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1.
Clin. transl. oncol. (Print) ; 17(7): 511-520, jul. 2015. ilus
Artigo em Inglês | IBECS | ID: ibc-138447

RESUMO

Introduction. In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal–epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124–131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. Materials and methods. Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. Results. We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40 % and C/EBPβ expression by 80 % in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40 % in GAL3ST2-treated cells. Conclusions. Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity (AU)


No disponible


Assuntos
Animais , Feminino , Camundongos , Células 3T3-L1/citologia , Células 3T3-L1/patologia , Galactose/análise , Galactose/isolamento & purificação , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/veterinária , Glândulas Mamárias Animais/citologia , Adipócitos/citologia , Adipócitos/patologia , Cromatografia , Cromatografia/veterinária , Western Blotting
2.
Clin Transl Oncol ; 17(7): 511-20, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25532485

RESUMO

INTRODUCTION: In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal-epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124-131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. MATERIALS AND METHODS: Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. RESULTS: We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40 % and C/EBPß expression by 80 % in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40 % in GAL3ST2-treated cells. CONCLUSIONS: Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity.


Assuntos
Adipócitos/metabolismo , Adipogenia , Glândulas Mamárias Animais/metabolismo , Sulfotransferases/metabolismo , Sulfurtransferases/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Células HEK293 , Humanos , Células MCF-7 , Glândulas Mamárias Animais/citologia , Camundongos , Células NIH 3T3 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/metabolismo
3.
Redox Rep ; 18(6): 210-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24112955

RESUMO

OBJECTIVES: Oxidative stress plays critical roles in the pathogeneses of diabetes, hypertension, and atherosclerosis, but its effect on fat accumulation is still unclear. In this study, we analyzed the role of the well-known antioxidant and a glutathione (GSH) precursor N-acetylcysteine (NAC) in fat accumulation and the expression of obesity-associated proteins. METHODS: We studied the effects of 10 µM NAC on obesity-related protein expression in cultured 3T3-L1 preadipocytes, which are able to differentiate into mature adipocytes and accumulate lipids. RESULTS: NAC treatment inhibited fat accumulation and reduced the expression of obesity-related proteins, including monoamine oxidase A, heat shock protein 70 (HSP70), aminoacylase -1 (ACY-1), and transketolase. DISCUSSION: Our results suggest that the effects of NAC on triglycerides (Tgs) and protein expression are correlated. In support of this, we showed that NAC treatment affected both the Tg synthesis pathway and the expression levels of proteins implicated in human obesity.


Assuntos
Acetilcisteína/farmacologia , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Amidoidrolases/biossíntese , Animais , Diferenciação Celular/fisiologia , Proteínas de Ligação a Ácido Graxo/biossíntese , Proteínas de Choque Térmico HSP72/biossíntese , Camundongos , Monoaminoxidase/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Transcetolase/biossíntese , Triglicerídeos/metabolismo
4.
Int J Obes (Lond) ; 34(3): 487-99, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20029374

RESUMO

CONTEXT: Very limited information is available regarding the function of human thyroid hormone responsive Spot 14 (human S14, hS14) in adipogenesis and human adiposity. OBJECTIVE: To evaluate hS14 levels during differentiation of human pre-adipocytes, in human fat depots and isolated fat cells. DESIGN: This was a cross-sectional study. SUBJECTS: A total of 161 omental (OM) and 87 subcutaneous (SC) adipose tissue samples obtained during elective surgical procedures from a population who varied widely in terms of obesity. MEASUREMENTS: hS14 gene expression and protein levels during adipogenesis were assessed by RT-PCR, western blot, and using an automated confocal imaging approach. RESULTS: hS14 gene expression levels were decreased in OM adipose tissue from overweight (-42.0%) and obese subjects (-56.5%) compared with lean subjects (P<0.05 and P<0.0001, respectively). hS14 mRNA (but not hS14-related) was inversely associated with obesity measures such as body mass index (P=0.001), percent fat mass (P=0.001), waist-to-hip ratio (P=0.020), and systolic blood pressure (P=0.031). hS14 gene expression and protein levels were up-regulated at the early stages of differentiation of human pre-adipocytes as well as for 3T3-L1 cells. That observation was most prominent in those individual cells exhibiting the more marked differentiation features. hS14 gene expression levels increased by approximately 45 000-fold in mature adipocytes. Increased hS14 levels were also found in stromal-vascular cells/pre-adipocytes (3.8-fold, P<0.05) and in adipose tissue samples (1.9-fold, P<0.0001) from SC compared with OM fat depots. CONCLUSIONS: These results suggest that hS14 is involved in human adipogenesis, but inversely related to obesity and OM fat accumulation.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Proteínas Nucleares/metabolismo , Obesidade/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipogenia/genética , Animais , Western Blotting , Diferenciação Celular/genética , Células Cultivadas , Estudos Transversais , Regulação para Baixo , Expressão Gênica , Humanos , Camundongos , Proteínas Nucleares/genética , Omento/metabolismo , Sobrepeso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Gordura Subcutânea/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Fatores de Transcrição/genética
5.
Rev. esp. investig. quir ; 10(1): 26-32, ene.-mar. 2007. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-87280

