RESUMEN
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing we uncovered the EYS gene as one of the most common genetic cause of autosomal recessive RP in northern Sweden accounting for at least 16%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. We also showed that two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. We conclude, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies.
Asunto(s)
Proteínas del Ojo/genética , Retinitis Pigmentosa/clasificación , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Estudios de Cohortes , Femenino , Efecto Fundador , Genes Recesivos , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Mutación , Empalme del ARN , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Retinitis Pigmentosa/genética , Suecia , Adulto JovenRESUMEN
BACKGROUND: Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intron mutation in the iron-sulfur cluster assembly (ISCU) gene. The mutation results in aberrant splicing, where part of the intron is retained in the final mRNA transcript, giving rise to a truncated nonfunctional ISCU protein. Using an ISCU mini-gene system, we have previously shown that PTBP1 can act as a repressor of the mis-splicing of ISCU, where overexpression of PTBP1 resulted in a decrease of the incorrect splicing. In this study, we wanted to, in more detail, analyze the role of PTBP1 in the regulation of endogenous ISCU mis-splicing. METHODS: Overexpression and knockdown of PTBP1 was performed in myoblasts from two HML patients and a healthy control. Quantification of ISCU mis-splicing was done by qRTPCR. Biotinylated ISCU RNA, representing wildtype and mutant intron sequence, was used in a pull-down assay with nuclear extracts from myoblasts. Levels of PTBP1 in human cell lines and mice tissues were analyzed by qRTPCR and western blot. RESULTS: PTBP1 overexpression in HML patient myoblasts resulted in a substantial decrease of ISCU mis-splicing while knockdown of PTBP1 resulted in a drastic increase. The effect could be observed in both patient and control myoblasts. We could also show that PTBP1 interacts with both the mutant and wild-type ISCU intron sequence, but with a higher affinity to the mutant sequence. Furthermore, low levels of PTBP1 among examined mouse tissues correlated with high levels of incorrect splicing of ISCU. CONCLUSION: Our results show that PTBP1 acts as a dominant repressor of ISCU mis-splicing. We also show an inverse correlation between the levels of PTBP1 and ISCU mis-splicing, suggesting that the high level of mis-splicing in the skeletal muscle is primarily due to the low levels of PTBP1.
Asunto(s)
Acidosis Láctica/congénito , Ribonucleoproteínas Nucleares Heterogéneas/genética , Proteínas Hierro-Azufre/genética , Enfermedades Musculares/congénito , Proteína de Unión al Tracto de Polipirimidina/genética , Empalme del ARN , Acidosis Láctica/genética , Animales , Células Cultivadas , Genes Dominantes , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteínas Hierro-Azufre/metabolismo , Ratones , Ratones Endogámicos CBA , Enfermedades Musculares/genética , Mioblastos/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Supresión GenéticaRESUMEN
PURPOSE: Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. METHODS: The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. RESULTS: We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. CONCLUSION: Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Intrones/genética , Degeneración Macular/congénito , Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN/genética , Adulto , Análisis Mutacional de ADN , Exones/genética , Femenino , Células HEK293/metabolismo , Humanos , Degeneración Macular/diagnóstico , Degeneración Macular/genética , Masculino , Mutación , Linaje , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/metabolismo , Enfermedad de Stargardt , TransfecciónRESUMEN
Hereditary myopathy with lactic acidosis (HML) is an autosomal recessive disease caused by an intronic one-base mutation in the iron-sulfur cluster assembly (ISCU) gene, resulting in aberrant splicing. The incorrectly spliced transcripts contain a 100 or 86 bp intron sequence encoding a non-functional ISCU protein, which leads to defects in several Fe-S containing proteins in the respiratory chain and the TCA cycle. The symptoms in HML are restricted to skeletal muscle, and it has been proposed that this effect is due to higher levels of incorrectly spliced ISCU in skeletal muscle compared with other energy-demanding tissues. In this study, we confirm that skeletal muscle contains the highest levels of incorrect ISCU splice variants compared with heart, brain, liver and kidney using a transgenic mouse model expressing human HML mutated ISCU. We also show that incorrect splicing occurs to a significantly higher extent in the slow-twitch soleus muscle compared with the gastrocnemius and quadriceps. The splicing factor serine/arginine-rich splicing factor 3 (SRSF3) was identified as a potential candidate for the slow fiber specific regulation of ISCU splicing since this factor was expressed at higher levels in the soleus compared to the gastrocnemius and quadriceps. We identified an interaction between SRSF3 and the ISCU transcript, and by overexpressing SRSF3 in human myoblasts we observed increased levels of incorrectly spliced ISCU, while knockdown of SRSF3 resulted in decreased levels. We therefore suggest that SRSF3 may participate in the regulation of the incorrect splicing of mutant ISCU and may, at least partially, explain the muscle-specific symptoms of HML.
