RESUMEN
BACKGROUND: The familial nonmedullary thyroid cancer (FNMTC) is suspected to be a Mendelian condition in up to 3-8% of thyroid cancers. The susceptibility chromosomal loci and genes of 95% of FNMTC cases remain to be characterized. The inheritance of FNMTC appears to be autosomal dominant with incomplete penetrance and variable expressivity. The finding of the causative gene of FNMTC and the identification of patients at risk that need genetic testing were our aim. METHODS: We analyzed by whole-exome sequencing patients and non-affected relatives of five families with at least two family members affected by papillary thyroid cancer, selecting for new or extremely rare variants with predicted pathogenic value. RESULTS: A family showed, in all three affected members, a new loss-of-function variant (frameshift deletion) in BROX gene at 1q41 that was absent from all internal and external databases. In a second family with three affected relatives, we found an additional new BROX variant. The smaller families presented no variants in BROX or in the other causative genes studied. CONCLUSIONS: BROX could be a new causative gene for FNMTC. Variants in BROX may result in the haploinsufficiency of a key gene involved in the morphogenesis of MVBs, in the endosomal sorting of cargo proteins, and in EGFR. Functional studies are needed to support this result. The thorough genomic analysis by NGS in all families with three or more affected members should become a routine approach to obtain a comprehensive genetic view and find confirmative second cases.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Haploinsuficiencia , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Adulto , Femenino , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Cáncer Papilar Tiroideo/etiología , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/etiología , Neoplasias de la Tiroides/metabolismoAsunto(s)
Síndrome de Gitelman , Guanina/análogos & derivados , Humanos , Hipopotasemia , Masculino , Persona de Mediana EdadRESUMEN
Primary autosomal recessive microcephaly (MCPH) is a developmental disorder characterized by prenatal onset of abnormal brain growth. MCPH occurs both alone and as part of a broad range of neurodevelopmental syndromes with or without cortical malformations and growth retardation. Here we report a consanguineous Moroccan family with two siblings affected by severe primary microcephaly, failure to thrive, congenital dermatitis and severe developmental delay. Brain magnetic resonance imaging showed lissencephaly of frontal lobes and periventricular heterotopia of the gray matter. We performed both Comparative Genomic Hybridization array and whole exome sequencing (WES) analyses of the kindred. No quantitative defects were detected. However, WES identified a new homozygous missense variation in the penultimate nucleotide of exon 23 of RTTN gene (c.2953A>G;pArg985Gly). cDNA sequencing revealed two abnormal spliced products, one lacking only exon 23 and the other lacking exons 22 and 23 (out-of-frame). RTTN is a protein involved in cilia structure and function. Homozygous mutations in RTTN gene have been described in bilateral diffuse isolated polymicrogyria and, more recently, in microcephalic primordial dwarfism (PD). We found a novel homozygous mutation in RTTN associated with microcephalic PD as well as complex brain malformations and congenital dermatitis, thus expanding the phenotypic spectrum of both RTTN-associated diseases and ciliary dysfunction.
Asunto(s)
Proteínas Portadoras/genética , Dermatitis/genética , Trastornos del Crecimiento/genética , Microcefalia/genética , Encéfalo/diagnóstico por imagen , Encéfalo/fisiopatología , Proteínas de Ciclo Celular , Hibridación Genómica Comparativa , Consanguinidad , Dermatitis/fisiopatología , Exones/genética , Femenino , Trastornos del Crecimiento/diagnóstico por imagen , Trastornos del Crecimiento/fisiopatología , Homocigoto , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Microcefalia/fisiopatología , Mutación , Linaje , FenotipoRESUMEN
A second genetic revolution is approaching thanks to next-generation DNA sequencing technologies. In the next few years, the 1,000$-genome sequencing promises to reveal every individual variation of DNA. There is, however, a major problem: the identification of thousands of nucleotide changes per individual with uncertain pathological meaning. This is also an ethical issue. In the middle, there is today the possibility to address the sequencing analysis of genetically heterogeneous disorders to selected groups of genes with defined mutation types. This will be cost-effective and safer. We assembled an easy-to manage overview of most Mendelian genes involved in myopathies, cardiomyopathies, and neuromyopathies. This was entirely put together using a number of open access web resources that are listed below. During this effort we realized that there are unexpected countless sources of data, but the confusion is huge. In some cases, we got lost in the validation of disease genes and in the difficulty to discriminate between polymorphisms and disease-causing alleles. In the table are the annotated genes, their associated disorders, genomic, mRNA and coding sizes. We also counted the number of pathological alleles so far reported and the percentage of single nucleotide mutations.
