RESUMEN
Emery-Dreifuss muscular dystrophy is characterized by the clinical triad of early onset contractures of elbows, Achilles tendons and spine, wasting and weakness with a predominantly humero-peroneal distribution and life-threatening cardiac conduction defects and/or cardiomyopathy. Two main types of inheritance have been described: the X-linked form is caused by mutations in the STA gene on locus Xq28 and the gene for the autosomal dominant form (LMNA gene) has been localized on chromosome 1q11-q23. Recently, mutations in this LMNA gene have been also found to be responsible for the less frequent autosomal recessive form of the disease. Although all forms share a similar clinical presentation, some differences appear to exist between them as has been described recently in a large number of patients. We present the first documented Spanish family genetically confirmed to have autosomal dominant Emery-Dreifuss muscular dystrophy. Clinical, pathological and genetic data are described. We emphasize the difficulties in diagnosis, especially in sporadic cases or young patients in whom the clinical picture is not completely established.
Asunto(s)
Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/patología , Tendón Calcáneo/patología , Adolescente , Biopsia , Cardiomiopatías/genética , Cardiomiopatías/patología , Niño , Contractura/genética , Contractura/patología , Articulación del Codo/patología , Salud de la Familia , Femenino , Genes Dominantes , Humanos , Laminas , Persona de Mediana Edad , Proteínas Nucleares/genética , Linaje , Polimorfismo Conformacional Retorcido-Simple , Columna Vertebral/patologíaRESUMEN
X-linked Emery-Dreifuss muscular dystrophy is usually caused by absence of the nuclear membrane protein, emerin, due to nonsense mutations or deletions, but a few missense mutations also exist. A pathogenic g993t mutation causes a Q133H change in the nuclear targeting region of emerin, but it may also reduce emerin levels by affecting mRNA splicing. We have introduced the g993t mutation by in vitro mutagenesis and studied the effect of Q133H on nuclear targeting by transfection of COS-7 cells. No qualitative or quantitative differences in nuclear targeting were observed between normal and mutant emerin. Quantitative BIAcore analysis showed no significant change in lamin A binding to emerin when the mutation was present. We conclude that Q133 is not essential for nuclear targeting of emerin or its interaction with lamin A. Reduced emerin levels due to altered splicing or defective interaction with an unidentified binding partner remain possible pathogenic mechanisms.
Asunto(s)
Proteínas de la Membrana/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutación Puntual , Timopoyetinas/genética , Técnicas Biosensibles , Lamina Tipo A , Laminas , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/etiología , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Timopoyetinas/metabolismoRESUMEN
Most pathogenic missense mutations in the lamin A/C gene identified so far cause autosomal-dominant dilated cardiomyopathy and/or Emery-Dreifuss muscular dystrophy. A few specific mutations, however, cause a disease with remarkably different clinical features: FPLD, or familial partial lipodystrophy (Dunnigan-type), which mainly affects adipose tissue. We have prepared lamin A with a known FPLD mutation (R482Q) by in vitro mutagenesis. Nuclear targeting of lamin A in transfected COS cells, human skeletal muscle cells or mouse adipocyte cell cultures (pre- and post-differentiation) was not detectably affected by the mutation. Quantitative in vitro measurements of lamin A interaction with emerin using a biosensor also showed no effect of the mutation. The results show that the loss of function of R482 in lamin A/C in FPLD does not involve loss of ability to form a nuclear lamina or to interact with the nuclear membrane protein, emerin.
Asunto(s)
Adipocitos/metabolismo , Núcleo Celular/metabolismo , Lipodistrofia/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares/genética , Timopoyetinas/metabolismo , Animales , Secuencia de Bases , Células COS , Cardiomiopatía Dilatada/genética , Cartilla de ADN , Lamina Tipo A , Laminas , Distrofias Musculares/genética , Proteínas Nucleares/metabolismoRESUMEN
Emerin is the protein of the inner nuclear membrane that is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. The autosomal dominant form of the disease is caused by mutations in the lamin A/C gene. Several lines of circumstantial evidence have suggested an interaction of emerin with lamins in the nuclear lamina but direct interaction between the two proteins has not yet been demonstrated. We now demonstrate direct interaction between recombinant emerin and lamin A molecules using biomolecular interaction analysis (BIA) and monoclonal antibodies. An emerin-lamin A interaction system may be related in function to the LAP2-lamin B system at the inner nuclear rim.
