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1.
Cell Mol Life Sci ; 78(16): 6017-6031, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34245311

RESUMO

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic ß-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proinsulina/metabolismo , Adolescente , Células Cultivadas , Cisteína/genética , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/genética , Dissulfetos/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Mutação de Sentido Incorreto/genética , Proinsulina/genética , Dobramento de Proteína
2.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198491

RESUMO

Rare germline pathogenic TP53 missense variants often predispose to a wide spectrum of tumors characterized by Li-Fraumeni syndrome (LFS) but a subset of variants is also seen in families with exclusively hereditary breast cancer (HBC) outcomes. We have developed a logistic regression model with the aim of predicting LFS and HBC outcomes, based on the predicted effects of individual TP53 variants on aspects of protein conformation. A total of 48 missense variants either unique for LFS (n = 24) or exclusively reported in HBC (n = 24) were included. LFS-variants were over-represented in residues tending to be buried in the core of the tertiary structure of TP53 (p = 0.0014). The favored logistic regression model describes disease outcome in terms of explanatory variables related to the surface or buried status of residues as well as their propensity to contribute to protein compactness or protein-protein interactions. Reduced, internally validated models discriminated well between LFS and HBC (C-statistic = 0.78-0.84; equivalent to the area under the ROC (receiver operating characteristic) curve), had a low risk for over-fitting and were well calibrated in relation to the known outcome risk. In conclusion, this study presents a phenotypic prediction model of LFS and HBC risk for germline TP53 missense variants, in an attempt to provide a complementary tool for future decision making and clinical handling.


Assuntos
Neoplasias da Mama/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Síndrome de Li-Fraumeni/genética , Mutação de Sentido Incorreto/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Modelos Logísticos , Análise Multivariada , Fenótipo , Conformação Proteica
3.
J Biol Chem ; 297(1): 100858, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097874

RESUMO

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-ß-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.


Assuntos
Córnea/ultraestrutura , Distrofias Hereditárias da Córnea/genética , Proteínas da Matriz Extracelular/genética , Agregados Proteicos/genética , Fator de Crescimento Transformador beta/genética , Amiloide/genética , Amiloide/ultraestrutura , Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Cristalografia por Raios X , Proteínas da Matriz Extracelular/ultraestrutura , Humanos , Mutação de Sentido Incorreto/genética , Multimerização Proteica/genética
4.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067798

RESUMO

In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the PANX1 gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.


Assuntos
Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Conexinas/genética , Conexinas/fisiologia , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Fenótipo , Sono/genética
5.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065616

RESUMO

We have performed 280 µs of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the frequently mutated proteins in lung cancer. The aim was to provide structural insight into the effects of these mutants, including a new class of ANCHOR (additionally NRF2-complexed hypomorph) mutant variants. Our work provides additional insight into the structural dynamics of mutants that could not be analyzed experimentally, painting a more complete picture of their mutagenic effects. Notably, blade-wise analysis of the Kelch domain points to stability as a possible target of cancer in KEAP1. Interestingly, structural analysis of the R470C ANCHOR mutant, the most prevalent missense mutation in KEAP1, revealed no significant change in structural stability or NRF2 binding site dynamics, possibly indicating an covalent modification as this mutant's mode of action.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Estabilidade Proteica
6.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063805

RESUMO

Large scale genome sequencing allowed the identification of a massive number of genetic variations, whose impact on human health is still unknown. In this review we analyze, by an in silico-based strategy, the impact of missense variants on cancer-related genes, whose effect on protein stability and function was experimentally determined. We collected a set of 164 variants from 11 proteins to analyze the impact of missense mutations at structural and functional levels, and to assess the performance of state-of-the-art methods (FoldX and Meta-SNP) for predicting protein stability change and pathogenicity. The result of our analysis shows that a combination of experimental data on protein stability and in silico pathogenicity predictions allowed the identification of a subset of variants with a high probability of having a deleterious phenotypic effect, as confirmed by the significant enrichment of the subset in variants annotated in the COSMIC database as putative cancer-driving variants. Our analysis suggests that the integration of experimental and computational approaches may contribute to evaluate the risk for complex disorders and develop more effective treatment strategies.


