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Contains fulltext : 251996.pdf (Publisher’s version ) (Open Access), We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Contains fulltext : 251996.pdf (Publisher’s version ) (Open Access), We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Contains fulltext : 208539.pdf (publisher's version ) (Closed access), Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.

RNA binding proteins are key players in posttranscriptional regulation and have been implicated in neurodevelopmental and neuropsychiatric disorders. Here, we report a significant burden of heterozygous, likely genedisrupting variants in CSDE1 (encoding a highly constrained RNA binding protein) among patients with autism and related neurodevelopmental disabilities. Analysis of 17 patients identifies common phenotypes including autism, intellectual disability, language and motor delay, seizures, macrocephaly, and variable ocular abnormalities. HITSCLIP revealed that Csde1binding targets are enriched in autismassociated gene sets, especially FMRP targets, and in neuronal development and synaptic plasticity–related pathways. Csde1 knockdown in primary mouse cortical neurons leads to an overgrowth of the neurites and abnormal dendritic spine morphology/synapse formation and impaired synaptic transmission, whereas mutant and knockdown experiments in Drosophila result in defects in synapse growth and synaptic transmission. Our study defines a new autismrelated syndrome and highlights the functional role of CSDE1 in synapse development and synaptic transmission.

Contains fulltext : 208539.pdf (publisher's version ) (Closed access), Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.

Contains fulltext : 193505.pdf (publisher's version ) (Closed access)

Contains fulltext : 193505.pdf (publisher's version ) (Closed access)

Contains fulltext : 193505.pdf (publisher's version ) (Closed access)

Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genomewide association study (GWAS) in 142,487 individuals from the UK Biobank to identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.

Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest ‘‘initiating’’ events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution., Computer Science, Electrical Engineering, Mathematics and Computer Science

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species., Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models., Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species., Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models., Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest ‘‘initiating’’ events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution., Computer Science, Electrical Engineering, Mathematics and Computer Science

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4-2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage-gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4-2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4-2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4-2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4-2 variants were as effective as wild-type NEDD4-2 in downregulating the VGSC subtype Na v1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4-2 with as yet unidentified accessory or target proteins. © 2007 Blackwell Publishing Ltd.

Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll- AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4-2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage-gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4-2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4-2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4-2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4-2 variants were as effective as wild-type NEDD4-2 in downregulating the VGSC subtype Na v1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4-2 with as yet unidentified accessory or target proteins. © 2007 Blackwell Publishing Ltd.

Scientific publication, Contains fulltext : 91522.pdf (publisher's version ) (Closed access)

Item does not contain fulltext

Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package., Mediamatics, Electrical Engineering, Mathematics and Computer Science

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data., Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.

Scientific publication, Contains fulltext : 91522.pdf (publisher's version ) (Closed access)

Contains fulltext : 248066.pdf (Publisher’s version ) (Closed access)

Scientific publication, Contains fulltext : 91522.pdf (publisher's version ) (Closed access)

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data., Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.

Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package., Mediamatics, Electrical Engineering, Mathematics and Computer Science

A combined electrochemical and biological system is described and the results presented of bench-scale tests on Se/nitrate waters and pilot-scale tests on As/nitrate waters carried out in an electro-biochemical reactor operated at about 23 degrees C. Site-indigenous microbes were employed and the nutrients consisted of a balanced mixture of molasses, yeast extract and phosphate. Higher nutrient levels and a microbial culture medium were used to establish the biofilms to the required density, and lower nutrient levels once stabilised operating conditions were reached. The bacteria in the system interact with the electrode through direct contact. The applied voltage reduces the system's oxidation-reduction potential in a controlled manner, eliminating the need for excess nutrients and reducing nutrient costs. The products of the process are elemental Se and elemental/sulphide As. The nitrates are converted to nitrogen gas, or ammonia which is readily utilised by the microbes. The system produces a more robust biofilm and increases contaminant transformation kinetics and removal efficiency. Contaminants are removed to low ppb levels., A combined electrochemical and biological system is described and the results presented of bench-scale tests on Se/nitrate waters and pilot-scale tests on As/nitrate waters carried out in an electro-biochemical reactor operated at about 23 degrees C. Site-indigenous microbes were employed and the nutrients consisted of a balanced mixture of molasses, yeast extract and phosphate. Higher nutrient levels and a microbial culture medium were used to establish the biofilms to the required density, and lower nutrient levels once stabilised operating conditions were reached. The bacteria in the system interact with the electrode through direct contact. The applied voltage reduces the system's oxidation-reduction potential in a controlled manner, eliminating the need for excess nutrients and reducing nutrient costs. The products of the process are elemental Se and elemental/sulphide As. The nitrates are converted to nitrogen gas, or ammonia which is readily utilised by the microbes. The system produces a more robust biofilm and increases contaminant transformation kinetics and removal efficiency. Contaminants are removed to low ppb levels.

