Burden analysis of missense variants in 1,330 disease-associated genes on 3D provides insights into the mutation effects

AbstractInterpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acid-substituting missense variants on protein structure and function being especially challenging. Here we evaluated the burden of amino acids affected in pathogenic variants (n=32,923) compared to the variants (n=164,915) from the general population in 1,330 disease-associated genes on forty protein features using over 14,000 experimentally-solved 3D structures. By analyzing the whole gene/variant set jointly, we identified 18 features associated with 3D mutational hotspots that are generally important for protein fitness and stability. Individual analyses performed for twenty-four protein functional classes further revealed 240 characteristics of mutational hotspots in total, including new associations recapitulating the sheer diversity across proteins essential structural regions. We demonstrated that the function-specific features of variants correspond to the readouts of mutagenesis experiments and positively correlate with clinically-interpreted pathogenic and benign missense variants. Finally, we made our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step towards translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the pathogenicity of variants in terms of the perturbed molecular mechanisms.

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Comprehensive characterization of amino acid positions in protein structures reveals molecular effect of missense variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002660117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28201-28211

Author(s):

Sumaiya Iqbal ◽

Eduardo Pérez-Palma ◽

Jakob B. Jespersen ◽

Patrick May ◽

David Hoksza ◽

...

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Molecular Mechanisms ◽

Amino Acid Level ◽

Protein Structures ◽

Point Mutations ◽

Independent Set ◽

Clinical Genetics ◽

Missense Variants ◽

The Impact

Interpretation of the colossal number of genetic variants identified from sequencing applications is one of the major bottlenecks in clinical genetics, with the inference of the effect of amino acid-substituting missense variations on protein structure and function being especially challenging. Here we characterize the three-dimensional (3D) amino acid positions affected in pathogenic and population variants from 1,330 disease-associated genes using over 14,000 experimentally solved human protein structures. By measuring the statistical burden of variations (i.e., point mutations) from all genes on 40 3D protein features, accounting for the structural, chemical, and functional context of the variations’ positions, we identify features that are generally associated with pathogenic and population missense variants. We then perform the same amino acid-level analysis individually for 24 protein functional classes, which reveals unique characteristics of the positions of the altered amino acids: We observe up to 46% divergence of the class-specific features from the general characteristics obtained by the analysis on all genes, which is consistent with the structural diversity of essential regions across different protein classes. We demonstrate that the function-specific 3D features of the variants match the readouts of mutagenesis experiments for BRCA1 and PTEN, and positively correlate with an independent set of clinically interpreted pathogenic and benign missense variants. Finally, we make our results available through a web server to foster accessibility and downstream research. Our findings represent a crucial step toward translational genetics, from highlighting the impact of mutations on protein structure to rationalizing the variants’ pathogenicity in terms of the perturbed molecular mechanisms.

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Mechanosensation and Mechanotransduction by Lymphatic Endothelial Cells Act as Important Regulators of Lymphatic Development and Function

International Journal of Molecular Sciences ◽

10.3390/ijms22083955 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3955

Author(s):

László Bálint ◽

Zoltán Jakus

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Fate ◽

Molecular Mechanisms ◽

Critical Role ◽

Mechanical Forces ◽

Lymphatic Endothelial Cells ◽

Lymphatic Development ◽

And Function ◽

The Impact

Our understanding of the function and development of the lymphatic system is expanding rapidly due to the identification of specific molecular markers and the availability of novel genetic approaches. In connection, it has been demonstrated that mechanical forces contribute to the endothelial cell fate commitment and play a critical role in influencing lymphatic endothelial cell shape and alignment by promoting sprouting, development, maturation of the lymphatic network, and coordinating lymphatic valve morphogenesis and the stabilization of lymphatic valves. However, the mechanosignaling and mechanotransduction pathways involved in these processes are poorly understood. Here, we provide an overview of the impact of mechanical forces on lymphatics and summarize the current understanding of the molecular mechanisms involved in the mechanosensation and mechanotransduction by lymphatic endothelial cells. We also discuss how these mechanosensitive pathways affect endothelial cell fate and regulate lymphatic development and function. A better understanding of these mechanisms may provide a deeper insight into the pathophysiology of various diseases associated with impaired lymphatic function, such as lymphedema and may eventually lead to the discovery of novel therapeutic targets for these conditions.