Assuntos
Mutação de Sentido Incorreto/genética , Neoplasias/genética , Biologia Computacional/métodos , Simulação por Computador , Humanos , Estabilidade Proteica , Proteínas/genética
7.
Front Immunol ; 12: 621503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995346

RESUMO

In common variable immunodeficiency (CVID), heterozygous damaging NFKB1 variants represent the most frequent monogenic cause. NFKB1 encodes the precursor p105, which undergoes proteasomal processing to generate the mature NF-κB transcription factor subunit p50. The majority of NFKB1 sequence changes comprises missense variants of uncertain significance (VUS), each requiring functional evaluation to assess causality, particularly in families with multiple affected members presenting with different phenotypes. In four affected members of a German family, all diagnosed with CVID, we identified a previously uncharacterized heterozygous NFKB1 missense variant (c.1049A>G; p.Tyr350Cys). The clinical phenotypes varied markedly regarding onset, frequency and severity of infections. Consistent immunologic findings were hypogammaglobulinemia with normal specific antibody response to protein- and polysaccharide-based vaccinations, reduced switched memory B cells and decreased lymphocyte proliferation upon stimulation with the B cell mitogen SAC. To assess the pathogenicity of the NFKB1 missense variant, we employed immunophenotyping and functional analyses in a routine in vitro cell culture model. Following site-directed mutagenesis to introduce the variant into overexpression vectors encoding EGFP-fused p105 or p50, we analyzed transiently transfected HEK293T cells by confocal imaging and Western blotting. The cytoplasmic p105-Tyr350Cys precursor gained only weak expression levels indicating accelerated decay. The missense change disabled processing of the precursor to prevent the generation of mutant p50. Unlike the wildtype p50, the overexpressed mutant p50-Tyr350Cys was also not sustainable and showed a conspicuous subnuclear mislocalization with accumulation in dense aggregates instead of a homogenous distribution. Electrophoretic mobility shift assays, fluorescence-based reporter gene analyses and co-transfection experiments however demonstrated, that the DNA-binding activity of p50-Tyr350Cys and the interaction with RelA(p65), IκBα and wildtype p50 were preserved. Mutation carriers had reduced p105 and p50 levels, indicating insufficient protein amounts as the most likely primary defect. In conclusion, the missense variant c.1049A>G caused a detrimental defect, preventing the persistent expression of both, the p105-Tyr350Cys precursor and the mature p50-Tyr350Cys. The variable clinical phenotypes among affected family members sharing an identical pathogenic NFKB1 variant support a disease mechanism provoked by a p105/p50 (haplo)insufficient condition.


Assuntos
Imunodeficiência de Variável Comum/genética , Mutação de Sentido Incorreto/genética , Subunidade p50 de NF-kappa B/genética , Alemanha , Células HEK293 , Haploinsuficiência , Heterozigoto , Humanos , Linhagem , Fenótipo , Ligação Proteica , Transporte Proteico , Proteólise
8.
Neurology ; 96(18): e2251-e2260, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34038384

RESUMO

OBJECTIVE: To identify the causative gene in a large unsolved family with genetic epilepsy with febrile seizures plus (GEFS+), we sequenced the genomes of family members, and then determined the contribution of the identified gene to the pathogenicity of epilepsies by examining sequencing data from 2,772 additional patients. METHODS: We performed whole genome sequencing of 3 members of a GEFS+ family. Subsequently, whole exome sequencing data from 1,165 patients with epilepsy from the Epi4K dataset and 1,329 Australian patients with epilepsy from the Epi25 dataset were interrogated. Targeted resequencing was performed on 278 patients with febrile seizures or GEFS+ phenotypes. Variants were validated and familial segregation examined by Sanger sequencing. RESULTS: Eight previously unreported missense variants were identified in SLC32A1, coding for the vesicular inhibitory amino acid cotransporter VGAT. Two variants cosegregated with the phenotype in 2 large GEFS+ families containing 8 and 10 affected individuals, respectively. Six further variants were identified in smaller families with GEFS+ or idiopathic generalized epilepsy (IGE). CONCLUSION: Missense variants in SLC32A1 cause GEFS+ and IGE. These variants are predicted to alter γ-aminobutyric acid (GABA) transport into synaptic vesicles, leading to altered neuronal inhibition. Examination of further epilepsy cohorts will determine the full genotype-phenotype spectrum associated with SLC32A1 variants.


