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Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future., Using an unbiased genome-wide CRISPR screen approach, Sanchez et al. identified modulators of endogenous tau protein. This study suggests that chromatin modifiers, neddylation, ubiquitination, and the mTOR pathways regulate overall levels of tau protein in neurons, which could help in future identification of therapeutics for neurodegenerative diseases.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function., Epidermal homeostasis requires long term stem cell function. Here, the authors apply transcriptional circuitry analysis based on integrated epigenomic profiling of primary human keratinocytes with high and low stem cell function to identify IRF2 as a negative regulator of stemness.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

Summary: Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses. : Recent outbreaks and lack of effective treatments against dengue and Zika virus have caused public concerns. Estoppey et al. have identified cavinafungin as exerting potent and selective antiviral activity by targeting the signal-binding cleft of the catalytic subunit of the endoplasmic reticulum signal peptidase. Keywords: Zika virus, dengue virus, cavinafungin, signal peptidase, SEC11A, SEC11, CRISPR/Cas9, chemogenomic profiling

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses., eLife digest Cancer is caused by cells growing and dividing uncontrollably as a result of mutations in certain genes. Many human lung cancers have a mutation in the gene that makes the protein EGFR. In healthy cells, EGFR allows a cell to respond to chemical signals that encourage healthy growth. In cancer, the altered EGFR is always on, which allows the cell to rapidly grow without any control, resulting in cancer. One approach to treating these cancers is with drugs that block the activity of mutant EGFR. Although these drugs have been very successful, they do not always succeed in completely treating the cancer. This is because over time the cancer cells can become resistant to the drug and start forming new tumors. One way that this can happen is if random mutations lead to changes in other proteins that make the drug less effective or stop it from accessing the EGFR proteins. However, it is unclear how other proteins in cancer cells affect the response to these EGFR inhibiting drugs. Now, Zeng et al. have used gene editing to systematically remove every protein from human lung cancer cells grown in the laboratory to see how this affects resistance to EGFR inhibitor treatment. This revealed that a number of different proteins could change how cancer cells responded to the drug. For instance, cells lacking the protein RIC8A were more sensitive to EGFR inhibitors and less likely to develop resistance. This is because loss of RIC8A turns down a key cell survival pathway in cancer cells. Whereas, cancer cells lacking the ARIH2 protein were able to produce more proteins that are needed for cancer cell growth, which resulted in them having increased resistance to EGFR inhibitors. The proteins identified in this study could be used to develop new drugs that improve the effectiveness of EGFR inhibitors. Understanding how cancer cells respond to EGFR inhibitor treatment could help determine how likely a patient is to develop resistance to these drugs.

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling., Axin is a scaffolding protein known for its role in Wnt signalling that can be marked with a variety of post-translational modifications. Here, Cong et al. demonstrate that USP7 de-ubiquinates Axin and that canonical Wnt signaling output can be increased with USP7 inhibitors.

YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/β-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy.

Scientific Reports, 7, ISSN:2045-2322

Summary Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein. DOI: http://dx.doi.org/10.7554/eLife.17290.001

Medulloblastomas are the most common malignant brain tumors in children1. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles2–5. Here, we utilized whole exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, novel findings in medulloblastoma. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma.

Some cases of familial frontotemporal dementia (FTD) leading to frontotemporal lobar degeneration (FTLD) are caused by mutations in tau on chromosome 17 (FTDP-17). Certain mutations alter the ratio between four (4R tau) and three (3R tau) repeat tau isoforms whereas cases with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) mainly have 4R tau brain pathology. We assessed tau mRNA and protein levels in frontal cortex from 15 sporadic FTLD, 21 PSP, 5 CBD, 15 Alzheimer's disease (AD) and 16 control brains. Moreover, we investigated the disease association and possible tau splicing effects of the tau H1 haplotype. Cases with FTLD and PSP had lower tau mRNA levels than control brains. When analyzing 4R tau and 3R tau mRNA separately, control subjects displayed a 4R tau/3R tau ratio of 0.48. Surprisingly, FTLD brains displayed a more elevated ratio (1.32) than PSP brains (1.12). Also, several FTLD and PSP cases had higher 4R tau/3R tau mRNA than FTDP-17 cases, included as reference tissues, and the ratio increase was seen regardless of underlying histopathology, i.e. both for tau-positive and tau-negative FTLD cases. Furthermore, total tau protein levels were slightly decreased in both FTLD and AD as compared to control subjects. Finally, we confirmed the association of tau H1 with PSP, but could not find any haplotype-related effect on tau exon 10 splicing. In conclusion, we demonstrated increased but largely variable 4R tau/3R tau mRNA ratios in FTLD and PSP cases, suggesting heterogeneous pathophysiological processes within these disorders.

