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Prader-Willi Syndrome (PWS) is a neurodevelopmental disorder caused by mutations affecting paternal chromosome 15q11-q13, and characterized by hypotonia, hyperphagia, impaired cognition, and behavioural problems. Psychotic illness is a challenging problem for individuals with PWS and has different rates of prevalence in distinct PWS genotypes. Previously, we demonstrated behavioural and cognitive endophenotypes of relevance to psychiatric illness in a mouse model for one of the associated PWS genotypes, namely PWS-IC, in which deletion of the imprinting centre leads to loss of paternally imprinted gene expression and over-expression of Ube3a. Here we examine the broader gene expression changes that are specific to the psychiatric endophenotypes seen in this model. To do this we compared the brain transcriptomic profile of the PWS-IC mouse to the PWS-cr model that carries a deletion of the PWS minimal critical interval spanning the snoRNA Snord116 and Ipw. Firstly, we examined the same behavioural and cognitive endophenotypes of relevance to psychiatric illness in the PWS-cr mice. Unlike the PWS-IC mice, PWS-cr exhibit no differences in locomotor activity, sensory-motor gating, and attention. RNA-seq analysis of neonatal whole brain tissue revealed a greater number of transcriptional changes between PWS-IC and wild-type littermates than between PWS-cr and wild-type littermates. Moreover, the differentially expressed genes in the PWS-IC brain were enriched for GWAS variants of episodes of psychotic illness but, interestingly, not schizophrenia. These data illustrate the molecular pathways that may underpin psychotic illness in PWS and have implications for potential therapeutic interventions.

The CNS has traditionally been considered an immune privileged site, but is now understood to have a system of immune surveillance, predominantly involving CD4+ T-cells. Identifying functional differences between CNS and blood CD4+ T-cells, therefore, have relevance to CNS immune surveillance as well as to neurological conditions, such as multiple sclerosis, in which CD4+ T-cells play a central role. Here, CD4+ T-cells were purified from CSF and blood from 21 patients with newly diagnosed treatment-naïve multiple sclerosis and 20 individuals with non-inflammatory disorders using fluorescence-activated cell sorting, and their transcriptomes were profiled by RNA sequencing. Paired comparisons between CD4+ T-cells from CSF and blood identified 5156 differentially expressed genes in controls and 4263 differentially expressed in multiple sclerosis patients at false discovery rate, Hrastelj et al. report the largest transcriptomic comparison of CSF and blood CD4+ T-cells to date. The study identifies thousands of significant genes, implicating cellular migration and cholesterol metabolism, as well as Th1, Th17 and effector memory function in immune surveillance of the CNS in multiple sclerosis and controls., Graphical Abstract Graphical Abstract

Maternal depression is associated with adverse child outcomes including antisocial behaviour (ASB). Prospective longitudinal studies have focused on the timing and cumulative exposure to maternal depression to further delineate the association and mechanisms of effect. The objective of this systematic review was to synthesise and evaluate the findings of longitudinal studies of maternal depression and offspring antisocial behaviour. Three databases were searched (Psychinfo, Web of Science, and Medline). Twenty of 5936 studies met inclusion criteria. Study quality was assessed using the Critical Appraisal Skills Programme criteria [Critical Appraisal Skills Programme (2017) CASP (cohort observation checklist). https://casp-uk.net/wpcontent/uploads/2018/01/CASP-Cohort-Study-Checklist.pdf]. Results of individual studies were highly varied, using diverse analytical approaches and not all studies explored the independent effects of different episodes. Only three studies examined hypothesised mechanisms. Prenatal, postnatal, and later episodes of depression were all predictive of antisocial outcomes. One particular time period of depression exposure did not emerge as more predictive of offspring ASB than another. However, measures of maternal depression after the perinatal period were limited and typically included a one-off assessment of mothers’ depressive symptoms that was concurrent to the assessment of offspring ASB. When cumulative exposure to maternal depression and specific timing effects were measured within the same study it was cumulative exposure that conferred the greatest risk for offspring ASB—particularly when this exposure began during the perinatal period. Findings are discussed in terms of limitations in the literature and highlight the need for future research to examine the biological and environmental mechanisms that underpin associations between maternal depression and offspring antisocial behaviour during different stages of development.