RESUMO

INTRODUCCIÓN: Los defectos óseos pueden ser debidos a anomalías congénitas, traumatismos, procesos infecciosos o tumorales,que a menudo se asocian a severos problemas funcionales y/o estéticos, que van a requerir un tratamiento complejo. El objetivo de este estudio fue comparar la formación de hueso nuevo tras la aplicación de una membrana reabsorbible y dos tipos de sustitutivos óseos. MATERIAL Y MÉTODO. Se utilizaron 24 ratas adultas machos tipo Wistar. Se crearon defectos circulares de 4 mm de diámetro en ambos lados de la mandíbula, y fueron divididos en 4 grupos: grupo I, grupo control con defectos vacíos; grupo II, los defectos fueron cubiertos con una membrana de hueso desmineralizado (Lambone®); grupo III, los defectos fueron rellenos con colágeno liofilizado bovino (Colloss®) y cubiertos con Lambone®; grupo IV, los defectos fueron rellenos con vidrio bioactivo (NovaBone®) y cubiertos con Lambone®. Los animales fueron sacrificados a las 3 y 6 semanas de la cirugía, realizándose un análisis radiológico e histológico. RESULTADOS. Los defectos control no mostraron formación ósea, apareciendo una reparación por tejido fibroso. La membrana de hueso utilizada de forma aislada, actuó como una barrera eficaz excluyendo los tejidos no osteogénicos, pero no se produjo reparación total del defecto en ningún caso. El grupo de Colloss® y membrana mostró una regeneración ósea completa del defecto a las 6 semanas. El grupo de NovaBone® y membrana no mostró formación ósea, apareciendo las partículas del biomaterial ocupando el defecto. CONCLUSIONES. La regeneración ósea fue significativamente mayor en los defectos rellenos con Colloss® y cubiertos con la membrana de Lambone®, comparado con los otros grupos experimentales (AU)


INTRODUCTION. Bony defects may be found as a result of congenital anomalies, trauma, neoplasms, or infectious conditions. uch conditions are often associated with severe funtional and esthetic problems. Corrective treatment is often complicated by limitations in tissue adaptations. The aim of this study was to compare the new bone formation following application of a bioabsorbable membrane and two types of bone substitutes. MATERIAL AND METHOD. In the present study, 24 4-month-old male Wistar rats were used. Standardized round through-and-through osseous defect (4 mm in diameter) was created similarly on both sides of the jaw and, were divided in four groups: group I, control defects were left empty; group II, the defects were covered with demineralized laminar bone membrane (Lambone®); group III, defects were filled with bovine bone collagen- protein extracts (Colloss®) and covered with Lambone®; group IV, defects were filled with bioactive glass (NovaBone®) and covered with Lambone®. Animals were killed 3 and 6 weeks after surgery, and healing of the bone defects was assessed by radiologic and histologic analysis. RESULTS. The control defects showed no bone formation, and the holes were filled with fibrous connective tissue. Bone membrane alone acted as an efficient barrier, excluding the nonosteogenic tissues, but new bone formation underneath the membrane was not full. The group of Colloss® and membrane showed complete healing after 6 weeks. The group of NovaBone® and membrane showed no bone formation, appearing the particles in the defect. CONCLUSION. A significantly higher percentage of bone regeneration was seen in the sites filled with Colloss® and covered with Lambone®, compared with the others experimental groups (AU)


