Pathogenic effects of Leu200Pro and Arg387His VRK1 protein variants on phosphorylation targets and H4K16 acetylation in distal hereditary motor neuropathy.

Rare recessive variants in the human VRK1 gene are associated with several motor neuron diseases (MND), such as amyotrophic lateral sclerosis, spinal muscular atrophy, or distal hereditary motor neuropathies (dHMN). A case with dHMN carrying two novel VRK1 gene variants, expressing Leu200Pro (L200P) and Arg387His (R387H) variant proteins, identified that these protein variants are functionally different. The Leu200Pro variant shares with several variants in the catalytic domain the loss of the kinase activity on different substrates, such as histones, p53, or coilin. However, the distal Arg387His variant and the distal Trp375* (W375X) chinese variant, both located at the end of the low complexity C-terminal region and proximal to the termination codon, retain their catalytic activity on some substrates, and mechanistically their functional impairment is different. The L200P variant, as well as most VRK1 pathogenic variants, impairs the phosphorylation of BAF and histone H4K16 acetylation, which are required for DNA attachment to the nuclear envelope and chromatin accessibility to DNA repair mechanisms, respectively. The R387H variant impairs phosphorylation of H2AX, an early step in different types of DNA damage responses. The functional variability of VRK1 protein variants and their different combinations are a likely contributor to the clinical phenotypic heterogeneity of motor neuron and neurological diseases associated with rare VRK1 pathogenic variants. KEY MESSAGES: VRK1 variants implicated in motor neuron diseases are functionally different. The L200P variant is kinase inactive, and the R387H variant is partially active. VRK1 variants alter H4K16 acetylation and loss of coilin and BAF phosphorylation. VRK1 variants alter Cajal bodies and DNA damage responses. VRK1 variant combination determines the neurological phenotype heterogeneity.

STAG2: Computational Analysis of Missense Variants Involved in Disease.

The human STAG2 protein is an essential component of the cohesin complex involved in cellular processes of gene expression, DNA repair, and genomic integrity. Somatic mutations in the STAG2 sequence have been associated with various types of cancer, while congenital variants have been linked to developmental disorders such as Mullegama-Klein-Martinez syndrome, X-linked holoprosencephaly-13, and Cornelia de Lange syndrome. In the cohesin complex, the direct interaction of STAG2 with DNA and with NIPBL, RAD21, and CTCF proteins has been described. The function of STAG2 within the complex is still unknown, but it is related to its DNA binding capacity and is modulated by its binding to the other three proteins. Every missense variant described for STAG2 is located in regions involved in one of these interactions. In the present work, we model the structure of 12 missense variants described for STAG2, as well as two other variants of NIPBl and two of RAD21 located at STAG2 interaction zone, and then analyze their behavior through molecular dynamic simulations, comparing them with the same simulation of the wild-type protein. This will allow the effects of variants to be rationalized at the atomic level and provide clues as to how STAG2 functions in the cohesin complex.

Structural and functional insights into GSDMB isoforms complex roles in pathogenesis.

SHADSGasdermins (GSDMs) have garnered significant scientific interest due to their protective and detrimental roles in innate immunity, host defense, inflammation, and cancer alongside with other pathologies. While GSDMs are mostly recognized as key effectors of a lytic type of pro-inflammatory cell death known as pyroptosis, they do also take part in other cell death processes (NETosis, secondary necrosis, or apoptosis) and exhibit cell-death independent functions depending on the cellular context. Among GSDMs, Gasdermin B (GSDMB) pyroptotic capacity has been a subject of conflicting findings in scientific literature even when its processing, and subsequent activation, by Granzyme A (GZMA) was decoded. Nevertheless, recent groundbreaking publications have shed light on the crucial role of alternative splicing in determining the pyroptotic capacity of GSDMB isoforms, which depends on the presence of exon 6-derived elements. This comprehensive review pays attention to the relevant structural differences among recently crystalized GSDMB isoforms. As a novelty, the structural aspects governing GSDMB isoform susceptibility to GZMA-mediated activation have been investigated. By elucidating the complex roles of GSDMB isoforms, this review aims to deepen the understanding of this multifunctional player and its potential implications in disease pathogenesis and therapeutic interventions. [Figure: see text].