Asunto(s)
Acidosis Láctica/congénito , Proteínas Hierro-Azufre/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/congénito , Empalme del ARN , Factores de Empalme Serina-Arginina/genética , Acidosis Láctica/genética , Acidosis Láctica/patología , Animales , Células Cultivadas , Humanos , Proteínas Hierro-Azufre/metabolismo , Ratones , Ratones Endogámicos CBA , Ratones Transgénicos , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutagénesis Sitio-Dirigida , Mioblastos/citología , Mioblastos/metabolismo , ARN/aislamiento & purificación , ARN/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Empalme Serina-Arginina/antagonistas & inhibidores , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU, which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39, and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.
Asunto(s)
Acidosis Láctica/genética , Intrones/genética , Proteínas Hierro-Azufre/genética , Enfermedades Musculares/genética , Empalme Alternativo/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Mutación , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-ArgininaRESUMEN
The primary dystonias are a genetically heterogeneous group of disorders that can be subdivided in pure dystonias, dystonia-plus syndromes, and paroxymal dystonia. Four pure autosomal dominant dystonia loci have been mapped to date, DYT1, 6, 7, and 13, with varying penetrance. We report the mapping of a novel locus for a late-onset form of pure torsion dystonia in a family from northern Sweden. The disease is inherited in an autosomal dominant manner with a penetrance that may be as high as 90%. The torsion dystonia locus in this family was mapped to chromosome 2q14.3-q21.3 using an Illumina linkage panel. We also confirmed the linkage, using ten tightly linked microsatellite markers in the region, giving a maximum LOD score of 5.59 for marker D2S1260. The disease-critical region is 3.6-8.9 Mb depending on the disease status of one individual carrying a centromeric recombination. Mutational analysis was performed on 22 genes in the disease-critical region, including all known and hypothetical genes in the smaller, 3.6-Mb region, but no disease-specific mutations were identified. Copy number variation analysis of the region did not reveal any deletions or duplications. In order to increase the chances of finding the disease gene, fine-mapping may be necessary to decrease the region of interest. This report will hopefully result in the identification of additional dystonia families with linkage to the same locus, and thereby, refinement of the disease critical region.
Asunto(s)
Cromosomas Humanos Par 2 , Distonía Muscular Deformante/genética , Sitios Genéticos , Penetrancia , Adolescente , Adulto , Edad de Inicio , Anciano , Mapeo Cromosómico , Cromosomas Humanos Par 2/genética , Distonía Muscular Deformante/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/genética , Linaje , Adulto JovenRESUMEN
Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.
Asunto(s)
Acidosis Láctica/genética , Empalme Alternativo , Proteínas Hierro-Azufre/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Acidosis Láctica/metabolismo , Adulto , Animales , Western Blotting , Encéfalo/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Proteínas Hierro-Azufre/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Esquelético/patología , Enfermedades Musculares/metabolismo , Miocardio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Adulto JovenRESUMEN
To evaluate pallidal DBS in a non-DYT1 form of hereditary dystonia. We present the results of pallidal DBS in a family with non-DYT1 dystonia where DYT5 to 17 was excluded. The dystonia is following an autosomal dominant pattern. Ten members had definite dystonia and five had dystonia with minor symptoms. Four patients received bilateral pallidal DBS. Mean age was 47 years. The patients were evaluated before surgery, and "on" stimulation after a mean of 2.5 years (range 1-3) using the Burke-Fahn-Marsden scale (BFM). Mean BFM score decreased by 79 % on stimulation, from 42.5 +/- 24 to 9 +/- 6.5 at the last evaluation. Cervical involvement improved by 89%. The 2 patients with oromandibular dystonia and blepharospasm demonstrated a reduction of 95% regarding these symptoms. The present study confirms the effectiveness of pallidal DBS in a new family with hereditary primary segmental and generalized dystonia.
