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1.
Gastroenterology ; 155(1): 118-129.e6, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29601828

RESUMO

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Proteínas Hedgehog/genética , Doença de Hirschsprung/genética , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteína Gli3 com Dedos de Zinco/genética , Animais , Células COS , Chlorocebus aethiops , Família , Feminino , Predisposição Genética para Doença , Variação Genética , Células HEK293 , Humanos , Masculino , Morfolinos , Países Baixos , Linhagem , Isoformas de Proteínas , Proto-Oncogene Mas , Análise de Sequência de DNA , Transdução de Sinais , Peixe-Zebra
2.
Hum Mol Genet ; 25(23): 5265-5275, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27702942

RESUMO

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.


Assuntos
Predisposição Genética para Doença , Doença de Hirschsprung/genética , Neuregulina-1/genética , Proteínas Proto-Oncogênicas c-ret/genética , Semaforina-3A/genética , Alelos , Povo Asiático/genética , Etnicidade/genética , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Doença de Hirschsprung/patologia , Humanos , Íntrons/genética , Masculino , Polimorfismo de Nucleotídeo Único , População Branca/genética
3.
Am J Hum Genet ; 96(4): 581-96, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25839327

RESUMO

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.


Assuntos
Epistasia Genética/genética , Predisposição Genética para Doença/genética , Variação Genética , Doença de Hirschsprung/genética , Proteínas Proto-Oncogênicas c-ret/genética , Semaforinas/genética , Animais , Sequência de Bases , Estudo de Associação Genômica Ampla , Camundongos , Dados de Sequência Molecular , Semaforinas/deficiência , Semaforinas/metabolismo , Análise de Sequência de DNA
4.
G3 (Bethesda) ; 5(1): 61-72, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25416705

RESUMO

Numerous linkage and association studies by our group and others have implicated DPYSL2 at 8p21.2 in schizophrenia. Here we explore DPYSL2 for functional variation that underlies these associations. We sequenced all 14 exons of DPYSL2 as well as 27 conserved noncoding regions at the locus in 137 cases and 151 controls. We identified 120 variants, eight of which we genotyped in an additional 729 cases and 1542 controls. Several were significantly associated with schizophrenia, including a three single-nucleotide polymorphism (SNP) haplotype in the proximal promoter, two SNPs in intron 1, and a polymorphic dinucleotide repeat in the 5'-untranslated region that alters sequences predicted to be involved in translational regulation by mammalian target of rapamycin signaling. The 3-SNP promoter haplotype and the sequence surrounding one of the intron 1 SNPs direct tissue-specific expression in the nervous systems of Zebrafish in a pattern consistent with the two endogenous dpysl2 paralogs. In addition, two SNP haplotypes over the coding exons and 3' end of DPYSL2 showed association with opposing sex-specific risks. These data suggest that these polymorphic, schizophrenia-associated sequences function as regulatory elements for DPYSL2 expression. In transient transfection assays, the high risk allele of the polymorphic dinucleotide repeat diminished reporter expression by 3- to 4-fold. Both the high- and low-risk alleles respond to allosteric mTOR inhibition by rapamycin until, at high drug levels, allelic differences are eliminated. Our results suggest that reduced transcription and mTOR-regulated translation of certain DPYSL2 isoforms increase the risk for schizophrenia.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Esquizofrenia/genética , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Córtex Cerebral/citologia , Éxons , Feminino , Células HEK293 , Haplótipos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Esquizofrenia/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Lobo Temporal/metabolismo , População Branca/genética , Adulto Jovem
5.
Mol Genet Genomics ; 288(11): 579-89, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23942840