Genetic variants could be useful as predictors of radiotherapy outcomes. Here we studied germline MRE11A variants in muscle invasive bladder cancer patients and found six rare variants and one SNP, rs1805363, to be associated with worse radiotherapy outcomes. If successfully validated in an independent radiotherapy cohort, this SNP could be used to select patients for bladder-conserving treatment., Background Muscle-invasive bladder cancer (MIBC) can be cured by radical radiotherapy (RT). We previously found tumour MRE11 expression to be predictive of survival following RT in MIBC, and this was independently validated in a separate institute. Here, we investigated germline MRE11A variants as possible predictors of RT outcomes in MIBC, using next-generation sequencing (NGS). Patients and methods The MRE11A gene was amplified in germline DNA from 186 prospectively recruited MIBC patients treated with RT and sequenced using bar-coded multiplexed NGS. Germline variants were analysed for associations with cancer-specific survival (CSS). For validation as a prognostic or predictive marker, rs1805363 was then genotyped in a cystectomy-treated MIBC cohort of 256 individuals. MRE11A mRNA isoform expression was measured in bladder cancer cell lines and primary tumour samples. Results Carriage of at least one of six (five novel) rare variants was associated with the worse RT outcome (hazard ratio [HR] 4.04, 95% confidence interval [95% CI] 1.42–11.51, P = 0.009). The single-nucleotide polymorphism (SNP), rs1805363 (minor allele frequency 11%), was also associated with worse CSS (per-allele HR 2.10, 95% CI 1.34–3.28, Ptrend = 0.001) following RT in MIBC, with a gene-dosage effect observed, but no effect seen on CSS in the cystectomy cohort (Ptrend = 0.89). Furthermore, rs1805363 influenced relative MRE11A isoform expression, with increased isoform 2 expression with carriage of the rs1805363 minor A allele. Conclusions Germline MRE11A SNP rs1805363 was predictive of RT, but not of cystectomy outcome in MIBC. If successfully validated in an independent RT-treated cohort, this SNP could be a useful clinical tool for selecting patients for bladder-conserving treatment.

The study examined the relation between reward and punishment sensitivity and antisocial behavior (ASB) in male adolescents. We compared Behavioral Inhibition/Behavioral Activation System (BIS/BAS) Scale scores in adolescent male offenders (n = 85) and non-offenders (n = 50), and explored the relation between BIS/BAS and measures associated with ASB (psychopathy, conduct problems and alcohol use) within the whole group of adolescents, and offending frequency in the offenders. Between group analyses indicated heightened BAS (reward sensitivity; specifically the drive to seek rewards) and lowered BIS (punishment sensitivity) in the offenders compared to the non-offenders. Regression analyses indicated that traits associated with reward seeking (BAS Drive and/or Fun Seeking) positively predicted psychopathic traits, conduct problems and alcohol use. In contrast, response to reward (BAS Reward Responsiveness) was negatively associated with psychopathy and conduct problems. Reduced punishment sensitivity (BIS) was associated with psychopathy only. The findings suggest that BAS reward traits are useful in understanding ASB and emphasize the importance of examining dimensions of reward processing in relation to different aspects of ASB in adolescents.