Asunto(s)
Estimulación Encefálica Profunda/métodos , Distonía Muscular Deformante/genética , Distonía Muscular Deformante/terapia , Salud de la Familia , Globo Pálido/fisiología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/genética , Índice de Severidad de la Enfermedad , SueciaRESUMEN
We have previously identified a homozygous missense (R221W) mutation in the NGFB gene in patients with loss of deep pain perception. NGF is important not only for the survival of sensory neurons but also for the sympathetic neurons and cholinergic neurons of the basal forebrain; however, it is the sensory neurons that are mainly affected in patients with mutant NGFB. In this report, we describe the effects of the mutation on the function of NGF protein and the molecular mechanisms that may underlie the pain insensitivity phenotype in these patients. We show that the mutant NGF has lost its ability to mediate differentiation of PC12 cells into a neuron-like phenotype. We also show that the inability of PC12 cells to differentiate is due to a markedly reduced secretion of mature R221W NGF. The R221W NGF is found mainly as proNGF, in contrast to wild-type NGF which is predominantly in the mature form in both undifferentiated and differentiated PC12 cells. The reduction in numbers of sensory fibers observed in the patients is therefore probably due to loss of trophic support as a result of drastically reduced secretion of NGF from the target organs. Taken together, these data show a clear decrease in the availability of mutant mature NGF and also an accumulation of proNGF in both neuronal and non-neuronal cells. The differential loss of NGF-dependent neurons in these patients, mainly affecting sensory neurons, may depend on differences in the roles of mature NGF and proNGF in different cells and tissues.
Asunto(s)
Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Neurogénesis , Precursores de Proteínas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Fibrinolisina/metabolismo , Furina/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Mutación Missense , Neuronas/metabolismo , Células PC12 , Dolor/genética , Ratas , Receptor trkA/metabolismoRESUMEN
We describe the mapping and identification of the gene for hereditary myopathy with lactic acidosis (HML). HML is characterized by low physical performance, resulting in physical exertion that causes early exhaustion, dyspnoea and palpitations. Using an autosomal recessive mode of inheritance, we mapped the trait to chromosome 12q23.3-24.11, with a maximum lod score of 5.26. The 1.6-Mb disease-critical region contained one obvious candidate gene-ISCU-specifying a protein involved in iron-sulphur cluster assembly. IscU is produced in two isoforms; one cytosolic and one mitochondrial, coded for by different splice variants of the ISCU gene. Mutational analysis of all exon and intron sequences as well as 1000 bp of the promoter of the ISCU gene revealed one intron mutation that was specific for the disease haplotype. The mutation is located in a region with homology to the interferon-stimulated response element (ISRE), but we could not see any effect of the mutation on expression levels in vitro or in vivo. We did, however, observe a drastic difference in the splicing pattern between patients and controls. In controls the mRNA was, as expected, mainly in the mitochondrial form, while in the patients a larger mRNA transcript was predominant. Sequencing of the product revealed that the mutation activates cryptic splice sites in intron 5 resulting in aberrant mRNA containing 100 bp of the intron. To conclude, our data strongly suggest that an intron mutation in the ISCU gene, leading to incorrectly spliced mRNA, is the cause of myopathy with lactic acidosis in this family.
