Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions.

RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene's importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Nonsynonymous Synonymous Variants Demand for a Paradigm Shift in Genetics.

Synonymous (also known as silent) variations are by definition not considered to change the coded protein. Still many variations in this category affect either protein abundance or properties. As this situation is confusing, we have recently introduced systematics for synonymous variations and those that may on the surface look like synonymous, but these may affect the coded protein in various ways. A new category, unsense variation, was introduced to describe variants that do not introduce a stop codon into the variation site, but which lead to different types of changes in the coded protein. Many of these variations lead to mRNA degradation and missing protein. Here, consequences of the systematics are discussed from the perspectives of variation annotation and interpretation, evolutionary calculations, nonsynonymous-to-synonymous substitution rates, phylogenetics and other evolutionary inferences that are based on the principle of (nearly) neutral synonymous variations. It may be necessary to reassess published results. Further, databases for synonymous variations and prediction methods for such variations should consider unsense variations. Thus, there is a need to evaluate and reflect principles of numerous aspects in genetics, ranging from variation naming and classification to evolutionary calculations.

PON-Fold: Prediction of Substitutions Affecting Protein Folding Rate.

Most proteins fold into characteristic three-dimensional structures. The rate of folding and unfolding varies widely and can be affected by variations in proteins. We developed a novel machine-learning-based method for the prediction of the folding rate effects of amino acid substitutions in two-state folding proteins. We collected a data set of experimentally defined folding rates for variants and used them to train a gradient boosting algorithm starting with 1161 features. Two predictors were designed. The three-class classifier had, in blind tests, specificity and sensitivity ranging from 0.324 to 0.419 and from 0.256 to 0.451, respectively. The other tool was a regression predictor that showed a Pearson correlation coefficient of 0.525. The error measures, mean absolute error and mean squared error, were 0.581 and 0.603, respectively. One of the previously presented tools could be used for comparison with the blind test data set, our method called PON-Fold showed superior performance on all used measures. The applicability of the tool was tested by predicting all possible substitutions in a protein domain. Predictions for different conformations of proteins, open and closed forms of a protein kinase, and apo and holo forms of an enzyme indicated that the choice of the structure had a large impact on the outcome. PON-Fold is freely available.

Systematic errors in annotations of truncations, loss-of-function and synonymous variants.

Description of genetic phenomena and variations requires exact language and concepts. Vast amounts of variation data are produced with next-generation sequencing pipelines. The obtained variations are automatically annotated, e.g., for their functional consequences. These tools and pipelines, along with systematic nomenclature, mainly work well, but there are still some problems in nomenclature, organization of some databases, misuse of concepts and certain practices. Therefore, systematic errors prevent correct annotation and often preclude further analysis of certain variation types. Problems and solutions are described for presumed protein truncations, variants that are claimed to be of loss-of-function based on the type of variation, and synonymous variants that are not synonymous and lead to sequence changes or to missing protein.

Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance.

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.

Individual Genetic Heterogeneity.

Genetic variation has been widely covered in literature, however, not from the perspective of an individual in any species. Here, a synthesis of genetic concepts and variations relevant for individual genetic constitution is provided. All the different levels of genetic information and variation are covered, ranging from whether an organism is unmixed or hybrid, has variations in genome, chromosomes, and more locally in DNA regions, to epigenetic variants or alterations in selfish genetic elements. Genetic constitution and heterogeneity of microbiota are highly relevant for health and wellbeing of an individual. Mutation rates vary widely for variation types, e.g., due to the sequence context. Genetic information guides numerous aspects in organisms. Types of inheritance, whether Mendelian or non-Mendelian, zygosity, sexual reproduction, and sex determination are covered. Functions of DNA and functional effects of variations are introduced, along with mechanism that reduce and modulate functional effects, including TARAR countermeasures and intraindividual genetic conflict. TARAR countermeasures for tolerance, avoidance, repair, attenuation, and resistance are essential for life, integrity of genetic information, and gene expression. The genetic composition, effects of variations, and their expression are considered also in diseases and personalized medicine. The text synthesizes knowledge and insight on individual genetic heterogeneity and organizes and systematizes the central concepts.

ProTstab2 for Prediction of Protein Thermal Stabilities.