Aims: The purpose of this research was to test and compare associations between participation in organised activities and indicators of hazardous drinking between young offenders and young non-offenders. Methods: Two groups of 13–18 year-old males were recruited in Cardiff, UK: 93 young offenders and 53 non-offenders from secondary schools matched on estimated IQ, sex and socioeconomic status. Indicators of hazardous drinking were measured using the Fast Alcohol Screening Test (FAST). Organised activity participation and externalising behaviour was measured by the Youth Self Report. The Wechsler Abbreviated Scale of Intelligence was also administered. Results: Young offenders participated in fewer organised activities and had higher FAST scores than non-offenders. Young offenders and non-offenders significantly differed on mean FAST scores if they participated in no organised activities but not if they participated in at least one team sport. Externalising behaviour problems were unrelated to participation in organised activities. Conclusions: Although young offenders were less likely to have participated in organised activities, for them, participation in a team sport was associated with less hazardous drinking. Vulnerable youths who might benefit most from sporting activities actually access them the least. Future research should identify the different barriers to participation that they face.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Background Recent sequencing studies have shown a small fraction of SZ risk comes from rare mutations. They have also advanced our understanding of SZ pathophysiology, with multiple studies implicating post-synaptic proteins, including the activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Thus far, only one gene, SETD1A, has been associated with SZ at genome-wide levels of significance. We aimed to identify novel SZ genes by sequencing 187 genes in 5,207 cases and 4,991 controls. Included among these genes are members of ARC and NMDAR complexes, as well as Voltage-Gated Sodium (VGSC) and Calcium Channels (VGCC), all of which have been previously implicated in SZ. Methods A total of 5,724 cases, 5,769 controls were selected for Ion Torrent sequencing of 187 candidate SZ genes. None of these samples have been included in previous SZ sequencing studies. After stringent quality control (low sequence quality, relatedness and ancestry), our sample consisted 5,207 cases and 4,991 controls. Gene-set and single-gene association statistics were generated using a Firth's penalised-likelihood logistic regression model. Results Cases had a significant excess of Loss-of-Function (LoF) mutations ( Discussion In a new, independent sequencing study of SZ, we found an excess of LoF alleles in cases in all 187 targeted genes, and provide independent support for the involvement of ARC and NMDAR post-synaptic protein complexes, and voltage-gated sodium channels, in SZ pathogenesis. We also provide further evidence for a shared genetic risk between SZ and DD. However, our study was underpowered to identify specific risk genes.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

Infantile myopathies with diaphragmatic paralysis are genetically heterogeneous, and clinical symptoms do not assist in differentiating between them. We used phased haplotype analysis with subsequent targeted exome sequencing to identify MEGF10 mutations in a previously unidentified type of infantile myopathy with diaphragmatic weakness, areflexia, respiratory distress and dysphagia. MEGF10 is highly expressed in activated satellite cells and regulates their proliferation as well as their differentiation and fusion into multinucleated myofibers, which are greatly reduced in muscle from individuals with early onset myopathy, areflexia, respiratory distress and dysphagia.

The atonal homolog 7 (ATOH7) gene encodes a transcription factor involved in determining the fate of retinal progenitor cells and is particularly required for optic nerve and ganglion cell development. Using a combination of autozygosity mapping and next generation sequencing, we have identified homozygous mutations in this gene, p.E49V and p.P18RfsX69, in two consanguineous families diagnosed with multiple ocular developmental defects, including severe vitreoretinal dysplasia, optic nerve hypoplasia, persistent fetal vasculature, microphthalmia, congenital cataracts, microcornea, corneal opacity and nystagmus. Most of these clinical features overlap with defects in the Norrin/beta-catenin signalling pathway that is characterized by dysgenesis of the retinal and hyaloid vasculature. Our findings document Mendelian mutations within ATOH7 and imply a role for this molecule in the development of structures at the front as well as the back of the eye. This work also provides further insights into the function of ATOH7, especially its importance in retinal vascular development and hyaloid regression.

Current methods for sequencing clonal populations of DNA molecules yield several gigabases of data per day, typically comprising reads of

Psychopathy is a serious personality disorder of which impulsivity is a key component. However, impulsivity is a multidimensional construct, with multiple approaches to measurement, and different measures may be differentially implicated in psychopathy. This study investigated the relationship between psychopathy as assessed by the Psychopathic Personality Inventory-Revised (PPI-R; Lilienfeld & Widows, 2005), a personality measure of impulsivity (Barratt Impulsiveness Scale-11), and four behavioural measures of impulsivity (GoStop Impulsivity Paradigm, Two Choice Impulsivity Paradigm, Delay Discounting Task, Iowa Gambling Task). A nonclinical sample (N = 80) was recruited from the local community to advance understanding of psychopathy in non-incarcerated samples. The results indicated that the personality measure of impulsivity was strongly correlated with the PPI-R, while the behavioural measures were either not correlated or only weakly correlated with the PPI-R. The results are discussed in terms of the multifaceted nature of impulsivity and the need for the further development of behavioural measures of impulsivity, given their importance in clinical assessment and intervention.

Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithelial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage.

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at-risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA-deficient breast cancers.

Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200-500 microM) compared with other mammals (3-120 microM). About 70% of daily urate disposal occurs via the kidneys, and in 5-25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7-5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

Age-related macular degeneration is the most common cause of blindness in Western populations. Susceptibility is influenced by age and by genetic and environmental factors. Complement activation is implicated in the pathogenesis.We tested for an association between age-related macular degeneration and 13 single-nucleotide polymorphisms (SNPs) spanning the complement genes C3 and C5 in case subjects and control subjects from the southeastern region of England. All subjects were examined by an ophthalmologist and had independent grading of fundus photographs to confirm their disease status. To test for replication of the most significant findings, we genotyped a set of Scottish cases and controls.The common functional polymorphism rs2230199 (Arg80Gly) in the C3 gene, corresponding to the electrophoretic variants C3S (slow) and C3F (fast), was strongly associated with age-related macular degeneration in both the English group (603 cases and 350 controls, P=5.9x10(-5)) and the Scottish group (244 cases and 351 controls, P=5.0x10(-5)). The odds ratio for age-related macular degeneration in C3 S/F heterozygotes as compared with S/S homozygotes was 1.7 (95% confidence interval [CI], 1.3 to 2.1); for F/F homozygotes, the odds ratio was 2.6 (95% CI, 1.6 to 4.1). The estimated population attributable risk for C3F was 22%.Complement C3 is important in the pathogenesis of age-related macular degeneration. This finding further underscores the influence of the complement pathway in the pathogenesis of this disease.

Dilp1 is a semi-dominant mouse mutation that causes dilated pupils when heterozygous and is lethal when homozygous. We report here that it is caused by a point mutation that introduces a stop codon close to the start of the coding sequence of the paired-like homeobox transcription factor Phox2b. Mice carrying a targeted allele of Phox2b also have dilated pupils and the two alleles do not complement. Phox2b is necessary for the development of the autonomic nervous system and when absent one of the consequences is that all parasympathetic ganglia fail to form. Constriction of the pupil is a parasympathetic response mediated by the ciliary ganglion and we find that in Phox2b heterozygous mutants it is highly atrophic. The development of other parasympathetic and sympathetic ganglia appears to be largely unaffected indicating that the ciliary ganglion is exquisitely sensitive to a reduction in dose of this transcription factor. PHOX2B has been implicated in human disease. Mutations, principally leading to polyalanine expansions within the protein, have been found in patients with congenital central hypoventilation syndrome (CCHS), the cardinal feature of which is an inability to breathe unassisted when asleep. Additionally, some CCHS patients have ocular abnormalities, including pupillary defects, although they principally have constricted rather than dilated pupils. The apparent phenotypic differences observed between mice carrying a loss-of-function mutation of Phox2b and CCHS patients indicate that PHOX2B mutations found in CCHS patients, all of which can produce proteins with intact DNA-binding domains, are gain-of-function mutations that alter rather than abolish protein function.

A large family with dominantly inherited rhegmatogenous retinal detachment, premature arthropathy, and development of phalangeal epiphyseal dysplasia, resulting in brachydactyly was linked to COL2A1, the gene encoding proalpha1(II) collagen. Mutational analysis of the gene by exon sequencing identified a novel mutation in the C-propeptide region of the molecule. The glycine to aspartic acid change occurred in a region that is highly conserved in all fibrillar collagen molecules. The resulting phenotype does not fit easily into pre-existing subgroups of the type II collagenopathies, which includes spondyloepiphyseal dysplasia, and the Kniest, Strudwick, and Stickler dysplasias.

