KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the ß-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

Transcriptional and spatiotemporal regulation of the dauer program.

Caenorhabditis elegans can enter a diapause stage called "dauer" when it senses that the environment is not suitable for development. This implies a detour from the typical developmental trajectory and requires a tight control of the developmental clock and a massive tissue remodeling. In the last decades, core components of the signaling pathways that govern the dauer development decision have been identified, but the tissues where they function for the acquisition of dauer-specific traits are still under intense study. Growing evidence demonstrates that these pathways engage in complex cross-talk and feedback loops. In this review, we summarize the current knowledge regarding the transcriptional regulation of the dauer program and the relevant tissues for its achievement. A better understanding of this process will provide insight on how developmental plasticity is achieved and how development decisions are under a robust regulation to ensure an all-or-nothing response. Furthermore, this developmental decision can also serve as a simplified model for relevant developmental disorders.Abbreviations: AID Auxin Induced Degron DA dafachronic acid Daf-c dauer formation constitutive Daf-d dauer formation defective DTC Distal Tip Cells ECM modified extracellular matrix GPCRs G protein-coupled receptors IIS insulin/IGF-1 signaling ILPs insulin-like peptides LBD Ligand Binding Domain PDL4 Post Dauer L4 TGF-ß transforming growth factor beta WT wild-type.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex.

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI's PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC's evolution and genetic diversity.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review.

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI’s PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC’s evolution and genetic diversity.

Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects.

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.

Curating the gnomAD database: Report of novel variants in the thyrogobulin gene using in silico bioinformatics algorithms.

Thyroglobulin (TG) is a large glycosylated protein of 2767 amino acids, secreted by the thyrocytes into the follicular lumen. It plays an essential role in the process of thyroid hormone synthesis. TG gene variants lead to permanent congenital hypothyroidism. In the present work, we report a detailed population and bioinformatic prediction analyses of the TG variants indexed in the Genome Aggregation Database (gnomAD). The results showed a clear predominance of nonsense variants in the European (Finnish), European (Non-Finnish) and Ashkenazi Jewish ethnic groups, whereas the splice site variants predominate in South Asian and African/African-American populations. In total, 282 novel TG variants were described (47 missense involving the wild-type cysteine residues, 177 missense located in the ChEL domain and 58 splice site variants) which were not reported in the literature and that would have deleterious effects in prediction programs. In the gnomAD population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:320. In conclusion, we provide an updated and curated reference source for the diagnosis of thyroid disease, mainly to congenital hypothyroidism due to TG deficiency. The identification and characterization of TG variants is undoubtedly a valuable approach to study the TG structure/function relations and an important tool for clinical diagnosis and genetic counseling.

SCN5A compound heterozygosity mutation in Brugada syndrome: Functional consequences and the implication for pharmacological treatment.

AIMS: SCN5A gene encodes the α-subunit of Nav1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Nav1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na+ channels in vitro. MATERIALS AND METHODS: The functional properties of human wild-type and Nav1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells. KEY FINDINGS: Both variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na+ current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na+ channel. SIGNIFICANCE: De novo compound heterozygosis mutations in SNC5A disrupt the Na+ macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na+ current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na+ channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Nav1.5.

Loss of STI1-mediated neuronal survival and differentiation in disease-associated mutations of prion protein.

Cellular prion protein (PrPC ) is widely expressed and displays a variety of well-described functions in the central nervous system (CNS). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain- or loss-of-function mutations to PrPC remain a matter for debate. Among the proteins described to interact with PrPC is Stress-inducible protein 1 (STI1), a co-chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrPC . In this work, we evaluated the impact of different PrPC pathogenic point mutations on signaling pathways induced by the STI1-PrPC interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase A (PKA) signaling triggered by STI1-PrPC engagement. A pathogenic mutant PrPC that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild-type PrPC . Also, a STI1-α7-nicotinic acetylcholine receptor-dependent cellular signaling was present in a PrPC mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild-type PrPC . These results point to a loss-of-function mechanism underlying the pathogenicity of PrPC mutations.

Role of the CHD7 chromatin remodeler protein in glioblastoma multiformePalavras-chave em inglês Cell motility / Papel do remodelador de cromatina CHD7 em glioblastoma multiforme