The stability of proteins is an essential property that has several biological implications. Knowledge about protein stability is important in many ways, ranging from protein purification and structure determination to stability in cells and biotechnological applications. Experimental determination of thermal stabilities has been tedious and available data have been limited. The introduction of limited proteolysis and mass spectrometry approaches has facilitated more extensive cellular protein stability data production. We collected melting temperature information for 34,913 proteins and developed a machine learning predictor, ProTstab2, by utilizing a gradient boosting algorithm after testing seven algorithms. The method performance was assessed on a blind test data set and showed a Pearson correlation coefficient of 0.753 and root mean square error of 7.005. Comparison to previous methods indicated that ProTstab2 had superior performance. The method is fast, so it was applied to predict and compare the stabilities of all proteins in human, mouse, and zebrafish proteomes for which experimental data were not determined. The tool is freely available.

When a Synonymous Variant Is Nonsynonymous.

Term synonymous variation is widely used, but frequently in a wrong or misleading meaning and context. Twenty three point eight % of possible nucleotide substitution types in the universal genetic code are for synonymous amino acid changes, but when these variants have a phenotype and functional effect, they are very seldom synonymous. Such variants may manifest changes at DNA, RNA and/or protein levels. Large numbers of variations are erroneously annotated as synonymous, which causes problems e.g., in clinical genetics and diagnosis of diseases. To facilitate precise communication, novel systematics and nomenclature are introduced for variants that when looking only at the genetic code seem like synonymous, but which have phenotypes. A new term, unsense variant is defined as a substitution in the mRNA coding region that affects gene expression and protein production without introducing a stop codon in the variation site. Such variants are common and need to be correctly annotated. Proper naming and annotation are important also to increase awareness of these variants and their consequences.

PON-All: Amino Acid Substitution Tolerance Predictor for All Organisms.

Genetic variations are investigated in human and many other organisms for many purposes (e.g., to aid in clinical diagnosis). Interpretation of the identified variations can be challenging. Although some dedicated prediction methods have been developed and some tools for human variants can also be used for other organisms, the performance and species range have been limited. We developed a novel variant pathogenicity/tolerance predictor for amino acid substitutions in any organism. The method, PON-All, is a machine learning tool trained on human, animal, and plant variants. Two versions are provided, one with Gene Ontology (GO) annotations and another without these details. GO annotations are not available or are partial for many organisms of interest. The methods provide predictions for three classes: pathogenic, benign, and variants of unknown significance. On the blind test, when using GO annotations, accuracy was 0.913 and MCC 0.827. When GO features were not used, accuracy was 0.856 and MCC 0.712. The performance is the best for human and plant variants and somewhat lower for animal variants because the number of known disease-causing variants in animals is rather small. The method was compared to several other tools and was found to have superior performance. PON-All is freely available at http://structure.bmc.lu.se/PON-All and http://8.133.174.28:8999/.

Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

Generic model for biological regulation.

A substantial portion of molecules in an organism are involved in regulation of a wide spectrum of biological processes. Several models have been presented for various forms of biological regulation, including gene expression regulation and physiological regulation; however, a generic model is missing. Recently a new unifying theory in biology, poikilosis, was presented.  Poikilosis indicates that all systems display intrinsic heterogeneity. The concept of poikilosis allowed development of a model for biological regulation applicable to all types of regulated systems. The perturbation-lagom-TATAR countermeasures-regulator (PLTR) model combines the effects of perturbation and lagom (allowed and sufficient extent of heterogeneity) in a system with tolerance, avoidance, repair, attenuation and resistance (TARAR) countermeasures, and possible regulators. There are three modes of regulation, two of which are lagom-related. In the first scenario, lagom is maintained, both intrinsic (passive) and active TARAR countermeasures can be involved. In the second mode, there is a shift from one lagom to another. In the third mode, reguland regulation, the regulated entity is the target of a regulatory shift, which is often irreversible or requires action of another regulator to return to original state. After the shift, the system enters to lagom maintenance mode, but at new lagom extent. The model is described and elaborated with examples and applications, including medicine and systems biology. Consequences of non-lagom extent of heterogeneity are introduced, along with a novel idea for therapy by reconstituting biological processes to lagom extent, even when the primary effect cannot be treated.

Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update).

