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. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. RESULTS: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. CONCLUSION: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.

Diverse mutational landscapes in human lymphocytes.

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, 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. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. 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. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

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). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.

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. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2nd-3rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.

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 cancer1. 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. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

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. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000-200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans.

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. Very high concentrations and a toxic health endangerment were assessed for lead and barium. The concentration of lead increased steadily with decreasing distance from the oil field from Rumbek (mean 2.8µg/g) to Koch (mean 18.7µg/g) and was there in the same range as in highly contaminated mining regions in Kosovo, China or Bolivia. The weighting materials in drilling muds barite (BaSO4) and galena (PbS) were considered to be the sources of drinking water pollution and high hair values. The high concentrations of lead and barium in hair demonstrate clearly the health risk caused by harmful deposition of toxic industrial waste but cannot be used for diagnosis of a chronic intoxication of the individuals.

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. There is currently no described immunoassay capable of specifically measuring GLP-19-36/7. DESIGN AND METHODS: We generated a monoclonal antibody specific for the N-terminal neoepitope of GLP-19-36/7. After affinity maturation, we paired this capture antibody with an anti-total GLP-1 monoclonal detection antibody to create a sandwich ELISA specific for GLP-19-36/7. RESULTS: The sandwich ELISA was highly specific for GLP-19-36/7 and did not recognize GLP-17-36 or GLP-17-37. The ELISA exhibited a broad dynamic range and a lower limit of detection (LLOD) of 3.17ng/L. In healthy volunteers, concentrations of GLP-19-36/7 increased dramatically in the postprandial state compared to the fasted state and were markedly elevated at both 30 and 120-minute postprandial time points. CONCLUSIONS: The optimization of an N-terminal-specific monoclonal antibody for GLP-19-36/7 enabled the development of a sensitive and specific sandwich ELISA assay capable of measuring physiological concentrations of GLP-19-36/7. This ELISA may have the potential to help expand our knowledge of GLP-1 biology.

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. In a validation cohort of 50 DM1 subjects, we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides to reduce levels of toxic RNA. RESULTS: Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue. INTERPRETATION: Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy.

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 ß-monooxygenase.

The enzyme tyramine ß-monooxygenase (TßM) 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. (2006) J. Biol. Chem. 281, 3013-3016]. A conserved tyrosine (Y216 in TßM) is positioned between the copper domains and is associated with Cu(H) (through an interaction with a Cu(H)-coordinating histidine). Mutations at Y216 (to W, I, and A) indicate little or no difference in electron paramagnetic resonance spectra, while X-ray absorption spectroscopy studies show only a very small decrease in distance between Cu(M) and its Met471 ligand in reduced enzyme. High-performance liquid chromatography assays demonstrate that turnover of substrate is complete with Y216W and Y216I, whereas Y216A undergoes a secondary inactivation that is linked to oxidation of ligands at Cu(M). Steady-state kinetic and isotope effect measurements were investigated. The significantly elevated K(m,Tyr) for Y216A, together with a very large (D)(k(cat)/K(m,Tyr)) of ~12, indicates a major impact on the binding of substrate at the Cu(M) site. The kinetic and isotopic parameters lead to estimated rate constants for C-H bond cleavage, dissociation of substrate from the Cu(M) site, and, in the case of Y216A, the rate of electron transfer (ET) from Cu(H) to Cu(M). These studies uncover a rate-limiting ET within the solvent-filled interface and lead to a paradigm shift in our understanding of the mononuclear dicopper monooxygenases.

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

Tyramine ß-monooxygenase (TßM), the insect homologue of dopamine ß-monooxygenase, is a neuroregulatory enzyme that catalyzes the ß-hydroxylation of tyramine to yield octopamine. Mutation of the methionine (Met) ligand to Cu(M) of TßM, Met471Cys, yielded a form of TßM that is catalytically active but susceptible to inactivation during turnover [Hess, C. R., Wu, Z., Ng, A., Gray, E. E., McGuirl, M. M., and Klinman, J. P. (2008) J. Am. Chem. Soc. 130, 11939-11944]. Further, although the wild-type (WT) enzyme undergoes coordination of Met471 to Cu(M) in its reduced form, the generation of Met471Cys almost completely eliminates this interaction [Hess, C. R., Klinman, J. P., and Blackburn, N. J. (2010) J. Biol. Inorg. Chem. 15, 1195-1207]. The aim of this study is to identify the chemical consequence of the poor ability of Cys to coordinate Cu(M). We show that Met471Cys TßM is ~5-fold more susceptible to inactivation than the WT enzyme in the presence of the cosubstrate/reductant ascorbate and that this process is not facilitated by the substrate tyramine. The resulting 50-fold smaller ratio for turnover to inactivation in the case of Met471Cys prevents full turnover of the substrate under all conditions examined. Liquid chromatography-tandem mass spectrometry analysis of proteolytic digests of inactivated Met471Cys TßM leads to the identification of cysteic acid at position 471. While both Met and Cys side chains are expected to be similarly subject to oxidative damage in proteins, the enhanced reactivity of Met471Cys toward solution oxidants in TßM is attributed to its weaker interaction with Cu(I)(M).

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 (nHe8). 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. METHODS: We analyzed 23 cases of de novo disease to determine the parental origin of SOX2 mutations and deletions using informative single nucleotide polymorphisms and a molecular haplotyping approach. We examined parental ages for SOX2 mutation and deletion cases, compared these with the general population, and adjusted for birth order. RESULTS: Although the majority of subjects had mutations or deletions that arose in the paternal germline (5/7 mutation and 5/8 deletion cases), there was no significant paternal bias for new mutations (binomial test, p=0.16) or deletions (binomial test, p=0.22). For both mutation and deletion cases, there was no significant association between any single nucleotide polymorphism allele and the mutant chromosome (p>0.05). Parents of the subjects with mutations were on average older at the birth of the affected child than the general population by 3.8 years (p=0.05) for mothers and 3.3 years (p=0.66) for fathers. Parents of the subjects with deletions were on average younger than the general population by 3.0 years (p=0.17) for mothers and 2.1 years (p=0.19) for fathers. Combining these data, the difference in pattern of parental age between the subjects with deletions and mutations was evident, with a difference of 6.5 years for mothers (p=0.05) and 5.0 years for fathers (p=0.22), with the mothers and fathers of subjects with mutations being older than the mothers and fathers of subjects with deletions. We observed that 14 of the 23 (61%) affected children were the first-born child to their mother, with 10/15 of the mutation cases (66%) and 4/8 deletion cases (50%) being first born. This is in comparison to 35% of births with isolated congenital anomalies overall who are first born (p=0.008). CONCLUSIONS: Sporadic SOX2 mutations and deletions arose in both the male and female germlines. In keeping with several genetic disorders, we found that SOX2 mutations were associated with older parental age and the difference was statistically significant for mothers (p=0.05), whereas, although not statistically significant, SOX2 deletion cases had younger parents. With the current sample size, there was no evidence that sequence variants in cis surrounding SOX2 confer susceptibility to either mutations or deletions.

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. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.

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. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the ß-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(•)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.

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. We determined that BMP7 is expressed in the developing eye, brain, and ear in human embryos in a manner consistent with the phenotype seen in our mutation cases. These data establish BMP7 as an important gene in human eye development, and suggest that BMP7 should be considered during clinical evaluation of individuals with developmental eye anomalies.

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. In contrast, only about half of mRNA-level changes can be attributed to loss of Mbnl1, indicating that CUG(exp) RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of Mbnl1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.