Assuntos
Epilepsia Generalizada/diagnóstico , Epilepsia Generalizada/genética , Variação Genética/genética , Mutação de Sentido Incorreto/genética , Convulsões Febris/diagnóstico , Convulsões Febris/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Feminino , Estudos de Associação Genética/métodos , Humanos , Masculino , Linhagem
11.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806565

RESUMO

Congenital fibrosis of the extraocular muscles (CFEOM) is a congenital cranial dysinnervation disorder caused by developmental abnormalities affecting cranial nerves/nuclei innervating the extraocular muscles. Autosomal dominant CFEOM arises from heterozygous missense mutations of KIF21A or TUBB3. Although spatiotemporal expression studies have shown KIF21A and TUBB3 expression in developing retinal ganglion cells, it is unclear whether dysinnervation extends beyond the oculomotor system. We aimed to investigate whether dysinnervation extends to the visual system by performing high-resolution optical coherence tomography (OCT) scans characterizing retinal ganglion cells within the optic nerve head and retina. Sixteen patients with CFEOM were screened for mutations in KIF21A, TUBB3, and TUBB2B. Six patients had apparent optic nerve hypoplasia. OCT showed neuro-retinal rim loss. Disc diameter, rim width, rim area, and peripapillary nerve fiber layer thickness were significantly reduced in CFEOM patients compared to controls (p < 0.005). Situs inversus of retinal vessels was seen in five patients. Our study provides evidence of structural optic nerve and retinal changes in CFEOM. We show for the first time that there are widespread retinal changes beyond the retinal ganglion cells in patients with CFEOM. This study shows that the phenotype in CFEOM extends beyond the motor nerves.


Assuntos
Fibrose/patologia , Músculos Oculomotores/patologia , Oftalmoplegia/patologia , Nervo Óptico/patologia , Retina/patologia , Adulto , Nervos Cranianos/patologia , Feminino , Fibrose/genética , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Oftalmoplegia/genética , Disco Óptico/patologia , Fenótipo , Células Ganglionares da Retina/patologia , Tomografia de Coerência Óptica/métodos , Adulto Jovem
12.
Commun Biol ; 4(1): 475, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846513

RESUMO

COVID-19 is a respiratory illness caused by a novel coronavirus called SARS-CoV-2. The viral spike (S) protein engages the human angiotensin-converting enzyme 2 (ACE2) receptor to invade host cells with ~10-15-fold higher affinity compared to SARS-CoV S-protein, making it highly infectious. Here, we assessed if ACE2 polymorphisms can alter host susceptibility to SARS-CoV-2 by affecting this interaction. We analyzed over 290,000 samples representing >400 population groups from public genomic datasets and identified multiple ACE2 protein-altering variants. Using reported structural data, we identified natural ACE2 variants that could potentially affect virus-host interaction and thereby alter host susceptibility. These include variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R that were predicted to increase susceptibility, while variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y were predicted to be protective variants that show decreased binding to S-protein. Using biochemical assays, we confirmed that K31R and E37K had decreased affinity, and K26R and T92I variants showed increased affinity for S-protein when compared to wildtype ACE2. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus suggesting that ACE2 variants can modulate susceptibility to SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Polimorfismo Genético , Receptores Virais/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
13.
Am J Hum Genet ; 108(5): 951-961, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33894126

RESUMO

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and ßIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and ßIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.


Assuntos
Agenesia do Corpo Caloso/genética , Cerebelo/anormalidades , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Adulto , Agenesia do Corpo Caloso/diagnóstico por imagem , Cerebelo/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Hidrolases/química , Hidrolases/genética , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/genética , Masculino , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Transtornos do Neurodesenvolvimento/diagnóstico por imagem , Tubulina (Proteína)/metabolismo , Adulto Jovem
14.
Nat Commun ; 12(1): 2107, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833240

RESUMO

Vacuolar H+-ATPases (V-ATPases) transport protons across cellular membranes to acidify various organelles. ATP6V0A1 encodes the a1-subunit of the V0 domain of V-ATPases, which is strongly expressed in neurons. However, its role in brain development is unknown. Here we report four individuals with developmental and epileptic encephalopathy with ATP6V0A1 variants: two individuals with a de novo missense variant (R741Q) and the other two individuals with biallelic variants comprising one almost complete loss-of-function variant and one missense variant (A512P and N534D). Lysosomal acidification is significantly impaired in cell lines expressing three missense ATP6V0A1 mutants. Homozygous mutant mice harboring human R741Q (Atp6v0a1R741Q) and A512P (Atp6v0a1A512P) variants show embryonic lethality and early postnatal mortality, respectively, suggesting that R741Q affects V-ATPase function more severely. Lysosomal dysfunction resulting in cell death, accumulated autophagosomes and lysosomes, reduced mTORC1 signaling and synaptic connectivity, and lowered neurotransmitter contents of synaptic vesicles are observed in the brains of Atp6v0a1A512P/A512P mice. These findings demonstrate the essential roles of ATP6V0A1/Atp6v0a1 in neuronal development in terms of integrity and connectivity of neurons in both humans and mice.