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe, recurrent bacterial and fungal infections. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is subsequently metabolized to hydrogen peroxide and other reactive oxygen species (ROS). These products are essential for intracellular killing of pathogens by phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages). The leukocyte NADPH oxidase is composed of five subunits, four of which are encoded by autosomal genes. These are CYBA, encoding p22phox, NCF1, encoding p47phox, NCF2, encoding p67phox and NCF4, encoding p40phox. This article lists all mutations identified in these genes in CGD patients. In addition, cytochrome b558 chaperone-1 (CYBC1), recently recognized as an essential chaperone protein for the expression of the X-linked NADPH oxidase component gp91phox (also called Nox2), is encoded by the autosomal gene CYBC1. Mutations in this gene also lead to CGD. Finally, RAC2, a small GTPase of the Rho family, is needed for activation of the NADPH oxidase, and mutations in the RAC2 gene therefore also induce CGD-like symptoms. Mutations in these last two genes are also listed in this article.

PON-Sol2: Prediction of Effects of Variants on Protein Solubility.

Genetic variations have a multitude of effects on proteins. A substantial number of variations affect protein-solvent interactions, either aggregation or solubility. Aggregation is often related to structural alterations, whereas solubilizable proteins in the solid phase can be made again soluble by dilution. Solubility is a central protein property and when reduced can lead to diseases. We developed a prediction method, PON-Sol2, to identify amino acid substitutions that increase, decrease, or have no effect on the protein solubility. The method is a machine learning tool utilizing gradient boosting algorithm and was trained on a large dataset of variants with different outcomes after the selection of features among a large number of tested properties. The method is fast and has high performance. The normalized correct prediction rate for three states is 0.656, and the normalized GC2 score is 0.312 in 10-fold cross-validation. The corresponding numbers in the blind test were 0.545 and 0.157. The performance was superior in comparison to previous methods. The PON-Sol2 predictor is freely available. It can be used to predict the solubility effects of variants for any organism, even in large-scale projects.

Structure-Function Relationships of Covalent and Non-Covalent BTK Inhibitors.

Low-molecular weight chemical compounds have a longstanding history as drugs. Target specificity and binding efficiency represent major obstacles for small molecules to become clinically relevant. Protein kinases are attractive cellular targets; however, they are challenging because they present one of the largest protein families and share structural similarities. Bruton tyrosine kinase (BTK), a cytoplasmic protein tyrosine kinase, has received much attention as a promising target for the treatment of B-cell malignancies and more recently autoimmune and inflammatory diseases. Here we describe the structural properties and binding modes of small-molecule BTK inhibitors, including irreversible and reversible inhibitors. Covalently binding compounds, such as ibrutinib, acalabrutinib and zanubrutinib, are discussed along with non-covalent inhibitors fenebrutinib and RN486. The focus of this review is on structure-function relationships.

Measuring and interpreting pervasive heterogeneity, poikilosis.

Measurements are widely used in science, engineering, industry, and trade. They form the basis for experimental scientific research, approach, and progress; however, their foundations are seldom thought or questioned. Recently poikilosis, pervasive heterogeneity ranging from subatomic level to biosphere, was introduced. Poikilosis makes single point measurements and estimates obsolete and irrelevant as measurands display intervals of magnitudes. Consideration of poikilosis requires new lines of thinking in experimental design, conduction of studies, data analysis and interpretation. Measurements of poikilosis must consider lagom, normal, variation extent. Measurements, measures, and measurands as well as the measuring systems and uncertainties are discussed from the perspective of poikilosis. New systematics is introduced for description of uncertainty in measurements and for types of experimental designs. Poikilosis-aware experimenting, data analysis and interpretation are discussed. Instructions are provided for how to measure lagom and non-lagom effects of poikilosis. Consideration of poikilosis can solve scientific controversies and enigmas and can allow novel insight into systems, processes, mechanisms, and reactions and their interpretation, understanding, and manipulation. Furthermore, it will increase reproducibility of measurements and studies.

Prognostic implications of troponin T variations in inherited cardiomyopathies using systems biology.

