scholarly journals Determining the impact of putative loss-of-function variants in protein-coding genes

2017 ◽  
Author(s):  
Suganthi Balasubramanian ◽  
Yao Fu ◽  
Mayur Pawashe ◽  
Patrick McGillivray ◽  
Mike Jin ◽  
...  
Keyword(s):  
De Novo ◽  
Mendelian Disease ◽  
Loss Of Function ◽  
Homozygous State ◽  
Driver Genes ◽  
Protein Coding ◽  
Disease Gene Discovery ◽  
Human Genes ◽  
Using Data ◽  
The Impact

AbstractVariants predicted to result in the loss of function (LoF) of human genes have attracted interest because of their clinical impact and surprising prevalence in healthy individuals. Here, we present ALoFT (Annotation of Loss-of-Function Transcripts), a method to annotate and predict the disease-causing potential of LoF variants. Using data from Mendelian disease-gene discovery projects, we show that ALoFT can distinguish between LoF variants deleterious as heterozygotes and those causing disease only in the homozygous state. Investigation of variants discovered in healthy populations suggests that each individual carries at least two heterozygous premature stop alleles that could potentially lead to disease if present as homozygotes. When applied to de novo pLoF variants in autism-affected families, ALoFT distinguishes between deleterious variants in patients and benign variants in unaffected siblings. Finally, analysis of somatic variants in > 6,500 cancer exomes shows that pLoF variants predicted to be deleterious by ALoFT are enriched in known driver genes.

scholarly journals Heterozygous gene truncation delineates the human haploinsufficient genome

2014 ◽  
Author(s):  
István Bartha ◽  
Antonio Rausell ◽  
Paul McLaren ◽  
Manuel Tardaguila ◽  
Pejman Mohammadi ◽  
...  
Keyword(s):  
De Novo ◽  
De Novo Mutation ◽  
Phenotypic Traits ◽  
Incomplete Penetrance ◽  
Heterozygous State ◽  
Loss Of Function ◽  
Protein Coding ◽  
Large Numbers ◽  
Human Genes ◽  
Compensatory Mutations

Sequencing projects have identified large numbers of rare stop-gain and frameshift variants in the human genome. As most of these are observed in the heterozygous state, they test a gene?s tolerance to haploinsufficiency and dominant loss of function. We analyzed the distribution of truncating variants across 16,260 protein coding autosomal genes in 11,546 individuals. We observed 39,893 truncating variants affecting 12,062 genes, which significantly differed from an expectation of 12,916 genes under a model of neutral de novo mutation (p<1E-4). Extrapolating this to increasing numbers of sequenced individuals, we estimate that 10.8% of human genes do not tolerate heterozygous truncating variants. An additional 10 to 15% of truncated genes may be rescued by incomplete penetrance or compensatory mutations, or because the truncating variants are of limited functional impact. The study of protein truncating variants delineates the essential genome and, more generally, identifies rare heterozygous variants as an unexplored source of diversity of phenotypic traits and diseases.

scholarly journals An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

2019 ◽  
Author(s):  
Thomas F. Martinez ◽  
Qian Chu ◽  
Cynthia Donaldson ◽  
Dan Tan ◽  
Maxim N. Shokhirev ◽  
...  
Keyword(s):  
De Novo ◽  
Transcriptome Assembly ◽  
Human Leukocyte ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
Proteomics Data ◽  
Protein Coding ◽  
Leukocyte Antigen ◽  
Human Genes ◽  
Small Open Reading Frames

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

scholarly journals Contribution of retrotransposition to developmental disorders

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Eugene J. Gardner ◽  
Elena Prigmore ◽  
Giuseppe Gallone ◽  
Petr Danecek ◽  
Kaitlin E. Samocha ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
De Novo ◽  
Purifying Selection ◽  
Selective Constraint ◽  
Protein Coding ◽  
Genome Wide ◽  
De Novo Gene ◽  
The Impact ◽  
Transcribed Sequences

Abstract Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient’s symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.

