scholarly journals Correction of copy number induced false positives in CRISPR screens

2017 ◽  
Author(s):  
Antoine de Weck ◽  
Javad Golji ◽  
Mike Jones ◽  
Joshua Korn ◽  
Eric Billy ◽  
...  
Keyword(s):  
False Positive ◽  
Copy Number ◽  
Additive Model ◽  
Drop Out ◽  
Systematic Effect ◽  
Loss Of Function ◽  
Dense Region ◽  
Cancer Models ◽  
General Additive Model ◽  
Genomic Regions

AbstractCell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70-80% decrease of false positive hits in regions of high copy number with either method.

scholarly journals False positive cell free DNA screening for microdeletions due to non-pathogenic copy number variants

2016 ◽  
Vol 36 (6) ◽  
pp. 584-586 ◽  
Author(s):  
Cheryl A. Mather ◽  
Zhongxia Qi ◽  
Arun P. Wiita
Keyword(s):  
False Positive ◽  
Copy Number ◽  
Copy Number Variants ◽  
Cell Free Dna ◽  
Free Dna

scholarly journals Gene discovery and functional assessment of rare copy-number variants in neurodevelopmental disorders

2014 ◽  
Author(s):  
Janani Iyer ◽  
Santhosh Girirajan
Keyword(s):  
Neurodevelopmental Disorders ◽  
Copy Number ◽  
Molecular Genetic ◽  
Copy Number Variants ◽  
Fruit Fly ◽  
Functional Evaluation ◽  
Rare Cnvs ◽  
Molecular Genetic Basis ◽  
Causal Genes ◽  
Genomic Regions

Rare copy-number variants (CNVs) are a significant cause of neurodevelopmental disorders. The sequence architecture of the human genome predisposes certain individuals to deletions and duplications within specific genomic regions. While assessment of individuals with different breakpoints has identified causal genes for certain rare CNVs, deriving gene-phenotype correlations for rare CNVs with similar breakpoints has been challenging. We present a comprehensive review of the literature related to genetic architecture that is predisposed to recurrent rearrangements, and functional evaluation of deletions, duplications, and candidate genes within rare CNV intervals using mouse, zebrafish, and fruit fly models. It is clear that phenotypic assessment and complete genetic evaluation of large cohorts of individuals carrying specific CNVs and functional evaluation using multiple animal models are necessary to understand the molecular genetic basis of neurodevelopmental disorders.

scholarly journals Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

2021 ◽  
Author(s):  
Sean Bergin ◽  
Fang Zhao ◽  
Adam P Ryan ◽  
Carolin A Müller ◽  
Conrad A Nieduszynski ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Copy Number ◽  
Candida Parapsilosis ◽  
Copy Number Variations ◽  
Loss Of Function ◽  
Adaptive Laboratory Evolution ◽  
Pathogenic Yeast ◽  
Lipid Asymmetry ◽  
Evolution Experiment ◽  
Almost All

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.

scholarly journals CNV-P: a machine-learning framework for predicting high confident copy number variations

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12564
Author(s):  
Taifu Wang ◽  
Jinghua Sun ◽  
Xiuqing Zhang ◽  
Wen-Jing Wang ◽  
Qing Zhou
Keyword(s):  
Machine Learning ◽  
False Positive ◽  
Copy Number ◽  
Genetic Disorders ◽  
Genetic Diseases ◽  
Basic Research ◽  
Read Depth ◽  
Copy Number Variations ◽  
Sequencing Data ◽  
Learning Framework

Background Copy-number variants (CNVs) have been recognized as one of the major causes of genetic disorders. Reliable detection of CNVs from genome sequencing data has been a strong demand for disease research. However, current software for detecting CNVs has high false-positive rates, which needs further improvement. Methods Here, we proposed a novel and post-processing approach for CNVs prediction (CNV-P), a machine-learning framework that could efficiently remove false-positive fragments from results of CNVs detecting tools. A series of CNVs signals such as read depth (RD), split reads (SR) and read pair (RP) around the putative CNV fragments were defined as features to train a classifier. Results The prediction results on several real biological datasets showed that our models could accurately classify the CNVs at over 90% precision rate and 85% recall rate, which greatly improves the performance of state-of-the-art algorithms. Furthermore, our results indicate that CNV-P is robust to different sizes of CNVs and the platforms of sequencing. Conclusions Our framework for classifying high-confident CNVs could improve both basic research and clinical diagnosis of genetic diseases.

Abstract 2723: Large Genomic Deletions in Plakophilin-2 are a Rare Cause of ARVD/C and ARVD/C-like Disease.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Jasper J van der Smagt ◽  
Moniek G Cox ◽  
Marcel R Nelen ◽  
J P van Tintelen ◽  
Marc M Entius ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Copy Number ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Task Force ◽  
Direct Sequencing ◽  
Null Alleles ◽  
Loss Of Function ◽  
Genomic Deletions ◽  
Large Deletions ◽  
Plakophilin 2

