Experiences with array-based sequence capture; toward clinical applications

Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions

Initial pen and field assessment of baits to use in oral rabies vaccination of Formosan ferret-badgers in response to the re-emergence of rabies in Taiwan

<div><p>Background</p><p>Taiwan had been considered rabies free since 1961, until a newly established wildlife disease surveillance program identified rabies virus transmission within the Formosan ferret-badger (<i>Melogale moschata subaurantiaca</i>) in 2013. Ferret-badgers occur throughout southern China and Southeast Asia, but their ecological niche is not well described.</p><p>Methodology/Principle findings</p><p>As an initial feasibility assessment for potential rabies control measures, field camera trapping and pen assessment of 6 oral rabies vaccine (ORV) baits were conducted in Taiwan in 2013. 46 camera nights were recorded; 6 Formosan ferret-badgers and 14 non-target mammals were sighted. No baits were consumed by ferret-badgers and 8 were consumed by non-target mammals. Penned ferret-badgers ingested 5 of the 18 offered baits. When pen and field trials were combined, and analyzed for palatability, ferret-badgers consumed 1 of 9 marshmallow baits (11.1%), 1 of 21 fishmeal baits (4.8%), 0 of 3 liver baits, and 3 of 3 fruit-flavored baits. It took an average of 261 minutes before ferret-badgers made oral contact with the non-fruit flavored baits, and 34 minutes for first contact with the fruit-based bait. Overall, ferret-badgers sought out the fruit baits 8 times faster, spent a greater proportion of time eating fruit baits, and were 7.5 times more likely to have ruptured the vaccine container of the fruit-based bait.</p><p>Conclusions/Significance</p><p>Ferret-badgers are now recognized as rabies reservoir species in China and Taiwan, through two independent ‘dog to ferret-badger’ host-shift events. Species of ferret-badgers can be found throughout Indochina, where they may be an unrecognized rabies reservoir. Findings from this initial study underscore the need for further captive and field investigations of fruit-based attractants or baits developed for small meso-carnivores. Non-target mammals’ competition for baits, ants, bait design, and dense tropical landscape represent potential challenges to effective ORV programs that will need to be considered in future studies.</p></div

Captive ferret badger feeding stations and consumption mechanics.

<p>Captive ferret badger feeding stations and consumption mechanics.</p

Three oral rabies vaccination bait constructs assessed for feasibility of use in ferret-badgers.

<p>Three oral rabies vaccination bait constructs assessed for feasibility of use in ferret-badgers.</p

Set-up used for testing the five bait types used in field trials.

<p>Cameras were set at dusk and retrieved in the morning. Data collected from images of ferret-badgers and non-target species were assessed on four criteria: 1) Animals were observed but had no interaction with baits; 2) Animals investigated the baits (attractiveness); 3) Animals had direct physical contact with the baits (attractiveness); 4) Animals were observed to have ingested the entire bait or there was evidence that the vaccine container was punctured (palatability).</p

Comparison of five oral rabies vaccines in four field sites, Taiwan 2013.

<p>Information was collected on whether the baits were investigated, contacted, ingested or removed, and ruptured.</p

Ferret-badger photographed at field camera site.

<p>Ferret-badger photographed at field camera site.</p

Comparison of 6 oral rabies vaccination baits in a pen trial, Taiwan 2013.

<p>Comparison of 6 oral rabies vaccination baits in a pen trial, Taiwan 2013.</p

Identification of a potential physiological precursor of aberrant cells in refractory coeliac disease type II

<p>Objective Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3(-)CD7(+)icCD3(+)phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells.</p><p>Design RCDII cell lines were compared with T-cell receptor positive (TCR+) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry.</p><p>Results RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3(-)CD7(+)icCD3(+) phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin(-)) CD7(+)CD127(-)CD34(-) phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin(-)CD7(+)CD127(-)CD34(-) subset was one of four subsets within the CD3(-)CD7(+)icCD3(+) population that could be distinguished on the basis of differential expression of CD56 and/or CD127.</p><p>Conclusion Our studies indicate that the CD3(-)CD7(+)icCD3(+) population is heterogeneous and reveal the existence of a Lin(-) subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma.</p>