Assuntos
Animais , Ratos , Regeneração Óssea/fisiologia , Mandíbula/fisiologia , Substitutos Ósseos/farmacocinética , Regeneração Tecidual Guiada/métodos , Desmineralização Patológica Óssea/cirurgia , Modelos Animais de Doenças , Membranas/fisiologia , Vidro , Colágeno/uso terapêutico
6.
Cell ; 106(3): 367-79, 2001 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-11509185

RESUMO

Anxiety disorders are complex and common psychiatric illnesses associated with considerable morbidity and social cost. We have studied the molecular basis of the cooccurrence of panic and phobic disorders with joint laxity. We have identified an interstitial duplication of human chromosome 15q24-26 (named DUP25), which is significantly associated with panic/agoraphobia/social phobia/joint laxity in families, and with panic disorder in nonfamilial cases. Mosaicism, different forms of DUP25 within the same family, and absence of segregation of 15q24-26 markers with DUP25 and the psychiatric phenotypes suggest a non-Mendelian mechanism of disease-causing mutation. We propose that DUP25, which is present in 7% control subjects, is a susceptibility factor for a clinical phenotype that includes panic and phobic disorders and joint laxity.


Assuntos
Cromossomos Humanos Par 15/genética , Duplicação Gênica , Predisposição Genética para Doença/genética , Instabilidade Articular/genética , Transtorno de Pânico/genética , Transtornos Fóbicos/genética , Polimorfismo Genético/genética , Adulto , Alelos , Feminino , Dosagem de Genes , Genes Duplicados/genética , Humanos , Hibridização in Situ Fluorescente , Interleucina-16/genética , Instabilidade Articular/complicações , Escore Lod , Masculino , Repetições de Microssatélites/genética , Mosaicismo/genética , Mutação/genética , Transtorno de Pânico/complicações , Linhagem , Penetrância , Transtornos Fóbicos/complicações , Mapeamento Físico do Cromossomo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Genome Res ; 11(1): 98-111, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11156619

RESUMO

Several cytogenetic alterations affect the distal part of the long arm of human chromosome 15, including recurrent rearrangements between 12p13 and 15q25, which cause congenital fibrosarcoma (CFS). We present here the construction of a BAC/PAC contig map that spans 2 Mb from the neurotrophin-3 receptor (NTRK3) gene region on 15q25.3 to the proximal end of the Bloom's syndrome region on 15q26.1, and the identification of a set of new chromosome 15 duplicons. The contig reveals the existence of several regions of sequence similarity with other chromosomes (6q, 7p, and 12p) and with other 15q cytogenetic bands (15q11-q13 and 15q24). One region of similarity maps on 15q11-q13, close to the Prader-Willi/Angelman syndromes (PWS/AS) imprinting center. The 12p similar sequence maps on 12p13, at a distance to the ets variant 6 (ETV6) gene that is equivalent on 15q26.1 to the distance to the NTRK3 gene. These two genes are the targets of the CFS recurrent translocations, suggesting that misalignments between these two chromosomes regions could facilitate recombination. The most striking similarity identified is based on a low copy repeat sequence, mainly present on human chromosome 15 (LCR15), which could be considered a newly recognized duplicon. At least 10 copies of this duplicon are present on chromosome 15, mainly on 15q24 and 15q26. One copy is located close to a HERC2 sequence on the distal end of the PWS/AS region, three around the lysyl oxidase-like (LOXL1) gene on 15q24, and three on 15q26, one of which close to the IQ motif containing GTPase-activating protein 1 (IQGAP1) gene on 15q26.1. These LCR15 span between 13 and 22 kb and contain high identities with the golgin-like protein (GLP) and the SH3 domain-containing protein (SH3P18) gene sequences and have the characteristics of duplicons. Because duplicons flank chromosome regions that are rearranged in human genomic disorders, the LCR15 described here could represent new elements of rearrangements affecting different regions of human chromosome 15q.


