RESUMO
Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.
Assuntos
Braquidactilia , Proteína Relacionada ao Hormônio Paratireóideo , Humanos , Proteína Relacionada ao Hormônio Paratireóideo/genética , Braquidactilia/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Metaloproteases , Proteínas ADAMRESUMO
Chromosomes occupy distinct interphase territories in the three-dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter-chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC-derived cell types by DNA-FISH We compared our findings in normal karyotypes with a three-generation family harboring a 2q37-deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 (HDAC4) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue-specific interaction bias at certain loci. These inter-chromosomal interactions were confirmed by Hi-C. Interestingly, the disease-related HDAC4 deletion resulted in displaced inter-chromosomal arrangements and altered interactions between the deletion-affected chromosome 2 and chromosome 12 and/or 17 in 2q37-deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis.
Assuntos
Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 2/genética , Deleção de Genes , Histona Desacetilases/genética , Proteínas Repressoras/genética , Translocação Genética/genética , Núcleo Celular/genética , Deleção Cromossômica , Humanos , Hibridização in Situ Fluorescente , Interfase/genética , Células-Tronco Mesenquimais/citologiaRESUMO
CD177 presents antigens in allo- and autoimmune diseases on the neutrophil surface. Individuals can be either CD177-deficient or harbor distinct CD177neg and CD177pos neutrophil subsets. We studied mechanisms controlling subset-restricted CD177 expression in bimodal individuals. CD177pos, but not CD177neg neutrophils, produced CD177 protein and mRNA. Haplotype analysis indicated a unique monoallelic CD177 expression pattern, where the offspring stably transcribed either the maternal or paternal allele. Hematopoietic stem cells expressed both CD177 alleles and silenced one copy during neutrophil differentiation. ChIP and reporter assays in HeLa cells with monoallelic CD177 expression showed that methylation reduced reporter activity, whereas demethylation caused biallelic CD177 expression. HeLa cell transfection with c-Jun and c-Fos increased CD177 mRNA. Importantly, CD177pos human neutrophils, but not CD177neg neutrophils, showed a euchromatic CD177 promoter, unmethylated CpGs, and c-Jun and c-Fos binding. We describe epigenetic mechanisms explaining the two distinct CD177 neutrophil subsets and a novel monoallelic CD177 expression pattern that does not follow classical random monoallelic expression or imprinting.
Assuntos
Perfilação da Expressão Gênica/métodos , Inativação Gênica , Isoantígenos/genética , Neutrófilos/metabolismo , Receptores de Superfície Celular/genética , Alelos , Sequência de Bases , Diferenciação Celular/genética , Ilhas de CpG/genética , Metilação de DNA , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HeLa , Células-Tronco Hematopoéticas/metabolismo , Humanos , Immunoblotting , Isoantígenos/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.
Assuntos
Braquidactilia/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Hipertensão/congênito , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Estudos de Casos e Controles , Diferenciação Celular , Criança , Feminino , Estudos de Associação Genética , Células HeLa , Humanos , Hipertensão/genética , Cinética , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Miócitos de Músculo Liso/fisiologia , LinhagemRESUMO
Long non-coding RNAs (lncRNAs) interact with the nuclear architecture and are involved in fundamental biological mechanisms, such as imprinting, histone-code regulation, gene activation, gene repression, lineage determination, and cell proliferation, all by regulating gene expression. Understanding the lncRNA regulation of transcriptional or post-transcriptional gene regulation expands our knowledge of disease. Several associations between altered lncRNA function and gene expression have been linked to clinical disease phenotypes. Early advances have been made in developing lncRNAs as biomarkers. Several mouse models reveal that human lncRNAs have very diverse functions. Their involvement in gene and genome regulation as well as disease underscores the importance of lncRNA-mediated regulatory networks. Because of their tissue-specific expression potential, their function as activators or repressors, and their selective targeting of genes, lncRNAs are of potential therapeutic interest. We review the regulatory mechanisms of lncRNAs, their major functional principles, and discuss their role in Mendelian disorders, cancer, cardiovascular disease, and neurological disorders.
