ClinSV: clinical grade structural and copy number variant detection from whole genome sequencing data.

Whole genome sequencing (WGS) has the potential to outperform clinical microarrays for the detection of structural variants (SV) including copy number variants (CNVs), but has been challenged by high false positive rates. Here we present ClinSV, a WGS based SV integration, annotation, prioritization, and visualization framework, which identified 99.8% of simulated pathogenic ClinVar CNVs > 10 kb and 11/11 pathogenic variants from matched microarrays. The false positive rate was low (1.5-4.5%) and reproducibility high (95-99%). In clinical practice, ClinSV identified reportable variants in 22 of 485 patients (4.7%) of which 35-63% were not detectable by current clinical microarray designs. ClinSV is available at https://github.com/KCCG/ClinSV .

Genomic diagnostics in polycystic kidney disease: an assessment of real-world use of whole-genome sequencing.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is common, with a prevalence of 1/1000 and predominantly caused by disease-causing variants in PKD1 or PKD2. Clinical diagnosis is usually by age-dependent imaging criteria, which is challenging in patients with atypical clinical features, without family history, or younger age. However, there is increasing need for definitive diagnosis of ADPKD with new treatments available. Sequencing is complicated by six pseudogenes that share 97% homology to PKD1 and by recently identified phenocopy genes. Whole-genome sequencing can definitively diagnose ADPKD, but requires validation for clinical use. We initially performed a validation study, in which 42 ADPKD patients underwent sequencing of PKD1 and PKD2 by both whole-genome and Sanger sequencing, using a blinded, cross-over method. Whole-genome sequencing identified all PKD1 and PKD2 germline pathogenic variants in the validation study (sensitivity and specificity 100%). Two mosaic variants outside pipeline thresholds were not detected. We then examined the first 144 samples referred to a clinically-accredited diagnostic laboratory for clinical whole-genome sequencing, with targeted-analysis to a polycystic kidney disease gene-panel. In this unselected, diagnostic cohort (71 males :73 females), the diagnostic rate was 70%, including a diagnostic rate of 81% in patients with typical ADPKD (98% with PKD1/PKD2 variants) and 60% in those with atypical features (56% PKD1/PKD2; 44% PKHD1/HNF1B/GANAB/ DNAJB11/PRKCSH/TSC2). Most patients with atypical disease did not have clinical features that predicted likelihood of a genetic diagnosis. These results suggest clinicians should consider diagnostic genomics as part of their assessment in polycystic kidney disease, particularly in atypical disease.

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation.

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

Adar3 Is Involved in Learning and Memory in Mice.

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

The RNA modification landscape in human disease.

RNA modifications have been historically considered as fine-tuning chemo-structural features of infrastructural RNAs, such as rRNAs, tRNAs, and snoRNAs. This view has changed dramatically in recent years, to a large extent as a result of systematic efforts to map and quantify various RNA modifications in a transcriptome-wide manner, revealing that RNA modifications are reversible, dynamically regulated, far more widespread than originally thought, and involved in major biological processes, including cell differentiation, sex determination, and stress responses. Here we summarize the state of knowledge and provide a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. With the advent of direct RNA-sequencing technologies, we expect that this catalog will help prioritize those RNA modifications for transcriptome-wide maps.

Analysis of steric effects in DamID profiling of transcription factor target genes.

DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion.

Seq and You Will Find.