Assuntos
Cromossomos Humanos Par 15/genética , Genes Duplicados , Sequência de Bases , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Mapeamento de Sequências Contíguas , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico
8.
Am J Hum Genet ; 68(1): 46-63, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11115377

RESUMO

Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of >50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.


Assuntos
Testes Genéticos , Mutação/genética , Rim Policístico Autossômico Dominante/diagnóstico , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Estudos de Coortes , Análise Mutacional de DNA/métodos , Primers do DNA , Enzimas de Restrição do DNA/metabolismo , Feminino , Ligação Genética/genética , Testes Genéticos/métodos , Análise Heteroduplex , Humanos , Masculino , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Sinais Direcionadores de Proteínas/genética , Análise de Sequência de DNA , Deleção de Sequência/genética , Canais de Cátion TRPP
9.
Cytogenet Cell Genet ; 88(1-2): 62-7, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10773667

RESUMO

The HMG box encodes a conserved DNA binding domain found in many proteins and is involved in the regulation of transcription and chromatin conformation. We describe HMG20A and HMG20B, two novel human HMG box-containing genes, discovered within the EURO-IMAGE Consortium full-length cDNA sequencing initiative. The predicted proteins encoded by these two genes are 48.4% identical (73.9% within the HMG domain). The HMG domain of both HMG20 proteins is most similar to that of yeast NHP6A (38% to 42%). Outside of this domain, HMG20 proteins lack any significant homology to other known proteins. We determined the genomic structure and expression pattern of HMG20A and HMG20B. Both genes have several alternative transcripts, expressed almost ubiquitously. HMG20A maps to chromosome 15q24 (near D15S1227) and HMG20B to 19p13.3 (between D19S209 and D19S216). The HMG20 genes define a distinct class of mammalian HMG box genes.


Assuntos
Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 19/genética , Proteínas de Ligação a DNA , Perfilação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Ubiquitinas/química , Ubiquitinas/genética , Processamento Alternativo/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Éxons/genética , Etiquetas de Sequências Expressas , Proteínas de Grupo de Alta Mobilidade/química , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Escore Lod , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência
10.
Tissue Antigens ; 55(2): 118-27, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10746783

RESUMO

CD84, a new member of the immunoglobulin superfamily, shows high homology with several molecules belonging to the CD2 family of differentiation antigens. By screening a peripheral blood leukocyte cDNA library four CD84 isoforms were obtained differing in their 3' sequence. A reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that these isoforms were normally found on leukocytes and a new isoform was identified. To establish the nature of the five isoforms obtained (CD84a, CD84b, CD84c, CD84d and CD84e) the genomic structure of the CD84 gene was determined. Our results show that it is composed of at least eight exons, with an exon coding for the 5' UTR and the leader peptide, two exons coding for each of the two immunoglobulin-like domains, an exon encoding the transmembrane portion and four exons coding for the cytoplasmic domains. The isoforms are generated by several mechanisms: alternative use of exons, reading frame shift, use of a cryptic splice site or absence of splicing. The differential expression of several potentially phosphorylatable residues on the different isoforms could be a way to regulate its possible activity in signal transduction.


Assuntos
Antígenos CD/genética , Glicoproteínas de Membrana , Sequência de Aminoácidos , Antígenos CD/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar , Expressão Gênica , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Família de Moléculas de Sinalização da Ativação Linfocitária
12.
Am J Hum Genet ; 60(6): 1399-410, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9199561

