Evaluation of the ASPYRE-Lung targeted variant panel: a rapid, low-input solution for non-small cell lung cancer biomarker testing and experience from three independent sites.

Background: Many patients with non-small cell lung cancer (NSCLC) lack access to highly effective approved targeted therapeutics due to multiple gaps in biomarker testing. Challenges in comprehensive molecular testing include complexities associated with the need to assess the presence of multiple variants, costs of running multiple sequential assays per sample, high assay quality control (QC) failure rates, clinical need for rapid turn-around time (TAT) to initiate therapy, and insufficient tissue samples. The ASPYRE-Lung NSCLC assay addresses gaps in multiplexed testing by simultaneously analyzing DNA and RNA, detecting 114 actionable genomic variants across 11 genes, consistent with current NSCLC treatment guidelines.

Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE.

Background: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns.

Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations.

Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA).

Timing the initiation of multiple myeloma.

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan.

The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals.

Population dynamics of normal human blood inferred from somatic mutations.

Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation.

High concentrations of lead and barium in hair of the rural population caused by water pollution in the Thar Jath oilfields in South Sudan.

In the oil fields of Thar Jath, South Sudan, increasing salinity of drinking water was observed together with human incompatibilities and rise in livestock mortalities. Hair analysis was used to characterize the toxic exposure of the population. Hair samples of volunteers from four communities with different distance from the center of the oil field (Koch 23km, n=24; Leer 50km, n=26; Nyal 110km, n=21; and Rumbek 220km, n=25) were analyzed for altogether 39 elements by inductively coupled plasma-mass spectrometry.

A dual-monoclonal, sandwich immunoassay specific for glucagon like peptide-19-36/7 (GLP-19-36/7).

Objective: Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by intestinal L-cells which stimulates glucose-dependent insulin secretion. GLP-1 is initially secreted as the active peptide GLP-17-36/7, but rapidly undergoes cleavage by dipeptidyl peptidase 4 (DPP4) to yield the inactive form, GLP-19-36/7. Despite a reduced affinity for the GLP-1 receptor, GLP-19-36/7 may have cardioprotective properties.

Splicing biomarkers of disease severity in myotonic dystrophy.

Objective: To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Methods: In a discovery cohort, we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects.

Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs.

We present an intramolecular reaction, Reflex™, to derive shorter, sequencer-ready, daughter polymerase chain reaction products from a pooled population of barcoded long-range polymerase chain reaction products, whilst still preserving the cognate DNA barcodes. Our Reflex workflow needs only a small number of primer extension steps to rapidly enable uniform sequence coverage of long contiguous sequence targets in large numbers of samples at low cost on desktop next-generation sequencers.

Interdomain long-range electron transfer becomes rate-limiting in the Y216A variant of tyramine β-monooxygenase.

The enzyme tyramine β-monooxygenase (TβM) belongs to a small eukaryotic family of physiologically important mononuclear dicopper monooxygenases. The properties of this family include noncoupled mononuclear copper centers ~11 Å apart, with Cu(M) performing C-H and O(2) activation and Cu(H) functioning as an electron storage site [Klinman, J. P.

Inactivation of Met471Cys tyramine β-monooxygenase results from site-specific cysteic acid formation.

Tyramine β-monooxygenase (TβM), the insect homologue of dopamine β-monooxygenase, is a neuroregulatory enzyme that catalyzes the β-hydroxylation of tyramine to yield octopamine. Mutation of the methionine (Met) ligand to Cu(M) of TβM, Met471Cys, yielded a form of TβM that is catalytically active but susceptible to inactivation during turnover [Hess, C. R.

Expansion of the Ligand Knowledge Base for Chelating P,P-Donor Ligands (LKB-PP).

We have expanded the ligand knowledge base for bidentate P,P- and P,N-donor ligands (LKB-PP, Organometallics2008, 31, 1372-1383) by 208 ligands and introduced an additional steric descriptor (nHe). This expanded knowledge base now captures information on 334 bidentate ligands and has been processed with principal component analysis (PCA) of the descriptors to produce a detailed map of bidentate ligand space, which better captures ligand variation and has been used for the analysis of ligand properties.

Parent-of-origin effects in SOX2 anophthalmia syndrome.

Purpose: Sex determining region Y (SRY)-box 2 (SOX2) anophthalmia syndrome is an autosomal dominant disorder manifesting as severe developmental eye malformations associated with brain, esophageal, genital, and kidney abnormalities. The syndrome is usually caused by de novo mutations or deletions in the transcription factor SOX2. To investigate any potential parental susceptibility factors, we set out to determine the parent of origin of the mutations or deletions, and following this, to determine if birth order or parental age were significant factors, as well as whether mutation susceptibility was related to any sequence variants in cis with the mutant allele.

A method for counting PCR template molecules with application to next-generation sequencing.

Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity.

Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I.

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor.

Bone morphogenetic protein 7 (BMP7) mutations are associated with variable ocular, brain, ear, palate, and skeletal anomalies.

Bone morphogenetic protein (BMP) signaling regulates a range of cellular processes and plays an important role in the specification and patterning of the early embryo. However, due to the functional redundancy of BMP ligands and receptors in tissues where they are coexpressed, relatively little is known about the role of individual BMP ligands in human disease. Here we report heterozygous variations in BMP7, including a frameshift, missense, and Kozak sequence mutation, in individuals with developmental eye anomalies and a range of systemic abnormalities, including developmental delay, deafness, scoliosis, and cleft palate.

Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy.

The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (Mbnl1) gene. Strong correlation in splicing changes for approximately 100 new Mbnl1-regulated exons indicates that loss of Mbnl1 explains >80% of the splicing pathology due to CUG(exp) RNA.

Seeing clearly: the dominant and recessive nature of FOXE3 in eye developmental anomalies.

FOXE3 is a lens-specific transcription factor with a highly conserved forkhead domain previously implicated in congenital primary aphakia and anterior segment dysgenesis. Here, we identify new recessive FOXE3 mutations causative for microphthalmia, sclerocornea, primary aphakia, and glaucoma in two extended consanguineous families by SNP array genotyping followed by a candidate gene approach. Following an additional screen of 236 subjects with developmental eye anomalies, we report two further novel heterozygous mutations segregating in a dominant fashion in two different families.

Reduced TFAP2A function causes variable optic fissure closure and retinal defects and sensitizes eye development to mutations in other morphogenetic regulators.

Mutations in the transcription factor encoding TFAP2A gene underlie branchio-oculo-facial syndrome (BOFS), a rare dominant disorder characterized by distinctive craniofacial, ocular, ectodermal and renal anomalies. To elucidate the range of ocular phenotypes caused by mutations in TFAP2A, we took three approaches. First, we screened a cohort of 37 highly selected individuals with severe ocular anomalies plus variable defects associated with BOFS for mutations or deletions in TFAP2A.

Reactive 4a-alkyl-4aH-carbazoles by catalytic dearomatisation, and their unusual dimerisation and dealkylation reactions.

Despite their intrinsic instability, 4a-alkyl-4aH-carbazoles can be generated by a catalytic dearomatisation process; their reactivity is demonstrated by facile dealkylation and highly unusual cyclodimerisation processes.