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Targeted protein degradation (TPD) allows cells to maintain a functional proteome and to rapidly adapt to changing conditions. Methods that repurpose TPD for the deactivation of specific proteins have demonstrated significant potential in therapeutic and research applications. Most of these methods are based on proteolysis targeting chimaeras (PROTACs) which link the protein target to an E3 ubiquitin ligase, resulting in the ubiquitin-based degradation of the target protein. In this study, we introduce a method for ubiquitin-independent TPD based on nanobody-conjugated plant ubiquitin regulatory X domain-containing (PUX) adaptor proteins. We show that the PUX-based NAnobody Degraders (P-NADs) can unfold a target protein through the Arabidopsis and human orthologues of the CDC48 unfoldase without the need for ubiquitination or initiating motifs. We demonstrate that P-NAD plasmids can be transfected into a human cell line, where the produced P-NADs use the endogenous CDC48 machinery for ubiquitin-independent TPD of a 143 kDa multidomain protein. Thus, P-NADs pave the road for ubiquitin-independent therapeutic TPD approaches. In addition, the modular P-NAD design combined with in vitro and cellular assays provide a versatile platform for elucidating functional aspects of CDC48-based TPD in plants and animals.
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Phenolic benzotriazole UV stabilizers (BUV) are commonly used additives in synthetic polymeric products, which constantly leak into the environment. They are persistent and bioaccumulative, and have been detected not only in fish, birds, and sea mammals, but also in humans, including breast milk samples. Several authorities including the European Chemical Agency already consider some BUVs as Substances of Very High Concern in need of further information, e.g. mechanistical studies and biomonitoring. In this study, we are addressing this need by investigating the effect of several BUVs on the activity of the human epidermal growth factor receptor (EGFR), an important regulator of cellular processes that has recently been identified as a cell-surface receptor for environmental organic chemicals. By combining in silico docking, mutant analyses, receptor binding and internalization assays, we demonstrate that BUVs, particularly the chlorinated variants, bind to the extracellular domain of EGFR and thereby prevent the binding of growth factors. Accordingly, BUVs can inhibit EGFR downstream events, such as ERK1/2 phosphorylation and DNA synthesis, in human keratinocytes. Our data establish EGFR as a plasma membrane receptor for BUVs, offering novel mechanistic insights into the biological effects induced by these widespread and persistent chemicals. The findings of this study may not only improve hazard assessment for BUVs, but also contribute to the development of novel EGFR-targeting drugs.
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Congenital insensitivity to pain is a rare human condition in which affected individuals do not experience pain throughout their lives. This study aimed to identify the molecular etiology of congenital insensitivity to pain in two Thai patients. Clinical, radiographic, histopathologic, immunohistochemical, and molecular studies were performed. Patients were found to have congenital insensitivity to pain, self-mutilation, acro-osteolysis, cornea scars, reduced temperature sensation, tooth agenesis, root maldevelopment, and underdeveloped maxilla and mandible. The skin biopsies revealed fewer axons, decreased vimentin expression, and absent neurofilament expression, indicating lack of dermal nerves. Whole exome and Sanger sequencing identified a rare homozygous variant c.4039C>T; p.Arg1347Cys in the plakin domain of Plec, a cytolinker protein. This p.Arg1347Cys variant is in the spectrin repeat 9 region of the plakin domain, a region not previously found to harbor pathogenic missense variants in other plectinopathies. The substitution with a cysteine is expected to decrease the stability of the spectrin repeat 9 unit of the plakin domain. Whole mount in situ hybridization and an immunohistochemical study suggested that Plec is important for the development of maxilla and mandible, cornea, and distal phalanges. Additionally, the presence of dental anomalies in these patients further supports the potential involvement of Plec in tooth development. This is the first report showing the association between the Plec variant and congenital insensitivity to pain in humans.
Assuntos
Homozigoto , Insensibilidade Congênita à Dor , Plectina , Humanos , Masculino , Plectina/genética , Plectina/metabolismo , Feminino , Insensibilidade Congênita à Dor/genética , Criança , Linhagem , Mutação de Sentido Incorreto , Sequenciamento do ExomaRESUMO
The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.
Assuntos
Sistemas CRISPR-Cas , Citosol , Humanos , Citosol/metabolismo , DNA/química , DNA/metabolismo , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Proteínas/química , Proteínas/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Células HeLa , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismoRESUMO
Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.
