RESUMO
N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.
Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Humanos , Chaperonas Moleculares/metabolismo , Lectinas/metabolismo , Polissacarídeos/química , Controle de QualidadeRESUMO
Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.
Assuntos
Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina , Animais , Células CHO , Cricetinae , Cricetulus , Dependovirus/genética , Terapia Genética , Hepatócitos/metabolismo , Humanos , Fibras Musculares Esqueléticas , Transdução Genética , alfa 1-Antitripsina/biossíntese , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed N-glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has N-glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway.
Assuntos
Antitrombina III/metabolismo , Controle de Qualidade , Serpinas/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetulus , Retículo Endoplasmático/metabolismo , HumanosRESUMO
Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly ß-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the protease-binding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.
Assuntos
Antitrombina III/química , Animais , Antitrombina III/genética , Células CHO , Cricetulus , Dissulfetos/química , Humanos , Mutação , Conformação Proteica , Dobramento de ProteínaRESUMO
Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, are often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7; hepatocytes; Chinese hamster ovarian cells (CHO), a common host to produce biologics; and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines.
Assuntos
Produtos Biológicos , Vacinas de mRNA , Animais , Cricetinae , Humanos , Hepatócitos , Fibras Musculares Esqueléticas , Células CHO , MamíferosRESUMO
Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N- to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α1-antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More ß-rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone ß-strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization.
RESUMO
Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcriptionquantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patientderived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5ethynyl2'deoxyuridine, lactate dehydrogenase release assay and calceinAM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/tratamento farmacológico , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Doxorrubicina/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased â¼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes.
RESUMO
Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.
Assuntos
Proteínas de Bactérias/genética , Temperatura Baixa , Escherichia coli/genética , Vetores Genéticos/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/metabolismo , Humanos , Marcação por Isótopo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMO
Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.
RESUMO
RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/metabolismo , Divisão Celular/fisiologia , Temperatura Baixa , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Substâncias Macromoleculares , Mutação , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Temperatura , Tripsina/metabolismoRESUMO
Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E.coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.
Assuntos
Proteínas de Escherichia coli/química , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/química , Ribossomos/química , Adaptação Fisiológica , Sequência de Aminoácidos , Temperatura Baixa , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fatores de Alongamento de Peptídeos/química , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Ribossômico 16S/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos , Choque , Fatores de Transcrição/química , Fatores de Elongação da TranscriçãoRESUMO
Oligochitosan, having an average molecular weight of 1000 Da and a degree of N-acetylation below 15%, can be obtained by either chemical or enzymic hydrolysis of chitosan. The present investigation demonstrated that oligochitosan can significantly increase the activity of inducible nitric oxide synthase (iNOS) and induce the synthesis of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in macrophages. Moreover, nuclear factor-kappaB (NF-kappaB) protein levels in nuclear extract are increased in response to oligochitosan. Blocking NF-kappaB with specific inhibitor results in decreased levels of NO and TNF-alpha. These results indicate that NF-kappaB plays a potential role in the induction of NO and TNF-alpha by oligochitosan in macrophages.
Assuntos
Quitina/análogos & derivados , Quitina/farmacologia , Quitosana/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Western Blotting , Sequência de Carboidratos , Linhagem Celular , Quitina/síntese química , Quitosana/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Oligossacarídeos , Pirrolidinas/metabolismo , Tiocarbamatos/metabolismoRESUMO
OBJECTIVE: To study the effect of polyamine biosynthesis inhibition on growth characteristics of human lung carcinoma cells and its correlation with the expression of human lung carcinoma associated antigen ALT-04ag gene. METHODS: The gene expression was detected by RT-PCR and immunocytochemical tests. The cell growth characteristics were studied by cell growth curves, morphological observation, FCM analysis and DNA electrophoresis. RESULTS: Human lung squamous carcinoma cells L78 treated with 5 mmol/L alpha-difluoromethylornithine (DFMO) for 5 days showed significant growth inhibition and apoptosis induction. The mRNA and protein expressions of ALT-04ag gene in the cells were downregulated, while these changes resulted from DFMO treatment were prevented by provision of DFMO along with exogenous putrescine. CONCLUSION: The effect of polyamine biosynthesis inhibition induced by DFMO restrains the growth characteristics and promotes apoptosis of human lung carcinoma L78 cells, which is associated with down regulation of ALT-04ag gene expression.
Assuntos
Antígenos de Neoplasias/genética , Eflornitina/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Antígenos de Neoplasias/biossíntese , Apoptose , Carcinoma de Células Escamosas/patologia , Divisão Celular , Regulação para Baixo , Humanos , Proteínas Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.
Assuntos
Doenças Fetais/genética , Queratina-9/genética , Ceratodermia Palmar e Plantar Epidermolítica/diagnóstico , Ceratodermia Palmar e Plantar Epidermolítica/genética , Mutação , Adulto , Amostra da Vilosidade Coriônica , Feminino , Doenças Fetais/diagnóstico , Humanos , Mutação de Sentido Incorreto , Linhagem , Gravidez , Diagnóstico Pré-NatalRESUMO
Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe.