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies.

Molecular Basis of the Schuurs-Hoeijmakers Syndrome: What We Know about the Gene and the PACS-1 Protein and Novel Therapeutic Approaches.

The Schuurs−Hoeijmakers syndrome (SHMS) or PACS1 Neurodevelopment Disorder (PACS1-NDD) is a rare autosomal dominant disease caused by mutations in the PACS1 gene. To date, only 87 patients have been reported and, surprisingly, most of them carry the same variant (c.607C>T; p.R203W). The most relevant clinical features of the syndrome include neurodevelopment delay, seizures or a recognizable facial phenotype. Moreover, some of these characteristics overlap with other syndromes, such as the PACS2 or Wdr37 syndromes. The encoded protein phosphofurin acid cluster sorting 1 (PACS-1) is able to bind to different client proteins and direct them to their subcellular final locations. Therefore, although its main function is protein trafficking, it could perform other roles related to its client proteins. In patients with PACS1-NDD, a gain-of-function or a dominant negative mechanism for the mutated protein has been suggested. This, together with the fact that most of the patients carry the same genetic variant, makes it a good candidate for novel therapeutic approaches directed to decreasing the toxic effect of the mutated protein. Some of these strategies include the use of antisense oligonucleotides (ASOs) or targeting of its client proteins.

Computational Modeling and Design of New Inhibitors of Carbapenemases: A Discussion from the EPIC Alliance Network.

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: "What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?" From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

A Novel Intragenic Duplication in the HDAC8 Gene Underlying a Case of Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a multisystemic genetic disorder characterized by distinctive facial features, growth retardation, and intellectual disability, as well as various systemic conditions. It is caused by genetic variants in genes related to the cohesin complex. Single-nucleotide variations are the best-known genetic cause of CdLS; however, copy number variants (CNVs) clearly underlie a substantial proportion of cases of the syndrome. The NIPBL gene was thought to be the locus within which clinically relevant CNVs contributed to CdLS. However, in the last few years, pathogenic CNVs have been identified in other genes such as HDAC8, RAD21, and SMC1A. Here, we studied an affected girl presenting with a classic CdLS phenotype heterozygous for a de novo ~32 kbp intragenic duplication affecting exon 10 of HDAC8. Molecular analyses revealed an alteration in the physiological splicing that included a 96 bp insertion between exons 9 and 10 of the main transcript of HDAC8. The aberrant transcript was predicted to generate a truncated protein whose accessibility to the active center was restricted, showing reduced ease of substrate entry into the mutated enzyme. Lastly, we conclude that the duplication is responsible for the patient's phenotype, highlighting the contribution of CNVs as a molecular cause underlying CdLS.

Neurodevelopmental Disorders Associated with PSD-95 and Its Interaction Partners.

The postsynaptic density (PSD) is a massive protein complex, critical for synaptic strength and plasticity in excitatory neurons. Here, the scaffolding protein PSD-95 plays a crucial role as it organizes key PSD components essential for synaptic signaling, development, and survival. Recently, variants in DLG4 encoding PSD-95 were found to cause a neurodevelopmental disorder with a variety of clinical features including intellectual disability, developmental delay, and epilepsy. Genetic variants in several of the interaction partners of PSD-95 are associated with similar phenotypes, suggesting that deficient PSD-95 may affect the interaction partners, explaining the overlapping symptoms. Here, we review the transmembrane interaction partners of PSD-95 and their association with neurodevelopmental disorders. We assess how the structural changes induced by DLG4 missense variants may disrupt or alter such protein-protein interactions, and we argue that the pathological effect of DLG4 variants is, at least partly, exerted indirectly through interaction partners of PSD-95. This review presents a direction for functional studies to elucidate the pathogenic mechanism of deficient PSD-95, providing clues for therapeutic strategies.

Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase.

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

Dysfunctional Homozygous VRK1-D263G Variant Impairs the Assembly of Cajal Bodies and DNA Damage Response in Hereditary Spastic Paraplegia.