Assuntos
Proteínas de Ciclo Celular , Deficiência Intelectual , Microcefalia , Linhagem , Humanos , Microcefalia/genética , Deficiência Intelectual/genética , Masculino , Feminino , Proteínas de Ciclo Celular/genética , Adulto , Proteínas Cromossômicas não Histona/genética , Proteínas do Tecido Nervoso/genética , Divisão Celular/genética , Mutação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Genômica , Adulto Jovem , Consanguinidade , Sequenciamento do Exoma , Homozigoto , Deficiências do Desenvolvimento/genética , Adolescente , Paquistão , Proteínas Associadas aos MicrotúbulosRESUMO
The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional "tertiary" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.
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Wiedemann-Rautenstrauch Syndrome (WRS; MIM 264090) is an extremely rare and highly heterogeneous syndrome that is inherited in a recessive fashion. The patients have hallmark features such as prenatal and postnatal growth retardation, short stature, a progeroid appearance, hypotonia, facial dysmorphology, hypomyelination leukodystrophy, and mental impairment. Biallelic disease-causing variants in the RNA polymerase III subunit A (POLR3A) have been associated with WRS. Here, we report the first identified cases of WRS syndrome with novel phenotypes in three consanguineous families (two Omani and one Saudi) characterized by biallelic variants in POLR3A. Using whole-exome sequencing, we identified one novel homozygous missense variant (NM_007055: c.2456C>T; p. Pro819Leu) in two Omani families and one novel homozygous variant (c.1895G>T; p Cys632Phe) in Saudi family that segregates with the disease in the POLR3A gene. In silico homology modeling of wild-type and mutated proteins revealed a substantial change in the structure and stability of both proteins, demonstrating a possible effect on function. By identifying the homozygous variants in the exon 14 and 18 of the POLR3A gene, our findings will contribute to a better understanding of the phenotype-genotype relationship and molecular etiology of WRS syndrome.
Assuntos
Progéria , Gravidez , Feminino , Humanos , Fenótipo , Progéria/genética , Retardo do Crescimento Fetal/genética , Mutação de Sentido Incorreto , Síndrome , RNA Polimerase III/genéticaRESUMO
Electronic immunosensors are indispensable tools for diagnostics, particularly in scenarios demanding immediate results. Conventionally, these sensors rely on the chemical immobilization of antibodies onto electrodes. However, globular proteins tend to adsorb and unfold on these surfaces. Therefore, self-assembled monolayers (SAMs) of thiolated alkyl molecules are commonly used for indirect gold-antibody coupling. Here, a limitation associated with SAMs is revealed, wherein they curtail the longevity of protein sensors, particularly when integrated into the state-of-the-art transducer of organic bioelectronics-the organic electrochemical transistor. The SpyDirect method is introduced, generating an ultrahigh-density array of oriented nanobody receptors stably linked to the gold electrode without any SAMs. It is accomplished by directly coupling cysteine-terminated and orientation-optimized spyTag peptides, onto which nanobody-spyCatcher fusion proteins are autocatalytically attached, yielding a dense and uniform biorecognition layer. The structure-guided design optimizes the conformation and packing of flexibly tethered nanobodies. This biolayer enhances shelf-life and reduces background noise in various complex media. SpyDirect functionalization is faster and easier than SAM-based methods and does not necessitate organic solvents, rendering the sensors eco-friendly, accessible, and amenable to scalability. SpyDirect represents a broadly applicable biofunctionalization method for enhancing the cost-effectiveness, sustainability, and longevity of electronic biosensors, all without compromising sensitivity.
Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Ouro/química , Eletrodos , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Anticorpos de Domínio Único/químicaRESUMO
BACKGROUND: Early infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. METHODS: Four families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. RESULTS: We present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). CONCLUSIONS: Our results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.
Assuntos
Encefalopatias , Epilepsia , Espasmos Infantis , Simportadores , Recém-Nascido , Humanos , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/genética , Espasmos Infantis/patologia , Epilepsia/genética , Encefalopatias/diagnóstico por imagem , Encefalopatias/genética , Mutação/genética , Síndrome , Ácido Cítrico , Simportadores/genéticaRESUMO
Congenital muscular dystrophies are a group of progressive disorders with wide range of symptoms associated with diverse cellular mechanisms. Recently, biallelic variants in GGPS1 were linked to a distinct autosomal recessive form of muscular dystrophy associated with hearing loss and ovarian insufficiency. In this report, we present a case of a young patient with a homozygous variant in GGPS1. The patient presented with only proximal muscle weakness, and elevated liver transaminases with spared hearing function. The hepatic involvement in this patient caused by a novel deleterious variant in the gene extends the phenotypic and genotypic spectrum of GGPS1 related muscular dystrophy.