We describe a family with a rapidly progressive neurodegenerative disorder characterized by amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) but with unusual neuropathologic features that include pallidoluysionigral degeneration. The proband presented with primary progressive aphasia that evolved into mutism. He subsequently developed dementia with mild disinhibition and parkinsonism and late in the disease showed evidence of motor neuron disease. Two other cases (the proband's mother and maternal uncle) had features of ALS exclusively. All 3 had a young onset (fourth decade) and rapid clinical course, with average time from onset of symptoms to death of 4 years. Postmortem neuropathologic examination of the proband and his uncle showed ALS changes and extensive pallidoluysionigral degeneration without neurofibrillary tangles, ubiquitin inclusions, or detectable abnormalities in the dentate nucleus of the cerebellum. Although this exceptional combination of neuropathologic features has been described in rare cases of sporadic ALS-FTD, no pedigrees have ever been reported. In 2 affected members of this family, we failed to identify mutations in genes associated with weakness, movement disorders, or dementia, including ALS, FTD, selected spinocerebellar ataxias, and Huntington disease. Thus, this disorder may represent a novel autosomal dominantly inherited and rapidly progressive neurodegenerative disorder with a spectrum of clinical presentations but common neuropathologic features.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

The cytoplasmic dynein-dynactin complex has been implicated in the aetiology of motor neuron degeneration in both mouse models and human forms of motor neuron disease. We have previously shown that mutations in the cytoplasmic dynein 1 heavy chain 1 gene (Dync1h1) are causal in a mouse model of late-onset motor neuron degeneration but have found no association of the homologous sites in human DYNC1H1 with human motor neuron disease. Here we extend these analyses to investigate the DYNC1H1 genomic locus to determine if it is associated with sporadic amyotrophic lateral sclerosis (ALS) in a northern European-derived population. Among the 16 single nucleotide polymorphisms (SNPs) we examined, just two SNPs (rs2251644 and rs941793) were sufficient to tag the majority of haplotypic variation (r2 > or = 0.85) and these were tested in a case-control association study with 266 North American sporadic ALS patients and 225 matched controls. We found no association between genetic variation at DYNC1H1 and sporadic ALS (rs2251644; p = 0.538, rs941793; p = 0.204, haplotype; p = 0.956). In addition we investigated patterns of diversity at DYNC1H1 in Japanese and Cameroonian populations to establish the evolutionary history for this gene and observed reduced genetic diversity in the northern Europeans suggestive of selection at this locus.

A reduced risk of Alzheimer’s disease (AD) associated with the apolipoprotein E (APOE) Ε4 allele is reported in populations of African origin. In order to clarify possible reasons for this, we examined the association between APOE genotype and early cognitive impairment in a community-based African Caribbean UK population aged 55–75 years. APOE genotype was available for 202 participants, 57 (28%) of whom were classified as having relative cognitive impairment on a battery of neuropsychological tests. Cognitive impairment was negatively associated with Ε2 and positively but more weakly associated with Ε4. Effects of both alleles increased markedly after age 70. The effect of Ε4 was increased in combination with hypertension, diabetes or lower educational attainment, but these factors did not influence Ε2 effects. Cholesterol and triglyceride levels partially explained effects of Ε2, but did not account for those of Ε4. A reduced association between Ε4 and later AD in populations of African origin is unlikely to be explained by reduced cognitive effects or by differential mortality. However, it may be accounted for by vascular comorbidity. The different patterns of association between Ε2 and Ε4 alleles suggest different pathways of effect.