Background: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer. Results: We show that although Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines. Conclusions: Paired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions., Mediamatics, Electrical Engineering, Mathematics and Computer Science

Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly cooccurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly cooccurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes., Mediamatics, Electrical Engineering, Mathematics and Computer Science

Background: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer. Results: We show that although Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines. Conclusions: Paired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions., Mediamatics, Electrical Engineering, Mathematics and Computer Science

Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly cooccurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly cooccurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes., Mediamatics, Electrical Engineering, Mathematics and Computer Science

A monitoring programme to determine the in situ behaviour of backfills and rockmass has been carried out for more than 3 years at six underground South African gold mines. Four types of backfill, classified tailings, dewatered full plant tailings, comminuted waste and cemented tailings are used. Two types of instrumentation have been developed to determine the in situ behaviour of backfills: hydraulic stress meters, and mechanical closure meters. The confined compression stress-strain behaviour of the uncemented fill was establshed; the comminuted waste is the stiffest type. The reduction of convergence during rock bursts in filled stopes is considerable compared with conventionally supported stopes. The closure rates are significantly lower in filled stopes than in unfilled stopes., A monitoring programme to determine the in situ behaviour of backfills and rockmass has been carried out for more than 3 years at six underground South African gold mines. Four types of backfill, classified tailings, dewatered full plant tailings, comminuted waste and cemented tailings are used. Two types of instrumentation have been developed to determine the in situ behaviour of backfills: hydraulic stress meters, and mechanical closure meters. The confined compression stress-strain behaviour of the uncemented fill was establshed; the comminuted waste is the stiffest type. The reduction of convergence during rock bursts in filled stopes is considerable compared with conventionally supported stopes. The closure rates are significantly lower in filled stopes than in unfilled stopes.

Reef stabilising pillars are commonly used as regional support on deep level South African gold mines where longwall mining layouts are adopted. A mine layout which uses concrete pillars in place of reef pillars has been considered from a rock mechanics, a cost and an implementation point of view. Energy release rates and average pillar stresses were used to assess the effectiveness of the mining and support layouts. The scheme is feasible but changes to the mining system will be necessary. The replacment of reef pillars with concrete will result in greater profits for the mines. These profits could be used to construct additional concrete pillars giving significant safety benefits., Reef stabilising pillars are commonly used as regional support on deep level South African gold mines where longwall mining layouts are adopted. A mine layout which uses concrete pillars in place of reef pillars has been considered from a rock mechanics, a cost and an implementation point of view. Energy release rates and average pillar stresses were used to assess the effectiveness of the mining and support layouts. The scheme is feasible but changes to the mining system will be necessary. The replacment of reef pillars with concrete will result in greater profits for the mines. These profits could be used to construct additional concrete pillars giving significant safety benefits.