Asunto(s)
Acidosis Láctica/genética , Cromosomas Humanos Par 12/genética , Proteínas Hierro-Azufre/genética , Enfermedades Musculares/genética , Empalme del ARN , Secuencia de Bases , Mapeo Cromosómico , Elementos de Facilitación Genéticos , Humanos , Intrones , Datos de Secuencia Molecular , Mutación , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SueciaRESUMEN
BACKGROUND: Congenital insensitivity to pain is a rare hereditary sensory neuropathy. PATIENTS: We present 6 patients from a family with a mutation in the nerve growth factor beta gene (NGFB). RESULTS: 3 patients were homozygous with a mutilating arthropathy starting early in life, and 3 patients were presumably heterozygous with a milder course starting in adulthood. All patients had normal mental abilities. In addition to absence of deep pain, the patients had impaired temperature sensation, but no autonomic deficiency. Sural nerve biopsies showed a moderate loss of A-delta fibres and a severe reduction in C fibers. Clinically, the disorder most often affected the lower extremities, with an insidious progressive joint swelling or a painless fracture, but the spine could also be involved with gross and unstable spondylolisthesis. Fracture healing was uneventful, but the arthropathy was progressive, eventually resulting in gross deformity and instability. When treating patients with congenital disorders such as this one, it is important to consider the slowly progressive nature of the disorder, and the orthopedic operations should therefore be planned from a long-term standpoint. Arthrodesis, limb lengthening and spinal decompression or fusion are the only elective procedures that seem reasonable. Fitting of orthosis for joint protection is also demanding. To delay the development of neuropathic arthropathy, patient education is essential but difficult in the very young. INTERPRETATION: The different expression between homo- and heterozygous subjects and the central role of nerve growth factor make this disease an interesting model system for studies of disease mechanisms and the molecular background to pain.
Asunto(s)
Fracturas Óseas , Artropatías , Factor de Crecimiento Nervioso/genética , Procedimientos Ortopédicos , Aparatos Ortopédicos , Insensibilidad Congénita al Dolor/genética , Adolescente , Adulto , Niño , Preescolar , Consanguinidad , Femenino , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/genética , Fracturas Óseas/cirugía , Heterocigoto , Homocigoto , Humanos , Artropatías/diagnóstico por imagen , Artropatías/genética , Artropatías/cirugía , Masculino , Mutación , Procedimientos Ortopédicos/métodos , Insensibilidad Congénita al Dolor/complicaciones , Insensibilidad Congénita al Dolor/cirugía , Linaje , Radiografía , Cicatrización de HeridasRESUMEN
Migraine is a complex, multifactorial disorder for which several loci have been identified in the human genome. We have previously reported linkage to a 10 Mb-region on chromosome 6p12.2-p21.1 in one large Swedish pedigree involving migraine with and without aura. To further investigate this candidate region, a dense set of single nucleotide polymorphic (SNP) markers was used for fine-mapping, decreasing the critical region to 8.5 Mb. Within this region, EFHC1 was recently identified as the disease gene for juvenile myoclonic epilepsy. Migraine and epilepsy has been suggested to share disease mechanisms and therefore EFHC1 is an excellent candidate gene for migraine in this family. Mutation analysis of the gene revealed a disease-segregating polymorphism in the promoter. Association analysis of the polymorphism in a case-control material did not support a role for this gene in migraine pathology. We therefore analyzed five additional candidate genes in the disease-critical region, including MEP1A, RHAG, IL17, SLC25A27 and TNFRSF21. In two of these genes, MEP1A and RHAG, we identified two novel polymorphisms associated with the disease haplotype. The combination of these polymorphisms could not be found in any control individuals, suggesting that they might be involved in genetic predisposition to migraine in this family.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas de Unión al Calcio/genética , Cromosomas Humanos Par 6/genética , Predisposición Genética a la Enfermedad/genética , Glicoproteínas de Membrana/genética , Metaloendopeptidasas/genética , Trastornos Migrañosos/genética , Aberraciones Cromosómicas , Análisis Mutacional de ADN , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad/epidemiología , Heterocigoto , Humanos , Incidencia , Masculino , Trastornos Migrañosos/epidemiología , Familia de Multigenes/genética , Linaje , Polimorfismo de Nucleótido Simple/genética , Suecia/epidemiologíaRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease primarily affecting the cerebellum, brainstem and retina. The disease is caused by a polyglutamine expansion in ataxin-7, a protein with