scholarly journals Quantifying gene selection in cancer through protein functional alteration bias

2019 ◽  
Vol 47 (13) ◽  
pp. 6642-6655 ◽  
Author(s):  
Nadav Brandes ◽  
Nathan Linial ◽  
Michal Linial
Keyword(s):  
Somatic Mutations ◽  
Gene Selection ◽  
De Novo ◽  
Cancer Genes ◽  
Driver Genes ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Machine Learning Model ◽  
Implicit And Explicit ◽  
False Discoveries

Abstract Compiling the catalogue of genes actively involved in cancer is an ongoing endeavor, with profound implications to the understanding and treatment of the disease. An abundance of computational methods have been developed to screening the genome for candidate driver genes based on genomic data of somatic mutations in tumors. Existing methods make many implicit and explicit assumptions about the distribution of random mutations. We present FABRIC, a new framework for quantifying the selection of genes in cancer by assessing the effects of de-novo somatic mutations on protein-coding genes. Using a machine-learning model, we quantified the functional effects of ∼3M somatic mutations extracted from over 10 000 human cancerous samples, and compared them against the effects of all possible single-nucleotide mutations in the coding human genome. We detected 593 protein-coding genes showing statistically significant bias towards harmful mutations. These genes, discovered without any prior knowledge, show an overwhelming overlap with known cancer genes, but also include many overlooked genes. FABRIC is designed to avoid false discoveries by comparing each gene to its own background model using rigorous statistics, making minimal assumptions about the distribution of random somatic mutations. The framework is an open-source project with a simple command-line interface.

scholarly journals LoFTK: a framework for fully automated calculation of predicted Loss-of-Function variants

2021 ◽  
Author(s):  
Abdulrahman Alasiri ◽  
Konrad J. Karczewski ◽  
Brian Cole ◽  
Bao-Li Loza ◽  
Jason H. Moore ◽  
...  
Keyword(s):  
Open Source Software ◽  
Compound Heterozygous ◽  
Summary Statistics ◽  
Loss Of Function ◽  
Healthy Individuals ◽  
High Confidence ◽  
Human Genes ◽  
Automated Prediction ◽  
Using Data ◽  
Motivation Loss

Motivation: Loss-of-Function (LoF) variants in human genes are important due to their impact on clinical phenotypes and frequent occurrence in the genomes of healthy individuals. Current approaches predict high-confidence LoF variants without identifying the specific genes or the number of copies they affect. Moreover, there is a lack of methods for detecting knockout genes caused by compound heterozygous (CH) LoF variants. Results: We have developed the Loss-of-Function ToolKit (LoFTK), which allows efficient and automated prediction of LoF variants from both genotyped and sequenced genomes. LoFTK enables the identification of genes that are inactive in one or two copies and provides summary statistics for downstream analyses. LoFTK can identify CH LoF variants, which result in LoF genes with two copies lost. Using data from parents and offspring we show that 96% of CH LoF genes predicted by LoFTK in the offspring have the respective alleles donated by each parent. Availability and implementation: LoFTK is an open source software and is freely available to non-commercial users at https://github.com/CirculatoryHealth/LoFTK

scholarly journals Human and mouse essentiality screens as a resource for disease gene discovery

2019 ◽  
Author(s):  
Pilar Cacheiro ◽  
Violeta Muñoz-Fuentes ◽  
Stephen A. Murray ◽  
Mary E. Dickinson ◽  
Maja Bucan ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Disease Gene ◽  
De Novo ◽  
Genetic Diseases ◽  
Gene Discovery ◽  
Tissue Expression ◽  
Human Cell Line ◽  
Disease Genes ◽  
Mendelian Disease ◽  
Disease Gene Discovery