Assuntos
Surdez , Dimetilaliltranstransferase , Perda Auditiva , Distrofias Musculares , Insuficiência Ovariana Primária , Feminino , Humanos , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Homozigoto , Dimetilaliltranstransferase/genética , Geraniltranstransferase/genética , Farnesiltranstransferase/genéticaRESUMO
The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.
Assuntos
COVID-19 , Sequenciamento por Nanoporos , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Teste para COVID-19 , Pandemias , Análise Custo-Benefício , Reprodutibilidade dos Testes , Técnicas de Laboratório Clínico/métodos , RNA Viral/genética , RNA Viral/análise , Sensibilidade e Especificidade , GenômicaRESUMO
Understanding how a gene variant affects protein function is important in life science, as it helps explain traits or dysfunctions in organisms. In a clinical setting, this understanding makes it possible to improve and personalize patient care. Bioinformatic tools often only assign a pathogenicity score, rather than providing information about the molecular basis for phenotypes. Experimental testing can furnish this information, but this is slow and costly and requires expertise and equipment not available in a clinical setting. Conversely, mapping a gene variant onto the three-dimensional (3D) protein structure provides a fast molecular assessment free of charge. Before 2021, this type of analysis was severely limited by the availability of experimentally determined 3D protein structures. Advances in artificial intelligence algorithms now allow confident prediction of protein structural features from sequence alone. The aim of the protocols presented here is to enable non-experts to use databases and online tools to investigate the molecular effect of a genetic variant. The Basic Protocol relies only on the online resources AlphaFold, Protein Structure Database, and UniProt. Alternate Protocols document the usage of the Protein Data Bank, SWISS-MODEL, ColabFold, and PyMOL for structure-based variant analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: 3D Mapping based on UniProt and AlphaFold Alternate Protocol 1: Using experimental models from the PDB Alternate Protocol 2: Using information from homology modeling with SWISS-MODEL Alternate Protocol 3: Predicting 3D structures with ColabFold Alternate Protocol 4: Structure visualization and analysis with PyMOL.
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Inteligência Artificial , Proteínas , Modelos Moleculares , Proteínas/química , Proteínas/genética , Algoritmos , Bases de Dados de ProteínasRESUMO
Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation.
Assuntos
Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Tirosina , Fosforilação , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Ligantes , Membrana CelularRESUMO
MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Arabidopsis/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Sistema de Sinalização das MAP Quinases , Arabidopsis/metabolismo , Imunidade Vegetal/genéticaRESUMO
Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Paraplegia Espástica Hereditária , Animais , Humanos , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Peixe-Zebra , Mutação , Neurônios Motores , Receptores do Fator Autócrino de Motilidade/genéticaRESUMO
A mesiodens is a supernumerary tooth located in the midline of the premaxilla. To investigate the genetic cause of mesiodens, clinical and radiographic examination were performed on 23 family members of a two-generation Hmong family. Whole exome sequencing (WES) or Sanger sequencing were performed in 22 family members and two unrelated Thai patients with mesiodens. WES in the Hmong family revealed a missense mutation (c.1807G>A;p.Glu603Lys) in PTPN23 in seven affected members and six unaffected members. The mode of inheritance was autosomal dominance with incomplete penetrance (53.84%). Two additional mutations in PTPN23, c.2248C>G;p.Pro750Ala and c.3298C>T;p.Arg1100Cys were identified in two unrelated patients with mesiodens. PTPN23 is a regulator of endosomal trafficking functioning to move activated membrane receptors, such as EGFR, from the endosomal sorting complex towards the ESCRT-III complex for multivesicular body biogenesis, lysosomal degradation, and subsequent downregulation of receptor signaling. Immunohistochemical study and RNAscope on developing mouse embryos showed broad expression of PTPN23 in oral tissues, while immunofluorescence showed that EGFR was specifically concentrated in the midline epithelium. Importantly, PTPN23 mutant protein was shown to have reduced phosphatase activity. In conclusion, mesiodens were associated with genetic variants in PTPN23, suggesting that mesiodens may form due to defects in endosomal trafficking, leading to disrupted midline signaling.