RESUMO

Mutation screening of the major autosomal dominant polycystic kidney disease gene (PKD1) has been complicated by the large transcript size (> 14 kb) and by reiteration of the genomic area encoding 75% of the protein on the same chromosome (the HG loci). The sequence similarity between the PKD1 and HG regions has precluded specific analysis of the duplicated region of PKD1, and consequently all previously described mutations map to the unique 3' region of PKD1. We have now developed a novel anchored reverse-transcription-PCR (RT-PCR) approach to specifically amplify duplicated regions of PKD1, employing one primer situated within the single-copy region and one within the reiterated area. This strategy has been incorporated in a mutation screen of 100 patients for more than half of the PKD1 exons (exons 22-46; 37% of the coding region), including 11 (exons 22-32) within the duplicated gene region, by use of the protein-truncation test (PTT). Sixty of these patients also were screened for missense changes, by use of the nonisotopic RNase cleavage assay (NIRCA), in exons 23-36. Eleven mutations have been identified, six within the duplicated region, and these consist of three stop mutations, three frameshifting deletions of a single nucleotide, two splicing defects, and three possible missense changes. Each mutation was detected in just one family (although one has been described elsewhere); no mutation hot spot was identified. The nature and distribution of mutations, plus the lack of a clear phenotype/genotype correlation, suggest that they may inactivate the molecule. RT-PCR/PTT proved to be a rapid and efficient method to detect PKD1 mutations (differentiating pathogenic changes from polymorphisms), and we recommend this procedure as a firstpass mutation screen in this disorder.


Assuntos
Mutação Puntual , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Sequências Repetitivas de Ácido Nucleico , Adulto , Idade de Início , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Éxons , Feminino , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Deleção de Sequência , Canais de Cátion TRPP , Transcrição Genética
13.
Hum Mol Genet ; 5(4): 539-42, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8845849

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most common single gene disorder resulting in renal failure. It is generally an adult onset disease, but rarely, cases of severe childhood polycystic disease arise in ADPKD families. The clear clinical anticipation in these pedigrees has led to the suggestion that the mutation may be an unstable trinucleotide repeat. We have now identified a nonsense mutation, Tyr3818Stop, in one such family (P117) within the major ADPKD gene, polycystic kidney disease 1 (PKD1). The mutation is shown to be a de novo change in the father, and of grandpaternal origin. PKD1 manifests as typical adult onset disease in the father, but is seen as severe disease, detected as enlarged polycystic kidneys in utero, in one of a pair of dizygotic twins; the other twin has the mutation but no evidence of cysts, consistent with an adult onset disease course. The finding of the same stable mutation associated with very different disease severity in this family indicates that phenotypic variation in PKD1 is not due to a dynamic mutation. It seems most likely that a small number of modifying factors may radically affect the course of disease in PKD1; identification of such factors will have important prognostic implications in this disorder.


Assuntos
Mutação Puntual , Rim Policístico Autossômico Dominante/genética , Adulto , Idade de Início , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Criança , DNA , Doenças em Gêmeos/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Rim Policístico Autossômico Dominante/congênito , Rim Policístico Autossômico Dominante/diagnóstico por imagem , Gêmeos Dizigóticos , Ultrassonografia
14.
Am J Hum Genet ; 58(1): 86-96, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8554072

RESUMO

Recently, the gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1 (polycystic kidney disease 1), has been fully characterized and shown to encode an integral membrane protein, polycystin, involved in cell-cell and/or cell-matrix interactions. Study of the PKD1 gene has been complicated because most of the gene lies in a genomic region reiterated several times elsewhere on the same chromosome, and consequently only seven mutations have been described so far. Here we report a systematic screen covering approximately 80% of the approximately 2.75 kb of translated transcript that is encoded by single-copy DNA. We have identified and characterized six novel mutations that, together with the previously described changes, amount to a detection rate of 10%-15% in the population studied. The newly described mutations are two deletions, an insertion of a T-nucleotide causing a frame shift, two single-base-pair substitutions resulting in premature stop codons, and a G-->C transversion that may be a missense mutation. These results have important implications for genetic diagnosis of PKD1 because they indicate that the majority of mutations lie within the duplicated area, which is difficult to study. The regions of polycystin removed in each mutation so far described are assessed for their functional significance; an area disrupted by two new small in-frame changes is highlighted. PKD1 mutations are contrasted with those in the PKD1/TSC2 contiguous-gene syndrome, and the likely mutational mechanism in PKD1 is considered.