BACKGROUND AND OBJECTIVES: To conduct a genetic and molecular functional study of a family with members affected of hereditary spastic paraplegia (HSP) of unknown origin and carrying a novel pathogenic vaccinia-related kinase 1 (VRK1) variant. METHODS: Whole-exome sequencing was performed in 2 patients, and their parents diagnosed with HSP. The novel VRK1 variant was detected by whole-exome sequencing, molecularly modeled and biochemically characterized in kinase assays. Functionally, we studied the role of this VRK1 variant in DNA damage response and its effect on the assembly of Cajal bodies (CBs). RESULTS: We have identified a very rare homozygous variant VRK1-D263G with a neurologic phenotype associated with HSP and moderate intellectual disability. The molecular modeling of this VRK1 variant protein predicted an alteration in the folding of a loop that interferes with the access to the kinase catalytic site. The VRK1-D263G variant is kinase inactive and does not phosphorylate histones H2AX and H3, transcription factors activating transcription factor 2 and p53, coilin needed for assembly of CBs, and p53 binding protein 1, a DNA repair protein. Functionally, this VRK1 variant protein impairs CB formation and the DNA damage response. DISCUSSION: This report expands the neurologic spectrum of neuromotor syndromes associated with a new and rare VRK1 variant, representing a novel pathogenic participant in complicated HSP and demonstrates that CBs and the DNA damage response are impaired in these patients.

Clinical relevance of postzygotic mosaicism in Cornelia de Lange syndrome and purifying selection of NIPBL variants in blood.

Postzygotic mosaicism (PZM) in NIPBL is a strong source of causality for Cornelia de Lange syndrome (CdLS) that can have major clinical implications. Here, we further delineate the role of somatic mosaicism in CdLS by describing a series of 11 unreported patients with mosaic disease-causing variants in NIPBL and performing a retrospective cohort study from a Spanish CdLS diagnostic center. By reviewing the literature and combining our findings with previously published data, we demonstrate a negative selection against somatic deleterious NIPBL variants in blood. Furthermore, the analysis of all reported cases indicates an unusual high prevalence of mosaicism in CdLS, occurring in 13.1% of patients with a positive molecular diagnosis. It is worth noting that most of the affected individuals with mosaicism have a clinical phenotype at least as severe as those with constitutive pathogenic variants. However, the type of genetic change does not vary between germline and somatic events and, even in the presence of mosaicism, missense substitutions are located preferentially within the HEAT repeat domain of NIPBL. In conclusion, the high prevalence of mosaicism in CdLS as well as the disparity in tissue distribution provide a novel orientation for the clinical management and genetic counselling of families.

Pathogenic convergence of CNVs in genes functionally associated to a severe neuromotor developmental delay syndrome.

BACKGROUND: Complex developmental encephalopathy syndromes might be the consequence of unknown genetic alterations that are likely to contribute to the full neurological phenotype as a consequence of pathogenic gene combinations. METHODS: To identify the additional genetic contribution to the neurological phenotype, we studied as a test case a boy, with a KCNQ2 exon-7 partial duplication, by single-nucleotide polymorphism (SNP) microarray to detect copy-number variations (CNVs). RESULTS: The proband presented a cerebral palsy like syndrome with a severe motor and developmental encephalopathy. The SNP array analysis detected in the proband several de novo CNVs, nine partial gene losses (LRRC55, PCDH9, NALCN, RYR3, ELAVL2, CDH13, ATP1A2, SLC17A5, ANO3), and two partial gene duplications (PCDH19, EFNA5). The biological functions of these genes are associated with ion channels such as calcium, chloride, sodium, and potassium with several membrane proteins implicated in neural cell-cell interactions, synaptic transmission, and axon guidance. Pathogenically, these functions can be associated to cerebral palsy, seizures, dystonia, epileptic crisis, and motor neuron dysfunction, all present in the patient. CONCLUSIONS: Severe motor and developmental encephalopathy syndromes of unknown origin can be the result of a phenotypic convergence by combination of several genetic alterations in genes whose physiological function contributes to the neurological pathogenic mechanism.

Novel Dominant KCNQ2 Exon 7 Partial In-Frame Duplication in a Complex Epileptic and Neurodevelopmental Delay Syndrome.

