The phenotypic and genotypic spectrum of individuals with mono- or biallelic ANK3 variants.

ANK3 encodes ankyrin-G, a protein involved in neuronal development and signaling. Alternative splicing gives rise to three ankyrin-G isoforms comprising different domains with distinct expression patterns. Mono- or biallelic ANK3 variants are associated with non-specific syndromic intellectual disability in 14 individuals (seven with monoallelic and seven with biallelic variants). In this study, we describe the clinical features of 13 additional individuals and review the data on a total of 27 individuals (16 individuals with monoallelic and 11 with biallelic ANK3 variants) and demonstrate that the phenotype for biallelic variants is more severe. The phenotypic features include language delay (92%), autism spectrum disorder (76%), intellectual disability (78%), hypotonia (65%), motor delay (68%), attention deficit disorder (ADD) or attention deficit hyperactivity disorder (ADHD) (57%), sleep disturbances (50%), aggressivity/self-injury (37.5%), and epilepsy (35%). A notable phenotypic difference was presence of ataxia in three individuals with biallelic variants, but in none of the individuals with monoallelic variants. While the majority of the monoallelic variants are predicted to result in a truncated protein, biallelic variants are almost exclusively missense. Moreover, mono- and biallelic variants appear to be localized differently across the three different ankyrin-G isoforms, suggesting isoform-specific pathological mechanisms.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

More Than One HMG-CoA Lyase: The Classical Mitochondrial Enzyme Plus the Peroxisomal and the Cytosolic Ones.

There are three human enzymes with HMG-CoA lyase activity that are able to synthesize ketone bodies in different subcellular compartments. The mitochondrial HMG-CoA lyase was the first to be described, and catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl-CoA, the common final step in ketogenesis and leucine catabolism. This protein is mainly expressed in the liver and its function is metabolic, since it produces ketone bodies as energetic fuels when glucose levels are low. Another isoform is encoded by the same gene for the mitochondrial HMG-CoA lyase (HMGCL), but it is located in peroxisomes. The last HMG-CoA lyase to be described is encoded by a different gene, HMGCLL1, and is located in the cytosolic side of the endoplasmic reticulum membrane. Some activity assays and tissue distribution of this enzyme have shown the brain and lung as key tissues for studying its function. Although the roles of the peroxisomal and cytosolic HMG-CoA lyases remain unknown, recent studies highlight the role of ketone bodies in metabolic remodeling, homeostasis, and signaling, providing new insights into the molecular and cellular function of these enzymes.

Human Mitochondrial HMG-CoA Synthase Deficiency: Role of Enzyme Dimerization Surface and Characterization of Three New Patients.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mitochondrial HMG-CoA synthase deficiency or mHS deficiency, OMIM #605911) is an inborn error of metabolism that affects ketone body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycemia and dicarboxylic aciduria. The diagnosis is difficult due to the relatively unspecific clinical and biochemical presentation, and fewer than 30 patients have been described. This work describes three new patients with mHS deficiency and two missense mutations c.334C>T (p.R112W) and c.430G>T (p.V144L) previously not reported. We developed a new method to express and measure the activity of the enzyme and in this work the study is extended to ten new missense variants including those of our patients. Enzymatic assays showed that three of the mutant proteins retained some but seven completely lacked activity. The identification of a patient homozygous for a mutation that retains 70% of enzyme activity opens the door to a new interpretation of the disease by demonstrating that a modest impairment of enzyme function can actually produce symptoms. This is also the first study employing molecular dynamics modelling of the enzyme mutations. We show that the correct maintenance of the dimerization surface is crucial for retaining the structure of the active center and therefore the activity of the enzyme.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay.

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg(2+). We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K(+) and Mg(2+) and was inhibited by Na(+). GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.

Special cases in Cornelia de Lange syndrome: The Spanish experience.

Cornelia de Lange Syndrome (CdLS) is an autosomal dominant (NIPBL, SMC3, and RAD21) or X-linked (SMC1A and HDAC8) disorder, characterized by distinctive craniofacial appearance, growth retardation, intellectual disability, and limb anomalies. In 2005, the Spanish CdLS Reference Center was started and now we have more than 270 cases in our database. In this special issue, we describe some of the unique or atypical patients studied by our group, whose clinical features have contributed to the expansion of the CdLS classical phenotype, helping clinicians to diagnose it. We include the case of a male with unilateral tibial hypoplasia and peroneal agenesis who had a mutation in NIPBL; we also describe one patient with a mutation in NIPBL and somatic mosaicism identified by new generation sequencing techniques; we also include one patient with CdLS and Turner syndrome; and last, an interesting patient with a duplication of the SMC1A gene. Finally, we make a short review of the splicing mutations we have found in NIPBL regarding the new knowledge on the physiological variants of the gene. © 2016 Wiley Periodicals, Inc.