Asunto(s)
Cardiomiopatías/genética , Enfermedades Musculares/genética , Enfermedades Neuromusculares/genética , Polineuropatías/genética , Análisis Mutacional de ADN , Variación Genética , Genoma Humano , Humanos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas Portadoras/genética , Proteínas de Drosophila , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Proteínas de Insectos/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleósido-Difosfato Quinasa , Sarcoma/genética , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Neoplasias de la Mama/patología , Células COS , Carcinoma/patología , Proteínas Portadoras/fisiología , División Celular , Femenino , Humanos , Proteínas de Insectos/fisiología , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Nucleósido Difosfato Quinasas NM23 , Metástasis de la Neoplasia , Monoéster Fosfórico Hidrolasas , ARN Neoplásico/biosíntesis , Sarcoma/patología , Factores de Transcripción/genéticaRESUMEN
The amino acid sequence of a novel tissue-and phase-specific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 +/- 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the C-terminus. They have thus been named [des-(Ala81) SNP1] and [des-(Lys80-Ala81) SNP2], respectively. The molecular weights are 9140.21 +/- 0.83 for [des-(Ala81) SNP1] and 9011 +/- 0.09 for [des-(Lys80-Ala81) SNP2].
Asunto(s)
Proteínas Nucleares/metabolismo , Oviductos/metabolismo , Proteínas de Reptiles , Secuencia de Aminoácidos , Animales , División Celular , Femenino , Lagartos , Espectrometría de Masas , Datos de Secuencia Molecular , Proteínas Nucleares/química , Oviductos/citología , Ratas , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Modifications in plasma amino acid patterns in cirrhotics are attributed to impaired liver function, being more evident in alcoholic than in viral cirrhosis. AIM: To evaluate whether diet influences plasma amino acid concentrations in different aetiological groups of cirrhotics. PATIENTS: Study population comprised 40 patients with cirrhosis (25 virus- and 15 alcohol-related], all Child A, and 30 healthy subjects (controls). METHOD: A food frequency and quality questionnaire was utilized to determine dietary history and alcohol intake. Nutritional status was evaluated by anthropometric method. Amino acids were determined, on venous blood samples, using a specific analyzer while cysteine was evaluated by fluorescent high power liquid chromatography RESULTS: The total daily intake of calories, proteins, lipids, and carbohydrates was similar in all individuals. Food quality distinguished the cirrhotics from the controls, but not the different aetiological groups of cirrhotics. Plasma cysteine levels were significantly lower, while aromatic amino acids and methionine were significantly higher, in all cirrhotics (p<0.001 and p<0.01, respectively, versus controls). The decrease in cysteine and the increase in other amino acids were more marked in alcoholics (p<0.01). CONCLUSIONS: Ethanol intake, but not diet, further enhances the changes in plasma aromatic amino acids, methionine and cysteine induced by impaired liver function in patients with cirrhosis, suggesting a direct interference of alcohol in their metabolism.
Asunto(s)
Aminoácidos/sangre , Dieta , Cirrosis Hepática/sangre , Adulto , Anciano , Femenino , Hepatitis Viral Humana/sangre , Humanos , Cirrosis Hepática Alcohólica/sangre , Masculino , Persona de Mediana Edad , Estado NutricionalRESUMEN
Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae, the causal agent of crown and root rot of tomato (Lycopersicon esculentum). The M(r) (10,349 +/- 1) of the purified protein, determined by ES-MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI-MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae, as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum (Capsicum annuum) and vegetable marrow (Cucurbita pepo) from P. capsici.
Asunto(s)
Proteínas Fúngicas/química , Phytophthora/química , Solanum lycopersicum , Secuencia de Aminoácidos , Cromatografía en Gel , Proteínas Fúngicas/aislamiento & purificación , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Phytophthora/patogenicidad , Enfermedades de las Plantas , Extractos Vegetales/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.
