ALBERT

Description: Motivation: Mapping of high-throughput sequencing data and other bulk sequence comparison applications have motivated a search for high-efficiency sequence alignment algorithms. The bit-parallel approach represents individual cells in an alignment scoring matrix as bits in computer words and emulates the calculation of scores by a series of logic operations composed of AND, OR, XOR, complement, shift and addition. Bit-parallelism has been successfully applied to the longest common subsequence (LCS) and edit-distance problems, producing fast algorithms in practice. Results: We have developed BitPAl, a bit-parallel algorithm for general, integer-scoring global alignment. Integer-scoring schemes assign integer weights for match, mismatch and insertion/deletion. The BitPAl method uses structural properties in the relationship between adjacent scores in the scoring matrix to construct classes of efficient algorithms, each designed for a particular set of weights. In timed tests, we show that BitPAl runs 7–25 times faster than a standard iterative algorithm. Availability and implementation: Source code is freely available for download at http://lobstah.bu.edu/BitPAl/BitPAl.html . BitPAl is implemented in C and runs on all major operating systems. Contact : jloving@bu.edu or yhernand@bu.edu or gbenson@bu.edu Supplementary information : Supplementary data are available at Bioinformatics online.

Description: : Next-generation sequencing (NGS) has a large potential in HIV diagnostics, and genotypic prediction models have been developed and successfully tested in the recent years. However, albeit being highly accurate, these computational models lack computational efficiency to reach their full potential. In this study, we demonstrate the use of graphics processing units (GPUs) in combination with a computational prediction model for HIV tropism. Our new model named gCUP, parallelized and optimized for GPU, is highly accurate and can classify 〉175 000 sequences per second on an NVIDIA GeForce GTX 460. The computational efficiency of our new model is the next step to enable NGS technologies to reach clinical significance in HIV diagnostics. Moreover, our approach is not limited to HIV tropism prediction, but can also be easily adapted to other settings, e.g. drug resistance prediction. Availability and implementation: The source code can be downloaded at http://www.heiderlab.de Contact: d.heider@wz-straubing.de

Description: : We present a new method to incrementally construct the FM-index for both short and long sequence reads, up to the size of a genome. It is the first algorithm that can build the index while implicitly sorting the sequences in the reverse (complement) lexicographical order without a separate sorting step. The implementation is among the fastest for indexing short reads and the only one that practically works for reads of averaged kilobases in length. Availability and implementation: https://github.com/lh3/ropebwt2 Contact: hengli@broadinstitute.org

Description: : AliView is an alignment viewer and editor designed to meet the requirements of next-generation sequencing era phylogenetic datasets. AliView handles alignments of unlimited size in the formats most commonly used, i.e. FASTA, Phylip, Nexus, Clustal and MSF. The intuitive graphical interface makes it easy to inspect, sort, delete, merge and realign sequences as part of the manual filtering process of large datasets. AliView also works as an easy-to-use alignment editor for small as well as large datasets. Availability and implementation: AliView is released as open-source software under the GNU General Public License, version 3.0 (GPLv3), and is available at GitHub ( www.github.com/AliView ). The program is cross-platform and extensively tested on Linux, Mac OS X and Windows systems. Downloads and help are available at http://ormbunkar.se/aliview Contact: anders.larsson@ebc.uu.se Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The ability to accurately read the order of nucleotides in DNA and RNA is fundamental for modern biology. Errors in next-generation sequencing can lead to many artifacts, from erroneous genome assemblies to mistaken inferences about RNA editing. Uneven coverage in datasets also contributes to false corrections. Result: We introduce Trowel, a massively parallelized and highly efficient error correction module for Illumina read data. Trowel both corrects erroneous base calls and boosts base qualities based on the k -mer spectrum. With high-quality k -mers and relevant base information, Trowel achieves high accuracy for different short read sequencing applications.The latency in the data path has been significantly reduced because of efficient data access and data structures. In performance evaluations, Trowel was highly competitive with other tools regardless of coverage, genome size read length and fragment size. Availability and implementation: Trowel is written in C++ and is provided under the General Public License v3.0 (GPLv3). It is available at http://trowel-ec.sourceforge.net . Contact: euncheon.lim@tue.mpg.de or weigel@tue.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : The application of protein–protein docking in large-scale interactome analysis is a major challenge in structural bioinformatics and requires huge computing resources. In this work, we present MEGADOCK 4.0, an FFT-based docking software that makes extensive use of recent heterogeneous supercomputers and shows powerful, scalable performance of 〉97% strong scaling. Availability and Implementation: MEGADOCK 4.0 is written in C++ with OpenMPI and NVIDIA CUDA 5.0 (or later) and is freely available to all academic and non-profit users at: http://www.bi.cs.titech.ac.jp/megadock . Contact: akiyama@cs.titech.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online

Description: Motivation: The identification of active transcriptional regulatory elements is crucial to understand regulatory networks driving cellular processes such as cell development and the onset of diseases. It has recently been shown that chromatin structure information, such as DNase I hypersensitivity (DHS) or histone modifications, significantly improves cell-specific predictions of transcription factor binding sites. However, no method has so far successfully combined both DHS and histone modification data to perform active binding site prediction. Results: We propose here a method based on hidden Markov models to integrate DHS and histone modifications occupancy for the detection of open chromatin regions and active binding sites. We have created a framework that includes treatment of genomic signals, model training and genome-wide application. In a comparative analysis, our method obtained a good trade-off between sensitivity versus specificity and superior area under the curve statistics than competing methods. Moreover, our technique does not require further training or sequence information to generate binding location predictions. Therefore, the method can be easily applied on new cell types and allow flexible downstream analysis such as de novo motif finding. Availability and implementation: Our framework is available as part of the Regulatory Genomics Toolbox. The software information and all benchmarking data are available at http://costalab.org/wp/dh-hmm . Contact: ivan.costa@rwth-aachen.de or eduardo.gusmao@rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: A proper target or marker is essential in any diagnosis (e.g. an infection or cancer). An ideal diagnostic target should be both conserved in and unique to the pathogen. Currently, these targets can only be identified manually, which is time-consuming and usually error-prone. Because of the increasingly frequent occurrences of emerging epidemics and multidrug-resistant ‘superbugs’, a rapid diagnostic target identification process is needed. Results: A new method that can identify uniquely conserved regions (UCRs) as candidate diagnostic targets for a selected group of organisms solely from their genomic sequences has been developed and successfully tested. Using a sequence-indexing algorithm to identify UCRs and a k -mer integer-mapping model for computational efficiency, this method has successfully identified UCRs within the bacteria domain for 15 test groups, including pathogenic, probiotic, commensal and extremophilic bacterial species or strains. Based on the identified UCRs, new diagnostic primer sets were designed, and their specificity and efficiency were tested by polymerase chain reaction amplifications from both pure isolates and samples containing mixed cultures. Availability and implementation: The UCRs identified for the 15 bacterial species are now freely available at http://ucr.synblex.com . The source code of the programs used in this study is accessible at http://ucr.synblex.com/bacterialIdSourceCode.d.zip Contact: yazhousun@synblex.com Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Motivation: A popular method for classification of protein domain movements apportions them into two main types: those with a ‘hinge’ mechanism and those with a ‘shear’ mechanism. The intuitive assignment of domain movements to these classes has limited the number of domain movements that can be classified in this way. Furthermore, whether intended or not, the term ‘shear’ is often interpreted to mean a relative translation of the domains. Results: Numbers of occurrences of four different types of residue contact changes between domains were optimally combined by logistic regression using the training set of domain movements intuitively classified as hinge and shear to produce a predictor for hinge and shear. This predictor was applied to give a 10-fold increase in the number of examples over the number previously available with a high degree of precision. It is shown that overall a relative translation of domains is rare, and that there is no difference between hinge and shear mechanisms in this respect. However, the shear set contains significantly more examples of domains having a relative twisting movement than the hinge set. The angle of rotation is also shown to be a good discriminator between the two mechanisms. Availability and implementation: Results are free to browse at http://www.cmp.uea.ac.uk/dyndom/interface/ . Contact: sjh@cmp.uea.ac.uk . Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Recent studies on human disease have revealed that aberrant interaction between proteins probably underlies a substantial number of human genetic diseases. This suggests a need to investigate disease inheritance mode using interaction, and based on which to refresh our conceptual understanding of a series of properties regarding inheritance mode of human disease. Results: We observed a strong correlation between the number of protein interactions and the likelihood of a gene causing any dominant diseases or multiple dominant diseases, whereas no correlation was observed between protein interaction and the likelihood of a gene causing recessive diseases. We found that dominant diseases are more likely to be associated with disruption of important interactions. These suggest inheritance mode should be understood using protein interaction. We therefore reviewed the previous studies and refined an interaction model of inheritance mode, and then confirmed that this model is largely reasonable using new evidences. With these findings, we found that the inheritance mode of human genetic diseases can be predicted using protein interaction. By integrating the systems biology perspectives with the classical disease genetics paradigm, our study provides some new insights into genotype–phenotype correlations. Contact: haodapeng@ems.hrbmu.edu.cn or biofomeng@hotmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. Availability and implementation: DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB Contact: avi.maayan@mssm.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Clinical and neuropathological similarities between dementia with Lewy bodies (DLB), Parkinson's and Alzheimer's diseases (PD and AD, respectively) suggest that these disorders may share etiology. To test this hypothesis, we have performed an association study of 54 genomic regions, previously implicated in PD or AD, in a large cohort of DLB cases and controls. The cohort comprised 788 DLB cases and 2624 controls. To minimize the issue of potential misdiagnosis, we have also performed the analysis including only neuropathologically proven DLB cases (667 cases). The results show that the APOE is a strong genetic risk factor for DLB, confirming previous findings, and that the SNCA and SCARB2 loci are also associated after a study-wise Bonferroni correction, although these have a different association profile than the associations reported for the same loci in PD. We have previously shown that the p.N370S variant in GBA is associated with DLB, which, together with the findings at the SCARB2 locus, suggests a role for lysosomal dysfunction in this disease. These results indicate that DLB has a unique genetic risk profile when compared with the two most common neurodegenerative diseases and that the lysosome may play an important role in the etiology of this disorder. We make all these data available.