Assuntos
Mutação , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Processamento Alternativo , Sequência de Bases , Primers do DNA , Éxons , Feminino , Mutação da Fase de Leitura , Humanos , Íntrons , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Deleção de Sequência , Canais de Cátion TRPP
15.
J Am Soc Nephrol ; 6(4): 1125-33, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8589278

RESUMO

The identification of the primary defect in autosomal dominant polycystic kidney disease (ADPKD) by biochemical methods has proved difficult because of the complexity of the cystic kidney. However, by the use of the genetic method of positional cloning, a gene accounting for approximately 85% of ADPKD, PKD1, has now been identified in the chromosome region 16p13.3. Its exact location was pinpointed because it was bisected by a chromosome translocation; members with the balanced exchange had PKD1. The PKD1 gene encodes an approximately 14-kb transcript, but full characterization was complicated, because most of the gene lies in a genomic region that is duplicated elsewhere on chromosome 16; the duplicate area encodes three genes with substantial homology to PKD1. At present, only seven mutations of PKD1 have been characterized and these are clustered in the nonduplicated, 3' end of the gene. However, a number of patients with large deletions of the PKD1 and adjacent tuberous sclerosis 2 (TSC2) genes, who have tuberous sclerosis and severe childhood-onset polycystic kidney disease, have also been described. Recently, the entire sequence of the PKD1 transcript and the genomic region containing the gene have been determined. The PKD1 gene covers approximately 52 kb of genomic DNA and is divided into 46 exons. The transcript is approximately 14.15 kb, and the predicted protein polycystin is 4302/3 amino acids with a calculated mass of approximately 460 kd. Polycystin contains several distinctive extracellular domains, including a flank-leucine rich repeat-flank domain, a C-type lectin, 16 approximately 85-amino-acid units that are similar to immunoglobulin repeats, four fibronectin Type III-related domains, and a low-density lipoprotein A domain. The C-terminal third of the protein has multiple hydrophobic regions, and modeling of this region suggests the presence of many transmembrane domains and a cytoplasmic C terminus. Hence, polycystin is probably an integral membrane protein with multiple extracellular domains that are involved in cell-cell and/or cell-matrix interactions. The ADPKD phenotype suggests that polycystin may play a role in cell-matrix communication, which is important for normal basement membrane production and for controlling cellular differentiation.


Assuntos
Mapeamento Cromossômico , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Clonagem Molecular , Genes , Humanos , Mutação , Proteínas/genética , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPP , Transcrição Genética , Translocação Genética
16.
Nat Genet ; 10(2): 151-60, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7663510

RESUMO

Characterization of the polycystic kidney disease 1 (PKD1) gene has been complicated by genomic rearrangements on chromosome 16. We have used an exon linking strategy, taking RNA from a cell line containing PKD1 but not the duplicate loci, to clone a cDNA contig of the entire transcript. The transcript consists of 14,148 bp (including a correction to the previously described C terminus), distributed among 46 exons spanning 52 kb. The predicted PKD1 protein, polycystin, is a glycoprotein with multiple transmembrane domains and a cytoplasmic C-tail. The N-terminal extracellular region of over 2,500 aa contains leucine-rich repeats, a C-type lectin, 16 immunoglobulin-like repeats and four type III fibronectin-related domains. Our results indicate that polycystin is an integral membrane protein involved in cell-cell/matrix interactions.


Assuntos
Glicoproteínas de Membrana/genética , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento Cromossômico , Cromossomos Humanos Par 16 , Clonagem Molecular , Simulação por Computador , DNA Complementar/análise , Fibronectinas/genética , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Rim Policístico Autossômico Dominante/química , Rim Policístico Autossômico Dominante/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Proteínas/química , Ratos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPP
17.
Hum Mol Genet ; 4(4): 569-74, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7633405

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease which frequently results in renal failure. The major ADPKD gene, polycystic kidney disease 1 (PKD1), has recently been identified. In an attempt to understand better the aetiology of this disorder we have searched for mutations in the PKD1 gene. Analysis of three regions in the 3' part of the gene has revealed two mutations that occur by a novel mechanism. Both mutations are deletions (of 18 or 20 bp) within the same 75 bp intron and although these deletions do not disrupt the splice donor or acceptor sites at the boundary of the intron, they nevertheless result in aberrant splicing. Two different transcripts are produced in each case; one includes the deleted intron while the other has a 66 bp deletion due to activation of a cryptic 5' splice site. No normal product is generated from the deleted gene. Aberrant splicing probably occurs because the deleted intron is too small for spliceosome assembly using the authentic splice sites; this mechanism has previously only been described from in vitro studies of vertebrate genes. A 9 bp direct repeat has been identified within the intron, which probably facilitated deletion by promoting misalignment of sequence. The possible phenotypic implications of producing more than one aberrant PKD1 transcript in these cases are discussed.