MEPSA: minimum energy pathway analysis for energy landscapes.

UNLABELLED: From conformational studies to atomistic descriptions of enzymatic reactions, potential and free energy landscapes can be used to describe biomolecular systems in detail. However, extracting the relevant data of complex 3D energy surfaces can sometimes be laborious. In this article, we present MEPSA (Minimum Energy Path Surface Analysis), a cross-platform user friendly tool for the analysis of energy landscapes from a transition state theory perspective. Some of its most relevant features are: identification of all the barriers and minima of the landscape at once, description of maxima edge profiles, detection of the lowest energy path connecting two minima and generation of transition state theory diagrams along these paths. In addition to a built-in plotting system, MEPSA can save most of the generated data into easily parseable text files, allowing more versatile uses of MEPSA's output such as the generation of molecular dynamics restraints from a calculated path. AVAILABILITY AND IMPLEMENTATION: MEPSA is freely available (under GPLv3 license) at: http://bioweb.cbm.uam.es/software/MEPSA/ CONTACT: pagomez@cbm.csic.es. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vivo functional and molecular characterization of the Penicillin-Binding Protein 4 (DacB) of Pseudomonas aeruginosa.

BACKGROUND: Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to ß-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of the Enterobacteriaceae family. This regulatory system also participates directly in peptidoglycan turnover and recycling. One of the regulatory mechanisms for AmpC expression, recently identified in clinical isolates, is the inactivation of LMM-PBP4 (Low-Molecular-Mass Penicillin-Binding Protein 4), a protein whose catalytic activity on natural substrates has remained uncharacterized until now. RESULTS: We carried out in vivo activity trials for LMM-PBP4 of Pseudomonas aeruginosa on macromolecular peptidoglycan of Escherichia coli and Pseudomonas aeruginosa. The results showed a decrease in the relative quantity of dimeric, trimeric and anhydrous units, and a smaller reduction in monomer disaccharide pentapeptide (M5) levels, validating the occurrence of D,D-carboxypeptidase and D,D-endopeptidase activities. Under conditions of induction for this protein and cefoxitin treatment, the reduction in M5 is not fully efficient, implying that LMM-PBP4 of Pseudomonas aeruginosa presents better behaviour as a D,D-endopeptidase. Kinetic evaluation of the direct D,D-peptidase activity of this protein on natural muropeptides M5 and D45 confirmed this bifunctionality and the greater affinity of LMM-PBP4 for its dimeric substrate. A three-dimensional model for the monomeric unit of LMM-PBP4 provided structural information which supports its catalytic performance. CONCLUSIONS: LMM-PBP4 of Pseudomonas aeruginosa is a bifunctional enzyme presenting both D,D-carboxypeptidase and D,D-endopeptidase activities; the D,D-endopeptidase function is predominant. Our study provides unprecedented functional and structural information which supports the proposal of this protein as a potential hydrolase-autolysin associated with peptidoglycan maturation and recycling. The fact that mutant PBP4 induces AmpC, may indicate that a putative muropeptide-subunit product of the DD-EPase activity of PBP4 could be a negative regulator of the pathway. This data contributes to understanding of the regulatory aspects of resistance to ß-lactam antibiotics in this bacterial model.

Torsion and curvature of FtsZ filaments.

FtsZ filaments participate in bacterial cell division, but it is still not clear how their dynamic polymerization and shape exert force on the underlying membrane. We present a theoretical description of individual filaments that incorporates information from molecular dynamic simulations. The structure of the crystallized Methanococcus jannaschii FtsZ dimer was used to model a FtsZ pentamer that showed a curvature and a twist. The estimated bending and torsion angles between monomers and their fluctuations were included in the theoretical description. The MD data also permitted positioning the curvature with respect to the protein coordinates and allowed us to explore the effect of the relative orientation of the preferred curvature with respect to the surface plane. We find that maximum tension is attained when filaments are firmly attached and oriented with their curvature perpendicular to the surface and that the twist serves as a valve to release or to tighten the tension exerted by the curved filaments on the membrane. The theoretical model also shows that the presence of torsion can explain the shape distribution of short filaments observed by Atomic Force Microscopy in previously published experiments. New experiments with FtsZ covalently attached to lipid membranes show that the filament on-plane curvature depends on lipid head charge, confirming the predicted monomer orientation effects. This new model underlines the fact that the combination of the three elements, filament curvature, twist and the strength and orientation of its surface attachment, can modulate the force exerted on the membrane during cell division.