Antisocial individuals have problems recognizing negative emotions (e.g. Marsh & Blair in Neuroscience and Biobehavioral Reviews 32:454–465, 2009); however, due to issues with sampling and different methods used, previous findings have been varied. Sixty-three male young offenders and 37 age-, IQ- and socio-economic status-matched male controls completed a facial emotion recognition task, which measures recognition of happiness, sadness, fear, anger, disgust, and surprise and neutral expressions across 4 emotional intensities. Conduct disorder (YSR), and psychopathic and callous/unemotional traits (YPI) were measured, and offenders’ offense data were taken from the Youth Offending Service’s case files. Relative to controls, offenders were significantly worse at identifying sadness, low intensity disgust and high intensity fear. A significant interaction for anger was also observed, with offenders showing reduced low- but increased high-intensity anger recognition in comparison with controls. Within the young offenders levels of conduct disorder and psychopathic traits explained variation in sadness and disgust recognition, whereas offense severity explained variation in anger recognition. These results suggest that antisocial youths show specific problems in recognizing negative emotions and support the use of targeted emotion recognition interventions for problematic behavior.

Massively parallel ("next generation") DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein-coding regions are enriched from patient genomic DNA, representing either the entire genome ("exome sequencing") or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user-friendly suite of programs that can interactively analyze, filter and screen data from enrichment-capture NGS data. These programs ("Agile Suite") are particularly suitable for small-scale gene discovery or for diagnostic analysis.

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.

Pachydermoperiostosis, or primary hypertrophic osteoarthropathy (PHO), is an inherited multisystem disorder, whose features closely mimic the reactive osteoarthropathy that commonly accompanies neoplastic and inflammatory pathologies. We previously described deficiency of the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (HPGD) as a cause of this condition, implicating elevated circulating prostaglandin E2 (PGE2) as causative of PHO, and perhaps also as the principal mediator of secondary HO. However, PHO is genetically heterogeneous. Here, we use whole-exome sequencing to identify recessive mutations of the prostaglandin transporter SLCO2A1, in individuals lacking HPGD mutations. We performed exome sequencing of four probands with severe PHO, followed by conventional mutation analysis of SLCO2A1 in nine others. Biallelic SLCO2A1 mutations were identified in 12 of the 13 families. Affected individuals had elevated urinary PGE2, but unlike HPGD-deficient patients, also excreted considerable quantities of the PGE2 metabolite, PGE-M. Clinical differences between the two groups were also identified, notably that SLCO2A1-deficient individuals have a high frequency of severe anemia due to myelofibrosis. These findings reinforce the key role of systemic or local prostaglandin excess as the stimulus to HO. They also suggest that the induction or maintenance of hematopoietic stem cells by prostaglandin may depend upon transporter activity. Hum Mutat 33:11751181, 2012. (c) 2012 Wiley Periodicals, Inc.

The X-linked gene filamin A (FIna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitro-sourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the FIna gene (Y2388X), leading to absence of the FIna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for FIna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.

Mice heterozygous for the N-ethyl-N-nitrosourea-induced Waved-5 (Wa5) mutation, isolated in a screen for dominant, visible mutations, exhibit a wavy coat similar to mice homozygous for the recessive Tgfa wa1 or Egfr wa2 alleles. In this study, we show that Wa5 is a new allele of Egfr (Egfr Wa5) containing a missense mutation within the coding region for the highly conserved DFG motif of the tyrosine kinase domain. In vivo analysis of placental development, modification of Apc Min tumorigenesis, and levels of EGF-dependent EGFR phosphorylation demonstrates that Egfr Wa5 functions as an antimorphic allele, recapitulating many abnormalities associated with reduced EGFR activity. Furthermore, Egfr wa5 enhances Egfr Wa2 compound or Tgfa tm1Dcl double mutants exposing additional EGFR-dependent phenotypes. In vitro characterization shows that the antimorphic property of Egfr Wa5 is caused by a kinase-dead receptor acting as a dominant negative.