Biological cyanide oxidation is a proven, economical technology for destroying cyanide in process and waste waters and spent heaps. Full-scale implementation usually gives treatment costs of less than US, Biological cyanide oxidation is a proven, economical technology for destroying cyanide in process and waste waters and spent heaps. Full-scale implementation usually gives treatment costs of less than US

Cu- and U-bearing ores and S deposits can produce selenate and/or selenite in waste waters at concentrations ranging from 3 micrograms/l to more than 12 mg/l. A site-specific test has been used to determine the applicability of a biological reduction process capable of reducing SeO4 and SeO3 to elemental Se, producing effluents with less than 20 micrograms/l of dissolved Se at a cost of no more than US, Cu- and U-bearing ores and S deposits can produce selenate and/or selenite in waste waters at concentrations ranging from 3 micrograms/l to more than 12 mg/l. A site-specific test has been used to determine the applicability of a biological reduction process capable of reducing SeO4 and SeO3 to elemental Se, producing effluents with less than 20 micrograms/l of dissolved Se at a cost of no more than US

Commonly-proposed methods for removing selenium and arsenic from waste waters involve precipitation with iron and alumina compounds. Other methods involve arsenic adsorption on activated carbon and selenium and arsenic adsorption on organic materials or clay, membrane filtration, ion exchange and precipitation as sulphides. The major water quality factors affecting removal by these methods are pH and Eh of feedwaters, contaminant and sulphate concentrations, and alkalinity. Oxidised metals such as selenium and arsenic are used as electron acceptors by microorganisms and can be fixed. The success of bioremediation for a particular site depends on the successful characterisation and optimisation of conditions with regard to co-contaminants, pH, microbial populations, redox potential, salinity, and carbon and nitrogen. Economical biotreatment systems can be effective for both high and low concentrations, with treatment costs of US, Commonly-proposed methods for removing selenium and arsenic from waste waters involve precipitation with iron and alumina compounds. Other methods involve arsenic adsorption on activated carbon and selenium and arsenic adsorption on organic materials or clay, membrane filtration, ion exchange and precipitation as sulphides. The major water quality factors affecting removal by these methods are pH and Eh of feedwaters, contaminant and sulphate concentrations, and alkalinity. Oxidised metals such as selenium and arsenic are used as electron acceptors by microorganisms and can be fixed. The success of bioremediation for a particular site depends on the successful characterisation and optimisation of conditions with regard to co-contaminants, pH, microbial populations, redox potential, salinity, and carbon and nitrogen. Economical biotreatment systems can be effective for both high and low concentrations, with treatment costs of US

Microbial oxidation of sulphide minerals under acidic conditions can produce over 1 000 times more acid rock drainage than chemical reaction alone. A specific mining site is being studied to characterise better the microbial populations involved in pyrite oxidation and their potential contribution to rock-pile weathering and to changes in stability/instability arising from the dissolution or deposition of various mineral species. Information on microbial populations in rock piles, soils and surface crust materials will be correlated with various geophysical and geochemical parameters. The focus of the study is on the influence of microbial populations on rock pile weathering processes at the surface and within piles, measurement of the changes in the microbial populations at the surface and within rock piles over time, and an examination of the relationship between the microbiology and geochemistry at particular locations to determine their combined effects on weathering processes and stability of the rock piles., Microbial oxidation of sulphide minerals under acidic conditions can produce over 1 000 times more acid rock drainage than chemical reaction alone. A specific mining site is being studied to characterise better the microbial populations involved in pyrite oxidation and their potential contribution to rock-pile weathering and to changes in stability/instability arising from the dissolution or deposition of various mineral species. Information on microbial populations in rock piles, soils and surface crust materials will be correlated with various geophysical and geochemical parameters. The focus of the study is on the influence of microbial populations on rock pile weathering processes at the surface and within piles, measurement of the changes in the microbial populations at the surface and within rock piles over time, and an examination of the relationship between the microbiology and geochemistry at particular locations to determine their combined effects on weathering processes and stability of the rock piles.