largely unknown function. To improve our knowledge of the expression and function of wild-type and expanded ataxin-7, we looked for alternative SCA7 transcripts in mice. We identified a murine SCA7 isoform (SCA7b) containing an uncharacterized exon homologous to the newly identified human exon 12b. Northern blot analysis revealed three exon 12b containing transcripts with molecular sizes of 7.5, 4.4 and 3.0 kb in mice. This contrasted with the situation in humans, where only one exon 12b-containing transcript was observed. Furthermore, Northern blot of the human 4.4 kb SCA7b isoform predominantly showed expression in the brain, while expression of both the murine 7.5-kb and the 4.4-kb transcripts were observed in several tissues including brain, heart, liver, kidney and testis. Quantitative real-time RT-PCR analysis revealed that in muscle and heart SCA7b is the predominant SCA7 isoform, while in brain equal levels of SCA7a and SCA7b was observed. Insertion of exon 12b into the murine SCA7 ORF resulted in a frame-shift that gave rise to an alternative ataxin-7 protein (ataxin-7b). The novel 58-amino acid C-terminus in ataxin-7b directed the protein to a more cytoplasmic location.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Ataxina-7 , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Exones , Mutación del Sistema de Lectura , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Sistemas de Lectura Abierta , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease primarily affecting the brainstem, retina and Purkinje cells of the cerebellum. The disease is caused by a polyglutamine expansion in ataxin-7, a protein found in two complexes TFTC and STAGA, involved in transcriptional regulation. Transcriptional dysregulation has been implicated in the pathology of several polyglutamine diseases. In this paper, we analyzed the effect of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP and the Purkinje cell expressed nuclear receptor RORalpha1. We could show that transcription mediated by both CBP and RORalpha1 was repressed by expanded ataxin-7. Interestingly, repression of transcription could also be observed with wild-type full-length ataxin-7, not only on CBP- and RORalpha1-mediated transcription, but also on basal transcription. The repression could be counteracted by inhibition of deacetylation, suggesting that ataxin-7 may act as a repressor of transcription by inhibiting the acetylation activity of TFTC and STAGA.
Asunto(s)
Cerebelo/metabolismo , Proteínas del Tejido Nervioso/genética , Elementos Reguladores de la Transcripción/genética , Ataxias Espinocerebelosas/genética , Acetilación , Ataxina-7 , Proteína de Unión a CREB/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cerebelo/patología , Cerebelo/fisiopatología , Niño , Histonas/metabolismo , Humanos , Masculino , Mutación/genética , Regiones Promotoras Genéticas/genética , Células de Purkinje/metabolismo , Células de Purkinje/patología , Proteínas Tirosina Quinasas Receptoras , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Receptores de Superficie Celular/genética , Proteínas Represoras/genética , Elementos Silenciadores Transcripcionales/genética , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/fisiopatología , Activación Transcripcional/genética , Expansión de Repetición de Trinucleótido/genética , Células Tumorales CultivadasRESUMEN
Identification of genes associated with pain insensitivity syndromes can increase the understanding of the pathways involved in pain and contribute to the understanding of how sensory pathways relate to other neurological functions. In this report we describe the mapping and identification of the gene responsible for loss of deep pain perception in a large family from northern Sweden. The loss of pain perception in this family is characterized by impairment in the sensing of deep pain and temperature but with normal mental abilities and with most other neurological responses intact. A severe reduction of unmyelinated nerve fibers and a moderate loss of thin myelinated nerve fibers are observed in the patients. Thus the cases in this study fall into the class of patients with loss of pain perception with underlying peripheral neuropathy. Clinically they best fit into HSAN V. Using a model of recessive inheritance we identified an 8.3 Mb region on chromosome 1p11.2-p13.2 shared by the affected individuals in the family. Analysis of functional candidate genes in the disease critical region revealed a mutation in the coding region of the nerve growth-factor beta (NGFB) gene specific for the disease haplotype. This NGF mutation seems to separate the effects of NGF involved in development of central nervous system functions such as mental abilities, from those involved in peripheral pain pathways. This mutation could therefore potentially provide an important tool to study different roles of NGF, and of pain control.