Assuntos
Epigênese Genética , RNA Longo não Codificante/fisiologia , Animais , Sequência de Bases , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismoRESUMO
Translocations are chromosomal rearrangements that are frequently associated with a variety of disease states and developmental disorders. We identified 2 families with brachydactyly type E (BDE) resulting from different translocations affecting chromosome 12p. Both translocations caused downregulation of the parathyroid hormone-like hormone (PTHLH) gene by disrupting the cis-regulatory landscape. Using chromosome conformation capturing, we identified a regulator on chromosome 12q that interacts in cis with PTHLH over a 24.4-megabase distance and in trans with the sex-determining region Y-box 9 (SOX9) gene on chromosome 17q. The element also harbored a long noncoding RNA (lncRNA). Silencing of the lncRNA, PTHLH, or SOX9 revealed a feedback mechanism involving an expression-dependent network in humans. In the BDE patients, the human lncRNA was upregulated by the disrupted chromosomal association. Moreover, the lncRNA occupancy at the PTHLH locus was reduced. Our results document what we believe to be a novel in cis- and in trans-acting DNA and lncRNA regulatory feedback element that is reciprocally regulated by coding genes. Furthermore, our findings provide a systematic and combinatorial view of how enhancers encoding lncRNAs may affect gene expression in normal development.
Assuntos
Braquidactilia , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 17 , Regulação da Expressão Gênica , Loci Gênicos , RNA Longo não Codificante , Translocação Genética , Animais , Braquidactilia/diagnóstico por imagem , Braquidactilia/genética , Braquidactilia/metabolismo , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/metabolismo , Feminino , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Radiografia , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genéticaRESUMO
Parathyroid hormone-like hormone (PTHLH) is an important chondrogenic regulator; however, the gene has not been directly linked to human disease. We studied a family with autosomal-dominant Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation with breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 and a disrupted KCNB2 on 8q13. We sequenced the BPs and identified a highly conserved Activator protein 1 (AP-1) motif on 12p11.2, together with a C-ets-1 motif translocated from 8q13. AP-1 and C-ets-1 bound in vitro and in vivo at the derivative chromosome 8 breakpoint [der(8) BP], but were differently enriched between the wild-type and BP allele. We differentiated fibroblasts from BDE patients into chondrogenic cells and found that PTHLH and its targets, ADAMTS-7 and ADAMTS-12 were downregulated along with impaired chondrogenic differentiation. We next used human and murine chondrocytes and observed that the AP-1 motif stimulated, whereas der(8) BP or C-ets-1 decreased, PTHLH promoter activity. These results are the first to identify a cis-directed PTHLH downregulation as primary cause of human chondrodysplasia.
Assuntos
Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 8/genética , Regulação para Baixo , Dedos/anormalidades , Proteína Relacionada ao Hormônio Paratireóideo/genética , Sequências Reguladoras de Ácido Nucleico/genética , Dedos do Pé/anormalidades , Translocação Genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS7 , Animais , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Humanos , CamundongosRESUMO
OBJECTIVE: Dysferlin (DYSF) gene mutations cause limb girdle muscular dystrophy type 2B and Miyoshi's myopathy. The consequences of DYSF mutations on protein structure are poorly understood. METHODS: The gene encoding dysferlin was sequenced in patients with suspected dysferlin-deficient muscular dystrophy. Muscle biopsy specimens were analyzed by histochemistry, immunohistochemistry, and electron microscopy. Antibodies against N-terminal dysferlin-peptides were raised. RESULTS: We found three families with muscular dystrophy caused by homozygous or compound heterozygous DYSF mutations featuring sarcolemmal and interstitial amyloid deposits. These mutations were all located in the N-terminal region of the protein. Dysferlin was a constituent of the amyloid deposits. INTERPRETATION: Limb girdle muscular dystrophy type 2B is the first muscular dystrophy associated with amyloidosis. Molecular treatment strategies will necessarily have to consider the presence of amyloidogenesis.
Assuntos
Amiloidose/genética , Amiloidose/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Amiloidose/diagnóstico , Disferlina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Estrutura Terciária de Proteína/genéticaRESUMO
Genomic imprinting is the epigenetic marking of gene subsets resulting in monoallelic or predominant expression of one of the two parental alleles according to their parental origin. We describe the systematic experimental verification of a prioritized 16 candidate imprinted gene set predicted by sequence-based bioinformatic analyses. We used Quantification of Allele-Specific Expression by Pyrosequencing (QUASEP) and discovered maternal-specific imprinted expression of the Kcnk9 gene as well as strain-dependent preferential expression of the Rarres1 gene in E11.5 (C57BL/6 x Cast/Ei)F1 and informative (C57BL/6 x Cast/Ei) x C57BL/6 backcross mouse embryos. For the remaining 14 candidate imprinted genes, we observed biallelic expression. In adult mouse tissues, we found that Kcnk9 expression was restricted to the brain and also was maternal-specific. QUASEP analysis of informative human fetal brain samples further demonstrated maternal-specific imprinted expression of the human KCNK9 orthologue. The CpG islands associated with the mouse and human Kcnk9/KCNK9 genes were not differentially methylated, but strongly hypomethylated. Thus, we speculate that mouse Kcnk9 imprinting may be regulated by the maternal germline differentially methylated region in Peg13, an imprinted non-coding RNA gene in close proximity to Kcnk9 on distal mouse chromosome 15. Our data have major implications for the proposed role of Kcnk9 in neurodevelopment, apoptosis and tumourigenesis, as well as for the efficiency of sequence-based bioinformatic predictions of novel imprinted genes.