The cardiac troponin T variations have often been used as an example of the application of clinical genotyping for prognostication and risk stratification measures for the management of patients with a family history of sudden cardiac death or familial cardiomyopathy. Given the disparity in patient outcomes and therapy options, we investigated the impact of variations on the intermolecular interactions across the thin filament complex as an example of an unbiased systems biology method to better define clinical prognosis to aid future management options. We present a novel unbiased dynamic model to define and analyse the functional, structural and physico-chemical consequences of genetic variations among the troponins. This was subsequently integrated with clinical data from accessible global multi-centre systematic reviews of familial cardiomyopathy cases from 106 articles of the literature: 136 disease-causing variations pertaining to 981 global clinical cases. Troponin T variations showed distinct pathogenic hotspots for dilated and hypertrophic cardiomyopathies; considering the causes of cardiovascular death separately, there was a worse survival in terms of sudden cardiac death for patients with a variation at regions 90-129 and 130-179 when compared to amino acids 1-89 and 200-288. Our data support variations among 90-130 as being a hotspot for sudden cardiac death and the region 131-179 for heart failure death/transplantation outcomes wherein the most common phenotype was dilated cardiomyopathy. Survival analysis into regions of high risk (regions 90-129 and 130-180) and low risk (regions 1-89 and 200-288) was significant for sudden cardiac death (p = 0.011) and for heart failure death/transplant (p = 0.028). Our integrative genomic, structural, model from genotype to clinical data integration has implications for enhancing clinical genomics methodologies to improve risk stratification.

Circulating plasma microRNAs in systemic sclerosis-associated pulmonary arterial hypertension.

OBJECTIVES: SSc-associated pulmonary arterial hypertension (SSc-APAH) is a late but devastating complication of SSc. Early identification of SSc-APAH may improve survival. We examined the role of circulating miRNAs in SSc-APAH. METHODS: Using quantitative RT-PCR the abundance of mature miRNAs in plasma was determined in 85 female patients with ACA-positive lcSSc. Twenty-two of the patients had SSc-APAH. Sixty-three SSc controls without PAH were matched for disease duration. Forty-six selected miRNA plasma levels were correlated with clinical data. Longitudinal samples were analysed from 14 SSc-APAH and 27 SSc patients. RESULTS: The disease duration was 12 years for the SSc-APAH patients and 12.7 years for the SSc controls. Plasma expression levels of 11 miRNAs were lower in patients with SSc-APAH. Four miRNAs displayed higher plasma levels in SSc-APAH patients compared with SSc controls. There was significant difference between groups for miR-20a-5p and miR-203a-3p when correcting for multiple comparisons (P = 0.002 for both). Receiver operating characteristics curve showed AUC = 0.69-0.83 for miR-21-5p and miR-20a-5p or their combination. miR-20a-5p and miR-203a-3p correlated inversely with NT-pro-Brain Natriuretic Protein levels (r = -0.42 and -0.47). Mixed effect model analysis could not identify any miRNAs as predictor of PAH development. However, miR-20a-5p plasma levels were lower in the longitudinal samples of SSc-APAH patients than in the SSc controls. CONCLUSIONS: Our study links expression levels of the circulating plasma miRNAs, especially miR-20a-5p and miR-203a-3p, to the occurrence of SSc-APAH in female patients with ACA-positive lcSSc.

TNF-α and α-synuclein fibrils differently regulate human astrocyte immune reactivity and impair mitochondrial respiration.

Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.

BTK gatekeeper residue variation combined with cysteine 481 substitution causes super-resistance to irreversible inhibitors acalabrutinib, ibrutinib and zanubrutinib.

Irreversible inhibitors of Bruton tyrosine kinase (BTK), pioneered by ibrutinib, have become breakthrough drugs in the treatment of leukemias and lymphomas. Resistance variants (mutations) occur, but in contrast to those identified for many other tyrosine kinase inhibitors, they affect less frequently the "gatekeeper" residue in the catalytic domain. In this study we carried out variation scanning by creating 11 substitutions at the gatekeeper amino acid, threonine 474 (T474). These variants were subsequently combined with replacement of the cysteine 481 residue to which irreversible inhibitors, such as ibrutinib, acalabrutinib and zanubrutinib, bind. We found that certain double mutants, such as threonine 474 to isoleucine (T474I) or methionine (T474M) combined with catalytically active cysteine 481 to serine (C481S), are insensitive to ≥16-fold the pharmacological serum concentration, and therefore defined as super-resistant to irreversible inhibitors. Conversely, reversible inhibitors showed a variable pattern, from resistance to no resistance, collectively demonstrating the structural constraints for different classes of inhibitors, which may affect their clinical application.