Asunto(s)
Proteínas de la Membrana/química , Proteínas Nucleares/química , Timopoyetinas/química , Anticuerpos Monoclonales , Sitios de Unión , Clonación Molecular , Humanos , Lamina Tipo A , Laminas , Distrofia Muscular de Emery-Dreifuss , Fragmentos de Péptidos/química , Proteínas Recombinantes/químicaRESUMEN
Emery-Dreifuss muscular dystrophy has some remarkably specific features, with only cardiac and skeletal tissues being affected. Equally remarkably, the disease is caused by mutations in widely expressed genes for the nuclear membrane/lamina proteins, emerin and lamin A/C. How do mutations in proteins at the heart of the cell lead to stiff joints and sudden heart failure? This and related questions are the subject of this review.
Asunto(s)
Huesos/fisiopatología , Corazón/fisiopatología , Distrofia Muscular de Emery-Dreifuss/patología , Distrofia Muscular de Emery-Dreifuss/fisiopatología , Proteínas Nucleares/metabolismo , Huesos/patología , Humanos , Lamina Tipo A , Laminas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutación/genética , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Timopoyetinas/genética , Timopoyetinas/metabolismoRESUMEN
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat which is expressed as a polyglutamine tract near the N-terminus of the gene product, huntingtin. N-terminal huntingtin fragments form intranuclear aggregates in HD patients and these may be involved in the pathogenesis. Monoclonal antibodies (mAbs) against three different regions of huntingtin (amino acids 997-1276, 1844-2131 and 2703-2911) have been produced and two of the epitopes have been identified using phage displayed peptide libraries. All mAbs reacted with 350 kDa huntingtin on Western blots and one mAb from each region was selected for further study by strong immunoreactivity with neurons in different regions of rabbit brain and by ability to immunoprecipitate native huntingtin. Subcellular fractionation and sucrose density centrifugation of rabbit brain extract showed that most of the huntingtin exists as a high molecular weight complex in the cytoplasm. Two outstanding problems have been addressed; the location of huntingtin in tissues outside the central nervous system and whether huntingtin is present in the nucleus of normal cells. We conclude that huntingtin is present at low levels in most non-neuronal cells though we have identified an interstitial cell type in skin with very high immunoreactivity. Using both immunolocalization and nuclear purification methods, we were unable to exclude the possibility that a small proportion of full-length huntingtin is present in the nucleus.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Enfermedad de Huntington/diagnóstico , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares/análisis , Proteínas Nucleares/inmunología , Animales , Bacteriófagos , Western Blotting , Química Encefálica/genética , Reacciones Cruzadas , Epítopos/inmunología , Biblioteca de Genes , Proteína Huntingtina , Enfermedad de Huntington/genética , Células de Langerhans/química , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Células Piramidales/química , Conejos , Proteínas Recombinantes de Fusión/inmunología , Repeticiones de TrinucleótidosRESUMEN
Emerin is a nuclear membrane protein which is missing or defective in Emery-Dreifuss muscular dystrophy (EDMD). It is one member of a family of lamina-associated proteins which includes LAP1, LAP2 and lamin B receptor (LBR). A panel of 16 monoclonal antibodies (mAbs) has been mapped to six specific sites throughout the emerin molecule using phage-displayed peptide libraries and has been used to localize emerin in human and rabbit heart. Several mAbs against different emerin epitopes did not recognize intercalated discs in the heart, though they recognized cardiomyocyte nuclei strongly, both at the rim and in intranuclear spots or channels. A polyclonal rabbit antiserum against emerin did recognize both nuclear membrane and intercalated discs but, after affinity purification against a pure-emerin band on a western blot, it stained only the nuclear membrane. These results would not be expected if immunostaining at intercalated discs were due to a product of the emerin gene and, therefore, cast some doubt upon the hypothesis that cardiac defects in EDMD are caused by absence of emerin from intercalated discs. Although emerin was abundant in the membranes of cardiomyocyte nuclei, it was absent from many non-myocyte cells in the heart. This distribution of emerin was similar to that of lamin A, a candidate gene for an autosomal form of EDMD. In contrast, lamin B1 was absent from cardiomyocyte nuclei, showing that lamin B1 is not essential for localization of emerin to the nuclear lamina. Lamin B1 is also almost completely absent from skeletal muscle nuclei. In EDMD, the additional absence of lamin B1 from heart and skeletal muscle nuclei which already lack emerin may offer an alternative explanation of why these tissues are particularly affected.