Asunto(s)
Genes de Plantas/genética , Genes Sintéticos , N-Glicosil Hidrolasas/genética , Proteínas de Plantas/genética , Proteínas Recombinantes/biosíntesis , Ribosomas/genética , Secuencia de Bases , Clonación Molecular , Activación Enzimática , Escherichia coli/genética , Vectores Genéticos , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/biosíntesis , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/farmacología , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
The primary structure has been determined for PD-S2, a new type 1 ribosome-inactivating protein (RIP), isolated from the seeds of Phytolacca dioica L. PD-S2 has 265 amino-acid residues, and a molecular mass of 29586 Da. The polypeptide chain contains four amino-acid residues more than PAP-S, a type-I RIP isolated from the seeds of the taxonomically related plant Phytolacca americana L. We have compared the amino-acid sequence of PD-S2 with those of two other RIPs with known three-dimensional structure: PAP-S and ricin A-chain (RTA), the active chain of the best known type-2 RIP. This analysis shows an identity of 76% and 33% with PAP-S and RTA respectively, and a similarity of 82% and 54%. Comparison with the PAP sequence, isolated from leaves of P. americana, shows an even higher identity (80%) and similarity (87%). Furthermore, the amino-acid residues reported in other RIPs to be invariant and participate in the definition of the active site (Tyr-76, Tyr-127, Glu-179, Arg-182 and Trp-211; PD-S2 numbering) are all present. Asn-74, Arg-138, Gln-175, and Glu-208 are also conserved, while Asn-209 is substituted by Glu, all residues located in the active-site cleft of RIPs (Tahirov, T.H., Lu, T.-H., Liaw, Y.-C., Chen, J.L. and Lin, J.Y. (1995) Crystal structure of abrin-a at 2.14 A, J. Mol. Biol. 250, 354-367). The polypeptide chain of PD-S2 contains two N-glycosylation sites at Asn-112 and Asn-120, the second of which appears to be linked to sugars. Like PAP-S, PD-S2 does not contain free sulfhydryl groups. The four cysteinyl residues of the two proteins have corresponding sequence positions, most likely with identical S-S pairing.
Asunto(s)
N-Glicosil Hidrolasas/química , Proteínas de Plantas/química , Semillas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Bromuro de Cianógeno , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Lectinas de Plantas , Proteínas de Plantas/aislamiento & purificación , Conformación Proteica , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas , Ricina/química , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.
Asunto(s)
Antivirales/farmacología , N-Glicosil Hidrolasas/metabolismo , Plantas/enzimología , Inhibidores de la Síntesis de la Proteína/farmacología , Células 3T3 , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/aislamiento & purificación , ADN/metabolismo , Femenino , Células HeLa , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Poli A/metabolismo , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , ARN Bacteriano/metabolismo , ARN Viral/metabolismo , Conejos , Ratas , Proteínas Inactivadoras de Ribosomas , Ribosomas , Células Tumorales CultivadasRESUMEN
A low-molecular-mass zinc-binding protein was purified from the eggs of the sea urchin Paracentrotus lividus using procedures that included gel-permeation and anion-exchange chromatography followed by HPLC. The primary structure of this protein was derived from the sequences of peptide fragments obtained by digestion with trypsin and thermolysin. The reconstructed sequence showed the presence of 20 cysteinyl residues, thus resembling that of a metallothionein. The Paracentrotus protein was most similar to the metallothionein of Strongylocentrotus purpuratus, another member of the order of Echinoida, living along the coast of the Pacific Ocean. However, the presence of non-conservative amino acid substitution, together with a deletion of two residues in the Strongylocentrotus metallothionein, make the similarity scores of the two sea urchin proteins lower than that of metallothioneins from vertebrates of the same order. In addition, the present data show that sea urchin metallothioneins display no homology with metallothioneins of any other species.
Asunto(s)
Equinodermos/química , Metalotioneína/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Metalotioneína/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Alineación de Secuencia , Termolisina , TripsinaRESUMEN
From the seeds of the Caryophyllaceae Saponaria ocymoides and Vaccaria pyramidata two proteins were purified which have the properties of the type-1 (single-chain) ribosome-inactivating proteins [reviewed by Barbieri, L., Battelli, M. G. & Stirpe, F. (1993) Ribosome-inactivating proteins from plants, Biochim. Biophys. Acta 1154, 237-282]. The proteins have molecular masses of 30.2 kDa (S. ocymoides) and 28.0 kDa (V. pyramidata) and pI greater than 9.5, their N-terminal amino acid sequences are similar to those of saporin-S6 and dianthin 30, ribosome-inactivating proteins from other Caryophyllaceae, and they partially cross-react with sera against these proteins. Both proteins inhibit protein synthesis by a rabbit-reticulocyte lysate with IC50 (concentrations giving 50% inhibition) below 10(-10) M, have a smaller effect on poly(U)-directed phenylalanine polymerisation by rat liver ribosomes (nanomolar IC50, approximately) and on protein synthesis by various cell lines (IC50 ranging from 4 nM to > 3000 nM) and possess rRNA N-glycosidase activity, releasing 1 mol adenine/ribosome.