The evolution of metazoans from their unicellular ancestors was one of the most important events in the history of life. However, the cellular and genetic changes that ultimately led to the evolution of multicellularity are not known. In this study, we describe an aggregative multicellular stage in the protist Capsaspora owczarzaki, a close unicellular relative of metazoans. Remarkably, transition to the aggregative stage is associated with significant upregulation of orthologs of genes known to establish multicellularity and tissue architecture in metazoans. We further observe transitions in regulated alternative splicing during the C. owczarzaki life cycle, including the deployment of an exon network associated with signaling, a feature of splicing regulation so far only observed in metazoans. Our results reveal the existence of a highly regulated aggregative stage in C. owczarzaki and further suggest that features of aggregative behavior in an ancestral protist may had been co-opted to develop some multicellular properties currently seen in metazoans. © Sebé-Pedrós et al., ASP is supported by a pregraduate Formacion Profesorado Universitario grant from MICINN. MI is supported by a postdoctoral fellowship from the Human Frontiers Science Program Organization. BJB acknowledges grant funding from the Canadian Institutes of Health Research.

We recently identified angiogenin (ANG) as a candidate susceptibility gene for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by adult-onset loss of motor neurons. We now report the finding of seven missense mutations in 15 individuals, of whom four had familial ALS and 11 apparently 'sporadic' ALS. Our findings provide further evidence that variations in hypoxia-inducible genes have an important role in motor neuron degeneration.

Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, Loa loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4 Mb genome of Loa loa, and the genome of the related filarial parasite Wuchereria bancrofti, and predict 14,907 Loa loa genes based on microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to several other nematode genomes, we demonstrate synteny among filariae but not with non-parasitic nematodes. The Loa loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for humans. Despite lacking Wolbachia, Loa loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role played by Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and non-parasitic nematodes.

Item does not contain fulltext The translation of "next-generation" sequencing directly to the clinic is still being assessed but has the potential for genetic diseases to reduce costs, advance accuracy, and point to unsuspected yet treatable conditions. To study its capability in the clinic, we performed whole-exome sequencing in 118 probands with a diagnosis of a pediatric-onset neurodevelopmental disease in which most known causes had been excluded. Twenty-two genes not previously identified as disease-causing were identified in this study (19% of cohort), further establishing exome sequencing as a useful tool for gene discovery. New genes identified included EXOC8 in Joubert syndrome and GFM2 in a patient with microcephaly, simplified gyral pattern, and insulin-dependent diabetes. Exome sequencing uncovered 10 probands (8% of cohort) with mutations in genes known to cause a disease different from the initial diagnosis. Upon further medical evaluation, these mutations were found to account for each proband's disease, leading to a change in diagnosis, some of which led to changes in patient management. Our data provide proof of principle that genomic strategies are useful in clarifying diagnosis in a proportion of patients with neurodevelopmental disorders.

De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8-40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling.

Distinguishing Ciliopathy Cilia were once thought to be evolutionary remnants, but structural defects reveal their importance in signaling pathways and human disease, such as Joubert syndrome. Either of the genes TMEM138 and TMEM216 can be found mutated in phenotypically indistinguishable ciliopathy patients. Interestingly, despite their lack of sequence homology, these genes have always been aligned in head-to-tail configuration during vertebrate evolution. The proteins expressed by these genes mark distinct tethered vesicles, which differentially carry ciliary proteins for assembly. Lee et al. (p. 966 , published online 26 January; see the Perspective by Chakravarti and Kapoor ) show that the coordinated expression of these adjacent genes depends upon a coevolved regulatory element in the noncoding intergenic region, which thus integrates the roles of both gene products. This discovery explains not only the indistinguishable pathogenesis of the patients' genotypes but also how the evolutionary clustering of genes unrelated in sequence may correlate with coordinated control of expression and function.

Pick's disease (PiD) is a rare neurodegenerative condition and is a member of a heterogeneous group of disorders known as tauopathies, so-called because of the predominantly neuronal aberrant tau accumulations found in these diseases. The tauopathy, familial frontotemporal dementia (FTD), is caused by mutations in the tau gene. Moreover, progressive supranuclear palsy (PSP) is associated with the tau H1 haplotype. In certain familial forms of FTD and in PSP the microtubule-binding four repeat tau isoform principally accumulates in neuropathological lesions. However, in PiD three repeat tau accumulations are found. We therefore investigated whether either the tau H1 or H2 haplotype was associated with PiD. Our results indicate a slight increase in H2H2 frequency in Pick's cases which is not statistically significant.

