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Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

Cutaneous squamous cell carcinoma (cSCC) is a common malignancy worldwide and the first as the cause of death from keratinocytic carcinomas. Around 5% of primary cSCCs metastasize, leading to a 5-year survival rate of only 11 %.This paper aims to investigate the proteome profile of primary and metastatic cSCC lesions for the identification of potential diagnostic biomarkers and molecular alterations.Liquid chromatography coupled with SWATH-MS workflow was used to analyse the proteome profile of formalin-fixed and paraffin-embedded samples of primary (n = 20) and metastatic cSCC (n = 25) lesions. Statistical and bioinformatics analysis was performed to identify differentially abundant proteins and molecular alterations between the lesions.A total of 5037 proteins were identified across the samples of which 19 proteins including ISG15, APOA1 and MARCKS with roles in metastasis were increased and 11 proteins including DMKN, APCS and CST6 decreased in metastatic cSCC lesions relative to the primary phenotypes (adj. p-value0.05). The proteomic data separated the lesions based on their histopathological diagnosis. Bioinformatics analysis revealed that cell migration, cell survival and immune response are likely activated, and apoptosis is inhibited in metastatic cSCC lesions, indicating increased lesion complexity as the disease progresses from primary to the metastatic phenotype. Two samples were reclassified after PCA analysis.SWATH-MS analysis identified important molecular changes between primary and metastatic cSCC samples. Exploring these findings further will allow their translation into the clinic for improved tumour diagnosis, staging and therapeutic intervention.

Polyandrous social insects such as the honey bee are prime candidates for parental manipulation of gene expression in offspring. Although there is good evidence for parent-of-origin effects in honey bees the epigenetic mechanisms that underlie these effects remain a mystery. Small RNA molecules such as miRNAs, piRNAs and siRNAs play important roles in transgenerational epigenetic inheritance and in the regulation of gene expression during development. Here we present the first characterisation of small RNAs present in honey bee reproductive tissues: ovaries, spermatheca, semen, fertilised and unfertilised eggs, and testes. We show that semen contains fewer piRNAs relative to eggs and ovaries, and that piRNAs and miRNAs which map antisense to genes involved in DNA regulation and developmental processes are differentially expressed between tissues. tRNA fragments are highly abundant in semen and have a similar profile to those seen in semen in other animals. Intriguingly we find abundant piRNAs that target the sex determination locus, suggesting that piRNAs may play a role in honey bee sex determination. We conclude that small RNAs play a fundamental role in honey bee gametogenesis and reproduction and provide a plausible mechanism for parent-of origin-effects on gene expression and reproductive physiology.

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation. Funding The European Commission FP7 NeurOmics project; CHDI Foundation; the Medical Research Council UK; the Brain Research Trust; and the Guarantors of Brain.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that are considered to be on the same disease spectrum because of overlapping genetic, pathological and clinical traits. Changes in serum proteins in FTD and ALS are poorly understood, and currently no definitive biomarkers exist for diagnosing or monitoring disease progression for either disease. Here we applied quantitative discovery proteomics to analyze protein changes in FTD (N = 72) and ALS (N = 28) patient serum compared to controls (N = 22). Twenty three proteins were significantly altered in FTD compared to controls (increased—APOL1, C3, CTSH, EIF5A, MYH2, S100A8, SUSD5, WDR1; decreased—C1S, C7, CILP2, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, IGHV1, ITIH2, PROS1, SHBG, UMOD, VASN) and 14 proteins were significantly altered in ALS compared to controls (increased—APOL1, CKM, CTSH, IGHG1, IGKC, MYH2; decreased—C7, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, SHBG). There was substantial overlap in the proteins that were altered in FTD and ALS. These results were validated using western blotting. Gene ontology tools were used to assess functional pathways potentially dysregulated in the two diseases, and calcium ion binding and innate immunity pathways were altered in both diseases. When put together, these results suggest significant overlap in pathophysiological peripheral changes in FTD and ALS. This study represents the first proteomics side-by-side comparison of serum changes in FTD and ALS, providing new insights into under-recognized perturbed pathways and an avenue for biomarker development for FTD and ALS.

Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of

Background High-throughput single-cell RNA-seq (scRNA-seq) is a powerful technology for studying gene expression variability in single cells; however, standard analysis approaches only consider the overall expression of each gene, masking additional heterogeneity that could exist through cell type-specific expression of alternative mRNA transcripts. Results Here we show that differential transcript usage (DTU) can be readily detected in data-sets generated from commonly used polyA-captured nanodroplet scRNA-seq technology. Our computational pipeline, Sierra, detects and quantifies polyadenylation sites in scRNA-seq data-sets, which are utilised to evaluate DTU between single-cell populations. We validate our approach by comparing cardiac cell populations derived from scRNA-seq to bulk RNA-seq of matched populations obtained through fluorescent activated cell sorting (FACS), demonstrating that we detect a significant overlap in cell-type DTU between the congruous data-sets. We further illustrate the utility of our method by detecting alternative transcript usage in human peripheral blood mononuclear cells (PBMCs), 3’UTR shortening in activated and proliferating cardiac fibroblasts from injured mouse hearts and finally by building an initial atlas of cell type-specific transcript usage across 12 mouse tissues. Conclusions We anticipate that Sierra will enable new avenues of transcriptional complexity and regulation to be explored in single-cell transcriptomic experiments. Sierra is available at https://github.com/VCCRI/Sierra .

Background Single-cell RNA-sequencing (scRNA-seq) is a fast emerging technology allowing global transcriptome profiling on the single cell level. Cell type identification from scRNA-seq data is a critical task in a variety of research such as developmental biology, cell reprogramming, and cancers. Typically, cell type identification relies on human inspection using a combination of prior biological knowledge (e.g. marker genes and morphology) and computational techniques (e.g. PCA and clustering). Due to the incompleteness of our current knowledge and the subjectivity involved in this process, a small amount of cells may be subject to mislabelling. Results Here, we propose a semi-supervised learning framework, named scReClassify, for ‘post hoc’ cell type identification from scRNA-seq datasets. Starting from an initial cell type annotation with potentially mislabelled cells, scReClassify first performs dimension reduction using PCA and next applies a semi-supervised learning method to learn and subsequently reclassify cells that are likely mislabelled initially to the most probable cell types. By using both simulated and real-world experimental datasets that profiled various tissues and biological systems, we demonstrate that scReClassify is able to accurately identify and reclassify misclassified cells to their correct cell types. Conclusions scReClassify can be used for scRNA-seq data as a post hoc cell type classification tool to fine-tune cell type annotations generated by any cell type classification procedure. It is implemented as an R package and is freely available from https://github.com/SydneyBioX/scReClassify

Background Differences in cell-type composition across subjects and conditions often carry biological significance. Recent advancements in single cell sequencing technologies enable cell-types to be identified at the single cell level, and as a result, cell-type composition of tissues can now be studied in exquisite detail. However, a number of challenges remain with cell-type composition analysis – none of the existing methods can identify cell-type perfectly and variability related to cell sampling exists in any single cell experiment. This necessitates the development of method for estimating uncertainty in cell-type composition. Results We developed a novel single cell differential composition (scDC) analysis method that performs differential cell-type composition analysis via bootstrap resampling. scDC captures the uncertainty associated with cell-type proportions of each subject via bias-corrected and accelerated bootstrap confidence intervals. We assessed the performance of our method using a number of simulated datasets and synthetic datasets curated from publicly available single cell datasets. In simulated datasets, scDC correctly recovered the true cell-type proportions. In synthetic datasets, the cell-type compositions returned by scDC were highly concordant with reference cell-type compositions from the original data. Since the majority of datasets tested in this study have only 2 to 5 subjects per condition, the addition of confidence intervals enabled better comparisons of compositional differences between subjects and across conditions. Conclusions scDC is a novel statistical method for performing differential cell-type composition analysis for scRNA-seq data. It uses bootstrap resampling to estimate the standard errors associated with cell-type proportion estimates and performs significance testing through GLM and GLMM models. We have made this method available to the scientific community as part of the scdney package (Single Cell Data Integrative Analysis) R package, available from https://github.com/SydneyBioX/scdney.