Description: Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.

Description: Immunoglobulin-like domain containing receptor 1 ( ILDR1 ) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1 -related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b -morphant zebrafish model. Ildr1b -morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na + /K + transporting, beta 2b polypeptide) in ildr1b -morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b -knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b -morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b ( cxcr4b ) and chemokine receptor 7b ( cxcr7b ) in posterior lateral line primordium of ildr1b -morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

Description: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by abnormal expansion of CAG repeats in the gene encoding huntingtin. Mutant huntingtin undergoes proteolytic processing and its N-terminal fragment containing polyglutamine repeat accumulates as inclusion not only in nucleus but also in cytoplasm and neuronal processes. Here, we demonstrate that removal of ubiquitin ligase Ube3a selectively from HD mice brain resulted in accelerated disease phenotype and shorter lifespan in comparison with HD mice. The deficiency of Ube3a in HD mice brain also caused significant increase in global aggregates load, and these aggregates were less ubiquitinated when compared with age-matched HD mice. These Ube3a -maternal deficient HD mice also showed drastic reduction of DARPP-32, a dopamine-regulated phoshphoprotein in their striatum. These results emphasize the crucial role of Ube3a in the progression of HD and its immense potential as therapeutic target.

Description: Parent-of-origin-specific expression at imprinted genes is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). This mechanism of gene regulation, where one element controls allelic expression of multiple genes, is not fully understood. Furthermore, the mechanism of gene dysregulation through ICR epimutations, such as loss or gain of DNA methylation, remains a mystery. We have used genetic mouse models to dissect ICR-mediated genetic and epigenetic regulation of imprinted gene expression. The H19/insulin-like growth factor 2 (Igf2) ICR has a multifunctional role including insulation, activation and repression. Microdeletions at the human H19/IGF2 ICR (IC1) are proposed to be responsible for IC1 epimutations associated with imprinting disorders such as Beckwith–Wiedemann syndrome (BWS). Here, we have generated and characterized a mouse model that mimics BWS microdeletions to define the role of the deleted sequence in establishing and maintaining epigenetic marks and imprinted expression at the H19/IGF2 locus. These mice carry a 1.3 kb deletion at the H19/Igf2 ICR [2,3] removing two of four CCCTC-binding factor (CTCF) sites and the intervening sequence, ~75% of the ICR. Surprisingly, the 2,3 deletion does not perturb DNA methylation at the ICR; however, it does disrupt imprinted expression. While repressive functions of the ICR are compromised by the deletion regardless of tissue type, insulator function is only disrupted in tissues of mesodermal origin where a significant amount of CTCF is poly(ADP-ribosyl)ated. These findings suggest that insulator activity of the H19/Igf2 ICR varies by cell type and may depend on cell-specific enhancers as well as posttranslational modifications of the insulator protein CTCF.

Description: Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1 , CCM2 or CCM3 . Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

Description: Genomic imprinting is the epigenetic process that results in monoallelic expression of genes depending on parental origin. These genes are known to be critical for placental development and fetal growth in mammals. Aberrant epigenetic profiles at imprinted loci, such as DNA methylation defects, are surprisingly rare in pregnancies with compromised fetal growth, while variations in transcriptional output from the expressed alleles of imprinted genes are more commonly reported in pregnancies complicated with intrauterine growth restriction (IUGR). To determine if PLAGL1 and HYMAI , two imprinted transcripts deregulated in Transient Neonatal Diabetes Mellitus, are involved in non-syndromic IUGR we compared the expression and DNA methylation levels in a large cohort of placental biopsies from IUGR and uneventful pregnancies. This revealed that despite appropriate maternal methylation at the shared PLAGL1 / HYMAI promoter, there was a loss of correlation between PLAGL1 and HYMAI expression in IUGR. This incongruity was due to higher HYMAI expression in IUGR gestations, coupled with PLAGL1 down-regulation in placentas from IUGR girls, but not boys. The PLAGL1 protein is a zinc-finger transcription factor that has been shown to be a master coordinator of a genetic growth network in mice. We observe PLAGL1 binding to the H19 / IGF2 shared enhancers in placentae, with significant correlations between PLAGL1 levels with H19 and IGF2 expression levels. In addition, PLAGL1 binding and expression also correlate with expression levels of metabolic regulator genes SLC2A4 , TCF4 and PPAR1 . Our results strongly suggest that fetal growth can be influenced by altered expression of the PLAGL1 gene network in human placenta.

Description: MicroRNAs (miRNAs) have emerged as a class of small, endogenous, regulatory RNAs that exhibit the ability to epigenetically modulate the translation of mRNAs into proteins. This feature enables them to control cell phenotypes and, consequently, modify cell function in a disease context. The role of inflammatory miRNAs in Alzheimer's disease (AD) and their ability to modulate glia responses are now beginning to be explored. In this study, we propose to disclose the functional role of miR-155, one of the most well studied immune-related miRNAs in AD-associated neuroinflammatory events, employing the 3xTg AD animal model. A strong upregulation of miR-155 levels was observed in the brain of 12-month-old 3xTg AD animals. This event occurred simultaneously with an increase of microglia and astrocyte activation, and before the appearance of extracellular Aβ aggregates, suggesting that less complex Aβ species, such as Aβ oligomers may contribute to early neuroinflammation. In addition, we investigated the contribution of miR-155 and the c-Jun transcription factor to the molecular mechanisms that underlie Aβ-mediated activation of glial cells. Our results suggest early miR-155 and c-Jun upregulation in the 3xTg AD mice, as well as in Aβ-activated microglia and astrocytes, thus contributing to the production of inflammatory mediators such as IL-6 and IFN-β. This effect is associated with a miR-155-dependent decrease of suppressor of cytokine signaling 1. Furthermore, since c-Jun silencing decreases the levels of miR-155 in Aβ-activated microglia and astrocytes, we propose that miR-155 targeting can constitute an interesting and promising approach to control neuroinflammation in AD.

Description: Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin ( HTT ) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT . Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient–derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh Q7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT.

Description: Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn -mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.

Description: Microphthalmia-associated transcription factor ( MITF ) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A , -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.

Description: The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Description: During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo . We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2 -deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.

Description: Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor β (TGFβ)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2 . Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1 cre/+ ; Tgfbr2 flox/flox (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFβ, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFβ aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFβ supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

Description: Pneumoconiosis is the most serious occupational disease in China and its leading cause is occupational silica exposure. Pneumoconiosis takes several years to develop depending on the exposure level of silica. However, individual variation in the susceptibility to pneumoconiosis has been observed among the subjects with similar exposure. We conducted a genome-wide screening with 710 999 single nucleotide polymorphisms (SNPs) in a cohort of 400 coal workers (202 cases and 198 exposed controls) for pneumoconiosis susceptible loci. Seven promising variants were evaluated in an independent cohort of 568 coal workers (323 cases and 245 exposed controls), followed by a second replication on 463 iron ore workers (167 cases and 296 exposed controls). By pooling all of the genome-wide association studies and replication stages together, we found a genome-wide significant ( P 〈 5.0 x 10 –8 ) association for rs73329476 ( P = 1.74 x 10 –8 , OR = 2.17, 95% CI = 1.66–2.85) and two additional replicated associations for rs4320486 ( P 〈 0.05) and rs117626015 ( P 〈 0.05) with combined P -values of 4.29 x 10 –6 and 5.05 x 10 –6 , respectively. In addition, the risk allele T of rs73329476 was significantly associated with lower mRNA expression levels of carboxypeptidase M ( CPM ) in total cellular RNA from whole blood of 156 healthy individuals ( P = 0.0252). The identified pneumoconiosis susceptibility loci may provide new insights into the pathogenesis of pneumoconiosis, and may also have some clinical utility for risk prediction for pneumoconiosis and high-risk population screening for workers with occupational silica exposure.

Description: Hearing function is known to be heritable, but few significant and reproducible associations of genetic variants have been identified to date in the adult population. In this study, genome-wide association results of hearing function from the G-EAR consortium and TwinsUK were used for meta-analysis. Hearing ability in eight population samples of Northern and Southern European ancestry ( n = 4591) and the Silk Road ( n = 348) was measured using pure-tone audiometry and summarized using principal component (PC) analysis. Genome-wide association analyses for PC1–3 were conducted separately in each sample assuming an additive model adjusted for age, sex and relatedness of subjects. Meta-analysis was performed using 2.3 million single-nucleotide polymorphisms (SNPs) tested against each of the three PCs of hearing ability in 4939 individuals. A single SNP lying in intron 6 of the salt-inducible kinase 3 ( SIK3 ) gene was found to be associated with hearing PC2 ( P = 3.7 x 10 –8 ) and further supported by whole-genome sequence in a subset. To determine the relevance of this gene in the ear, expression of the Sik3 protein was studied in mouse cochlea of different ages. Sik3 was expressed in murine hair cells during early development and in cells of the spiral ganglion during early development and adulthood. Our results suggest a developmental role of Sik3 in hearing and may be required for the maintenance of adult auditory function.