The human genome sequence is freely available, nearly complete and is providing a foundation of research opportunities that are overturning our current understanding of human biology. The advent of next generation sequencing has revolutionized the way we can interrogate the genome and its transcriptional products and how we analyze, diagnose, monitor and even treat human disease. Personal genetic profiles are increasing dramatically in medical value as researchers accumulate more and more knowledge about the interaction between genetic and environmental factors that contribute to the onset of common disorders. As the cost of sequencing plummets, whole genome sequencing of individuals is becoming a reality and the field of personalized genomic medicine is rapidly developing. Now there is great need for accurate annotation of all functionally important sequences in the human genome and the variations within them that contribute to health and disease. The vast majority of our genome gives rise to RNA transcripts. This extraordinarily versatile molecule not only encodes protein information but also has great structural dynamics and plasticity, capacity for DNA/RNA/protein interactions and catalytic activity. It is a key regulator of biological networks with clear links to human disease and a more comprehensive understanding of its function is needed to maximise its use in medical practice. This review focuses on the complexity of our genome and the impact of sequencing technologies in understanding its many products and functions in health and disease.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets.

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Long noncoding RNAs in cardiac development and pathophysiology.

Heart function requires sophisticated regulatory networks to orchestrate organ development, physiological responses, and environmental adaptation. Until recently, it was thought that these regulatory networks are composed solely of protein-mediated transcriptional control and signaling systems; consequently, it was thought that cardiac disease involves perturbation of these systems. However, it is becoming evident that RNA, long considered to function primarily as the platform for protein production, may in fact play a major role in most, if not all, aspects of gene regulation, especially the epigenetic processes that underpin organogenesis. These include not only well-validated classes of regulatory RNAs, such as microRNAs, but also tens of thousands of long noncoding RNAs that are differentially expressed across the entire genome of humans and other animals. Here, we review this emerging landscape, summarizing what is known about their functions and their role in cardiac biology, and provide a toolkit to assist in exploring this previously hidden layer of gene regulation that may underpin heart adaptation and complex heart diseases.

Decoding the non-coding RNAs in Alzheimer's disease.

Non-coding RNAs (ncRNAs) are integral components of biological networks with fundamental roles in regulating gene expression. They can integrate sequence information from the DNA code, epigenetic regulation and functions of multimeric protein complexes to potentially determine the epigenetic status and transcriptional network in any given cell. Humans potentially contain more ncRNAs than any other species, especially in the brain, where they may well play a significant role in human development and cognitive ability. This review discusses their emerging role in Alzheimer's disease (AD), a human pathological condition characterized by the progressive impairment of cognitive functions. We discuss the complexity of the ncRNA world and how this is reflected in the regulation of the amyloid precursor protein and Tau, two proteins with central functions in AD. By understanding this intricate regulatory network, there is hope for a better understanding of disease mechanisms and ultimately developing diagnostic and therapeutic tools.

Target gene repression mediated by miRNAs miR-181c and miR-9 both of which are down-regulated by amyloid-ß.

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that are essential for normal brain development and function. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer's disease (AD) that is characterized by amyloid-ß (Aß) and tau deposition in brain. How deregulated miRNAs contribute to AD is not understood, as their dysfunction could be both a cause and a consequence of disease. To address this question we had previously profiled miRNAs in models of AD. This identified miR-9 and -181c as being down-regulated by Aß in hippocampal cultures. Interestingly, there was a remarkable overlap with those miRNAs that are deregulated in Aß-depositing APP23 transgenic mice and in human AD tissue. While the Aß precursor protein APP itself is a target of miRNA regulation, the challenge resides in identifying further targets. Here, we expand the repertoire of miRNA target genes by identifying the 3' untranslated regions (3' UTRs) of TGFBI, TRIM2, SIRT1 and BTBD3 as being repressed by miR-9 and -181c, either alone or in combination. Taken together, our study identifies putative target genes of miRNAs miR-9 and 181c, which may function in brain homeostasis and disease pathogenesis.

MicroRNA networks surrounding APP and amyloid-ß metabolism--implications for Alzheimer's disease.