Asunto(s)
Proteínas de la Membrana/análisis , Distrofias Musculares/metabolismo , Miocardio/química , Proteínas Nucleares/análisis , Timopoyetinas/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Humanos , Inmunohistoquímica , Lamina Tipo A , Lamina Tipo B , Laminas , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Datos de Secuencia Molecular , Distrofias Musculares/patología , Miocardio/citología , Conejos , Ratas , Homología de Secuencia de Aminoácido , Timopoyetinas/inmunologíaRESUMEN
Emerin is a nuclear membrane protein which is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. We have previously suggested that emerin is a member of a family of type II integral membrane proteins which associate with the nuclear lamina and which include lamina-associated proteins and the lamin B receptor. We now show that emerin in COS cells is not restricted to the nuclear rim but is also found at intranuclear sites, where it colocalizes with nuclear lamins B1, B2 and A/C. During mitosis, emerin is dispersed throughout the cell and then participates in the reconstitution of membranes around the daughter nuclei. Although emerin and lamins do not remain colocalized during mitosis, they all show some association with the midbody of the mitotic spindle.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Timopoyetinas/metabolismo , Animales , Anticuerpos Monoclonales , Células COS , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Humanos , Interfase , Lamina Tipo B , Laminas , Proteínas de la Membrana/genética , Mitosis , Distrofias Musculares/genética , Huso Acromático/metabolismo , Timopoyetinas/genéticaRESUMEN
Emery-Dreifuss muscular dystrophy is an X-linked neuromuscular disorder caused by defects in the STA gene on Xq28, which codes for a nuclear protein named emerin. Affected patients usually present in early adolescence with scapulo-peroneal muscle weakness and wasting, and contractures of the tendo Achilles, elbows and paraspinal muscles, resulting in spine rigidity. We present here a case of Emery-Dreifuss muscular dystrophy with an unusually severe, early presentation. He presented at 2.5 years with predominantly proximal weakness and mild equinovarus deformity of the right foot. Serum creatine kinase activity was elevated (1994 IU/I) and a muscle biopsy at the age of 4 years showed marked dystrophic abnormalities. Normal expression of dystrophin, and no detectable deletion in the corresponding gene, excluded a diagnosis of Duchenne muscular dystrophy. Similarly, normal expression of alpha-sarcoglycan made a limb-girdle muscular dystrophy caused by a defect in a sarcoglycan unlikely. Several years later, examination of the proband's maternal cousin, aged 14 years, suggested Emery-Dreifuss muscular dystrophy. This was confirmed in both affected boys by the absence of emerin in muscle and leucocytes, and identification of a mutation in exon 4 of the STA gene. Carrier status in both mothers was also confirmed by mutational and protein analysis. Emery-Dreifuss muscular dystrophy should therefore be considered in the differential diagnosis of cases of early onset muscular dystrophy, even in the absence of the typical clinical features.
Asunto(s)
Ligamiento Genético , Distrofias Musculares/genética , Cromosoma X , Edad de Inicio , Biopsia , Preescolar , Diagnóstico Diferencial , Humanos , Leucocitos/patología , Masculino , Proteínas de la Membrana/genética , Músculos/patología , Distrofias Musculares/clasificación , Distrofias Musculares/patología , Distrofia Muscular de Emery-Dreifuss , Mutación , Proteínas Nucleares , Linaje , Piel/patología , Timopoyetinas/genéticaRESUMEN
Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (<5% of normal) due to skewed X-inactivation. In the three autosomal cases of EDMD, emerin was normal on western blots of blood cells, which suggests that autosomal EDMD is not caused by indirect reduction of emerin levels.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Distrofias Musculares/genética , Mutación/genética , Timopoyetinas/biosíntesis , Timopoyetinas/genética , Adolescente , Adulto , Línea Celular Transformada , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Linfocitos , Masculino , Persona de Mediana Edad , Distrofias Musculares/metabolismo , Distrofia Muscular de Emery-Dreifuss , Proteínas Nucleares , Linaje , Cromosoma X/genéticaRESUMEN
The X-linked form of Emery-Dreifuss muscular dystrophy (EDMD) was recently shown to be due to mutations in the STA gene on chromosome Xq28. We have demonstrated a simple test for the diagnosis of this condition, looking for altered expression of the protein, emerin, in leucocytes and skin with a monoclonal antibody. Full-length emerin is completely absent in affected boys from the EDMD families studied. The method has also enabled identification of a female carrier of the disease by reduced levels of the protein on the leucocyte Western blot and a mosaic pattern of expression by immunofluorescence microscopy of the skin biopsy.