Description: Genome instability, epigenetic remodelling and structural chromosomal rearrangements are hallmarks of cancer. However, the coordinated epigenetic effects of constitutional chromosomal rearrangements that disrupt genes associated with congenital neurodevelopmental diseases are poorly understood. To understand the genetic–epigenetic interplay at breakpoints of chromosomal translocations disrupting CG-rich loci, we quantified epigenetic modifications at DLGAP4 ( SAPAP4), a key post-synaptic density 95 (PSD95) associated gene, truncated by the chromosome translocation t(8;20)(p12;q11.23), co-segregating with cerebellar ataxia in a five-generation family. We report significant epigenetic remodelling of the DLGAP4 locus triggered by the t(8;20)(p12;q11.23) translocation and leading to dysregulation of DLGAP4 expression in affected carriers. Disruption of DLGAP4 results in monoallelic hypermethylation of the truncated DLGAP4 promoter CpG island. This induced hypermethylation is maintained in somatic cells of carriers across several generations in a t(8;20) dependent-manner however, is erased in the germ cells of the translocation carriers. Subsequently, chromatin remodelling of the locus-perturbed monoallelic expression of DLGAP4 mRNAs and non-coding RNAs in haploid cells having the translocation. Our results provide new mechanistic insight into the way a balanced chromosomal rearrangement associated with a neurodevelopmental disorder perturbs allele-specific epigenetic mechanisms at breakpoints leading to the deregulation of the truncated locus.

Description: We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G〉A, p.Arg394His). KCNH7 c.1181G〉A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G〉A had the highest enrichment among individuals with bipolar spectrum disorder ( 2 = 7.3) and the strongest family-based association with bipolar 1 ( P = 0.021), bipolar spectrum ( P = 0.031) and any major affective disorder ( P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Description: Complex III (cytochrome bc 1 ) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c . Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83 , hereafter named UQCC3 , to be the ortholog of the fungal complex III assembly factor CBP4 . We describe a homozygous c.59T〉A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T〉A mutation has a causal role in complex III deficiency.

Description: Mutations in the photoreceptor-specific gene peripherin-2 ( PRPH-2 , also known as retinal degeneration slow/RDS) cause incurable retinal degeneration with a high degree of phenotypic variability. Patient phenotypes range from retinitis pigmentosa to various forms of macular and pattern dystrophy. Macular and pattern dystrophy in particular are associated with complex, poorly understood disease mechanisms, as severe vision loss is often associated both with defects in the photoreceptors, as well as the choroid and retinal pigment epithelium (RPE). Since there is currently no satisfactory model to study pattern dystrophy disease mechanisms, we generated a knockin mouse model expressing an RDS pattern dystrophy mutation, Y141C. Y141C mice exhibited clinical signs similar to those in patients including late-onset fundus abnormalities characteristic of RPE and choroidal defects and electroretinogram defects. Ultrastructural examination indicated that disc formation was initiated by the Y141C protein, but proper sizing and alignment of discs required wild-type RDS. The biochemical mechanism underlying these abnormalities was tied to defects in the normal process of RDS oligomerization which is required for proper RDS function. Y141C-RDS formed strikingly abnormal disulfide-linked complexes which were localized to the outer segment (OS) where they impaired the formation of proper OS structure. These data support a model of pattern dystrophy wherein a primary molecular defect occurring in all photoreceptors leads to secondary sequellae in adjacent tissues, an outcome which leads to macular vision loss. An understanding of the role of RDS in the interplay between these tissues significantly enhances our understanding of RDS-associated pathobiology and our ability to design rational treatment strategies.

Description: Motivation : Structural variation is common in human and cancer genomes. High-throughput DNA sequencing has enabled genome-scale surveys of structural variation. However, the short reads produced by these technologies limit the study of complex variants, particularly those involving repetitive regions. Recent ‘third-generation’ sequencing technologies provide single-molecule templates and longer sequencing reads, but at the cost of higher per-nucleotide error rates. Results : We present MultiBreak-SV, an algorithm to detect structural variants (SVs) from single molecule sequencing data, paired read sequencing data, or a combination of sequencing data from different platforms. We demonstrate that combining low-coverage third-generation data from Pacific Biosciences (PacBio) with high-coverage paired read data is advantageous on simulated chromosomes. We apply MultiBreak-SV to PacBio data from four human fosmids and show that it detects known SVs with high sensitivity and specificity. Finally, we perform a whole-genome analysis on PacBio data from a complete hydatidiform mole cell line and predict 1002 high-probability SVs, over half of which are confirmed by an Illumina-based assembly. Availability and implementation : MultiBreak-SV is available at http://compbio.cs.brown.edu/software/ . Contact : annaritz@vt.edu or braphael@cs.brown.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Insertions play an important role in genome evolution. However, such variants are difficult to detect from short-read sequencing data, especially when they exceed the paired-end insert size. Many approaches have been proposed to call short insertion variants based on paired-end mapping. However, there remains a lack of practical methods to detect and assemble long variants. Results: We propose here an original method, called M ind T he G ap , for the integrated detection and assembly of insertion variants from re-sequencing data. Importantly, it is designed to call insertions of any size, whether they are novel or duplicated, homozygous or heterozygous in the donor genome. M ind T he G ap uses an efficient k -mer-based method to detect insertion sites in a reference genome, and subsequently assemble them from the donor reads. M ind T he G ap showed high recall and precision on simulated datasets of various genome complexities. When applied to real Caenorhabditis elegans and human NA12878 datasets, M ind T he G ap detected and correctly assembled insertions 〉1 kb, using at most 14 GB of memory. Availability and implementation: http://mindthegap.genouest.org Contact: guillaume.rizk@inria.fr or claire.lemaitre@inria.fr

Description: Motivation: Most tumor samples are a heterogeneous mixture of cells, including admixture by normal (non-cancerous) cells and subpopulations of cancerous cells with different complements of somatic aberrations. This intra-tumor heterogeneity complicates the analysis of somatic aberrations in DNA sequencing data from tumor samples. Results: We describe an algorithm called THetA2 that infers the composition of a tumor sample—including not only tumor purity but also the number and content of tumor subpopulations—directly from both whole-genome (WGS) and whole-exome (WXS) high-throughput DNA sequencing data. This algorithm builds on our earlier Tumor Heterogeneity Analysis (THetA) algorithm in several important directions. These include improved ability to analyze highly rearranged genomes using a variety of data types: both WGS sequencing (including low ~7 x coverage) and WXS sequencing. We apply our improved THetA2 algorithm to WGS (including low-pass) and WXS sequence data from 18 samples from The Cancer Genome Atlas (TCGA). We find that the improved algorithm is substantially faster and identifies numerous tumor samples containing subclonal populations in the TCGA data, including in one highly rearranged sample for which other tumor purity estimation algorithms were unable to estimate tumor purity. Availability and implementation: An implementation of THetA2 is available at http://compbio.cs.brown.edu/software Contact: layla@cs.brown.edu or braphael@brown.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas . In Drosophila , we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left–right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates.

Description: Trisomy 21 (Down syndrome, DS) is the most common genetic cause of developmental cognitive deficits, and the so-called Down syndrome critical region (DSCR) has been proposed as a major determinant of this phenotype. The regions on human chromosome 21 (Hsa21) are syntenically conserved on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. DSCR is conserved between the Cbr1 and Fam3b genes on Mmu16. Ts65Dn mice carry three copies of ~100 Hsa21 gene orthologs on Mmu16 and exhibited impairments in the Morris water maze and hippocampal long-term potentiation (LTP). Converting the Cbr1 - Fam3b region back to two copies in Ts65Dn mice rescued these phenotypes. In this study, we performed similar conversion of the Cbr1 - Fam3b region in Dp(16)1Yey /+ mice that is triplicated for all ~115 Hsa21 gene orthologs on Mmu16, which also resulted in the restoration of the wild-type phenotypes in the Morris water maze and hippocampal LTP. However, converting the Cbr1-Fam3b region back to two copies in a complete model, Dp(10)1Yey/+ ; Dp(16)1Yey/+ ; Dp(17)1Yey /+, failed to yield the similar phenotypic restorations. But, surprisingly, converting both the Cbr1 - Fam3b region and the Hsa21 orthologous region on Mmu17 back to two copies in the complete model did completely restore these phenotypes to the wild-type levels. Our results demonstrated that the Hsa21 orthologous region on Mmu17 is a major determinant of DS-related developmental cognitive deficits. Therefore, the inclusion of the three copies of this Hsa21 orthologous region in mouse models is necessary for unraveling the mechanism underlying DS-associated developmental cognitive deficits and for developing effective interventions for this clinical manifestation.

Description: Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

Description: The hallmark of Alzheimer's disease (AD) pathology is an accumulation of amyloid β (Aβ) and phosphorylated tau, which are encoded by the amyloid precursor protein (APP) and microtubule-associated protein tau ( MAPT ) genes, respectively. Less than 5% of all AD cases are familial in nature, i.e. caused by mutations in APP , PSEN1 or PSEN2 . Almost all mutations found in them are related to an overproduction of Aβ 1–42 , which is prone to aggregation. While these genes are mutation free, their function, or those of related genes, could be compromised in sporadic AD as well. In this study, pyrosequencing analysis of post-mortem brains revealed aberrant CpG methylation in APP , MAPT and GSK3B genes of the AD brain. These changes were further evaluated by a newly developed in vitro -specific DNA methylation system, which in turn highlighted an enhanced expression of APP and MAPT . Cell nucleus sorting of post-mortem brains revealed that the methylation changes of APP and MAPT occurred in both neuronal and non-neuronal cells, whereas GSK3B was abnormally methylated in non-neuronal cells. Further analysis revealed an association between abnormal APP CpG methylation and apolipoprotein E 4 allele ( APOE 4)-negative cases. The presence of a small number of highly methylated neurons among normal neurons contribute to the methylation difference in APP and MAPT CpGs, thus abnormally methylated cells could compromise the neural circuit and/or serve as ‘seed cells’ for abnormal protein propagation. Our results provide a link between familial AD genes and sporadic neuropathology, thus emphasizing an epigenetic pathomechanism for sporadic AD.