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that function as "fine-tuning" tools of gene expression in development and tissue homeostasis. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer's disease (AD) that is characterized by both amyloid-ß (Aß) and tau deposition in brain. A key challenge remains in determining how changes in miRNA profiles translate into biological function in a physiological and pathological context. The key lies in identifying specific target genes for deregulated miRNAs and understanding which pathogenic factors trigger their deregulation. Here we review the literature about the intricate network of miRNAs surrounding the regulation of the amyloid precursor protein (APP) from which Aß is derived by proteolytic cleavage. Normal brain function is highly sensitive to any changes in APP metabolism and miRNAs function at several steps to ensure that the correct APP end product is produced and in the right form and abundance. Disruptions in this miRNA regulatory network may therefore alter Aß production, which in turn can affect miRNA expression.

Neuronal microRNA deregulation in response to Alzheimer's disease amyloid-beta.

Normal brain development and function depends on microRNA (miRNA) networks to fine tune the balance between the transcriptome and proteome of the cell. These small non-coding RNA regulators are highly enriched in brain where they play key roles in neuronal development, plasticity and disease. In neurodegenerative disorders such as Alzheimer's disease (AD), brain miRNA profiles are altered; thus miRNA dysfunction could be both a cause and a consequence of disease. Our study dissects the complexity of human AD pathology, and addresses the hypothesis that amyloid-beta (Abeta) itself, a known causative factor of AD, causes neuronal miRNA deregulation, which could contribute to the pathomechanisms of AD. We used sensitive TaqMan low density miRNA arrays (TLDA) on murine primary hippocampal cultures to show that about half of all miRNAs tested were down-regulated in response to Abeta peptides. Time-course assays of neuronal Abeta treatments show that Abeta is in fact a powerful regulator of miRNA levels as the response of certain mature miRNAs is extremely rapid. Bioinformatic analysis predicts that the deregulated miRNAs are likely to affect target genes present in prominent neuronal pathways known to be disrupted in AD. Remarkably, we also found that the miRNA deregulation in hippocampal cultures was paralleled in vivo by a deregulation in the hippocampus of Abeta42-depositing APP23 mice, at the onset of Abeta plaque formation. In addition, the miRNA deregulation in hippocampal cultures and APP23 hippocampus overlaps with those obtained in human AD studies. Taken together, our findings suggest that neuronal miRNA deregulation in response to an insult by Abeta may be an important factor contributing to the cascade of events leading to AD.

Arabidopsis MSI1 is required for negative regulation of the response to drought stress.

Arabidopsis MSI1 has fundamental functions in plant development. MSI1 is a subunit of Polycomb group protein complexes and Chromatin assembly factor 1, and it interacts with the Retinoblastoma-related protein 1. Altered levels of MSI1 result in pleiotropic phenotypes, reflecting the complexity of MSI1 protein functions. In order to uncover additional functions of MSI1, we performed transcriptional profiling of wild-type and plants with highly reduced MSI1 levels (msi1-cs). Surprisingly, the known functions of MSI1 could only account for a minor part of the transcriptional changes in msi1-cs plants. One of the most striking unexpected observations was the up-regulation of a subset of ABA-responsive genes eliciting the response to drought and salt stress. We report that MSI1 can bind to the chromatin of the drought-inducible downstream target RD20 and suggest a new role for MSI1 in the negative regulation of the Arabidopsis drought-stress response.

Alzheimer's disease selective vulnerability and modeling in transgenic mice.

Neurodegenerative diseases are characterized by 'hot spots' of degeneration. The regions of primary vulnerability vary between different neurodegenerative diseases. Within these regions, some neurons are lost whereas others that are morphologically indiscriminate survive. The enigma of this selective vulnerability is tightly linked to two fundamental problems in the neurosciences. First, it is not understood how many neuronal cell types make up the mammalian brain; estimates are in the order of more than a thousand. Second, the mechanisms by which some nerve cells undergo functional impairment followed by degeneration while others do not, remain elusive. Understanding the basis for this selective vulnerability has significant implications for understanding the pathogenesis of disease and for developing treatments. Here, we review what is known about selective vulnerability in Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. We suggest, since transgenic animal models of disease reproduce aspects of selective vulnerability, that these models offer a valuable system for future investigations into the physiological basis of selective vulnerability.