ABSTRACTAlthough genomic sequencing has been transformative in the study of rare genetic diseases, identifying causal variants remains a considerable challenge that can be addressed in part by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from the comprehensive viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and from human cell line essentiality screens. We propose a novel, cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing characteristics in the biological processes they regulate, tissue expression levels and human mutation rates. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented in the developmental lethal category, representing genes not essential for cell survival but required for organism development. Exploiting this finding, we have screened developmental disorder cases from three independent disease sequencing consortia and identified potentially pathogenic, de novo variants shared in different patients for several developmental lethal genes that have not previously been associated with rare disease. We therefore propose FUSIL as an efficient resource for disease gene discovery.

scholarly journals A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009889
Author(s):  
Amandine Bonnet ◽  
Carole Chaput ◽  
Noé Palmic ◽  
Benoit Palancade ◽  
Pascale Lesage
Keyword(s):  
Transcriptional Control ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Ltr Retrotransposons ◽  
Loss Of Function ◽  
Protein Coding ◽  
Multiple Copies ◽  
Ty1 Retrotransposition ◽  
Pore Complexes ◽  
The Impact

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposon under tight transcriptional control.

P1230Impact of regulated junctophilin-2 clustering at axial tubule junctions on atrial excitation-contraction coupling and therapeutic implications

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
S Brandenburg ◽  
J Pawlowitz ◽  
B Eikenbusch ◽  
T Kohl ◽  
S Sossalla ◽  
...  
Keyword(s):  
Left Atrial ◽  
De Novo ◽  
Electron Tomography ◽  
Pressure Overload ◽  
Aortic Pressure ◽  
Subcellular Localisation ◽  
Loss Of Function ◽  
Therapeutic Implications ◽  
Ec Coupling ◽  
The Impact

Abstract Objectives Atrial dysfunction is highly prevalent and known to significantly aggravate heart failure. While rapid excitation-contraction (EC) coupling depends on axial tubule junctions in atrial myocytes (AMs), the mechanisms leading to atrial loss-of-function remain unclear. Junctophilin-2 (JP2), a tail-anchored protein of the sarcoplasmic reticulum, stabilizes the integrity of ventricular Ca2+ release units, which is disrupted in ventricular myocytes by reduced JP2 expression or proteolysis. Here we aim to characterize the abundance and subcellular localisation of JP2 in AMs, to assess the impact of decreased JP2 expression on atrial remodelling, and to investigate the potential to correct JP2 expression and atrial dysfunction. Results We identified 5-fold lower JP2 levels in atrial compared to ventricular tissue in mouse and human hearts by SDS-PAGE. Surprisingly, in AMs, this resulted in subcellular expression of large JP2 clusters at axial tubule junctions together with highly phosphorylated ryanodine receptor (RyR2) channels visualized by STED superresolution microscopy. Importantly, left atrial hypertrophy induced by aortic pressure overload led to an additional strong decrease in JP2 expression compared to sham control, disrupted junctional RyR2 clustering and EC-coupling. This loss-of-function mechanism was confirmed by conditional shRNA-mediated JP2 knockdown. Quantitative image analysis after atrial JP2 knockdown showed a 50% decrease in area overlap between RyR2 and JP2 in AMs (JP2 knockdown 0.03±0.003 μm2 vs. control 0.06±0.004 μm2, p<0.001), and a ∼2-fold increased Ca2+ spark frequency, consistent with decreased left atrial fractional shortening (JP2 knockdown 12.9±0.8% vs. control 16.5±0.9%, p<0.01). Whereas atrial-ventricular dysfunction due to aortic pressure overload resulted in 40% mortality, additional JP2 knockdown exacerbated mortality to 100% (n: 10 control vs. 9 JP2 knockdown mice). In contrast, transgenic JP2 overexpressor mice showed greatly improved atrial contractility without mortality after induced aortic pressure overload (n: 21 control vs. 16 JP2 overexpressor mice). JP2-OE not only augmented atrial RyR2-clustering, but induced the de-novo biogenesis of large poly-adic junctional membrane complexes, which were resolved by STED microscopy via high-resolution cholesterol-based membrane staining in live AMs and electron tomography. Conclusions Nanoscale imaging identifies a new subcellular mechanism of significantly limited atrial JP2 protein expression in large clusters at axial tubule junctions. In atrial hypertrophy, JP2 is further decreased with junctional RyR2 cluster disruption leading to impaired Ca2+ release and decreased contractility. Importantly, JP2 overexpression effectively protected from atrial dysfunction, providing a novel therapeutic rationale for atrial cardiomyopathies.