Description: Acute lymphoblastic leukemia (ALL) accounts for ~25% of pediatric malignancies. Of interest, the incidence of ALL is observed ~20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP . We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ~30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1 -rearranged ALL (both overall P 〈 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected ( P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls.

Description: Utrophin is a potential therapeutic target for the fatal muscle disease, Duchenne muscular dystrophy (DMD). In adult skeletal muscle, utrophin is restricted to the neuromuscular and myotendinous junctions and can compensate for dystrophin loss in mdx mice, a mouse model of DMD, but requires sarcolemmal localization. NFATc1-mediated transcription regulates utrophin expression and the LIM protein, FHL1 which promotes muscle hypertrophy, is a transcriptional activator of NFATc1. By generating mdx /FHL1-transgenic mice, we demonstrate that FHL1 potentiates NFATc1 activation of utrophin to ameliorate the dystrophic pathology. Transgenic FHL1 expression increased sarcolemmal membrane stability, reduced muscle degeneration, decreased inflammation and conferred protection from contraction-induced injury in mdx mice. Significantly, FHL1 expression also reduced progressive muscle degeneration and fibrosis in the diaphragm of aged mdx mice. FHL1 enhanced NFATc1 activation of the utrophin promoter and increased sarcolemmal expression of utrophin in muscles of mdx mice, directing the assembly of a substitute utrophin–glycoprotein complex, and revealing a novel FHL1-NFATc1-utrophin signaling axis that can functionally compensate for dystrophin.

Description: DNA methylation and hydroxymethylation have been implicated in normal development and differentiation, but our knowledge is limited about the genome-wide distribution of 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC) during cellular differentiation. Using an in vitro model system of gradual differentiation of human embryonic stem (hES) cells into ventral midbrain-type neural precursor cells and terminally into dopamine neurons, we observed dramatic genome-wide changes in 5 mC and 5 hmC patterns during lineage commitment. The 5 hmC pattern was dynamic in promoters, exons and enhancers. DNA hydroxymethylation within the gene body was associated with gene activation. The neurogenesis-related genes NOTCH1 , RGMA and AKT1 acquired 5 hmC in the gene body and were up-regulated during differentiation. DNA methylation in the promoter was associated with gene repression. The pluripotency-related genes POU5F1 , ZFP42 and HMGA1 acquired 5 mC in their promoters and were down-regulated during differentiation. Promoter methylation also acted as a locking mechanism to maintain gene silencing. The mesoderm development-related genes NKX2-8 , TNFSF11 and NFATC1 acquired promoter methylation during neural differentiation even though they were already silenced in hES cells. Our findings will help elucidate the molecular mechanisms underlying lineage-specific differentiation of pluripotent stem cells during human embryonic development.

Description: This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1 –/– ) was found. Majority of Tie2-Cre/Tsc1 –/– embryos died at embryonic day 14.5 in utero . Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1 –/– embryos. Endothelial cells in Tsc1 -deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

Description: The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal β- N -acetylhexosaminidase system. Accumulation of β-hexosaminidases A and B substrates is presumed to cause this fatal condition. An authentic mouse model of Sandhoff disease (SD) with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described. We have shown that expression of β-hexosaminidase by intracranial delivery of recombinant adeno-associated viral vectors to young adult SD mice can prevent many features of the disease and extends lifespan. To investigate the nature of the neurological injury in GM2 gangliosidosis and the extent of its reversibility, we have examined the evolution of disease in the SD mouse; we have moreover explored the effects of gene transfer delivered at key times during the course of the illness. Here we report greatly increased survival only when the therapeutic genes are expressed either before the disease is apparent or during its early manifestations. However, irrespective of when treatment was administered, widespread and abundant expression of β-hexosaminidase with consequent clearance of glycoconjugates, α-synuclein and ubiquitinated proteins, and abrogation of inflammatory responses and neuronal loss was observed. We also show that defects in myelination occur in early life and cannot be easily resolved when treatment is given to the adult brain. These results indicate that there is a limited temporal opportunity in which function and survival can be improved—but regardless of resolution of the cardinal pathological features of GM2 gangliosidosis, a point is reached when functional deterioration and death cannot be prevented.

Description: The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimer's disease and Huntington's disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila . Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

Description: Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs), suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7—a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis—causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7, we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1, an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development, functions not previously associated with members of the NLRP family.

Description: Hexanucleotide repeat expansions within the C9orf72 gene are the most important genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The difficulty of developing a precise method to determine the expansion size has hampered the study of possible correlations between the hexanucleotide repeat number and clinical phenotype. Here we characterize, through a new non-radioactive Southern blot protocol, the expansion size range in a series of 38 ALS and 22 FTD heterozygous carriers of 〉30 copies of the repeat. Maximum, median and modal hexanucleotide repeat number were higher in ALS patients than in FTD patients ( P 〈 0.05 in all comparisons). A higher median number of repeats correlated with a bigger range of repeat sizes (Spearman's = 0.743, P = 1.05 x 10 –11 ). We did not find any correlation between age of onset or disease duration with the repeat size in neither ALS nor FTD mutation carriers. Clinical presentation (bulbar or spinal) in ALS patients did not correlate either with the repeat length. We finally analyzed two families with affected and unaffected repeat expansion carriers, compared the size of the repeat expansion between two monozygotic (MZ) twins (one affected of ALS and the other unaffected), and examined the expansion size in two different tissues (cerebellum and peripheral blood) belonging to the same FTD patient. The results suggested that the length of the C9orf72 repeat varies between family members, including MZ twins, and among different tissues from the same individual.

Description: Genome-wide association studies (GWAS) have uncovered many genetic associations for cardiovascular disease (CVD). However, data are limited regarding causal genetic variants within implicated loci. We sought to identify regulatory variants ( cis- and trans -eQTLs) affecting expression levels of 93 genes selected by their proximity to SNPs with significant associations in prior GWAS for CVD traits. Expression levels were measured by qRT–PCR in leukocytes from 1846 Framingham Heart Study participants. An additive genetic model was applied to 2.5 million imputed SNPs for each gene. Approximately 45% of genes ( N = 38) harbored at least one cis -eSNP after a regional multiple-test adjustment. Applying a more rigorous significance threshold ( P 〈 5 x 10 –8 ), we found the expression level of 10 genes was significantly associated with more than one cis -eSNP. The top cis -eSNPs for 7 of these 10 genes exhibited moderate-to-strong association with ≥1 CVD clinical phenotypes. Several eSNPs or proxy SNPs ( r 2 = 1) were replicated by other eQTL studies. After adjusting for the lead GWAS SNPs for the 10 genes, expression variances explained by top cis -eSNPs were attenuated markedly for LPL , FADS2 and C6orf184 , suggesting a shared genetic basis for the GWAS and expression trait . A significant association between cis -eSNPs, gene expression and lipid levels was discovered for LPL and C6orf184 . In conclusion, strong cis -acting variants are localized within nearly half of the GWAS loci studied, with particularly strong evidence for a regulatory role of the top GWAS SNP for expression of LPL , FADS2 and C6orf184 .

Description: The worldwide burden of tuberculosis (TB) remains an enormous problem, and is particularly severe in the admixed South African Coloured (SAC) population residing in the Western Cape. Despite evidence from twin studies suggesting a strong genetic component to TB resistance, only a few loci have been identified to date. In this work, we conduct a genome-wide association study (GWAS), meta-analysis and trans-ethnic fine mapping to attempt the replication of previously identified TB susceptibility loci. Our GWAS results confirm the WT1 chr11 susceptibility locus (rs2057178: odds ratio = 0.62, P = 2.71e –06 ) previously identified by Thye et al ., but fail to replicate previously identified polymorphisms in the TLR8 gene and locus 18q11.2. Our study demonstrates that the genetic contribution to TB risk varies between continental populations, and illustrates the value of including admixed populations in studies of TB risk and other complex phenotypes. Our evaluation of local ancestry based on the real and simulated data demonstrates that case-only admixture mapping is currently impractical in multi-way admixed populations, such as the SAC, due to spurious deviations in average local ancestry generated by current local ancestry inference methods. This study provides insights into identifying disease genes and ancestry-specific disease risk in multi-way admixed populations.

Description: Obesity is a major public health problem with strong genetic determination. Multiple genetic variants have been implicated for obesity by conducting genome-wide association (GWA) studies, primarily focused on body mass index (BMI). Fat body mass (FBM) is phenotypically more homogeneous than BMI and is more appropriate for obesity research; however, relatively few studies have been conducted on FBM. Aiming to identify variants associated with obesity, we carried out meta-analyses of seven GWA studies for BMI-related traits including FBM, and followed these analyses by de novo replication. The discovery cohorts consisted of 21 969 individuals from diverse ethnic populations and a total of over 4 million genotyped or imputed SNPs. The de novo replication cohorts consisted of 6663 subjects from two independent samples. To complement individual SNP-based association analyses, we also carried out gene-based GWA analyses in which all variations within a gene were considered jointly. Individual SNP-based association analyses identified a novel locus 1q21 [ rs2230061 , CTSS (Cathepsin S)] that was associated with FBM after the adjustment of lean body mass (LBM) ( P = 3.57 x 10 –8 ) at the genome-wide significance level. Gene-based association analyses identified a novel gene NLK (nemo-like kinase) in 17q11 that was significantly associated with FBM adjusted by LBM. In addition, we confirmed three previously reported obesity susceptibility loci: 16q12 [ rs62033400 , P = 1.97 x 10 –14 , FTO (fat mass and obesity associated)], 18q22 [ rs6567160 , P = 8.09 x 10 –19 , MC4R (melanocortin 4 receptor)] and 2p25 [ rs939583 , P = 1.07 x 10 –7 , TMEM18 (transmembrane protein 18)]. We also found that rs6567160 may exert pleiotropic effects to both FBM and LBM. Our results provide additional insights into the molecular genetic basis of obesity and may provide future targets for effective prevention and therapeutic intervention.