Is tau aggregation toxic or protective: a sensible question in the absence of sensitive methods?

In Alzheimer's disease brain, the microtubule-associated protein tau detaches from the microtubules, pathologically interacts with cellular proteins, and eventually forms insoluble aggregates that also bind and trap a myriad of proteins. As these proteins are depleted from the cellular pool, they are unavailable for physiological functions. Thus elevated tau levels are pathogenic, even in the absence of tau aggregation. Whereas it is reasonable to assume that tau aggregation is toxic during late stages of disease, the question arises whether early in disease it may be protective. This question can be addressed in tau transgenic animal models in which tau aggregation has been correlated with behavioral impairment. We discuss ways of how tau aggregation is monitored in these mice and what the detection limits are of these methods. We conclude that new tools are needed to measure the different stages of tau aggregation.

Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response.

BACKGROUND: Alzheimer's disease (AD) is characterized by beta-amyloid (Abeta) peptide-containing plaques and tau-containing neurofibrillary tangles. By intracerebral injection of Abeta(42), both pathologies have been combined in P301L tau mutant mice. Furthermore, in cell culture, Abeta(42) induces tau aggregation. While both Abeta(42) and mutant tau cause neuronal dysfunction, their modes of action are only vaguely understood. METHODS: To determine which processes are disrupted by Abeta(42) and/or P301L mutant tau, we used transcriptomic and proteomic techniques followed by functional validation and analysis of human AD tissue. RESULTS: Our transcriptomic study in the SH-SY5Y cell culture system revealed that Abeta(42) and P301L tau expression independently affect genes controlling the cell cycle and cell proliferation. Proteomics applied to Abeta(42)-treated P301L tau-expressing SH-SY5Y cells and the amygdala of Abeta(42)-injected P301L transgenic mice revealed that a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. Among the proteins identified was valosin-containing protein (VCP), a component of the quality control system during endoplasmic reticulum stress. Mutations in VCP have recently been linked to frontotemporal dementia. CONCLUSION: Our data support the mitosis failure hypothesis that claims that aberrant cell cycle reentry of postmitotic neurons induces apoptosis. Furthermore, our data underline a role of Abeta(42) in the stress response associated with protein folding.

An update on the toxicity of Abeta in Alzheimer's disease.

Alzheimer's disease is characterized histopathologically by deposition of insoluble forms of the peptide Abeta and the protein tau in brain. Abeta is the principal component of amyloid plaques and tau of neurofibrillary tangles. Familial cases of AD are associated with causal mutations in the gene encoding the amyloid precursor protein, APP, from which the amyloidogenic Abeta peptide is derived, and this supports a role for Abeta in disease. Abeta can promote tau pathology and at the same time its toxicity is also tau-dependent. Abeta can adopt different conformations including soluble oligomers and insoluble fibrillar species present in plaques. We discuss which of these conformations exert toxicity, highlight molecular pathways involved and discuss what has been learned by applying functional genomics.

A decade of tau transgenic animal models and beyond.

The first tau transgenic mouse model was established more than a decade ago. Since then, much has been learned about the role of tau in Alzheimer's disease and related disorders. Animal models, both in vertebrates and invertebrates, were significantly improved and refined as a result of the identification of pathogenic mutations in Tau in human cases of frontotemporal dementia. They have been instrumental for dissecting the cross-talk between tau and the second hallmark lesion of Alzheimer's disease, the Abeta peptide-containing amyloid plaque. We discuss how the tau models have been used to unravel the pathophysiology of Alzheimer's disease, to search for disease modifiers and to develop novel treatment strategies. While tau has received less attention than Abeta, it is rapidly acquiring a more prominent position and the emerging view is one of a synergistic action of Abeta and tau in Alzheimer's disease. Moreover, the existence of a number of neurodegenerative diseases with tau pathology in the absence of extracellular deposits underscores the relevance of research on tau.