The Kinship Theory of Genomic Imprinting (KTGI) posits that, in species where females mate with multiple males, there is selection for a male to enhance the reproductive success of his offspring at the expense of other males and his mating partner. Reciprocal crosses between honey bee subspecies show parent-of-origin effects for reproductive traits, suggesting that males modify the expression of genes related to female function in their female offspring. This effect is likely to be greater in the Cape honey bee (Apis mellifera capensis), because a male's daughters have the unique ability to produce female offspring that can develop into reproductive workers or the next queen without mating. We generated reciprocal crosses between Capensis and another subspecies and used RNA-seq to identify transcripts that are over- or underexpressed in the embryos, depending on the parental origin of the gene. As predicted, 21 genes showed expression bias towards the Capensis father's allele in colonies with a Capensis father, with no such bias in the reciprocal cross. A further six genes showed a consistent bias towards expression of the father's allele across all eight colonies examined, regardless of the direction of the cross. Consistent with predictions of the KTGI, six of the 21 genes are associated with female reproduction. No gene consistently showed overexpression of the maternal allele.

Concerted examination of multiple collections of single-cell RNA sequencing (RNA-seq) data promises further biological insights that cannot be uncovered with individual datasets. Here we present scMerge, an algorithm that integrates multiple single-cell RNA-seq datasets using factor analysis of stably expressed genes and pseudoreplicates across datasets. Using a large collection of public datasets, we benchmark scMerge against published methods and demonstrate that it consistently provides improved cell type separation by removing unwanted factors; scMerge can also enhance biological discovery through robust data integration, which we show through the inference of development trajectory in a liver dataset collection.

We report the results of a volume-limited survey using the Australia Telescope Compact Array to search for transient and quiescent radio emission from 15 southern hemisphere ultracool dwarfs. We detect radio emission from 2MASSW J0004348-404405 increasing the number of radio loud ultracool dwarfs to 22. We also observe radio emission from 2MASS J10481463-3956062 and 2MASSI J0339352-352544, two sources with previous radio detections. The radio emission from the three detected sources shows no variability or flare emission. Modelling this quiescent emission we find that it is consistent with optically thin gyrosynchrotron emission from a magnetosphere with an emitting region radius of (1 - 2)$R_*$, magnetic field inclination 20$^{\circ}$ - 80$^{\circ}$, field strength $\sim$10 - 200 G, and power-law electron density $\sim$10$^4$ - 10$^8$ cm$^{-3}$. Additionally, we place upper limits on four ultracool dwarfs with no previous radio observations. This increases the number of ultracool dwarfs studied at radio frequencies to 222. Analysing general trends of the radio emission for this sample of 15 sources, we find that the radio activity increases for later spectral types and more rapidly rotating objects. Furthermore, comparing the ratio of the radio to X-ray luminosities for these sources, we find 2MASS J10481463-3956062 and 2MASSI J0339352-352544 violate the Guedel-Benz relation by more than two orders of magnitude., 10 pages, 7 figures

The use of massively parallel sequencing of maternal cfDNA for non-invasive prenatal testing (NIPT) of aneuploidy is widely available. Recently, the scope of testing has increased to include selected subchromosomal abnormalities, but the number of samples reported has been small. We developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma. The same read depth used in our standard pipeline for aneuploidy NIPT detected 15/18 (83%) samples with pathogenic rearrangements > 6 Mb but only 2/10 samples with rearrangements < 6 Mb, unless they were maternally inherited. There were two false-positive calls in 534 samples with no known subchromosomal abnormalities (specificity 99.6%). Using higher read depths, we detected 29/31 fetal subchromosomal abnormalities, including the three samples with maternally inherited microduplications. We conclude that test sensitivity is a function of the fetal fraction, read depth, and size of the fetal CNV and that at least one of the two false negatives is due to a low fetal fraction. The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in test sensitivity, which currently has implications for this technique’s future as a clinical diagnostic test. Furthermore, to be effective, NIPT must be able to detect chromosomal rearrangements across the whole genome for a very low false-positive rate. Because standard NIPT can only detect the majority of larger (>6 Mb) chromosomal rearrangements and requires knowledge of fetal fraction, we consider that it is not yet ready for routine clinical implementation.