Description: Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 x 10 –4 ) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 x 10 –4 ) among carriers of missense variants in CHRNB3 . Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms ( P = 7.4 x 10 –5 , β = 2.04) and alcohol dependence symptoms ( P = 2.6 x 10 –4 , β = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence ( P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence.

Description: Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 ( AIPL1 ) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Description: In the large-quantity production of α2,3- and α2,6-sialyllactose (Neu5Ac(α2,3)Galβ1,4Glc (3'-SL) and Neu5Ac(α2,6)Galβ1,4Glc (6'-SL)) using sialyltransferases (STs), there are major hurdles to overcome for further improvement in yield and productivity of the enzyme reactions. Specifically, Pasteurella multocida α2,3-sialyltransferase (α2,3PST) forms a by-product to a certain extent, owing to its multifunctional activity at pH below 7.0, and Photobacterium damselae α2,6-sialyltransferase (α2,6PdST) shows relatively low ST activity. In this study, α2,3PST and α2,6PdST were successfully engineered using a hybrid approach that combines rational design with site-saturation mutagenesis. Narrowly focused on the substrate-binding pocket of the STs, putative functional residues were selected by multiple sequence alignment and alanine scanning, and subsequently subjected to site-saturation mutagenesis. In the case of α2,3PST, R313N single mutation improved its activity slightly (by a factor of 1.5), and further improvement was obtained by making the double mutants (R313N/T265S and R313H/T265S) resulting in an overall 2-fold improvement in its specific α2,3 ST activity, which is mainly caused by the increase in k cat . It was revealed that the R313 mutations to N, D, Y, H or T greatly reduced the α2,6 ST side-reaction activity of α2,3PST at below pH 7.0. In the case of α2,6PdST, single-mutation L433S/T and double-mutation I411T/L433T exhibited 3- and 5-fold enhancement of the α2,6 ST-specific activity compared with the wild-type, respectively, via increase in k cat values. Our results show a very good model system for enhancing ST activity and demonstrate that the generated mutants could be used efficiently for the mass production of 3'-SL and 6'-SL with enhanced productivity and yield.

Description: Transcriptional dysregulation has been proposed to play a major role in the pathology of Huntington's disease (HD). However, the mechanisms that cause selective downregulation of target genes remain unknown. Previous studies have shown that mutant huntingtin (Htt) protein interacts with a number of transcription factors thereby altering transcription. Here we report that Htt directly interacts with methyl-CpG binding protein 2 (MeCP2) in mouse and cellular models of HD using complimentary biochemical and Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer approaches. Htt–MeCP2 interactions are enhanced in the presence of the expanded polyglutamine (polyQ) tract and are stronger in the nucleus compared with the cytoplasm. Furthermore, we find increased binding of MeCP2 to the promoter of brain-derived neurotrophic factor (BDNF), a gene that is downregulated in HD, in the presence of mutant Htt. Finally, decreasing MeCP2 levels in mutant Htt-expressing cells using siRNA increases BDNF levels, suggesting that MeCP2 downregulates BDNF expression in HD. Taken together, these findings suggest that aberrant interactions between Htt and MeCP2 contribute to transcriptional dysregulation in HD.

Description: In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreER T2 and Sohlh1-CreER T2 inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ~3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo , we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan.

Description: Genome-wide association studies (GWASs) have shown that approximately 60 genetic variants influence the risk of developing multiple sclerosis (MS). Our aim was to identify the cell types in which these variants are active. We used available data on MS-associated single nucleotide polymorphisms (SNPs) and deoxyribonuclease I hypersensitive sites (DHSs) from 112 different cell types. Genomic intervals were tested for overlap using the Genomic Hyperbrowser. The expression profile of the genes located nearby MS-associated SNPs was assessed using the software GRAIL (Gene Relationships Across Implicated Loci). Genomic regions associated with MS were significantly enriched for a number of immune DHSs and in particular T helper (Th) 1, Th17, CD8+ cytotoxic T cells, CD19+ B cells and CD56+ natural killer (NK) cells (enrichment = 2.34, 2.19, 2.27, 2.05 and 1.95, respectively; P 〈 0.0001 for all of them). Similar results were obtained when genomic regions with suggestive association with MS and additional immune-mediated traits were investigated. Several new candidate MS-associated genes located within regions of suggestive association were identified by GRAIL ( CARD11, FCRL2, CHST12, SYK, TCF7, SOCS1, NFKBIZ and NPAS1 ). Genetic data indicate that Th1, Th17, cytotoxic T, B and NK cells play a prominent role in the etiology of MS. Regions with confirmed and suggestive association have a similar immunological profile, indicating that many SNPs truly influencing the risk of MS actually fail to reach genome-wide significance. Finally, similar cell types are involved in the etiology of other immune-mediated diseases.

Description: Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.

Description: Friedreich's ataxia (FRDA), the most common hereditary ataxia, is characterized by progressive degeneration of the central and peripheral nervous system, hypertrophic cardiomyopathy and a high risk of diabetes. FRDA is caused by abnormally low levels of frataxin, a highly conserved mitochondrial protein. Drosophila has been previously successfully used to model FRDA in various cell types, including neurons and glial cells. Here, we report the development of a Drosophila cardiac model of FRDA. In vivo heart imaging revealed profound impairments in heart function in frataxin-depleted Drosophila, including a strong increase in end-systolic and end-diastolic diameters and a decrease in fractional shortening (FS). These features, reminiscent of pathological phenotypes in humans, are fully rescued by complementation with human frataxin, suggesting conserved cardiac functions of frataxin between the two organisms. Oxidative stress is not a major factor of heart impairment in frataxin-depleted flies, suggesting the involvement of other pathological mechanisms notably mitochondrial respiratory chain (MRC) dysfunction. Accordingly, we report that methylene blue (MB), a compound known to act as an alternative electron carrier that bypasses mitochondrial complexes I-III, was able to prevent heart dysfunction. MB also partially rescued the phenotype when administered post-symptomatically. Analysis of MB derivatives demonstrates that only compounds with electron carrier properties are able to prevent the heart phenotype. Thus MB, a compound already used for several clinical applications, appears promising for the treatment of the heart dysfunctions that are a major cause of death of FRDA patients. This work provides the grounds for further evaluation of MB action in mammals.

Description: Using a Drosophila model of Alzheimer's disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci ( P 〈 10 –4 ) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr , the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9 , as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.

Description: Spinal muscular atrophy (SMA) is characterized by the selective loss of spinal motor neurons owing to reduced levels of survival motor neuron (Smn) protein. In addition to its well-established role in assembling constituents of the spliceosome, diverse cellular functions have been proposed for Smn, but the reason why low levels of this widely expressed protein result in selective motor neuron pathology is still debated. In longitudinal studies of exon-level changes in SMA mouse model tissues, designed to determine the contribution of splicing dysfunction to the disease, we have previously shown that a generalized defect in splicing is unlikely to play a causative role in SMA. Nevertheless, we identified a small subset of genes that were alternatively spliced in the spinal cord compared with control mice before symptom onset, indicating a possible mechanistic role in disease. Here, we have performed functional studies of one of these genes, chondrolectin ( Chodl ), known to be highly expressed in motor neurons and important for correct motor axon outgrowth in zebrafish. Using in vitro and in vivo models of SMA, we demonstrate altered expression of Chodl in SMA mouse spinal motor neurons, show that Chodl has distinct effects on cell survival and neurite outgrowth and that increasing the expression of chodl can rescue motor neuron outgrowth defects in Smn-depleted zebrafish. Our findings thus link the dysregulation of Chodl to the pathophysiology of motor neuron degeneration in SMA.

Description: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a severe decline of memory performance. A widely studied AD mouse model is the APPswe/PSEN1E9 (APP/PS1) strain, as mice exhibit amyloid plaques as well as impaired memory capacities. To test whether restoring synaptic plasticity and decreasing β-amyloid load by Parkin could represent a potential therapeutic target for AD, we crossed APP/PS1 transgenic mice with transgenic mice overexpressing the ubiquitin ligase Parkin and analyzed offspring properties. Overexpression of Parkin in APP/PS1 transgenic mice restored activity-dependent synaptic plasticity and rescued behavioral abnormalities. Moreover, overexpression of Parkin was associated with down-regulation of APP protein expression, decreased β-amyloid load and reduced inflammation. Our data suggest that Parkin could be a promising target for AD therapy.

Description: Gene amplification is a common phenomenon in malignant neoplasms of all types. One mechanism behind increased gene copy number is the formation of ring chromosomes. Such structures are mitotically unstable and during tumor progression they accumulate material from many different parts of the genome. Hence, their content varies considerably between and within tumors. Partly due to this extensive variation, the genetic content of many ring-containing tumors remains poorly characterized. Ring chromosomes are particularly prevalent in specific subtypes of sarcoma. Here, we have combined fluorescence in situ hybridization (FISH), global genomic copy number and gene expression data on ring-containing soft tissue sarcomas and show that they harbor two fundamentally different types of ring chromosome: MDM2 -positive and MDM2 -negative rings. While the former are often found in an otherwise normal chromosome complement, the latter seem to arise in the context of general chromosomal instability. In line with this, sarcomas with MDM2 -negative rings commonly show complete loss of either CDKN2A or RB1 —both known to be important for genome integrity. Sarcomas with MDM2 -positive rings instead show co-amplification of a variety of potential driver oncogenes. More than 100 different genes were found to be involved, many of which are known to induce cell growth, promote proliferation or inhibit apoptosis. Several of the amplified and overexpressed genes constitute potential drug targets.