Assuntos
Encefalopatias/genética , Encéfalo/crescimento & desenvolvimento , Neurônios/fisiologia , Neurotransmissores/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Autofagossomos/patologia , Mapeamento Encefálico/métodos , Catepsina D/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Mutação com Perda de Função/genética , Lisossomos/patologia , Imageamento por Ressonância Magnética/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , Neurônios/citologia , Vesículas Sinápticas/patologia
15.
Biomed Res Int ; 2021: 6685840, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33884270

RESUMO

SARS-CoV-2 coronavirus uses for entry to human host cells a SARS-CoV receptor of the angiotensin-converting enzyme (ACE2) that catalyzes the conversion of angiotensin II into angiotensin (1-7). To understand the effect of ACE2 missense variants on protein structure, stability, and function, various bioinformatics tools were used including SIFT, PANTHER, PROVEAN, PolyPhen2.0, I. Mutant Suite, MUpro, SWISS-MODEL, Project HOPE, ModPred, QMEAN, ConSurf, and STRING. All twelve ACE2 nsSNPs were analyzed. Six ACE2 high-risk pathogenic nsSNPs (D427Y, R514G, R708W, R710C, R716C, and R768W) were found to be the most damaging by at least six software tools (cumulative score between 6 and 7) and exert deleterious effect on the ACE2 protein structure and likely function. Additionally, they revealed high conservation, less stability, and having a role in posttranslation modifications such a proteolytic cleavage or ADP-ribosylation. This in silico analysis provides information about functional nucleotide variants that have an impact on the ACE2 protein structure and function and therefore susceptibility to SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Algoritmos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Biologia Computacional , Simulação por Computador , Humanos , Polimorfismo de Nucleotídeo Único/genética , SARS-CoV-2/química , SARS-CoV-2/metabolismo
16.
Genet Test Mol Biomarkers ; 25(3): 218-226, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33734897

RESUMO

Background: Muscular dystrophies are a heterogeneous group of inherited disorders that cannot be diagnosed clinically due to overlapping clinical phenotypes. Whole-exome sequencing is considered as the diagnostic strategy of choice in these cases. In this study we aimed to determine the mutational spectrum of multiplex ligation-dependent probe amplification (MLPA)-negative muscular dystrophy patients in Pakistan using whole-exome sequencing. Subsequently the mutations identified via WES were used to screen additional dystrophinopathy patients by Sanger sequencing. Materials and Methods: DNA extracted from the peripheral blood of three MLPA-negative muscular dystrophy patients was sent for whole-exome sequencing. The identified variants in these 3 patients were then checked in 18 dystrophinopathy patients using Sanger sequencing. Results: Four missense variants and one nonsense variant in the Duchenne muscular dystrophy (DMD) gene were detected. WES diagnosed a DMD patient carrying a nonsense variant c.4375C>T (rs398123953) who can benefit from Ataluren therapy. The other two patients carried missense variant (c.572G>T) in the YARS2 gene (rs11539445) labeling them as patients of MLASA (myopathy, lactic acidosis, and sideroblastic anemia). The identified missense and nonsense variants in the DMD gene were detected in 18 clinically diagnosed dystrophinopathy patients using Sanger sequencing. Three missense variants were detected in our cohort of 18 dystrophinopathy patients. One missense variant c.3406A>T (rs3827462) and a nonsense variant c.4375C>T (rs398123953) were not detected in our cohort of 18 dystrophinopathy patients. Conclusions: Whole-exome sequencing identified a nonsense variant in Pakistani muscular dystrophy patients, which is amenable to treatment by Ataluren and a missense variant in YARS2 gene responsible for causing MLASA.


Assuntos
Distrofina/genética , Distrofias Musculares/genética , Adolescente , Alelos , Criança , Códon sem Sentido/genética , Éxons/genética , Feminino , Frequência do Gene/genética , Testes Genéticos/métodos , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Distrofias Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Mutação/genética , Mutação de Sentido Incorreto/genética , Paquistão/epidemiologia , Sequenciamento Completo do Exoma/métodos
17.
Am J Med Genet A ; 185(6): 1836-1840, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33650182