Complex neurodevelopmental syndromes frequently have an unknown etiology, in which genetic factors play a pathogenic role. This study utilizes whole-exome sequencing (WES) to examine four members of a family with a son presenting, since birth, with epileptic-like crises, combined with cerebral palsy, severe neuromotor and developmental delay, dystonic tetraparexia, axonal motor affectation, and hyper-excitability of unknown origin. The WES study detected within the patient a de novo heterozygous in-frame duplication of thirty-six nucleotides within exon 7 of the human KCNQ2 gene. This insertion duplicates the first twelve amino acids of the calmodulin binding site I. Molecular dynamics simulations of this KCNQ2 peptide duplication, modelled on the 3D structure of the KCNQ2 protein, suggest that the duplication may lead to the dysregulation of calcium inhibition of this protein function.

VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy.

BACKGROUND: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. PATIENT AND METHODS: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. RESULTS: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1. CONCLUSION: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.

Molecular Characterization of Tc964, A Novel Antigenic Protein from Trypanosoma cruzi.

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.

Delineation of phenotypes and genotypes related to cohesin structural protein RAD21.

RAD21 encodes a key component of the cohesin complex, and variants in RAD21 have been associated with Cornelia de Lange Syndrome (CdLS). Limited information on phenotypes attributable to RAD21 variants and genotype-phenotype relationships is currently published. We gathered a series of 49 individuals from 33 families with RAD21 alterations [24 different intragenic sequence variants (2 recurrent), 7 unique microdeletions], including 24 hitherto unpublished cases. We evaluated consequences of 12 intragenic variants by protein modelling and molecular dynamic studies. Full clinical information was available for 29 individuals. Their phenotype is an attenuated CdLS phenotype compared to that caused by variants in NIPBL or SMC1A for facial morphology, limb anomalies, and especially for cognition and behavior. In the 20 individuals with limited clinical information, additional phenotypes include Mungan syndrome (in patients with biallelic variants) and holoprosencephaly, with or without CdLS characteristics. We describe several additional cases with phenotypes including sclerocornea, in which involvement of the RAD21 variant is uncertain. Variants were frequently familial, and genotype-phenotype analyses demonstrated striking interfamilial and intrafamilial variability. Careful phenotyping is essential in interpreting consequences of RAD21 variants, and protein modeling and dynamics can be helpful in determining pathogenicity. The current study should be helpful when counseling families with a RAD21 variation.

Evaluating Face2Gene as a Tool to Identify Cornelia de Lange Syndrome by Facial Phenotypes.

Characteristic or classic phenotype of Cornelia de Lange syndrome (CdLS) is associated with a recognisable facial pattern. However, the heterogeneity in causal genes and the presence of overlapping syndromes have made it increasingly difficult to diagnose only by clinical features. DeepGestalt technology, and its app Face2Gene, is having a growing impact on the diagnosis and management of genetic diseases by analysing the features of affected individuals. Here, we performed a phenotypic study on a cohort of 49 individuals harbouring causative variants in known CdLS genes in order to evaluate Face2Gene utility and sensitivity in the clinical diagnosis of CdLS. Based on the profile images of patients, a diagnosis of CdLS was within the top five predicted syndromes for 97.9% of our cases and even listed as first prediction for 83.7%. The age of patients did not seem to affect the prediction accuracy, whereas our results indicate a correlation between the clinical score and affected genes. Furthermore, each gene presents a different pattern recognition that may be used to develop new neural networks with the goal of separating different genetic subtypes in CdLS. Overall, we conclude that computer-assisted image analysis based on deep learning could support the clinical diagnosis of CdLS.

MEPSAnd: minimum energy path surface analysis over n-dimensional surfaces.

SUMMARY: n-dimensional energy surfaces are becoming computationally accessible, yet interpreting their information is not straightforward. We present minimum energy path surface analysis over n-dimensional surfaces (MEPSAnd), an open source GUI-based program that natively calculates minimum energy paths across energy surfaces of any number of dimensions. Among other features, MEPSAnd can compute the path through lowest barriers and automatically provide a set of alternative paths. MEPSAnd offers distinct plotting solutions as well as direct python scripting. AVAILABILITY AND IMPLEMENTATION: MEPSAnd is freely available (under GPLv3 license) at: http://bioweb.cbm.uam.es/software/MEPSAnd/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.