Description: Rett syndrome (RTT) is one of the most prevalent female mental disorders. De novo mutations in methyl CpG-binding protein 2 (MeCP2) are a major cause of RTT. MeCP2 regulates gene expression as a transcription regulator as well as through long-range chromatin interaction. Because MeCP2 is present on the X chromosome, RTT is manifested in an X-linked dominant manner. Investigation using murine MeCP2 null models and post-mortem human brain tissues has contributed to understanding the molecular and physiological function of MeCP2. In addition, RTT models using human induced pluripotent stem cells derived from RTT patients (RTT-iPSCs) provide novel resources to elucidate the regulatory mechanism of MeCP2. Previously, we obtained clones of female RTT-iPSCs that express either wild-type or mutant MECP2 due to the inactivation of one X chromosome. Reactivation of the X chromosome also allowed us to have RTT-iPSCs that express both wild-type and mutant MECP2 . Using these unique pluripotent stem cells, we investigated the regulation of gene expression by MeCP2 in pluripotent stem cells by transcriptome analysis. We found that MeCP2 regulates genes encoding mitochondrial membrane proteins. In addition, loss of function in MeCP2 results in de-repression of genes on the inactive X chromosome. Furthermore, we showed that each mutation in MECP2 affects a partly different set of genes. These studies suggest that fundamental cellular physiology is affected by mutations in MECP2 from early development, and that a therapeutic approach targeting to unique forms of mutant MeCP2 is needed.

Description: Motivation: We have recently characterized an instance of alternative splicing that differs from the canonical gene transcript by deletion of a length of sequence not divisible by three, but where translation can be rescued by an alternative start codon. This results in a predicted protein in which the amino terminus differs markedly in sequence from the known protein product(s), as it is translated from an alternative reading frame. Automated pipelines have annotated thousands of splice variants but have overlooked these protein isoforms, leading to them being underrepresented in current databases. Results: Here we describe 1849 human and 733 mouse transcripts that can be transcribed from an alternate ATG. Of these, 〉80% have not been annotated previously. Those conserved between human and mouse genomes (and hence under likely evolutionary selection) are identified. We provide mass spectroscopy evidence for translation of selected transcripts. Of the described splice variants, only one has previously been studied in detail and converted the encoded protein from an activator of cell-function to a suppressor, demonstrating that these splice variants can result in profound functional change. We investigate the potential functional effects of this splicing using a variety of bioinformatic tools. The 2582 variants we describe are involved in a wide variety of biological processes, and therefore open many new avenues of research. Contact: aude.fahrer@anu.edu.au Supplementary Inforation: Supplementary data are available at Bioinformatics online.

Description: Motivation : High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results : We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent–daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. Availability : The R package absfilter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter Contact : sebastian.waszak@epfl.ch or bart.deplancke@epfl.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation : Recently, investigators have proposed state-of-the-art Identity-by-descent (IBD) mapping methods to detect IBD segments between purportedly unrelated individuals. The IBD information can then be used for association testing in genetic association studies. One approach for this IBD association testing strategy is to test for excessive IBD between pairs of cases (‘pairwise method’). However, this approach is inefficient because it requires a large number of permutations. Moreover, a limited number of permutations define a lower bound for P -values, which makes fine-mapping of associated regions difficult because, in practice, a much larger genomic region is implicated than the region that is actually associated. Results: In this article, we introduce a new pairwise method ‘Fast-Pairwise’. Fast-Pairwise uses importance sampling to improve efficiency and enable approximation of extremely small P -values. Fast-Pairwise method takes only days to complete a genome-wide scan. In the application to the WTCCC type 1 diabetes data, Fast-Pairwise successfully fine-maps a known human leukocyte antigen gene that is known to cause the disease. Availability: Fast-Pairwise is publicly available at: http://genetics.cs.ucla.edu/graphibd . Contact: eeskin@cs.ucla.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Measurements are commonly taken from two phenotypes to build a classifier, where the number of data points from each class is predetermined, not random. In this ‘separate sampling’ scenario, the data cannot be used to estimate the class prior probabilities. Moreover, predetermined class sizes can severely degrade classifier performance, even for large samples. Results: We employ simulations using both synthetic and real data to show the detrimental effect of separate sampling on a variety of classification rules. We establish propositions related to the effect on the expected classifier error owing to a sampling ratio different from the population class ratio. From these we derive a sample-based minimax sampling ratio and provide an algorithm for approximating it from the data. We also extend to arbitrary distributions the classical population-based Anderson linear discriminant analysis minimax sampling ratio derived from the discriminant form of the Bayes classifier. Availability: All the codes for synthetic data and real data examples are written in MATLAB. A function called mmratio, whose output is an approximation of the minimax sampling ratio of a given dataset, is also written in MATLAB. All the codes are available at: http://gsp.tamu.edu/Publications/supplementary/shahrokh13b . Contact: edward@ece.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation:  Expression vectors used in different biotechnology applications are designed with domain-specific rules. For instance, promoters, origins of replication or homologous recombination sites are host-specific. Similarly, chromosomal integration or viral delivery of an expression cassette imposes specific structural constraints. As de novo gene synthesis and synthetic biology methods permeate many biotechnology specialties, the design of application-specific expression vectors becomes the new norm. In this context, it is desirable to formalize vector design strategies applicable in different domains. Results:  Using the design of constructs to express genes in the chloroplast of Chlamydomonas reinhardtii as an example, we show that a vector design strategy can be formalized as a domain-specific language. We have developed a graphical editor of context-free grammars usable by biologists without prior exposure to language theory. This environment makes it possible for biologists to iteratively improve their design strategies throughout the course of a project. It is also possible to ensure that vectors designed with early iterations of the language are consistent with the latest iteration of the language. Availability and implementation:  The context-free grammar editor is part of the GenoCAD application. A public instance of GenoCAD is available at http://www.genocad.org . GenoCAD source code is available from SourceForge and licensed under the Apache v2.0 open source license. Contact:   peccoud@vt.edu Supplementary Information:   Supplementary data are available at Bioinformatics online.

Description: Motivation: Homology search methods are dominated by the central paradigm that sequence similarity is a proxy for common ancestry and, by extension, functional similarity. For determining sequence similarity in proteins, most widely used methods use models of sequence evolution and compare amino-acid strings in search for conserved linear stretches. Probabilistic models or sequence profiles capture the position-specific variation in an alignment of homologous sequences and can identify conserved motifs or domains. While profile-based search methods are generally more accurate than simple sequence comparison methods, they tend to be computationally more demanding. In recent years, several methods have emerged that perform protein similarity searches based on domain composition. However, few methods have considered the linear arrangements of domains when conducting similarity searches, despite strong evidence that domain order can harbour considerable functional and evolutionary signal. Results: Here, we introduce an alignment scheme that uses a classical dynamic programming approach to the global alignment of domains. We illustrate that representing proteins as strings of domains (domain arrangements) and comparing these strings globally allows for a both fast and sensitive homology search. Further, we demonstrate that the presented methods complement existing methods by finding similar proteins missed by popular amino-acid–based comparison methods. Availability: An implementation of the presented algorithms, a web-based interface as well as a command-line program for batch searching against the UniProt database can be found at http://rads.uni-muenster.de . Furthermore, we provide a JAVA API for programmatic access to domain-string–based search methods. Contact: terrapon.nicolas@gmail.com or ebb@uni-muenster.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: DNA enrichment followed by sequencing is a versatile tool in molecular biology, with a wide variety of applications including genome-wide analysis of epigenetic marks and mechanisms. A common requirement of these diverse applications is a comparison of read coverage between experimental conditions. The amount of samples generated for such comparisons ranges from few replicates to hundreds of samples per condition for epigenome-wide association studies. Consequently, there is an urgent need for software that allows for fast and simple processing and comparison of sequencing data derived from enriched DNA. Results: Here, we present a major update of the R/Bioconductor package MEDIPS, which allows for an arbitrary number of replicates per group and integrates sophisticated statistical methods for the detection of differential coverage between experimental conditions. Our approach can be applied to a diversity of quantitative sequencing data. In addition, our update adds novel functionality to MEDIPS, including correlation analysis between samples, and takes advantage of Bioconductor’s annotation databases to facilitate annotation of specific genomic regions. Availability and implementation: The latest version of MEDIPS is available as version 1.12.0 and part of Bioconductor 2.13. The package comes with a manual containing detailed description of its functionality and is available at http://www.bioconductor.org . Contact: lienhard@molgen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation:  Most methods for estimating differential expression from RNA-seq are based on statistics that compare normalized read counts between treatment classes. Unfortunately, reads are in general too short to be mapped unambiguously to features of interest, such as genes, isoforms or haplotype-specific isoforms. There are methods for estimating expression levels that account for this source of ambiguity. However, the uncertainty is not generally accounted for in downstream analysis of gene expression experiments. Moreover, at the individual transcript level, it can sometimes be too large to allow useful comparisons between treatment groups. Results:  In this article we make two proposals that improve the power, specificity and versatility of expression analysis using RNA-seq data. First, we present a Bayesian method for model selection that accounts for read mapping ambiguities using random effects. This polytomous model selection approach can be used to identify many interesting patterns of gene expression and is not confined to detecting differential expression between two groups. For illustration, we use our method to detect imprinting, different types of regulatory divergence in cis and in trans and differential isoform usage, but many other applications are possible. Second, we present a novel collapsing algorithm for grouping transcripts into inferential units that exploits the posterior correlation between transcript expression levels. The aggregate expression levels of these units can be estimated with useful levels of uncertainty. Our algorithm can improve the precision of expression estimates when uncertainty is large with only a small reduction in biological resolution. Availability and implementation:  We have implemented our software in the mmdiff and mmcollapse multithreaded C++ programs as part of the open-source MMSEQ package, available on https://github.com/eturro/mmseq . Contact:   et341@cam.ac.uk Supplementary information:   Supplementary data are available at Bioinformatics online.