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Compound heterozygous mutations in SHQ1 have been associated with a rare and severe neurological disorder characterized by global developmental delay (GDD), cerebellar degeneration coupled with seizures, and early-onset dystonia. Currently, only five affected individuals have been documented in the literature. Here, we report three children from two unrelated families harboring a homozygous variant in the gene but with a milder phenotype than previously described. The patients had GDD and seizures. Magnetic resonance imaging analyses revealed diffuse white matter hypomyelination. Sanger sequencing confirmed the whole-exome sequencing results and revealed full segregation of the missense variant (SHQ1:c.833 T > C; p.I278T) in both families. We performed a comprehensive in silico analysis using different prediction classifiers and structural modeling of the variant. Our findings demonstrate that this novel homozygous variant in SHQ1 is likely to be pathogenic and leads to the clinical features observed in our patients.
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BACKGROUND: Phosphodiesterase 10A (PDE10A) controls body movements by regulating cyclic adenosine monophosphate signaling in the basal ganglia. Two classes of PDE10A variants are reported with distinctive genotype-phenotype correlation. The autosomal recessive mutations in the GAF-A and catalytic domains are associated with compromised membrane localization, and manifest with infantile onset chorea, developmental, and cognition delay with normal brain MRI. Conversely, autosomal dominant mutations in the GAF-B domain cause protein aggregates which results in childhood onset chorea in the context of normal cognition and development, with striatal lesions. METHODS: Phenotypic characteristics of affected individuals with PDE10A mutations belonging to a single family were recorded. In addition, Sanger sequencing and in silico analysis were used to identify the mutations. Homozygosity mapping was applied together with whole exome sequencing. RESULTS: Four individuals from a consanguineous family affected with PDE10A mutations were observed for up to 40 years. Although these individuals displayed a clinical phenotype attributed to the recessive GAF-A mutations, they revealed a bi-allelic GAF-B mutation (c.883G > A:p. D295 N; p.Asp295Asn) that was segregated with all affected individuals. In addition to chorea, we observed peculiar foot deformities and pronounced social phobia, with normal brain MRI. In silico structural analysis suggested that the GAF-B mutation blocked allosteric PDE10A activation. The resulting lack of PDE10A activity likely phenocopies GAF-A mutations, and this is achieved through a distinct mechanism. CONCLUSIONS: Collectively, our findings demonstrate the association of recessive and dominant phenotypes of known variants, and further expands the genotype-phenotype landscape of PDE10A-associated movement disorders.
Assuntos
Coreia , Transtornos dos Movimentos , Humanos , Coreia/genética , Diester Fosfórico Hidrolases/genética , Genótipo , FenótipoRESUMO
Heterozygous pathogenic variants in DNM1 are linked to an autosomal dominant form of epileptic encephalopathy. Recently, homozygous loss-of-function variants in DNM1 were reported to cause an autosomal recessive form of developmental and epileptic encephalopathy in unrelated patients. Here, we investigated a singleton from a first-degree cousin marriage who presented with facial dysmorphism, global developmental delay, seizure disorder, and nystagmus. To identify the involvement of any likely genetic cause, diagnostic clinical exome sequencing was performed. Comprehensive filtering revealed a single plausible candidate variant in DNM1. Sanger sequencing of the trio, the patient, and her parents, confirmed the full segregation of the variant. The variant is a deletion leading to a premature stop codon and is predicted to cause a protein truncation. Structural modeling implicated a complete loss of function of the Dynamin 1 (DNM1). Such mutation is predicted to impair the nucleotide binding, dimer formation, and GTPase activity of DNM1. Our study expands the phenotypic spectrum of pathogenic homozygous loss-of-function variants in DNM1.
Assuntos
Epilepsia Generalizada , Epilepsia , Feminino , Humanos , Dinamina I/genética , Epilepsia/genética , Homozigoto , MutaçãoRESUMO
The genetic architecture of mitochondrial disease continues to expand and currently exceeds more than 350 disease-causing genes. Bi-allelic variants in RTN4IP1, also known as Optic Atrophy-10 (OPA10), lead to early-onset recessive optic neuropathy, atrophy, and encephalopathy in the afflicted patients. The gene is known to encode a mitochondrial ubiquinol oxidoreductase that interacts with reticulon 4 and is thought to be a mitochondrial antioxidant NADPH oxidoreductase. Here, we describe two unrelated consanguineous families from the northern region of Saudi Arabia harboring a missense variant (RTN4IP1:NM_032730.5; c.475GAssuntos
Encefalopatias
, Atrofia Óptica
, Antioxidantes
, Proteínas de Transporte/genética
, Humanos
, Proteínas Mitocondriais/genética
, Mutação/genética
, NADP/genética
, Atrofia Óptica/genética
, Oxirredutases/genética
, Arábia Saudita