Introduction: Plakophilin-2 (PKP2) is the major genetic determinant of ARVD/C in the Netherlands. Mutations have been found in 43 out of 82 index cases (52%), diagnosed in accordance with the 1994 Task Force criteria (TFC). A total of 15 different mutations were detected, most of them nonsense (7), frameshift (2) and splice mutations (3), suggesting haploinsufficiency (loss of function) of PKP2 as the predominant mechanism in the etiology of ARVD/C. Since many of these mutations are probably functional null alleles, it was hypothesized that large deletions within - or encompassing - the PKP2 gene can also predispose to ARVD/C. Aim: to determine whether large deletions in PKP2 underlie apparently PKP2 negative ARVD/C cases. Methods: a multiple ligation-dependent probe amplification kit was developed to determine copy number of all 14 exons of the PKP2 gene. In total 120 patients were included that had been referred for PKP2 testing, and that had tested negative with direct sequencing or denaturing gradient gel electrophoresis. Twenty-six patients had definite ARVD/C (TFC: ≥ 4 pts), 11 patients scored 3 pts and 83 patients scored less than 3 pts or had insufficient clinical data. Results: a single deletion was detected encompassing the first 4 exons and promoter region of the PKP2 gene. The patient, a 35 year old male, had experienced near-syncope during exercise. Mild dilation of the right ventricle (1 pt), negative T-waves in V1-V5 (1 pt) and short VT’s with LBBB morphology during exercise testing were found (1 pt). There were frequent PVC’s. The father also carried the deletion. A third degree paternal relative died suddenly at age 28 during exercise. Conclusion: large genomic deletions of PKP2, that go unnoticed with sequencing, can cause ARVD/C-like disease. Comprehensive testing of PKP2 should include assessment of copy number. However, the frequency of large deletions seems to be low. Since the detected deletion includes the promoter and the transcription initiation site, no protein can be derived from the affected allele. This patient demonstrates that complete loss of one PKP2 allele does not necessarily lead to more severe ARVD/C. This is in keeping with the concept of haploinsufficiency and the assumption that many PKP2 mutations cause functional null alleles.

Basic principles of genetic disease

ESC CardioMed ◽  
2018 ◽  
pp. 669-671
Author(s):  
Eric Schulze-Bahr
Keyword(s):  
Genetic Disease ◽  
Copy Number ◽  
Copy Number Variants ◽  
Single Nucleotide Variants ◽  
Individual Genome ◽  
Base Pairs ◽  
Single Nucleotide ◽  
Basic Principles ◽  
Bona Fide ◽  
Genomic Regions

The human genome consists of approximately 3 billion (3 × 109) base pairs of DNA (around 20,000 genes), organized as 23 chromosomes (diploid parental set), and a small mitochondrial genome (37 genes, including 13 proteins; 16,589 base pairs) of maternal origin. Most human genetic variation is natural, that is, common or rare (minor allele frequency >0.1%) and does not cause disease—apart from every true disease-causing (bona fide) mutation each individual genome harbours more than 3.5 million single nucleotide variants (including >10,000 non-synonymous changes causing amino acid substitutions) and 200–300 large structural or copy number variants (insertions/deletions, up to several thousands of base-pairs) that are non-disease-causing variations and scattered throughout coding and non-coding genomic regions.

scholarly journals Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting

2005 ◽  
Vol 25 (8) ◽  
pp. 3076-3086 ◽  
Author(s):  
Flávio S. J. de Souza ◽  
Andrea M. Santangelo ◽  
Viviana Bumaschny ◽  
María Elena Avale ◽  
James L. Smart ◽  
...  
Keyword(s):  
Energy Homeostasis ◽  
Significant Quantitative Trait Locus ◽  
Phylogenetic Footprinting ◽  
Transcriptional Unit ◽  
Human Populations ◽  
Chromosome 2 ◽  
Loss Of Function ◽  
Trait Locus ◽  
Genomic Regions ◽  
Arcuate Neurons

ABSTRACT The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity.

scholarly journals Silencing of Euchromatic Transposable Elements as a Consequence of Nuclear Lamina Dysfunction

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 625
Author(s):  
Valeria Cavaliere ◽  
Giovanna Lattanzi ◽  
Davide Andrenacci
Keyword(s):  
Transposable Elements ◽  
Genome Instability ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Rna Degradation ◽  
Loss Of Function ◽  
Heterochromatin Formation ◽  
Repressive Effect ◽  
Regulatory Effects ◽  
Genomic Regions

Transposable elements (TEs) are mobile genomic sequences that are normally repressed to avoid proliferation and genome instability. Gene silencing mechanisms repress TEs by RNA degradation or heterochromatin formation. Heterochromatin maintenance is therefore important to keep TEs silent. Loss of heterochromatic domains has been linked to lamin mutations, which have also been associated with derepression of TEs. In fact, lamins are structural components of the nuclear lamina (NL), which is considered a pivotal structure in the maintenance of heterochromatin domains at the nuclear periphery in a silent state. Here, we show that a lethal phenotype associated with Lamin loss-of-function mutations is influenced by Drosophila gypsy retrotransposons located in euchromatic regions, suggesting that NL dysfunction has also effects on active TEs located in euchromatic loci. In fact, expression analysis of different long terminal repeat (LTR) retrotransposons and of one non-LTR retrotransposon located near active genes shows that Lamin inactivation determines the silencing of euchromatic TEs. Furthermore, we show that the silencing effect on euchromatic TEs spreads to the neighboring genomic regions, with a repressive effect on nearby genes. We propose that NL dysfunction may have opposed regulatory effects on TEs that depend on their localization in active or repressed regions of the genome.

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