Description: Motivation:  Nucleotide sequence data are being produced at an ever increasing rate. Clustering such sequences by similarity is often an essential first step in their analysis—intended to reduce redundancy, define gene families or suggest taxonomic units. Exact clustering algorithms, such as hierarchical clustering, scale relatively poorly in terms of run time and memory usage, yet they are desirable because heuristic shortcuts taken during clustering might have unintended consequences in later analysis steps. Results:  Here we present HPC-CLUST, a highly optimized software pipeline that can cluster large numbers of pre-aligned DNA sequences by running on distributed computing hardware. It allocates both memory and computing resources efficiently, and can process more than a million sequences in a few hours on a small cluster. Availability and implementation:  Source code and binaries are freely available at http://meringlab.org/software/hpc-clust/ ; the pipeline is implemented in C++ and uses the Message Passing Interface (MPI) standard for distributed computing. Contact:  mering@imls.uzh.ch Supplementary Information:  Supplementary data are available at Bioinformatics online.

Description: : High-throughput technologies have led to an explosion of genomic data available for automated analysis. The consequent possibility to simultaneously sample multiple layers of variation along the gene expression flow requires computational methods integrating raw information from different ‘-omics’. It has been recently demonstrated that translational control is a widespread phenomenon, with profound and still underestimated regulation capabilities. Although detecting changes in the levels of total messenger RNAs (mRNAs; the transcriptome), of polysomally loaded mRNAs (the translatome) and of proteins (the proteome) is experimentally feasible in a high-throughput way, the integration of these levels is still far from being robustly approached. Here we introduce tRanslatome, a new R/Bioconductor package, which is a complete platform for the simultaneous pairwise analysis of transcriptome, translatome and proteome data. The package includes most of the available statistical methods developed for the analysis of high-throughput data, allowing the parallel comparison of differentially expressed genes and the corresponding differentially enriched biological themes. Notably, it also enables the prediction of translational regulatory elements on mRNA sequences. The utility of this tool is demonstrated with two case studies. Availability and implementation: tRanslatome is available in Bioconductor. Contact : t.tebaldi@unitn.it Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : DoMosaics is an application that unifies protein domain annotation, domain arrangement analysis and visualization in a single tool. It simplifies the analysis of protein families by consolidating disjunct procedures based on often inconvenient command-line applications and complex analysis tools. It provides a simple user interface with access to domain annotation services such as InterProScan or a local HMMER installation, and can be used to compare, analyze and visualize the evolution of domain architectures. Availability and implementation: DoMosaics is licensed under the Apache License, Version 2.0, and binaries can be freely obtained from www.domosaics.net . Contact: radmoore@uni-muenster.de or e.bornberg@uni-muenster.de

Description: Motivation: A common problem in understanding a biochemical system is to infer its correct structure or topology. This topology consists of all relevant state variables—usually molecules and their interactions. Here we present a method called topological augmentation to infer this structure in a statistically rigorous and systematic way from prior knowledge and experimental data. Results: Topological augmentation starts from a simple model that is unable to explain the experimental data and augments its topology by adding new terms that capture the experimental behavior. This process is guided by representing the uncertainty in the model topology through stochastic differential equations whose trajectories contain information about missing model parts. We first apply this semiautomatic procedure to a pharmacokinetic model. This example illustrates that a global sampling of the parameter space is critical for inferring a correct model structure. We also use our method to improve our understanding of glutamine transport in yeast. This analysis shows that transport dynamics is determined by glutamine permeases with two different kinds of kinetics. Topological augmentation can not only be applied to biochemical systems, but also to any system that can be described by ordinary differential equations. Availability and implementation: Matlab code and examples are available at: http://www.csb.ethz.ch/tools/index . Contact: mikael.sunnaker@bsse.ethz.ch ; andreas.wagner@ieu.uzh.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Assembling and/or producing integrated knowledge of sequence features continues to be an onerous and redundant task despite a large number of existing resources. We have developed SeqDepot—a novel database that focuses solely on two primary goals: (i) assimilating known primary sequences with predicted feature data and (ii) providing the most simple and straightforward means to procure and readily use this information. Access to 〉28.5 million sequences and 300 million features is provided through a well-documented and flexible RESTful interface that supports fetching specific data subsets, bulk queries, visualization and searching by MD5 digests or external database identifiers. We have also developed an HTML5/JavaScript web application exemplifying how to interact with SeqDepot and Perl/Python scripts for use with local processing pipelines. Availability: Freely available on the web at http://seqdepot.net/ . REST access via http://seqdepot.net/api/v1 . Database files and scripts may be downloaded from http://seqdepot.net/download . Contact: ulrich.luke+sci@gmail.com

Description: Motivation: Microarray data analysis is often applied to characterize disease populations by identifying individual genes linked to the disease. In recent years, efforts have shifted to focus on sets of genes known to perform related biological functions (i.e. in the same pathways). Evaluating gene sets reduces the need to correct for false positives in multiple hypothesis testing. However, pathways are often large, and genes in the same pathway that do not contribute to the disease can cause a method to miss the pathway. In addition, large pathways may not give much insight to the cause of the disease. Moreover, when such a method is applied independently to two datasets of the same disease phenotypes, the two resulting lists of significant pathways often have low agreement. Results: We present a powerful method, PFSNet, that identifies smaller parts of pathways (which we call subnetworks), and show that significant subnetworks (and the genes therein) discovered by PFSNet are up to 51% (64%) more consistent across independent datasets of the same disease phenotypes, even for datasets based on different platforms, than previously published methods. We further show that those methods which initially declared some large pathways to be insignificant would declare subnetworks detected by PFSNet in those large pathways to be significant, if they were given those subnetworks as input instead of the entire large pathways. Availability: http://compbio.ddns.comp.nus.edu.sg:8080/pfsnet/ Contact: kevinl@comp.nus.edu.sg Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: :  Pathway Commons is a resource permitting simultaneous queries of multiple pathway databases. However, there is no standard mechanism for using these data (stored in BioPAX format) to annotate and build quantitative mathematical models. Therefore, we developed a new module within the virtual cell modeling and simulation software. It provides pathway data retrieval and visualization and enables automatic creation of executable network models directly from qualitative connections between pathway nodes. Availability and implementation:  Available at Virtual Cell ( http://vcell.org/ ). Application runs on all major platforms and does not require registration for use on the user’s computer. Tutorials and video are available at user guide page. Contact:   vcell_support@uchc.edu

Description: : myChEMBL is a completely open platform, which combines public domain bioactivity data with open source database and cheminformatics technologies. myChEMBL consists of a Linux (Ubuntu) Virtual Machine featuring a PostgreSQL schema with the latest version of the ChEMBL database, as well as the latest RDKit cheminformatics libraries. In addition, a self-contained web interface is available, which can be modified and improved according to user specifications. Availability and implementation: The VM is available at: ftp://ftp.ebi.ac.uk/pub/databases/chembl/VM/myChEMBL/current . The web interface and web services code is available at: https://github.com/rochoa85/myChEMBL . Contact: jpo@ebi.ac.uk

Description: Motivation: The identification of cell cycle-regulated genes through the cyclicity of messenger RNAs in genome-wide studies is a difficult task due to the presence of internal and external noise in microarray data. Moreover, the analysis is also complicated by the loss of synchrony occurring in cell cycle experiments, which often results in additional background noise. Results: To overcome these problems, here we propose the LEON (LEarning and OptimizatioN) algorithm, able to characterize the ‘cyclicity degree’ of a gene expression time profile using a two-step cascade procedure. The first step identifies a potentially cyclic behavior by means of a Support Vector Machine trained with a reliable set of positive and negative examples. The second step selects those genes having peak timing consistency along two cell cycles by means of a non-linear optimization technique using radial basis functions. To prove the effectiveness of our combined approach, we use recently published human fibroblasts cell cycle data and, performing in vivo experiments, we demonstrate that our computational strategy is able not only to confirm well-known cell cycle-regulated genes, but also to predict not yet identified ones. Availability and implementation: All scripts for implementation can be obtained on request. Contact: lorenzo.farina@uniroma1.it or gurtner@ifo.it Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: RNA-seq technology has been widely adopted as an attractive alternative to microarray-based methods to study global gene expression. However, robust statistical tools to analyze these complex datasets are still lacking. By grouping genes with similar expression profiles across treatments, cluster analysis provides insight into gene functions and networks, and hence is an important technique for RNA-seq data analysis. Results: In this manuscript, we derive clustering algorithms based on appropriate probability models for RNA-seq data. An expectation-maximization algorithm and another two stochastic versions of expectation-maximization algorithms are described. In addition, a strategy for initialization based on likelihood is proposed to improve the clustering algorithms. Moreover, we present a model-based hybrid-hierarchical clustering method to generate a tree structure that allows visualization of relationships among clusters as well as flexibility of choosing the number of clusters. Results from both simulation studies and analysis of a maize RNA-seq dataset show that our proposed methods provide better clustering results than alternative methods such as the K-means algorithm and hierarchical clustering methods that are not based on probability models. Availability and implementation: An R package, MBCluster.Seq, has been developed to implement our proposed algorithms. This R package provides fast computation and is publicly available at http://www.r-project.org . Contact: sy@swufe.edu.cn ; pliu@iastate.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Motivation:  Modern biomedical and epidemiological studies often measure hundreds or thousands of biomarkers, such as gene expression or metabolite levels. Although there is an extensive statistical literature on adjusting for ‘multiple comparisons’ when testing whether these biomarkers are directly associated with a disease, testing whether they are biological mediators between a known risk factor and a disease requires a more complex null hypothesis, thus offering additional methodological challenges. Results:  We propose a permutation approach that tests multiple putative mediators and controls the family wise error rate. We demonstrate that, unlike when testing direct associations, replacing the Bonferroni correction with a permutation approach that focuses on the maximum of the test statistics can significantly improve the power to detect mediators even when all biomarkers are independent. Through simulations, we show the power of our method is 2–5 x larger than the power achieved by Bonferroni correction. Finally, we apply our permutation test to a case-control study of dietary risk factors and colorectal adenoma to show that, of 149 test metabolites, docosahexaenoate is a possible mediator between fish consumption and decreased colorectal adenoma risk. Availability and implementation:  R-package included in online Supplementary Material. Contact:   joshua.sampson@nih.gov Supplementary information:   Supplementary materials are available at Bioinformatics online.