Asunto(s)
Factor de Crecimiento Nervioso/genética , Insensibilidad Congénita al Dolor/genética , Dolor/genética , Adolescente , Adulto , Animales , Bovinos , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Cobayas , Humanos , Masculino , Ratones , Linaje , Estructura Secundaria de Proteína , RatasRESUMEN
We have identified a large family in the northern part of Sweden with multiple cases of autoimmune diseases, namely type 1 diabetes (T1D), Graves' disease (GD) and Hashimoto's thyroiditis (HT). The family members affected by any of these diseases share a region of 2.4 Mb that comprises among others the CTLA4 gene. We determined that all affected members of the family shared the HLA susceptibility haplotype (DR4-DQA1*0301-DQB1*0302). Analysis of genetic interaction conditioning for HLA haplotype provided strong evidence that the critical region which includes the CTLA4 gene acts together with the HLA locus on the etiology of disease (lodscore 4.20 (theta=0.0). The study of this family allowed us to: (1) reinforce a number of reports on linkage and association of the CTLA4 region to T1D and AITD; (2) demonstrate that a single haplotypic variant in this region constitutes an etiological factor to disease susceptibility in T1D, GD and HT; (3) reveal a strong genetic interaction of the CTLA4 and HLA loci in the genetic architecture of autoimmune disease; (4) emphasise the value of large pedigrees drawn from isolated populations as tools to single out the effect of individual loci in the etiology of complex diseases.
Asunto(s)
Antígenos de Diferenciación/genética , Autoinmunidad/genética , Diabetes Mellitus Tipo 1/genética , Enfermedad de Graves/genética , Inmunoconjugados , Tiroiditis Autoinmune/genética , Abatacept , Antígenos CD , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos CD28/genética , Antígeno CTLA-4 , Diabetes Mellitus Tipo 1/etiología , Ligamiento Genético , Marcadores Genéticos , Enfermedad de Graves/etiología , Antígenos HLA/genética , Antígenos HLA/inmunología , Haplotipos/genética , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles , Escala de Lod , Mutación , Linaje , Suecia , Tiroiditis Autoinmune/etiologíaRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein ataxin-7, a protein of unknown function. In order to analyze the expression pattern of wild type ataxin-7 in detail, the murine SCA7 gene homolog was cloned and the expression pattern in mice analyzed. The SCA7 mouse and human gene exhibit a high degree of identity at both DNA (88.2%) and protein (88.7%) level. The CAG repeat region, known to be polymorphic in man, is conserved in mouse but contained only five repeats in all mouse strains analyzed. The arrestin homology domain and the nuclear localization signal found in human ataxin-7 is also conserved in the murine homolog. Expression of ataxin-7 was detected during mouse embryonic development and in all adult mouse tissues examined by northern and western blots. In brain, immunohistological staining revealed an ataxin-7 expression pattern similar to that in human, with ataxin-7 expression in cerebellum, several brainstem nuclei, cerebral cortex and hippocampus. Our data show high conservation of ataxin-7 both structurally and at the level of expression, suggesting a conserved role for the protein in mice and humans.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Ataxina-7 , Northern Blotting , Western Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Embrión de Mamíferos/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Testículo/metabolismoRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder primarily affecting the cerebellum, brain stem and retina. The disease is caused by an expanded polyglutamine tract in the protein ataxin-7. In this study we analyzed the expression pattern of ataxin-7 in CNS and non-CNS tissue from three SCA7 patients and age-matched controls. SCA7 is a rare autosomal dominant disorder, limiting the number of patients available for analysis. We therefore compiled data on ataxin-7 expression from all SCA7 patients (n=5) and controls (n=7) published to date, and compared with the results obtained in this study. Expression of ataxin-7 was found in neurons throughout the CNS and was highly abundant in Purkinje cells of the cerebellum, in regions of the hippocampus and in cerebral cortex. Ataxin-7 expression was not restricted to regions of pathology, and there were no apparent regional differences in ataxin-7 expression patterns between patients and controls. The subcellular distribution of ataxin-7 was primarily nuclear in all brain regions studied. In cerebellar Purkinje cells, however, differences in subcellular distribution of ataxin-7 were observed between patients and controls of different ages. Here we provide an increased understanding of the distribution of ataxin-7, and the possible implication of subcellular localization of this protein on disease pathology is discussed.