We have carried out a genome-wide screen for novel N-ethyl-N-nitrosourea-induced mutations that give rise to eye and vision abnormalities in the mouse and have identified 25 inherited phenotypes that affect all parts of the eye. A combination of genetic mapping, complementation and molecular analysis revealed that 14 of these are mutations in genes previously identified to play a role in eye pathophysiology, namely Pax6, Mitf, Egfr and Pde6b. Many of the others are located in genomic regions lacking candidate genes and these define new loci. Four of the mutants display a similar phenotype of dilated pupils but do not appear to be allelic, and at least two of these are embryonic lethal when homozygous. This collection of eye mutations will be valuable for understanding gene function, for dissecting protein function and as models of human eye disease.

Introduction Hepatitis C virus (HCV) is the most common cause of hepatocellular cancer (HCC) in the western world. HCV is an RNA virus that does not integrate with human DNA and so the oncogenic mechanisms of HCV remain unclear. Next generation sequencing (NGS) provides a flexible platform and generates large amounts of data at a relatively small time and constantly reducing costs. The role of viral infection is well established in the aetiology of a wide range of tumours. In this study we investigate DNA of HCV driven HCC for the possibility of integration of all known viral genomes. Methods Bar-coded DNA libraries from 41 samples of various stages of development of HCC from 6 different patients were sequenced in parallel using NGS. One to two million 74bp reads per genome were generated. The reads were aligned to all known viral genomes downloaded from the National Center for Biotechnology Information using Burrows-Wheeler Aligner (BWA). Reads with mapping scores of Results Six test samples mapped to unique sequences from Human herpesvirus 6. The test samples included a single HCC and 5 pre-malignant nodules from 2 different patients. Six test samples mapped to unique sequences of Human Adenovirus (6/41). The test samples in this case included 4 HCCS and a 2 premalignant nodules from 2 different patients. A single dysplastic nodule mapped to Human papillomavirus. Conclusion DNA from HCV driven HCC was searched for all viral genome sequences only Human Herpes 6, Human Adenovirus and Human pappilomavirus were found in a small number of cases. Further studies are needed to understand their relation to HCV hepatocarcinogenesis. Disclosure of Interest None Declared.

Underlying chromosomal copy number variation is responsible for 10–15% of fetal structural abnormalities, but even in the presence of a normal karyotype neonatal outcome can be poor. Current whole genome prenatal analysis is limited to G-banded karyotyping. Prenatal microarrays have yet to be fully established, but detection rates of up to 5.8% are reported.1 Potential disadvantages of arrays include the reliance on nucleic acid hybridisation, where fluctuations may increase experimental noise and reduce reproducibility.2 Prenatal material is commonly of low quantity and quality, characteristics which inhibit performance of microarrays. We have developed an alternative method of copy number diagnosis using Next Generation Sequencing (CNV-Seq). Massively-parallel sequencing of test and control individuals is performed using the Illumina Genome Analyser platform. Millions of genomic reads are aligned to the reference sequence and analysed using custom-designed segmentation algorithms. Comparison with similarly-segmented normal controls is then performed for accurate variant detection. Analysis of platform performance showed 100% detection rate in 10 prenatal samples with known chromosomal abnormalities ranging in size from submicroscopic microdeletion syndromes to aneuploidy. Multiplexing of up to 15 samples is possible for cost benefits without loss of sensitivity. Depth of coverage required is fractions of that reported previously.3 Comparison with array CGH data supports the hypothesis that CNV-Seq offers advantages over both conventional karyotyping and DNA microarrays. We suggest that this approach is unique in its potential to offer a single platform solution for genetic analysis, allowing detection of a range of abnormalities from single point mutations to balanced rearrangements and aneuploidy.

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

PURPOSE. To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa. METHODS. Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visualtracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis. RESULTS. Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b rd1 mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles. CONCLUSIONS. These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b rd1 mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa. (Invest Ophthalmol Vis Sci. 2005;46:3443‐3450)

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.