High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

Background

Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

Methods

Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.

Results

Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.

Conclusions

These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.     

The STK33-Linked SNP rs4929949 Is Associated with Obesity and BMI in Two Independent Cohorts of Swedish and Greek Children

Recent genome wide association studies (GWAS) have identified a locus on chromosome 11p15.5, closely associated with serine/threonine kinase 33 (STK33), to be associated with body mass. STK33, a relatively understudied protein, has been linked to KRAS mutation-driven cancers and explored as a potential antineoplastic drug target. The strongest association with body mass observed at this loci in GWAS was rs4929949, a single nucleotide polymorphism located within intron 1 of the gene encoding STK33. The functional implications of rs4929949 or related variants have not been explored as of yet. We have genotyped rs4929949 in two cohorts, an obesity case-control cohort of 991 Swedish children, and a cross-sectional cohort of 2308 Greek school children. We found that the minor allele of rs4929949 was associated with obesity in the cohort of Swedish children and adolescents (OR = 1.199 (95%CI: 1.002–1.434), p = 0.047), and with body mass in the cross-sectional cohort of Greek children (β = 0.08147 (95% CI: 0.1345–0.1618), p = 0.021). We observe the effects of rs4929949 on body mass to be detectable already at adolescence. Subsequent analysis did not detect any association of rs4929949 to phenotypic measurements describing body adiposity or to metabolic factors such as insulin levels, triglycerides and insulin resistance (HOMA).     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.     

Phylogenetic relationships of Microdontinae (Diptera: Syrphidae) based on molecular and morphological characters

The intrasubfamilial classification of Microdontinae Rondani (Diptera: Syrphidae) has been a challenge: until recently more than 300 out of more than 400 valid species names were classified in Microdon Meigen. We present phylogenetic analyses of molecular and morphological characters (both separate and combined) of Microdontinae. The morphological dataset contains 174 characters, scored for 189 taxa (9 outgroup), representing all 43 presently recognized genera and several subgenera and species groups. The molecular dataset, representing 90 ingroup species of 28 genera, comprises sequences of five partitions in total from the mitochondrial gene COI and the nuclear ribosomal genes 18S and 28S. We test the sister‐group relationship of Spheginobaccha with the other Microdontinae, attempt to elucidate phylogenetic relationships within the Microdontinae and discuss uncertainties in the classification of Microdontinae. Trees based on molecular characters alone are poorly resolved, but combined data are better resolved. Support for many deeper nodes is low, and placement of such nodes differs between parsimony and Bayesian analyses. However, Spheginobaccha is recovered as highly supported sister group in both. Both analyses agree on the early branching of Mixogaster, Schizoceratomyia, Afromicrodon and Paramicrodon. The taxonomical rank in relation to the other Syrphidae is discussed briefly. An additional analysis based on morphological characters only, including all 189 taxa, used implied weighting. A range of weighting strengths (k‐values) is applied, chosen such that values of character fit of the resulting trees are divided into regular intervals. Results of this analysis are used for discussing the phylogenetic relationships of genera unrepresented in the molecular dataset.     

Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats

Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K⁺ (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.     

Multi‐generational long‐distance migration of insects: studying the painted lady butterfly in the Western Palaearctic

Long‐range, seasonal migration is a widespread phenomenon among insects, allowing them to track and exploit abundant but ephemeral resources over vast geographical areas. However, the basic patterns of how species shift across multiple locations and seasons are unknown in most cases, even though migrant species comprise an important component of the temperate‐zone biota. The painted lady butterfly Vanessa cardui is such an example; a cosmopolitan continuously‐brooded species which migrates each year between Africa and Europe, sometimes in enormous numbers. The migration of 2009 was one of the most impressive recorded, and thousands of observations were collected through citizen science programmes and systematic entomological surveys, such as high altitude insect‐monitoring radar and ground‐based butterfly monitoring schemes. Here we use V. cardui as a model species to better understand insect migration in the Western Palaearctic, and we capitalise on the complementary data sources available for this iconic butterfly. The migratory cycle in this species involves six generations, encompassing a latitudinal shift of thousands of kilometres (up to 60 degrees of latitude). The cycle comprises an annual poleward advance of the populations in spring followed by an equatorward return movement in autumn, with returning individuals potentially flying thousands of kilometres. We show that many long‐distance migrants take advantage of favourable winds, moving downwind at high elevation (from some tens of metres from the ground to altitudes over 1000 m), pointing at strong similarities in the flight strategies used by V. cardui and other migrant Lepidoptera. Our results reveal the highly successful strategy that has evolved in these insects, and provide a useful framework for a better understanding of long‐distance seasonal migration in the temperate regions worldwide.     

Thanetian gastropods from the Mesopotamian high folded zone in northern Iraq

An assemblage of gastropods from the Thanetian of the Kolosh Formation from the Zakho region in northern Iraq is documented for the first time. The ten species represent the families Campanilidae Douvillé 1904, Potamididae H. & A. Adams 1854, Batillariidae Thiele 1929, Thiaridae Gill 1871, Pachychilidae Fischer & Crosse 1892, Cerithiidae Fleming 1822 and Pseudolividae de Gregorio 1880, suggesting a littoral to shallow sublittoral depositional environment. Six of the species are new and five are formally described as new species. At least seven species are also known from the Thanetian and/or Early Ypresian of the Ankara region in Turkey. Only a single species occurs also in the Paleocene of the Paris Basin. No relation to Paleocene and Eocene faunas of Pakistan and India is detectable. This points to a considerably bioprovincialism along the northern coast of the Tethys. Consequently we suppose the existence of an Anatolian Province in the Thanetian/Ypresian Mediterranean Region of the Tethys Realm, represented by rather homogeneous mollusc faunas from western Turkey and northern Iraq. Campanile zakhoense nov. sp., Pyrazopsis hexagonpyramidalis nov. sp., Pachymelania islamogluae nov. sp., “Faunus” dominicii nov. sp. and Pseudoaluco mesopotamicus nov. sp. are introduced as new species. Varicipotamides Pacaud & Harzhauser nov. nom. is proposed as the replacement name for Exechestoma Cossmann (1889) non Brandt (1837).