Experience and impact of alopecia in patients (pts) with HER2+ metastatic breast cancer (MBC) in the SystHERs registry.

2017 ◽  
Vol 35 (8_suppl) ◽  
pp. 239-239
Author(s):  
Ginny Mason ◽  
Musa Mayer ◽  
Sandra M. Swain ◽  
Peter Kaufman ◽  
Debu Tripathy ◽  
...  
Keyword(s):  
Hair Loss ◽  
Systemic Chemotherapy ◽  
De Novo ◽  
Psychological Impact ◽  
Metastatic Breast ◽  
Instrument Measuring ◽  
Visceral Metastases ◽  
Using Data ◽  
Metastatic Sites ◽  
The Impact

239 Background: Although alopecia is common with systemic chemotherapy, its psychological impact is often overlooked in treatment (tx) decisions. We sought to better understand the impact of alopecia using data from SystHERs, an observational registry of pts with HER2+ MBC. Methods: SystHERs enrolled pts ≥ 18 years within 6 mos of an MBC diagnosis. Pt-reported outcomes are assessed quarterly. The Alopecia Patient Assessment (APA, Genentech) is a validated instrument measuring the incidence of hair loss in the prior 3 mos and the impact of hair loss in the prior 7 days. The APA includes 5 items scored on a 5-point scale (total range: 1–25). Higher scores indicate greater impact. Pts completed the APA within 30–90 days of initial MBC tx. Subgroups with no hair loss, hair loss without impact, or impactful hair loss were compared descriptively. Results: As of June 3, 2016, 591 of 976 pts were eligible for this analysis. Of these, 175 (30%) reported no hair loss, 123 (21%) reported hair loss without impact, and 293 (50%) reported impactful hair loss within 30–90 days of initial MBC tx. Respectively, 23% (41/175), 82% (101/123), and 79% (231/293) were on active chemotherapy, most commonly docetaxel (39% [16/41], 66% [67/101], and 62% [144/231]) and paclitaxel (5% [2/41], 27% [27/101], and 23% [54/231]). Median time from initial chemotherapy tx to APA was 55, 70, and 64 days. Of all pts with no hair loss, 39% had de novo MBC vs 56% of pts with hair loss but no impact and 55% with impactful hair loss. Relative to the no-hair-loss and hair-loss-but-no-impact subgroups, the subgroup with impactful hair loss was younger (median 57 and 59 vs 54 years, respectively) and had more pts with ≥ 2 metastatic sites (51% and 54% vs 63%) and visceral metastases (59% and 63% vs 69%). The median total APA score for pts with impactful hair loss was 8.8 (IQR 7.0–12.5). Conclusions: Of pts reporting hair loss, 79–82% were receiving systemic chemotherapy. Pts with impactful hair loss may have had greater disease burden. Alopecia impacted self-image and embarrassment more than work or social functioning. Clinical trial information: NCT01615068. [Table: see text]

scholarly journals Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis

2014 ◽  
Vol 112 (2) ◽  
pp. 400-405 ◽  
Author(s):  
Martha S. Field ◽  
Elena Kamynina ◽  
David Watkins ◽  
David S. Rosenblatt ◽  
Patrick J. Stover
Keyword(s):  
De Novo ◽  
Megaloblastic Anemia ◽  
Nucleotide Metabolism ◽  
Loss Of Function ◽  
Methionine Biosynthesis ◽  
De Novo Purine Biosynthesis ◽  
Methylenetetrahydrofolate Dehydrogenase ◽  
One Carbon Metabolism ◽  
Purine Nucleotide Metabolism ◽  
The Impact

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

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