Description: After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N -glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N -glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.

Description: Neisseria meningitidis serogroups A, B, C, Y, W135 and X are responsible for most cases of meningococcal meningitis. Neisseria meningitidis serogroup X has recently emerged as a contributor to outbreaks of disease in Africa, but there is currently no vaccine against serogroup X. Understanding of the biosynthesis of the serogroup X capsular polysaccharide would provide useful tools for vaccine production. The serogroup X polysaccharide is a homopolymer of (α1-〉4)-linked N -acetylglucosamine (GlcNAc)-1-phosphate. It has been shown that the gene cluster xcb ABC encodes synthesis of this polysaccharide. The xcbA gene product has significant homology with sac B, which is responsible for synthesis of the Neisseria serogroup A capsular polysaccharide, an (α1-〉6)- N -acetylmannosamine-1-phosphate homopolymer. The xcbA protein also shares homology with the catalytic domain of human N -acetylglucosamine-1-phosphoryltransferase, a key enzyme in the mannose-6-phosphate receptor pathway. In this study, we show that xcbA in the appropriate background is sufficient for the synthesis of N. meningitidis serogroup X polysaccharide. By ELISA we detected polysaccharide in fractions of Escherichia coli expressing the xcbA gene. We isolated polysaccharide from an E. coli strain expressing XcbA and demonstrated that this polysaccharide has a 13 C-NMR spectrum identical to that of polysaccharide isolated from N. meningitidis Group X. We also demonstrate that the purified XcbA protein is an N -acetylglucosamine-1-phosphotransferase that transfers N -acetylglucosamine-1-phosphate from UDP-GlcNAc to the 4-hydroxyl of an N -acetylglucosamine-1-phosphate oligosaccharide. Oligosaccharides fluorescently labeled at the aglycon are extended by XcbA only after the 4-phosphate occupying the non-reducing GlcNAc has been removed. The minimum size of fluorescent acceptors is a trisaccharide.

Description: The invasion of host cells by the intracellular protozoan Trypanosoma cruzi requires interactions with host cell molecules, and the replication of the parasite requires escape from a parasitophorous vacuole into the host cell cytosol. Galectin-3, a member of β-galactosidase-binding lectin family, has numerous extracellular and intracellular functions. In this study, we investigated the role of galectin-3 during the invasion and intracellular trafficking of T. cruzi extracellular amastigotes (EAs). Endogenous galectin-3 from mouse peritoneal macrophages accumulated around the pathogen during cell invasion by EAs. In addition, galectin-3 accumulated around parasites after their escape from the parasitophorous vacuole. Thus, galectin-3 behaved as a novel marker of phagolysosome lysis during the infection of host cells by T. cruzi .

Description: Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery–Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

Description: RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo .

Description: Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.

Description: PRESENILIN1 ( PSEN1 ) is the major locus for mutations causing familial Alzheimer's disease (FAD) and is also mutated in Pick disease of brain, familial acne inversa and dilated cardiomyopathy. It is a critical facilitator of Notch signalling and many other signalling pathways and protein cleavage events including production of the Amyloidβ (Aβ) peptide from the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). We previously reported that interference with splicing of transcripts of the zebrafish orthologue of PSEN1 creates dominant negative effects on Notch signalling. Here, we extend this work to show that various truncations of human PSEN1 (or zebrafish Psen1) protein have starkly differential effects on Notch signalling and cleavage of zebrafish Appa (a paralogue of human APP). Different truncations can suppress or stimulate Notch signalling but not Appa cleavage and vice versa. The G183V mutation possibly causing Pick disease causes production of aberrant transcripts truncating the open reading frame after exon 5 sequence. We show that the truncated protein potentially translated from these transcripts avidly incorporates into very stable Psen1-dependent higher molecular weight complexes and suppresses cleavage of Appa but not Notch signalling. In contrast, the truncated protein potentially produced by the P242LfsX11 acne inversa mutation has no effect on Appa cleavage but, unexpectedly, enhances Notch signalling. Our results suggest novel hypotheses for the pathological mechanisms underlying these diseases and illustrate the importance of investigating the function of dominant mutations at physiologically relevant expression levels and in the normally heterozygous state in which they cause human disease rather than in isolation from healthy alleles.

Description: Parkinson's disease (PD) has a number of known genetic risk factors. Clinical and epidemiological studies have suggested the existence of intermediate factors that may be associated with additional risk of PD. We construct genetic risk profiles for additional epidemiological and clinical factors using known genome-wide association studies (GWAS) loci related to these specific phenotypes to estimate genetic comorbidity in a systematic review. We identify genetic risk profiles based on GWAS variants associated with schizophrenia and Crohn's disease as significantly associated with risk of PD. Conditional analyses adjusting for SNPs near loci associated with PD and schizophrenia or PD and Crohn's disease suggest that spatially overlapping loci associated with schizophrenia and PD account for most of the shared comorbidity, while variation outside of known proximal loci shared by PD and Crohn's disease accounts for their shared genetic comorbidity. We examine brain methylation and expression signatures proximal to schizophrenia and Crohn's disease loci to infer functional changes in the brain associated with the variants contributing to genetic comorbidity. We compare our results with a systematic review of epidemiological literature, while the findings are dissimilar to a degree; marginal genetic associations corroborate the directionality of associations across genetic and epidemiological data. We show a strong genetically defined level of comorbidity between PD and Crohn's disease as well as between PD and schizophrenia, with likely functional consequences of associated variants occurring in brain.

Description: Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.

Description: Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is an important event in aggregate formation. The A30P mutation is known for its fast oligomerization, but slow fibrillization rate. Despite its toxicity to neurons, mechanisms involved in either clearance or conversion of A30P alpha-synuclein from its soluble state into insoluble fibrils and their effects in vivo are poorly understood. Synphilin-1 is present in Lewy bodies, interacting with alpha-synuclein in vivo and in vitro and promotes its sequestration into aggresomes, which are thought to act as cytoprotective agents facilitating protein degradation. We therefore crossed animals overexpressing A30P alpha-synuclein with synphilin-1 transgenic mice to analyze its impact on aggregation, protein clearance and phenotype progression. We observed that co-expression of synphilin-1 mildly delayed the motor phenotype caused by A30P alpha-synuclein. Additionally, the presence of N- and C-terminal truncated alpha-synuclein species and fibrils were strongly reduced in double-transgenic mice when compared with single-transgenic A30P mice. Insolubility of mutant A30P and formation of aggresomes was still detectable in aged double-transgenic mice, paralleled by an increase of ubiquitinated proteins and high autophagic activity. Hence, this study supports the notion that co-expression of synphilin-1 promotes formation of autophagic-susceptible aggresomes and consecutively the degradation of human A30P alpha-synuclein. Notably, although synphilin-1 overexpression significantly reduced formation of fibrils and astrogliosis in aged animals, a similar phenotype is present in single- and double-transgenic mice suggesting additional neurotoxic processes in disease progression.

Description: Motivation: For samples of unrelated individuals, we propose a general analysis framework in which hundred thousands of genetic loci can be tested simultaneously for association with complex phenotypes. The approach is built on spatial-clustering methodology, assuming that genetic loci that are associated with the target phenotype cluster in certain genomic regions. In contrast to standard methodology for multilocus analysis, which has focused on the dimension reduction of the data, our multilocus association-clustering test profits from the availability of large numbers of genetic loci by detecting clusters of loci that are associated with the phenotype. Results: The approach is computationally fast and powerful, enabling the simultaneous association testing of large genomic regions. Even the entire genome or certain chromosomes can be tested simultaneously. Using simulation studies, the properties of the approach are evaluated. In an application to a genome-wide association study for chronic obstructive pulmonary disease, we illustrate the practical relevance of the proposed method by simultaneously testing all genotyped loci of the genome-wide association study and by testing each chromosome individually. Our findings suggest that statistical methodology that incorporates spatial-clustering information will be especially useful in whole-genome sequencing studies in which millions or billions of base pairs are recorded and grouped by genomic regions or genes, and are tested jointly for association. Availability and implementation: Implementation of the approach is available upon request. Contact : daq412@mail.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Neisseria meningitidis ( Nm ) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is Nm X for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of Nm X called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the Nm X capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with Nm X-specific antibodies. Moreover, the chemical identity of in vitro produced Nm X polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the Nm X CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.