Joubert syndrome (JBTS), related disorders (JSRDs) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and CORS2 (JBTS2) loci are allelic and caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. Individuals with CORS2 frequently had nephronophthisis and polydactyly, and two affected individuals conformed to the oro-facio-digital type VI phenotype, whereas skeletal dysplasia was common in fetuses affected by MKS. A single G218T mutation (R73L in the protein) was identified in all cases of Ashkenazi Jewish descent (n = 10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in mutant fibroblasts or after knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 formed a complex with Meckelin, which is encoded by a gene also mutated in JSRDs and MKS. Disruption of tmem216 expression in zebrafish caused gastrulation defects similar to those in other ciliary morphants. These data implicate a new family of proteins in the ciliopathies and further support allelism between ciliopathy disorders.

Open Access Poster presentation P09-20 LB. Ultra-deep sequencing of full-length HIV-1 genomes identifies rapid viral evolution during acute infection MR Henn1, C Boutwell3, N Lennon1, K Power3, C Malboeuf1, P Charlebois1, A Gladden3, J Levin1, M Casali1, L Philips3, A Berlin1, A Berical3, R Erlich1, S Anderson1, H Streeck3, M Kemper3, E Ryan1, Y Wang3, L Green1, K Axten3, Z Brumme3, C Brumme3, C Russ1, E Rosenberg3, H Jessen2, M Altfeld3, C Nusbaum1, B Walker3, B Birren1 and TM Allen*3

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; 12-23% are associated with mutations in the Cu/Zn superoxide dismutase gene (SOD1). All ALS-linked SOD1 mutations present with a dominant pattern of inheritance apart from the aspartate to alanine mutation in exon 4 (D90A). This mutation has been observed in dominant, recessive and apparently sporadically cases. SOD1(D90A/D90A) ALS cases have a very slow disease progression (>10 years), raising the hypothesis that modifier genes linked to SOD1 ameliorate the phenotype of recessively inherited SOD1(D90A/D90A) mutations. Previous sequence analysis of a conserved haplotype region around the SOD1 gene did not reveal any functional polymorphisms within known coding or putative regulatory regions. In the current study we expanded the previous analyses by sequencing the entire SOD1 conserved haplotypic region. Although many polymorphisms were identified, none of these variants explain the slowly progressive phenotype observed in patients with recessive SOD1(D90A) mutations. This study disproves the hypothesis that there is a tightly linked genetic protective factor specifically located close to the SOD1 gene in SOD1(D90A) mediated ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder. Ten percent of cases are inherited; most involve unidentified genes. We report here 13 mutations in the fused in sarcoma/translated in liposarcoma ( FUS/TLS ) gene on chromosome 16 that were specific for familial ALS. The FUS/TLS protein binds to RNA, functions in diverse processes, and is normally located predominantly in the nucleus. In contrast, the mutant forms of FUS/TLS accumulated in the cytoplasm of neurons, a pathology that is similar to that of the gene TAR DNA-binding protein 43 ( TDP43 ), whose mutations also cause ALS. Neuronal cytoplasmic protein aggregation and defective RNA metabolism thus appear to be common pathogenic mechanisms involved in ALS and possibly in other neurodegenerative disorders.

VPS54 is a component of the Golgi-associated retrograde protein (GARP) complex of vesicle sorting proteins. A missense mutation of Vps54 is responsible for motor neuron disease in the wobbler mouse, but the human gene on chromosome 2p14-15 has not been evaluated as a disease gene. We completely sequenced the 22 coding exons from 96 individuals with sporadic ALS, 96 individuals with familial ALS, and 96 controls. Twenty-one novel SNPs were identified. The non-synonymous variant, T360A, was observed in one patient and 0/910 controls. Several polymorphic non-synonymous SNPs were also observed in patients and controls. These initial data suggest that mutations in VPS54 are not a major cause of ALS.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS. Approximately 20% of cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). In North America, SOD1(A4V) is the most common SOD1 mutation. Carriers of the SOD1(A4V) mutation share a common phenotype with rapid disease progression and death on average occurring at 1.4 years (versus 3-5 years with other dominant SOD1 mutations). Previous studies of SOD1(A4V) carriers identified a common haplotype around the SOD1 locus, suggesting a common founder for most SOD1(A4V) patients. In the current study we sequenced the entire common haplotypic region around SOD1 to test the hypothesis that polymorphisms in either previously undescribed coding regions or non-coding regions around SOD1 are responsible for the more aggressive phenotype in SOD1(A4V)-mediated ALS. We narrowed the conserved region around the SOD1 gene in SOD1(A4V) ALS to 2.8Kb and identified five novel SNPs therein. None of these variants was specifically found in all SOD1(A4V) patients. It therefore appears likely that the aggressive nature of the SOD1(A4V) mutation is not a result of a modifying factor within the region around the SOD1 gene. Founder analysis estimates that the A4V mutation occurred 540 generations (approximately 12,000 years) ago (95% CI 480-700). The conserved minimal haplotype is statistically more similar to Asian than European population DNA sets, suggesting that the A4V mutation arose in native Asian-Americans who reached the Americas through the Bering Strait.