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Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

Genetics in Medicine ◽

10.1038/s41436-021-01158-1 ◽

2021 ◽

Author(s):

Paolo Zanoni ◽

Katharina Steindl ◽

Deepanwita Sengupta ◽

Pascal Joset ◽

Angela Bahr ◽

...

Keyword(s):

Loss Of Function ◽

Mild Phenotype ◽

Psychological Issues ◽

Missense Variants ◽

Mild Developmental Delay ◽

Pathogenic Variants ◽

In Silico Modeling ◽

And Function ◽

Methylation Activity

Abstract Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

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Insights into the Cellular and Molecular Mechanisms That Govern the Fracture-Healing Process: A Narrative Review

Journal of Clinical Medicine ◽

10.3390/jcm10163554 ◽

2021 ◽

Vol 10 (16) ◽

pp. 3554

Author(s):

Dionysios J. Papachristou ◽

Stavros Georgopoulos ◽

Peter V. Giannoudis ◽

Elias Panagiotopoulos

Keyword(s):

Fracture Healing ◽

Bone Repair ◽

Molecular Mechanisms ◽

Current Knowledge ◽

Healing Process ◽

Bone Architecture ◽

Multi Stage ◽

Stage Process ◽

And Function ◽

The Impact

Fracture-healing is a complex multi-stage process that usually progresses flawlessly, resulting in restoration of bone architecture and function. Regrettably, however, a considerable number of fractures fail to heal, resulting in delayed unions or non-unions. This may significantly impact several aspects of a patient’s life. Not surprisingly, in the past few years, a substantial amount of research and number of clinical studies have been designed, aiming at shedding light into the cellular and molecular mechanisms that regulate fracture-healing. Herein, we present the current knowledge on the pathobiology of the fracture-healing process. In addition, the role of skeletal cells and the impact of marrow adipose tissue on bone repair is discussed. Unveiling the pathogenetic mechanisms that govern the fracture-healing process may lead to the development of novel, smarter, and more effective therapeutic strategies for the treatment of fractures, especially of those with large bone defects.

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Interferon β-Mediated Protective Functions of Microglia in Central Nervous System Autoimmunity

International Journal of Molecular Sciences ◽

10.3390/ijms20010190 ◽

2019 ◽

Vol 20 (1) ◽

pp. 190 ◽

Cited By ~ 7

Author(s):

Stefanie Scheu ◽

Shafaqat Ali ◽

Ritu Mann-Nüttel ◽

Lisa Richter ◽

Volker Arolt ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Molecular Mechanisms ◽

Chronic Inflammatory Disease ◽

Clinical Severity ◽

Interferon Β ◽

Treatment Regimens ◽

Cns Autoimmunity ◽

And Function ◽

The Impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination and axonal damage. It often affects young adults and can lead to neurological disability. Interferon β (IFNβ) preparations represent widely used treatment regimens for patients with relapsing-remitting MS (RRMS) with therapeutic efficacy in reducing disease progression and frequency of acute exacerbations. In mice, IFNβ therapy has been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS while genetic deletion of IFNβ or its receptor augments clinical severity of disease. However, the complex mechanism of action of IFNβ in CNS autoimmunity has not been fully elucidated. Here, we review our current understanding of the origin, phenotype, and function of microglia and CNS immigrating macrophages in the pathogenesis of MS and EAE. In addition, we highlight the emerging roles of microglia as IFNβ-producing cells and vice versa the impact of IFNβ on microglia in CNS autoimmunity. We finally discuss recent progress in unraveling the underlying molecular mechanisms of IFNβ-mediated effects in EAE.

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Pathogenic Effect of GDAP1 Gene Mutations in a Yeast Model

Genes ◽

10.3390/genes11030310 ◽

2020 ◽

Vol 11 (3) ◽

pp. 310 ◽

Cited By ~ 1

Author(s):

Weronika Rzepnikowska ◽

Joanna Kaminska ◽

Dagmara Kabzińska ◽

Andrzej Kochański

Keyword(s):