Background: Huntington’s disease (HD) and myotonic dystrophy type 1 (DM1) are dominantly inherited diseases caused respectively by CAG and CTG repeat expansions. In both, inherited repeat length correlates positively with severity and negatively with onset. Expanded repeats are unstable through the germline, and somatically, likely contributing to onset and progression. Genetic: variation in MSH3, a DNA mismatch repair protein, is associated with slower HD progression and a lower rate of somatic expansion in DM1. The lead SNP in a GWAS of HD progression, rs557874766, is located in a polymorphic 9 bp tandem repeat in MSH3 exon 1. Msh3 inactivation in animal models of HD and DM1 is known to prevent repeat expansion and ameliorate disease phenotype. Aim: Determine the exact genotype of the MSH3 exon 1 repeat and conduct genotype-phenotype analysis in HD and DM1. Methods/techniques: We conducted next-generation sequencing of the MSH3 repeat region in cohorts of 218 HD and 200 DM1 patients. RNA-Seq was used to quantify whole blood MSH3 expression in HD subjects. In a transcriptome-wide association study (TWAS), MSH3 expression from human prefrontal cortex was imputed into HD GWAS data. Results/outcome: Rs557874766 was found to be an alignment artefact due to the sequence complexity of varying numbers of 9 bp tandem repeats. Individuals who appeared to have the rs557874766 minor allele, associated with delayed HD onset, had a three-repeat variant (allele frequency 26%). Fourteen MSH3 repeat alleles were observed, with the commonest containing six repeats and corresponding to the reference sequence. Each copy of the three-repeat allele slows HD progression by 0.34 UHDRS motor units and 0.1 functional capacity units per year (p=2.18E-7), and delays onset by 2.08 years (p=0.037). In DM1 each three-repeat allele correlates with a lower rate of somatic expansion (p=0.02) and later age of onset (p=0.04). Three-repeat homozygotes had significantly lower MSH3 expression in blood (p=0.028). The TWAS identified significant association of decreased MSH3 with slow progression (p=2.52E-6). Conclusion: A three-repeat variant in MSH3 exon 1 is associated with reduced MSH3 expression and slower progression in HD, reduced rate of somatic expansion in DM1 and delayed onset in both diseases. Genetic variation reducing MSH3 expression may reduce somatic expansion, delay onset and slow progression. Therapies reducing MSH3 could ameliorate several repeat expansion diseases.

Concerted examination of multiple collections of single cell RNA-Seq (scRNA-Seq) data promises further biological insights that cannot be uncovered with individual datasets. However, such integrative analyses are challenging and require sophisticated methodologies. To enable effective interrogation of multiple scRNA-Seq datasets, we have developed a novel algorithm, named scMerge, that removes unwanted variation by combining stably expressed genes and utilizing pseudo-replicates across datasets. Analysis of large collections of publicly available datasets demonstrates that scMerge performs well in multiple scenarios and enhances biological discovery, including inferring cell developmental trajectories.

TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages. Keywords: ALS; cryptic exon; skiptic exon; splicing; TDP-43

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10(-11) for rs4733781; P = 1.0 × 10(-10) for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis-infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB.

Devoy et al. develop the first mouse model to fully recapitulate human FUS-ALS, as defined by midlife-onset progressive degeneration of motor neurons with dominant inheritance. A toxic gain of function occurs in the absence of FUS protein aggregation, involving disturbance of ribosomes and mitochondria at the endoplasmic reticulum., Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease—with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.