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, rendering the transcriptional machinery available to the condensed genomic DNA. Due to this central role in regulating gene transcription, deregulation of these molecular machines may lead to severe perturbations in the normal cell functions. Loss-of-function mutations in the CHD7 gene, a member of the chromodomain helicase DNA-binding (CHD) family, are the major cause of the CHARGE syndrome in humans. The disease is characterized by a variety of congenital anomalies, including malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. In this context, several studies have already shown the importance of CHD7 for proper function of the neural stem cells (NSCs). Interestingly, we found that CHD7 mRNA levels are upregulated in gliomas, when compared to normal brain tissue, therefore, we hypothesized that CHD7 might have a role in the pathogenesis of these tumors. To investigate the possible oncogenic role of CHD7 in glioblastoma (GBM), we adopted gain- and loss-of-function approaches in adherent GBM cell lines. Using CRISPR_Cas9 genome editing, we found that CHD7 deletion suppresses anchorage-independent growth and reduces spheroid invasion in human LN-229 cells. Moreover, deletion of CHD7 delayed tumor growth and improved overall survival in an orthotopic xenograft glioma mouse model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 cells was found to increase cell motility and invasiveness in vitro and LN-428 tumor growth in vivo. RNAseq analysis showed that alterations of CHD7 expression levels promote changes in several molecular pathways and modulate critical genes associated with cell adhesion and locomotion. However, the mechanisms underlying the effects of CHD7 overexpression in glioma tissue are still not understood. Here, we also generated recombinant plasmid with functional CHD7 promoter activity reported by luciferase assay. This powerful tool should enable future studies to determine the direct targeting relationship between different signal transduction pathways and CHD7 geneexpression. In summary, our findings indicate that GBM cells expressing a high level of CHD7 may exist and contribute to tumor infiltration and recurrence. Further studies should warrant important clinical-translational implications of our findings for GBM treatment

As proteínas remodeladoras de cromatina exercem importante papel, promovendo modificações dinâmicas na arquitetura da cromatina e dando acesso à maquinaria transcricional ao DNA genômico condensado. Devido à esta função central na regulação da transcrição gênica, a desregulação dessas máquinas moleculares pode levar a perturbações graves na função normal das células. Assim, por exemplo, mutações do tipo perda de função no gene CHD7, um membro da família "chromodomain helicase DNA-binding" (CHD), são a principal causa da síndrome de CHARGE em humanos. A doença é caracterizada por uma variedade de anomalias congênitas, incluindo malformações das estruturas craniofaciais, sistema nervoso periférico, orelhas, olhos e coração. Neste contexto, vários estudos já mostraram a importância da proteína CHD7 para o funcionamento normal de células-tronco neurais (NSCs). Curiosamente, descobrimos que os níveis de mRNA de CHD7 estão mais fortemente expressos em gliomas, quando comparados ao tecido cerebral normal, portanto, nós hipotetizamos que CHD7 poderia ter um papel na patogênese desses tumores. Para investigar o possível papel oncogênico de CHD7 em glioblastoma (GBM), utilizamos enfoques de ganho e perda de função em linhagens celulares aderentes de GBM. Utilizando a técnica de CRISPR_Cas9 para edição do genoma, demonstramos que a deleção do gene CHD7 suprime o crescimento independente de ancoragem e reduz a invasão de esferóides em células LN-229 humanas de GBM. Além disso, a deleção de CHD7 reduziu o crescimento do tumor e melhorou a sobrevida em modelo de injeção ortotópica xenográfica em camundongo. Por outro lado, verificou-se que a super-expressão ectópica de CHD7 nas células LN-428 e A172 aumenta não só a motilidade celular e a capacidade de invasão in vitro, mas, também, o crescimento do tumor de LN-428 in vivo. A análise de RNA-seq mostrou que o nocauteamento da sequência codificadora de CHD7 e sua super-expressão promovem alterações em diversas vias moleculares, modulando genes críticosassociados à adesão e locomoção celular. No entanto, os mecanismos subjacentes aos efeitos da super-expressão de CHD7 em tecidos de glioma ainda não são compreendidos. Neste trabalho, geramos um plasmídeo recombinante contendo um fragmento da região promotora de CHD7, o qual se mostrou funcional em ensaios de luciferase. Esta ferramenta permitirá que estudos futuros possam identificar a relação direta entre as diferentes vias de transdução de sinal e a expressão do gene CHD7. Em resumo, nossos achados indicam que células de GBM expressando um alto nível de CHD7 podem existir e contribuir para a infiltração e recorrência do tumor. Estudos posteriores deverão avaliar as possíveis implicações dos resultados apresentados neste trabalho para a translação clínica no tratamento de pacientes com GBM

Characterization of specific allosteric effects of the Na+ channel ß1 subunit on the Nav1.4 isoform.

The mechanism of inactivation of mammalian voltage-gated Na+ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the ß1 subunit. Navß1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na+ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNav1.4 (skeletal muscle isoform of the rat Na+ channel) on the α-ß1 functional interaction and showed for the first time that the modulation of ß1 is independent of the IFM motif. We found that: (1) Nav1.4 channels that lack the IFM inactivation particle can undergo a "C-type-like inactivation" albeit in an ultraslow gating mode; (2) ß1 can significantly accelerate the inactivation of Nav1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the ß1 subunit that disrupt the α-ß1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na+ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting ß subunits to treat pathologies associated to Na+ current dysfunction.

Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.