Gene Mutations ◽

Causative Mutation ◽

Mitochondrial Morphology ◽

Clinical Genetics ◽

Sequence Variant ◽

Sequence Variants ◽

Pathogenic Variants ◽

Pathogenic Effect ◽

And Function ◽

Rare Sequence

The question of whether a newly identified sequence variant is truly a causative mutation is a central problem of modern clinical genetics. In the current era of massive sequencing, there is an urgent need to develop new tools for assessing the pathogenic effect of new sequence variants. In Charcot-Marie-Tooth disorders (CMT) with their extreme genetic heterogeneity and relatively homogenous clinical presentation, addressing the pathogenic effect of rare sequence variants within 80 CMT genes is extremely challenging. The presence of multiple rare sequence variants within a single CMT-affected patient makes selection for the strongest one, the truly causative mutation, a challenging issue. In the present study we propose a new yeast-based model to evaluate the pathogenic effect of rare sequence variants found within the one of the CMT-associated genes, GDAP1. In our approach, the wild-type and pathogenic variants of human GDAP1 gene were expressed in yeast. Then, a growth rate and mitochondrial morphology and function of GDAP1-expressing strains were studied. Also, the mutant GDAP1 proteins localization and functionality were assessed in yeast. We have shown, that GDAP1 was not only stably expressed but also functional in yeast cell, as it influenced morphology and function of mitochondria and altered the growth of a mutant yeast strain. What is more, the various GDAP1 pathogenic sequence variants caused the specific for them effect in the tests we performed. Thus, the proposed model is suitable for validating the pathogenic effect of known GDAP1 mutations and may be used for testing of unknown sequence variants found in CMT patients.

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A stochastic roadmap method to model protein structural transitions

Robotica ◽

10.1017/s0263574715001058 ◽

2015 ◽

Vol 34 (8) ◽

pp. 1705-1733 ◽

Cited By ~ 8

Author(s):

Kevin Molloy ◽

Rudy Clausen ◽

Amarda Shehu

Keyword(s):

Protein Structure ◽

Molecular Basis ◽

Structure And Function ◽

Structural Transitions ◽

Simulation Framework ◽

Human Disorders ◽

Model Protein ◽

And Function ◽

The Impact

SUMMARYEvidence is emerging that the role of protein structure in disease needs to be rethought. Sequence mutations in proteins are often found to affect the rate at which a protein switches between structures. Modeling structural transitions in wildtype and variant proteins is central to understanding the molecular basis of disease. This paper investigates an efficient algorithmic realization of the stochastic roadmap simulation framework to model structural transitions in wildtype and variants of proteins implicated in human disorders. Our results indicate that the algorithm is able to extract useful information on the impact of mutations on protein structure and function.

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New pathogenic variants and insights into pathogenic mechanisms in GRK1-related Oguchi disease

10.1101/2020.02.20.936880 ◽

2020 ◽

Author(s):

James A. Poulter ◽

Molly S. C. Gravett ◽

Rachel L. Taylor ◽

Kaoru Fujinami ◽

Julie De Zaeytijd ◽

...

Keyword(s):

Protein Function ◽

Kinase Domain ◽

Missense Variants ◽

Pathogenic Variants ◽

Oguchi Disease ◽

Whole Exome ◽

Novel Variants ◽

Variation Database ◽

Biallelic Mutations ◽

The Impact

AbstractPurposeBiallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify pathogenic GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity.MethodsPatients’ genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Pathogenic variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools.ResultsWe identified eleven previously unpublished cases with biallelic pathogenic GRK1 variants, including seven novel variants, and reviewed all GRK1 pathogenic variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. Additionally, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate pathogenic from non-pathogenic variants.ConclusionWe identified new GRK1 pathogenic variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function, giving new insights into the mechanisms of pathogenicity. All pathogenic GRK1 variants described to date have been collated into a Leiden Open Variation Database (http://dna2.leeds.ac.uk/GRK1_LOVD/genes/GRK1).

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A CNTNAP1 Missense Variant Is Associated with Canine Laryngeal Paralysis and Polyneuropathy

Genes ◽

10.3390/genes11121426 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1426

Author(s):

Anna Letko ◽

Katie M. Minor ◽

Steven G. Friedenberg ◽

G. Diane Shelton ◽

Jill Pesayco Salvador ◽

...

Keyword(s):

Molecular Mechanisms ◽

Missense Variant ◽

Nerve Biopsy ◽

Vocal Cord Palsy ◽

Severe Respiratory Distress ◽

Laryngeal Paralysis ◽

Pathogenic Variants ◽

Homozygous Mutant ◽

The Impact ◽

Saint Bernard

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

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