There is widespread transcriptional dysregulation in Huntington’s disease (HD) brain, but analysis is inevitably limited by advanced disease and postmortem changes. However, mutant HTT is ubiquitously expressed and acts systemically, meaning blood, which is readily available and contains cells that are dysfunctional in HD, could act as a surrogate for brain tissue. We conducted an RNA-Seq transcriptomic analysis using whole blood from two HD cohorts, and performed gene set enrichment analysis using public databases and weighted correlation network analysis modules from HD and control brain datasets. We identified dysregulated gene sets in blood that replicated in the independent cohorts, correlated with disease severity, corresponded to the most significantly dysregulated modules in the HD caudate, the most prominently affected brain region, and significantly overlapped with the transcriptional signature of HD myeloid cells. High-throughput sequencing technologies and use of gene sets likely surmounted the limitations of previously inconsistent HD blood expression studies. Our results suggest transcription is disrupted in peripheral cells in HD through mechanisms that parallel those in brain. Immune upregulation in HD overlapped with Alzheimer’s disease, suggesting a common pathogenic mechanism involving macrophage phagocytosis and microglial synaptic pruning, and raises the potential for shared therapeutic approaches.

Our UK National Health Service regional genetics laboratory offers NIPD for autosomal dominant and de novo conditions (achondroplasia, thanataphoric dysplasia, Apert syndrome), paternal mutation exclusion for cystic fibrosis and a range of bespoke tests. NIPD avoids the risks associated with invasive testing, making prenatal diagnosis more accessible to families at high genetic risk. However, the challenge remains in offering definitive diagnosis for autosomal recessive diseases, which is complicated by the predominance of the maternal mutant allele in the cell-free DNA sample and thus requires a variety of different approaches. Validation and diagnostic implementation for NIPD of congenital adrenal hyperplasia (CAH) is further complicated by presence of a pseudogene that requires a different approach. We have used an assay targeting approximately 6700 heterozygous SNPs around the CAH gene (CYP21A2) to construct the high-risk parental haplotypes and tested this approach in five cases, showing that inheritance of the parental alleles can be correctly identified using NIPD. We are evaluating various measures of the fetal fraction to help determine inheritance of parental mutations. We are currently exploring the utility of an NIPD multi-disorder panel for autosomal recessive disease, to make testing more widely applicable to families with a variety of serious genetic conditions.

Our UK National Health Service regional genetics laboratory offers NIPD for autosomal dominant and de novo conditions (achondroplasia, thanataphoric dysplasia, Apert syndrome), paternal mutation exclusion for cystic fibrosis and a range of bespoke tests. NIPD avoids the risks associated with invasive testing, making prenatal diagnosis more accessible to families at high genetic risk. However, the challenge remains in offering definitive diagnosis for autosomal recessive diseases, which is complicated by the predominance of the maternal mutant allele in the cell-free DNA sample and thus requires a variety of different approaches. Validation and diagnostic implementation for NIPD of congenital adrenal hyperplasia (CAH) is further complicated by presence of a pseudogene that requires a different approach. We have used an assay targeting approximately 6700 heterozygous SNPs around the CAH gene (CYP21A2) to construct the high-risk parental haplotypes and tested this approach in five cases, showing that inheritance of the parental alleles can be correctly identified using NIPD. We are evaluating various measures of the fetal fraction to help determine inheritance of parental mutations. We are currently exploring the utility of an NIPD multi-disorder panel for autosomal recessive disease, to make testing more widely applicable to families with a variety of serious genetic conditions.