Assuntos
Rim Policístico Autossômico Dominante/genética , Processamento de RNA/genética , Deleção de Sequência , Adulto , Idoso , Sequência de Bases , DNA Complementar , Humanos , Íntrons , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo
18.
Hum Mol Genet ; 4 Spec No: 1745-9, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8541874

RESUMO

Using a positional cloning approach the major autosomal dominant polycystic kidney disease (ADPKD) gene (PKD1) has been identified on chromosome 16: a disease associated chromosome translocation was instrumental in its identification. Study of the PKD1 gene has been complicated because most of the gene lies in a genomic region reiterated elsewhere on the same chromosome. The duplicate area contains three genes which share substantial homology with PKD1 and generate polyadenylated transcripts. Most PKD1 mutations have so far been detected in the single copy, 3' end of the gene, but a group of patients with deletion of PKD1 and the adjacent TSC2 gene, which have severe infantile polycystic kidney disease, have also been characterised. The full length transcript of PKD1 (approximately 14 kb) has now been cloned and is predicted to encode a protein, polycystin, of 4302/3 aa. Polycystin contains multiple extracellular domains including leucine rich repeats, a C-type lectin, immunoglobulin and fibronectin type III-like domains and has a C terminal region which is likely associated with the membrane. These homologies indicate that polycystin is a cell-cell/matrix interaction protein.


Assuntos
Cromossomos Humanos Par 16 , Rim Policístico Autossômico Dominante/genética , Sequência de Bases , Mapeamento Cromossômico , Humanos , Dados de Sequência Molecular , Proteínas/genética , Canais de Cátion TRPP , Translocação Genética
19.
Am J Hum Genet ; 56(1): 248-53, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7825585

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder. At least two distinct forms of ADPKD are now well defined. In approximately 86% of affected European families, a gene defect localized to 16p13.3 was responsible for ADPKD, while a second locus has been recently localized to 4q13-q23 as candidate for the disease in the remaining families. We present confirmation of linkage to microsatellite markers on chromosome 4q in eight Spanish families with ADPKD, in which the disease was not linked to 16p13.3. By linkage analysis with marker D4S423, a maximum lod score of 9.03 at a recombination fraction of .00 was obtained. Multipoint linkage analysis, as well as a study of recombinant haplotypes, placed the PKD2 locus between D4S1542 and D4S1563, thereby defining a genetic interval of approximately 1 cM. The refined map will serve as a genetic framework for additional genetic and physical mapping of the region and will improve the accuracy of presymptomatic diagnosis of PKD2.


Assuntos
Cromossomos Humanos Par 4 , Genes Dominantes , Rim Policístico Autossômico Dominante/genética , Adulto , Idoso , Alelos , Sequência de Bases , Mapeamento Cromossômico , DNA Satélite/genética , Feminino , Humanos , Escore Lod , Linfócitos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Recombinação Genética , Espanha
20.
Nat Genet ; 8(4): 328-32, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7894481

RESUMO

Major genes which cause tuberous sclerosis (TSC) and autosomal dominant polycystic kidney disease (ADPKD), known as TSC2 and PKD1 respectively, lie immediately adjacent to each other on chromosome 16p. Renal cysts are often found in TSC, but a specific renal phenotype, distinguished by the severity and infantile presentation of the cystic changes, is seen in a small proportion of cases. We have identified large deletions disrupting TSC2 and PKD1 in each of six such cases studied. Analysis of the deletions indicates that they inactivate PKD1, in contrast to the mutations reported in ADPKD patients, where in each case abnormal transcripts have been detected.


Assuntos
Deleção de Genes , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Pré-Escolar , Cromossomos Humanos Par 16 , Primers do DNA , Eletroforese em Gel de Campo Pulsado , Humanos , Lactente , Pessoa de Meia-Idade , Dados de Sequência Molecular , Canais de Cátion TRPP , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor
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