Assuntos
Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/genética , Análise de Sequência de DNA/métodos , Adulto , Sequência de Aminoácidos , Povo Asiático/genética , Éxons , Testes Genéticos , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Reação em Cadeia da Polimerase , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Danon disease is a rare X-linked lysosomal storage disease characterized by hypertrophic cardiomyopathy, myopathy and mental retardation, and is due to a primary defect in lysosome-associated membrane protein-2 (LAMP 2). More than 26 mutations in the LAMP2 gene have been described, including a small number of de novo mutations, some of which are suspected to be caused by germline mosaicism. Here, we describe the first molecularly documented evidence of somatic mosaicism for a LAMP2 mutation, identified in the asymptomatic mother of a boy with Danon disease caused by the frameshift mutation c.808dupG (p.A270Gfx3) within exon 6. In addition, in order to gain insight into the possible explanation for the mother's lack of phenotype, the level of somatic mosaicism and the X-chromosome inactivation pattern were investigated. This study provides new insight into the causes of phenotypic variability in female mutation-carriers and underlines the importance of parental molecular testing for accurate genetic counseling for Danon disease.
Assuntos
Mutação da Fase de Leitura , Doença de Depósito de Glicogênio Tipo IIb/genética , Proteínas de Membrana Lisossomal/genética , Mosaicismo , Adolescente , Sequência de Aminoácidos , Povo Asiático/genética , Sequência de Bases , China , DNA/genética , Feminino , Heterozigoto , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Masculino , Dados de Sequência Molecular , Inativação do Cromossomo XRESUMO
Epidermolytic palmoplantar keratoderma (EPPK) is generally associated with dominant-negative mutations of the Keratin 9 gene (KRT9), and rarely with the Keratin 1 gene (KRT1). To date, a myriad of mutations has been reported with a high frequency of codon 163 mutations within the first exon of KRT9 in different populations. Notably, a distinct phenotypic heterogeneity, digital mutilation, was found recently in a 58-year-old female Japanese EPPK patient with p.R163W. Here, we report the most common mutation, c.C487T (p.R163W) of KRT9, in two large EPPK pedigrees from southeast China. The arginine residue in peptide position 163 remains almost constant in at least 47 intermediate filament proteins ranging from snail to human. A substitution in arginine alters both the charge and shape of the 1A rod domain and disrupts the function of the helix initiation motif of keratins, finally compromising the integrity of filaments and weakening their stability in the epidermis of palms and soles. We summarize the clinical symptoms of EPPK in Chinese and show that knuckle pads are associated with KRT9 mutations. We suggest that the frequency of p.R163W in Chinese EPPK patients (31.03%) is consistent with that in the general population (29.33%), and that codon 163 is truly a hotspot mutational site of KRT9.
Assuntos
Povo Asiático/genética , Queratina-9/genética , Ceratodermia Palmar e Plantar Epidermolítica/genética , Mutação/genética , Adulto , Arginina/genética , Feminino , Pé/patologia , Genes Dominantes , Mãos/patologia , Humanos , Ceratodermia Palmar e Plantar Epidermolítica/diagnóstico , Ceratodermia Palmar e Plantar Epidermolítica/patologia , Masculino , Linhagem , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genéticaRESUMO
BACKGROUND: Mutations in the KRT6A or KRT16 gene cause pachyonychia congenita type 1 (PC-1), while mutations in KRT16 or KRT6C underlie focal palmoplantar keratoderma (FPPK). A new classification system of PC has been adopted based on the mutated gene. PC rarely presents the symptoms of diffuse plantar keratoderma. Mutation in the tail domain of keratins is rarely reported. PC combined with fissured tongue has never been described. OBJECTIVES: To investigate the genotype-phenotype correlations between clinical features and gene mutational sites in two unrelated southern Chinese PC pedigrees (one family presented with specific fissured tongue, the other with diffuse plantar keratoderma). MATERIALS & METHODS: The whole coding regions of the KRT6A/KRT16/KRT17/KRT6B genes were amplified and directly sequenced to detect the mutation. To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of a patient was extracted and reverse-transcribed to cDNA for sequence analysis. RESULTS: Two novel de novo mutations, a splice acceptor site variant IVS8-2A>C (p.S487FfsX72) in KRT6A and a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16, were found separately in the two PC families. CONCLUSION: Genotype-phenotype correlations among PC patients with codon-125 mutation in KRT16 were established, while the phenotypes caused by the IVS8-2A>C mutation in KRT6A need further studies to confirm the rare feature of fissured tongue.
Assuntos
Queratina-16/genética , Queratina-6/genética , Ceratodermia Palmar e Plantar/genética , Mutação , Paquioníquia Congênita/genética , Língua Fissurada/genética , Adulto , Povo Asiático/genética , Feminino , Humanos , Ceratodermia Palmar e Plantar/complicações , Paquioníquia Congênita/complicações , Linhagem , Fenótipo , Língua Fissurada/complicaçõesRESUMO
BACKGROUND: Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families. METHODS: Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed. RESULTS: The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period. CONCLUSIONS: Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.