Huntington9s disease is caused by a CAG repeat expansion; ∼60% onset variability is accounted for by age and CAG but studies point to ≥40% residual heritability. We aimed to define accurate stable measures of disease progression in Huntington9s, and use these to identify genetic modifiers. Disease progression offers advantages over age-at-motor onset (AAO) as an analytical phenotype. The availability of high quality longitudinal data in TRACK-HD enabled us to define rate of progression accurately. We find AAO and progression are correlated, but no evidence of distinct phenotypic clusters: imaging, cognitive, and quantitative-motor measures progress in parallel. 216 TRACK-HD subjects were genotyped; imputation then QC generated 9,938,174 variants. Using mixed-linear models we tested for association with disease progression and identified two genome-wide significant signals (P=8.5×10−8 and 4.66×10−8), one associated with a gene implicated in HD mouse and cell studies. To validate these findings we developed a cross-sectional measure of disease progression (“CSP”), andused this to stratify the EHDN Registry cohort. 1,773 subjects with valid phenotype were genotyped. The peak associated with the previously implicated gene was replicated with P=1.4×10−5. Furthermore we found an association (P=2.90×10−7) between the CSP score and a Chromosome 15 locus previously associated with AAO.

Background While the central nervous system is considered to be the primary site of Huntington’s disease (HD) pathology, patients exhibit a wide range of peripheral abnormalities. Indeed, there is much interest in the systemic innate immune system as a modifier of disease progression, with HD myeloid cells exhibiting numerous functional abnormalities, but the molecular mechanisms underpinning these changes remain only partially understood. Aim To investigate alterations in transcriptional regulation that are associated with HD myeloid cell hyper-reactivity. Methods RNA deep-sequencing was performed for whole transcriptome analysis of primary monocytes isolated from HD patients and control subjects cultured with and without a proinflammatory stimulus and various bioinformatic analyses were carried out. ChIP-seq was undertaken to identify changes in histone modifications or transcription factor binding that are associated with altered transcription. Results Significant transcriptional differences in HD monocytes in their basal, unstimulated state were observed, including elevated expression of genes encoding proinflammatory cytokines. That such differences are observed under basal conditions contrasts with previous studies that have required stimulation to elicit phenotypic abnormalities. Pathway analysis of differentially expressed genes reveals an enrichment of genes associated with innate immunity and the inflammatory response, while analysis of upstream regulators suggests that abnormal activation of particular intracellular signalling pathways plays a major causative role in mediating this transcriptional dysregulation. The precise mechanism by which mutant huntingtin exerts these effects may reside in particular histone modifications and/or altered transcription factor binding to increase the expression of specific sets of genes. Conclusions HD myeloid cells are intrinsically abnormal, with a proinflammatory phenotype in the absence of stimulation that is consistent with a priming effect of mutant huntingtin such that an exaggerated inflammatory response is produced when a stimulus is encountered. This is associated with transcriptional dysregulation, which may be a key mechanism causing innate immune dysfunction in Huntington’s disease.

Background Huntington’s disease is caused by a CAG repeat expansion; ~60% onset variability is accounted for by age and CAG but studies point to ≥40% residual heritability. Disease progression offers advantages over age-at-motor onset (AAO) as an analytical phenotype which include use of multiple variables and time-points, and less inter-rater subjectivity. Aims To define accurate stable measures of disease progression in Huntington’s, and use these to identify genetic modifiers. Methods The availability of high quality longitudinal and multivariate data in TRACK-HD enabled us to define rate of progression accurately in 218 subjects. These subjects were then genotyped; imputation then QC generated 9,938,174 variants in 216 subjects. Using mixed-linear models we tested for association with disease progression. To validate our findings we developed a cross-sectional measure of disease progression, and used this to stratify the EHDN REGISTRY cohort. 1,773 subjects with valid phenotype were genotyped, mixed-linear models were used to determine association with cross sectional disease progression. Results We find AAO and progression are correlated, but no evidence of distinct phenotypic clusters: imaging, cognitive, and quantitative-motor measures progress in parallel. In our TRACK-HD analysis we identify one significant association (P = 1.92 × 10 −8 ) and one association approaching significance in a gene previously implicated in HD mouse and cell studies (P = 5.82 × 10 −8 ). The TRACK-HD peak in a previously HD-implicated gene was replicated with P = 1.39 × 10 −5 in the REGISTRY analysis. Furthermore we found an association (P = 2.90 × 10 −7 ) between the cross sectional progression score and a Chromosome 15 locus previously associated with AAO.