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). In North America, SOD1(A4V) is the most common SOD1 mutation. A4V ALS cases typically have a very short survival (1-1.5 years versus 3-5 years for other dominant SOD1 mutations). A recent study of A4V carriers identified a common haplotype around the SOD1 locus, suggesting the hypothesis that genetic variations within the haplotypic region might accelerate the course of A4V cases. By contrast, SOD1(D90A/D90A) ALS cases have a very slow progression (10 years), raising the reciprocal hypothesis that modifier genes linked to SOD1 ameliorate the phenotype of recessively inherited SOD1(D90A/D90A) mutations. In the present study, DNA sequencing of four genes within the haplotypic region shared in A4V and D90A ALS patients revealed 15 novel variants, but none result in changes in amino acid sequences specifically associated with SOD1(D90A/D90A) or SOD1(A4V) ALS. We conclude that mutations within coding regions of genes around the SOD1 locus are not responsible for the more aggressive and more benign natures of the SOD1(A4V) and SOD1(D90A/D90A) mutations, respectively.

Glycogen synthase kinase-3beta (GSK3beta) is a target of lithium as well as sodium valproate, both of which are effective mood stabilizing prophylatics/treatments for bipolar disorder, a highly heritable psychiatric disorder. Though it is not clear whether the mood stabilizing effects of these drugs act directly through GSK3beta, it is a good candidate for mediating at least part of lithium's action, and is an aetiological candidate gene for the disease itself. Recently, a potential locus for bipolar disorder was reported on chromosome 3q, close to 3q13.37 where GSK3beta maps. We conducted an association study to test the hypothesis that polymorphism of GSK3beta is involved in susceptibility to bipolar disorder by examining association between GSK3beta-gene polymorphisms and bipolar disorder. Of the five polymorphisms we examined, three were very rare in the study population and were not examined further. Neither of the remaining two polymorphisms we examined showed association with bipolar disorder. Thus, it is unlikely that the GSK3beta-gene is a risk factor for bipolar disorder in our sample, but we cannot exclude the gene completely as other unknown polymorphisms in the gene may increase susceptibility.

BackgroundMutations in tau have been found in a group of related disorders including the frontal lobe dementias.AimsTo describe the clinical features and molecular pathology changes in a single case of a patient with frontal lobe dementia.MethodA case report was compiled from neuropathological reports and genomic and gene expression analyses.ResultsA case with a splice-site mutation resulting in a typical frontotemporal clinical and neuropathological phenotype was found. Gene expression analysis suggests differential expression of isoforms of tau in regions in the brain.ConclusionsFrontotemporal dementia can result from gene mutations that alter splicing and expression of tau.

Several studies have suggested an association between polymorphisms and an extended haplotype of the microtubule associated protein tau gene and Alzheimer's disease (AD) in synergy with apolipoprotein E (APOE) epsilon 4 status. However these findings have not been consistently replicated. We investigated the role of the tau haplotype in AD by conducting an association study as well as a meta-analysis of all the studies conducted to date. We examined six polymorphisms known to be in the extended tau haplotypes, one in exon 7 and five in and around exon 9 in 200 late onset AD and 189 control samples. All the polymorphisms examined fell into the recognised tau haplotypes. There was no statistical significant association with any of the polymorphisms and late onset AD. Stratification of data by APOE epsilon 4 status also produced no strongly significant association. The meta-analysis showed no significant differences between AD cases and controls, however stratification of data by APOE epsilon 4 status showed a small significant decrease in the H1 haplotype in AD before correction for multiple testing.

High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000-140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8-2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.