions in AS occur on the maternally inherited chromosome 15, and deletions in PWS occur on the paternally derived chromosome 15. This observation has led to the suggestion that one or more genes in this region show differential expression dependent on parental origin (genetic imprinting). No genes of known function have previously been mapped to this region. We show here that the gene encoding the GABAA (gamma-aminobutyric acid) receptor beta 3 subunit maps to the AS/PWS region. Deletion of this gene (GABRB3) was found in AS and PWS patients with interstitial cytogenetic deletions. Evidence of beta 3 gene deletion was also found in an AS patient with an unbalanced 13;15 translocation but not in a PWS patient with an unbalanced 9;15 translocation. The localization of this receptor gene to the AS/PWS region suggests a possible role of the inhibitory neurotransmitter GABA in the pathogenesis of one or both of these syndromes.     

The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.     

Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non‐endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.     

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.     

Analysis of commercial proanthocyanidins. Part 1: the chemical composition of quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood extract

Quebracho (Schinopsis lorentzii and Schinopsis balansae) extract is an important source of natural polymers for leather tanning and adhesive manufacturing. We combined established phyto- and synthetic chemistry perspectives with electrospray mass spectrometry experiments to prove that quebracho proanthocyanidin polymers consist of an homologous series of flavan-3-ol based oligomers. The starter unit is always catechin which is angularly bonded to fisetinidol extender units. By comparison of the MS(2) fragmentation spectra of the oligomer with product ion scans of authentic catechin and robinetinidol samples, we proved that quebracho extract contains no robinetinidol, as is often reported. Quebracho proanthocyanidins have acid resistant interflavanyl bonds, due to the absence of 5-OH groups in fisetinidol, and the aDP cannot be determined via conventional thiolysis and phloroglucinolysis. We used the MS data to estimate a conservative (minimum value) aDP of 3.1.     

Paucity of V-D-D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and TdT+/+ mice

CDR3 regions containing two D segments, or containing the footprints of V(H) replacement events, have been reported in both mice and humans. However, the 12-23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for V(H) replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential V(H) replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the "cryptic heptamer" but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and V(H) replacement are rare occurrences in the mouse.     

Towards a comprehensive structural variation map of an individual human genome

Background

Several genomes have now been sequenced, with millions of genetic variants annotated. While significant progress has been made in mapping single nucleotide polymorphisms (SNPs) and small (<10 bp) insertion/deletions (indels), the annotation of larger structural variants has been less comprehensive. It is still unclear to what extent a typical genome differs from the reference assembly, and the analysis of the genomes sequenced to date have shown varying results for copy number variation (CNV) and inversions.

Results

We have combined computational re-analysis of existing whole genome sequence data with novel microarray-based analysis, and detect 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing of the first published personal genome. We estimate a total non-SNP variation content of 48.8 Mb in a single genome. Our results indicate that this genome differs from the consensus reference sequence by approximately 1.2% when considering indels/CNVs, 0.1% by SNPs and approximately 0.3% by inversions. The structural variants impact 4,867 genes, and >24% of structural variants would not be imputed by SNP-association.

Conclusions

Our results indicate that a large number of structural variants have been unreported in the individual genomes published to date. This significant extent and complexity of structural variants, as well as the growing recognition of their medical relevance, necessitate they be actively studied in health-related analyses of personal genomes. The new catalogue of structural variants generated for this genome provides a crucial resource for future comparison studies.     

Developing a simplified consent form for biobanking

Background

Consent forms have lengthened over time and become harder for participants to understand. We sought to demonstrate the feasibility of creating a simplified consent form for biobanking that comprises the minimum information necessary to meet ethical and regulatory requirements. We then gathered preliminary data concerning its content from hypothetical biobank participants.

Methodology/Principal Findings

We followed basic principles of plain-language writing and incorporated into a 2-page form (not including the signature page) those elements of information required by federal regulations and recommended by best practice guidelines for biobanking. We then recruited diabetes patients from community-based practices and randomized half (n = 56) to read the 2-page form, first on paper and then a second time on a tablet computer. Participants were encouraged to use “More information” buttons on the electronic version whenever they had questions or desired further information. These buttons led to a series of “Frequently Asked Questions” (FAQs) that contained additional detailed information. Participants were asked to identify specific sentences in the FAQs they thought would be important if they were considering taking part in a biorepository. On average, participants identified 7 FAQ sentences as important (mean 6.6, SD 14.7, range: 0–71). No one sentence was highlighted by a majority of participants; further, 34 (60.7%) participants did not highlight any FAQ sentences.

Conclusions

Our preliminary findings suggest that our 2-page form contains the information that most prospective participants identify as important. Combining simplified forms with supplemental material for those participants who desire more information could help minimize consent form length and complexity, allowing the most substantively material information to be better highlighted and enabling potential participants to read the form and ask questions more effectively.