Asunto(s)
Ligamiento Genético , Pruebas Inmunológicas , Leucocitos/metabolismo , Proteínas de la Membrana/metabolismo , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Piel/metabolismo , Timopoyetinas/metabolismo , Cromosoma X , Adolescente , Anticuerpos Monoclonales , Femenino , Técnica del Anticuerpo Fluorescente , Tamización de Portadores Genéticos , Humanos , Masculino , Distrofia Muscular de Emery-Dreifuss , Proteínas Nucleares , LinajeRESUMEN
AIM: To develop a DNA-based diagnostic test for adrenoleukodystrophy (ALD) in a large New Zealand family. METHODS: Mutation screening of the X chromosome-linked ALD gene was undertaken by direct sequencing of PCR amplified products encompassing defined exons of the ALD gene. The identification of a mutation led to the development of a simple restriction enzyme digestion protocol of a PCR amplified product to identify those individuals with the mutation. RESULTS: A nonsense mutation, resulting in deduced premature termination of translation of the ALD gene product, was detected in exon 4 of the ALD gene in an affected male. This mutation was found in three obligate gene carriers in the same ALD family. A DNA-based test was established to identify this mutation by Bgl II digestion of a PCR amplified product encompassing exons 3 and 4 of the ALD gene. The DNA-based test was applied to a chorionic villus sampling for prenatal diagnosis. CONCLUSIONS: A simple DNA-based test has been developed for ALD in a large New Zealand family. This test provides a rapid means of determining carrier status and for undertaking prenatal diagnosis for ALD in this family.
Asunto(s)
Adrenoleucodistrofia/genética , ADN/genética , Adolescente , Adrenoleucodistrofia/diagnóstico , Adulto , Niño , Preescolar , Muestra de la Vellosidad Coriónica , Codón sin Sentido/genética , ADN/análisis , Análisis Mutacional de ADN , Exones/genética , Femenino , Amplificación de Genes , Ligamiento Genético/genética , Pruebas Genéticas , Heterocigoto , Humanos , Masculino , Mutación/genética , Nueva Zelanda , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas/genética , Transcripción Genética/genética , Cromosoma X/genéticaRESUMEN
We have previously shown that urinary trypsin inhibitor (UTI), also known and bikunin, was mitogenic for human fibroblasts at low concentrations, and growth-inhibitory at higher concentrations, and have identified high- and low-affinity cellular binding sites for this protein. We have now investigated fibroblast proteins which interact with bikunin. Bikunin binds to proteins of about 50K and 250K. The simplest interpretation, is that the 50K protein may be a proteinase which is also the low-affinity bikunin binding site, involved in growth inhibition, and that the larger protein may be responsible for the mitogenic response to bikunin. Inhibitors of intracellular calcium mobilisation also inhibit the mitogenic response to bikunin, and by the measurement of the efflux of pre-loaded 45Ca2+, bikunin at mitogenic concentrations can be shown to stimulate calcium mobilization.
Asunto(s)
Glicoproteínas/orina , Glicoproteínas de Membrana , Inhibidores de Proteasas/orina , Inhibidor de la Tripsina de Soja de Kunitz , Inhibidores de Tripsina/orina , Autorradiografía , División Celular/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Mitógenos , Neomicina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100,000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Distrofias Musculares/metabolismo , Proteínas Nucleares/metabolismo , Timopoyetinas/metabolismo , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Núcleo Celular/metabolismo , Mapeo Epitopo , Feto/metabolismo , Humanos , Proteínas de la Membrana/genética , Músculos/metabolismo , Distrofias Musculares/patología , Proteínas Nucleares/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Timopoyetinas/genéticaRESUMEN
The possibility that a growth-related proteinase may act by degrading a negative growth regulatory protein has been investigated. Proteinase inhibitors which inhibit the enzyme also enhance the accumulation of the growth regulator by human fibroblasts. The negative growth regulator shows a similar specificity of inhibition of cellular growth to inhibitors of the growth-related proteinase.