Background In Huntington’s disease (HD) mutant HTT is ubiquitously expressed and has systemic effects. Availability of brain tissue is limited and postmortem samples are from advanced disease. Blood, however, is readily available, can be studied longitudinally, and contains cells shown to be dysfunctional in HD. There is marked transcriptional dysregulation in HD brain. Differential expression has also been described in muscle and blood, though changes are inconsistent and poorly replicate CNS findings, questioning their biological relevance. Aims and methods We used RNA-Seq to analyse the transcriptome of whole blood in two separate cohorts comprising a total of 186 HD subjects and 49 controls. Results Though individual transcripts were not significantly differentially expressed in either cohort, gene set enrichment analysis applied to publicly-available pathway databases and HD and control brain co-expression modules showed a significant overlap in dysregulated pathways between the cohorts. In particular, immune pathways were upregulated in both cohorts. Notably, modules previously shown to be significantly dysregulated in HD caudate were also significantly dysregulated in the same direction in both blood cohorts, as well as an independent cortex expression dataset. In addition, we identified overlapping immune upregulation in HD and Alzheimer’s disease (AD), suggesting these two distinct neurodegenerative diseases share some common pathogenic mechanisms. Conclusions We have therefore shown that transcriptional dysregulation in key pathways in HD blood parallels changes found in brain. This overlaps with the transcriptional signature in AD, raising the potential for shared therapeutic approaches. Furthermore, analysing pathways may overcome the cellular and technical heterogeneity that prevent the blood transcriptome replicating changes in the brain transcriptome at a single-gene level.

Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes. This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high. Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications. Citation Format: Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3639.

Innate immune activation beyond the central nervous system is emerging as a vital component of the pathogenesis of neurodegeneration. Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The systemic innate immune system is thought to act as a modifier of disease progression; however, the molecular mechanisms remain only partially understood. Here we use RNA-sequencing to perform whole transcriptome analysis of primary monocytes from thirty manifest HD patients and thirty-three control subjects, cultured with and without a proinflammatory stimulus. In contrast with previous studies that have required stimulation to elicit phenotypic abnormalities, we demonstrate significant transcriptional differences in HD monocytes in their basal, unstimulated state. This includes previously undetected increased resting expression of genes encoding numerous proinflammatory cytokines, such as IL6. Further pathway analysis revealed widespread resting enrichment of proinflammatory functional gene sets, while upstream regulator analysis coupled with Western blotting suggests that abnormal basal activation of the NFOEB pathway plays a key role in mediating these transcriptional changes. That HD myeloid cells have a proinflammatory phenotype in the absence of stimulation is consistent with a priming effect of mutant huntingtin, whereby basal dysfunction leads to an exaggerated inflammatory response once a stimulus is encountered. These data advance our understanding of mutant huntingtin pathogenesis, establish resting myeloid cells as a key source of HD immune dysfunction, and further demonstrate the importance of systemic immunity in the potential treatment of HD and the wider study of neurodegeneration.

In the current era of large surveys and massive data sets, autoclassification of astrophysical sources using intelligent algorithms is becoming increasingly important. In this paper we present the catalog of variable sources in the Third XMM-Newton Serendipitous Source catalog (3XMM) autoclassified using the Random Forest machine learning algorithm. We used a sample of manually classified variable sources from the second data release of the XMM-Newton catalogs (2XMMi-DR2) to train the classifier, obtaining an accuracy of ~92%. We also evaluated the effectiveness of identifying spurious detections using a sample of spurious sources, achieving an accuracy of ~95%. Manual investigation of a random sample of classified sources confirmed these accuracy levels and showed that the Random Forest machine learning algorithm is highly effective at automatically classifying 3XMM sources. Here we present the catalog of classified 3XMM variable sources. We also present three previously unidentified unusual sources that were flagged as outlier sources by the algorithm: a new candidate supergiant fast X-ray transient, a 400 s X-ray pulsar, and an eclipsing 5 hr binary system coincident with a known Cepheid.