RESUMO

Some mammalian genes contain both major and minor introns, the splicing of which require distinctive major and minor spliceosomes, respectively; these genes are referred to as minor intron containing-genes. RNPC3 (RNA-binding domain-containing protein 3) is one of the proteins that are unique to the minor spliceosome U11/U12 di-snRNP. Only two families with biallelic pathogenic variants in the RNPC3 gene encoding the protein have been reported so far, and the affected members in both families had proportional short stature. While the affected members of the originally identified family did not have intellectual disability, the patients from the other family exhibited intellectual disability. Here, we report on a patient with severe primordial microcephalic dwarfism and intellectual disability who carried compound heterozygous variants in RNPC3 (NM_017619.3): c.261dup, p.Leu88Thrfs*11 and c.1228T>G, p.Phe410Val. The single nucleotide substitution c.1228T>G had a very high predictive score for pathogenicity: the p.Phe410 residue is highly conserved down to fish. Based on ACMG (American College of Medical Genetics and Genomics) guideline, this non-synonymous variant was scored as likely pathogenic. This documentation of yet another patient with biallelic RNPC3 variants exhibiting intellectual disability lends further support to the notion that intellectual disability is a key feature of the spectrum of RNPC3-related disorders.


Assuntos
Predisposição Genética para Doença , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/patologia , Íntrons/genética , Mutação de Sentido Incorreto/genética , Spliceossomos/genética , Adulto Jovem
19.
Hum Genet ; 140(7): 1031-1043, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33689014

RESUMO

Cilia and flagella are formed around an evolutionary conserved microtubule-based axoneme and are required for fluid and mucus clearance, tissue homeostasis, cell differentiation and movement. The formation and maintenance of cilia and flagella require bidirectional transit of proteins along the axonemal microtubules, a process called intraflagellar transport (IFT). In humans, IFT defects contribute to a large group of systemic diseases, called ciliopathies, which often display overlapping phenotypes. By performing exome sequencing of a cohort of 167 non-syndromic infertile men displaying multiple morphological abnormalities of the sperm flagellum (MMAF) we identified two unrelated patients carrying a homozygous missense variant adjacent to a splice donor consensus site of IFT74 (c.256G > A;p.Gly86Ser). IFT74 encodes for a core component of the IFT machinery that is essential for the anterograde transport of tubulin. We demonstrate that this missense variant affects IFT74 mRNA splicing and induces the production of at least two distinct mutant proteins with abnormal subcellular localization along the sperm flagellum. Importantly, while IFT74 deficiency was previously implicated in two cases of Bardet-Biedl syndrome, a pleiotropic ciliopathy with variable expressivity, our data indicate that this missense mutation only results in primary male infertility due to MMAF, with no other clinical features. Taken together, our data indicate that the nature of the mutation adds a level of complexity to the clinical manifestations of ciliary dysfunction, thus contributing to the expanding phenotypical spectrum of ciliopathies.


Assuntos
Astenozoospermia/genética , Síndrome de Bardet-Biedl/genética , Proteínas do Citoesqueleto/genética , Flagelos/genética , Infertilidade Masculina/genética , Mutação de Sentido Incorreto/genética , Tubulina (Proteína)/genética , Animais , Axonema/genética , Cílios/genética , Homozigoto , Humanos , Masculino , Transporte Proteico/genética , Sítios de Splice de RNA/genética , Cauda do Espermatozoide/fisiologia , Sequenciamento Completo do Exoma/métodos
20.
Am J Med Genet A ; 185(6): 1883-1887, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33779033

RESUMO

Noonan syndrome (NS) is an autosomal dominant condition with variable expressivity most commonly due to a germline pathogenic variant in PTPN11, which encodes the protein tyrosine phosphatase SHP-2. Gain-of-function variants in PTPN11 are known to promote oncogenic behavior in affected tissues. We report the clinical description of a young adult male presenting with relapsing ganglioneuromas, dysmorphic features, cardiac abnormalities, and multiple lentigines, strongly suspicious for NS. Solid tumor testing identified the recurrent pathogenic c.922G>A (p.Asn308Asp) in PTPN11. Proband and parental blood sampling testing confirmed c.922G>A as a de novo germline alteration. Comprehensive literature review of solid tumors specifically associated to PTPN11, indicates that this is the first documentation of ganglioneuroma and its clinical recurrence after resection in conjunction with a genetically confirmed NS diagnosis. The findings in our patient further extend the list of neuroblastic and neural crest-derived neoplasms associated with this condition.


Assuntos
Ganglioneuroma/genética , Cardiopatias Congênitas/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Ganglioneuroma/patologia , Predisposição Genética para Doença , Cardiopatias Congênitas/patologia , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Recidiva Local de Neoplasia/genética , Síndrome de Noonan/patologia , Fenótipo , Adulto Jovem
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