The weathering of coal mine waste rock releases Fe, Ca, sulphate and associated trace elements such as Se. Elevated concentrations of Se are common in coal-mining environments in British Columbia, and a major concern is the potential for its bioaccumulation in aquatic food chains. Conventional membrane and ion exchange treatments produce concentrated brine streams which are difficult to treat or require disposal. Conventional biotreatment systems use excess nutrients to provide the required electrons to compensate for inefficient and variable electron availability, as well as to adjust reactor chemistry. EBR technology provides a controllable and consistent supply of useable electrons to the microbes directly at a low voltage potential of 1-3 V and low current, reduces the required nutrient addition and does not produce excess solids or biomass. Laboratory EBR bench-scale and on-site pilot-scale systems were used to treat five coal-mine waste waters. Influent Se concentrations were in the range 35-531 microg/l. Se treatment targets ranged from 5 to 10 microg/l, and mean Se concentrations in the EBR effluents were 0.5-1.4 microg/l, with sulphate concentrations also being reduced. On-site tests with average influent water temperatures of 14 to a low of 1 degrees C showed the effectiveness of EBR at low temperatures., The weathering of coal mine waste rock releases Fe, Ca, sulphate and associated trace elements such as Se. Elevated concentrations of Se are common in coal-mining environments in British Columbia, and a major concern is the potential for its bioaccumulation in aquatic food chains. Conventional membrane and ion exchange treatments produce concentrated brine streams which are difficult to treat or require disposal. Conventional biotreatment systems use excess nutrients to provide the required electrons to compensate for inefficient and variable electron availability, as well as to adjust reactor chemistry. EBR technology provides a controllable and consistent supply of useable electrons to the microbes directly at a low voltage potential of 1-3 V and low current, reduces the required nutrient addition and does not produce excess solids or biomass. Laboratory EBR bench-scale and on-site pilot-scale systems were used to treat five coal-mine waste waters. Influent Se concentrations were in the range 35-531 microg/l. Se treatment targets ranged from 5 to 10 microg/l, and mean Se concentrations in the EBR effluents were 0.5-1.4 microg/l, with sulphate concentrations also being reduced. On-site tests with average influent water temperatures of 14 to a low of 1 degrees C showed the effectiveness of EBR at low temperatures.

The results of a rock-engineering monitoring programme carried out at West Driefontein gold mine, where dewatered and classified tailings have been used as backfills for local and regional support, are described. The in situ behaviour of these backfills and the surrounding rock mass were determined, and it was found that the backfill used on this mine provided significant regional support. The underground observations also showed less rockburst damage in backfilled panels than in unfilled panels, provided that the backfill had been placed properly in the stopes., The results of a rock-engineering monitoring programme carried out at West Driefontein gold mine, where dewatered and classified tailings have been used as backfills for local and regional support, are described. The in situ behaviour of these backfills and the surrounding rock mass were determined, and it was found that the backfill used on this mine provided significant regional support. The underground observations also showed less rockburst damage in backfilled panels than in unfilled panels, provided that the backfill had been placed properly in the stopes.

In situ measurements and numerical modelling based on elastic theory showed that backfill stresses are considerably higher than originally thought. This has led to a change in understanding of rockmass behaviour. Previous work on the behaviour of the rockmass is described, possible reasons for the high stresses measured in backfill are discussed, and the rockmass mechanisms possibly generating these stresses are identified. It is concluded that the joints and the geological stratigraphy can lead to increased elastic convergence and that the inelastic behaviour of the rockmass is also partly responsible for the increased closure measured in backfill. The use of an in situ modulus is suggested as an interim solution until a new computer model that allows for joints, different layers and inelastic closures becomes available., In situ measurements and numerical modelling based on elastic theory showed that backfill stresses are considerably higher than originally thought. This has led to a change in understanding of rockmass behaviour. Previous work on the behaviour of the rockmass is described, possible reasons for the high stresses measured in backfill are discussed, and the rockmass mechanisms possibly generating these stresses are identified. It is concluded that the joints and the geological stratigraphy can lead to increased elastic convergence and that the inelastic behaviour of the rockmass is also partly responsible for the increased closure measured in backfill. The use of an in situ modulus is suggested as an interim solution until a new computer model that allows for joints, different layers and inelastic closures becomes available.