Assuntos
Impressão Genômica , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Biologia Computacional , Ilhas de CpG , Metilação de DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , Canais de Potássio/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Análise de Sequência de DNARESUMO
We screened a white population for single nucleotide polymorphisms (SNPs) in five long QT syndrome genes, namely, KCNQ1 (LQT1), HERG (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6). We found 35 SNPs, 10 of which have not been previously described. Ten SNPs were in KCNE1, six in HERG, eight in KCNQ1, four in KCNE2, and seven in SCN5A. Four SNPs were associated with QTc interval in our 141 subjects, one in KCNE1, one in KCNE2, and two in SCN5A. Two of these SNPs have not been described. We conclude that these five long QT syndrome genes contain common variants, some of which are associated with QTc interval in normal persons. We suggest that analysis of these SNPs in a much larger cohort would enable establishment of common haplotypes that are associated with QTc. These haplotypes could facilitate prediction of arrhythmia risk in the general population.
Assuntos
Síndrome do QT Longo/genética , Polimorfismo de Nucleotídeo Único , Estudos de Coortes , Análise Mutacional de DNA , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Testes Genéticos , Genótipo , Haplótipos , Humanos , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Canal de Sódio Disparado por Voltagem NAV1.5 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Fatores de Risco , Canais de Sódio/genética , Estudos em Gêmeos como AssuntoRESUMO
We developed a two-in-one, polymerase chain reaction (PCR)-based method with a specific amplification step and a universal amplification step in one tube to screen for the presence of DNA variations. The method relies on fluorescence-labeled artificial nonhuman sequences for mutation detection. To document utility, we applied this method as a high-throughput capillary single-strand conformation polymorphism screening system to identify 30 mutations in the low-density lipoprotein receptor gene. The sensitivity of mutant allele detection compared to wild-type allele detection was 93%. We conclude that the "two-in-one PCR" is sensitive, simple, and cost effective.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Receptores de LDL/genética , Animais , Análise Mutacional de DNA , Primers do DNA , Eletroforese Capilar , Corantes Fluorescentes , Variação Genética , Reprodutibilidade dos Testes , Coloração e RotulagemAssuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Processamento Alternativo/genética , Hiperlipoproteinemia Tipo II/genética , RNA Mensageiro/genética , Sequência de Bases , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Genes Recessivos/genética , Humanos , Mutação , Deleção de Sequência , Síria , Transfecção , Células Tumorais CultivadasRESUMO
We studied a Syrian family with 3 children who had low-density lipoprotein cholesterol (LDL) concentrations of 13.3, 12.2, and 8.6 mmol/L, respectively. Three other siblings and the parents all had LDL values <4.52 mmol/L, suggesting an autosomal-recessive mode of inheritance. The extended pedigree had 66 additional persons with normal LDL values. A genome-wide scan in the core family with 427 markers showed support for linkage on both chromosomes 1 and 13. Markers on chromosome 1 revealed a 3.07 multipoint LOD score between 1p36.1-p35, an 18-cM interval. Surprisingly, we also found linkage to 13q22-q32, a 14-cM interval, with a 3.08 LOD score. We had identified this locus earlier as containing a gene strongly influencing LDL in another Arab family with autosomal-dominant familial hypercholesterolemia and in normal dizygotic twins. We found evidence for an interaction between these loci. We next genotyped our twin panel and confirmed linkage of the 1p36.1-p35 locus to LDL (P<0.002) in this normal population. Elucidation of ARH, the LDL receptor adaptor protein at chromosome 1p35, caused us to sequence that gene. We first identified the genomic structure of ARH gene and then sequenced the gene in our family. We found an intron 1 acceptor splice-site mutation. This mutation was not found in any other family members, in 31 nonrelated Syrian persons, or in 30 Germans. Our results underscore the importance of ARH on chromosome 1 and the chromosome 13q locus to LDL, not only in families with unusual illnesses, but also to the general population.