RESUMO

LMX1A and LMX1B encode two closely related members of the LIM homeobox family of transcription factors. These genes play significant, and frequently overlapping, roles in the development of many structures in the nervous system, including the cerebellum, hindbrain, spinal cord roof plate, sensory systems and dopaminergic midbrain neurons. Little is known about the cis-acting regulatory elements (REs) that dictate their temporal and spatial expression or about the regulatory landscape surrounding them. The availability of comparative sequence data and the advent of genomic technologies such as ChIP-seq have revolutionized our capacity to identify regulatory sequences like enhancers. Despite this wealth of data, the vast majority of loci lack any significant in vivo functional exploration of their non-coding regions. We have completed a significant functional screen of conserved non-coding sequences (putative REs) scattered across these critical human loci, assaying the temporal and spatial control using zebrafish transgenesis. We first identify and describe the LMX1A paralogs lmx1a and lmx1a-like, comparing their expression during embryogenesis with that in mammals, along with lmx1ba and lmx1bb genes. Consistent with their prominent neuronal expression, 47/71 sequences selected within and flanking LMX1A and LMX1B exert spatial control of reporter expression in the central nervous system (CNS) of mosaic zebrafish embryos. Upon germline transmission, we identify CNS reporter expression in multiple independent founders for 22 constructs (LMX1A, n = 17; LMX1B, n = 5). The identified enhancers display significant overlap in their spatial control and represent only a fraction of the conserved non-coding sequences at these critical genes. Our data reveal the abundance of regulatory instruction located near these developmentally important genes.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genômica , Proteínas com Homeodomínio LIM/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência Conservada , Embrião não Mamífero , Genes Reporter , Loci Gênicos , Humanos , Hibridização In Situ , Proteínas com Homeodomínio LIM/metabolismo , Mesencéfalo/citologia , Mesencéfalo/embriologia , Especificidade de Órgãos , Rombencéfalo/citologia , Rombencéfalo/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
Genome Res ; 22(11): 2278-89, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22759862

RESUMO

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes.


Assuntos
Elementos Facilitadores Genéticos , Rombencéfalo/metabolismo , Análise de Sequência de DNA/métodos , Transcrição Gênica , Algoritmos , Animais , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Rombencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra
7.
PLoS One ; 5(12): e14346, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21179557

RESUMO

BACKGROUND: Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants) that affect MPZ expression. METHODOLOGY: We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants. PRINCIPAL FINDINGS: Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. CONCLUSIONS: This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.


Assuntos
Elementos Facilitadores Genéticos , Proteína P0 da Mielina/genética , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Variação Genética , Humanos , Técnicas In Vitro , Camundongos , Mutação , Proteína P0 da Mielina/metabolismo , Ratos , Fatores de Transcrição SOXE/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Fatores de Transcrição/metabolismo , Peixe-Zebra
8.
Hum Mol Genet ; 19(18): 3642-51, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20621975

RESUMO

Goldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo. To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Neurogênese , Serpinas/metabolismo , Estatmina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Linhagem Celular , Células Cultivadas , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Modelos Animais de Doenças , Células HeLa , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microtúbulos , Células NIH 3T3 , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Ligação Proteica , Ratos , Serpinas/genética , Estatmina/genética , Proteínas de Peixe-Zebra/genética
9.
Am J Hum Genet ; 87(1): 60-74, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20598273

RESUMO

The major gene for Hirschsprung disease (HSCR) encodes the receptor tyrosine kinase RET. In a study of 690 European- and 192 Chinese-descent probands and their parents or controls, we demonstrate the ubiquity of a >4-fold susceptibility from a C-->T allele (rs2435357: p = 3.9 x 10(-43) in European ancestry; p = 1.1 x 10(-21) in Chinese samples) that probably arose once within the intronic RET enhancer MCS+9.7. With in vitro assays, we now show that the T variant disrupts a SOX10 binding site within MCS+9.7 that compromises RET transactivation. The T allele, with a control frequency of 20%-30%/47% and case frequency of 54%-62%/88% in European/Chinese-ancestry individuals, is involved in all forms of HSCR. It is marginally associated with proband gender (p = 0.13) and significantly so with length of aganglionosis (p = 7.6 x 10(-5)) and familiality (p = 6.2 x 10(-4)). The enhancer variant is more frequent in the common forms of male, short-segment, and simplex families whereas multiple, rare, coding mutations are the norm in the less common and more severe forms of female, long-segment, and multiplex families. The T variant also increases penetrance in patients with rare RET coding mutations. Thus, both rare and common mutations, individually and together, make contributions to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggests a "cellular-recessive" genetic model where both RET alleles' function is compromised. The RET allelic series, and its genotype-phenotype correlations, shows that success in variant identification in complex disorders may strongly depend on which patients are studied.


Assuntos
Doença de Hirschsprung/genética , Proteínas Proto-Oncogênicas c-ret/genética , Povo Asiático , Sequência de Bases , Estudos de Casos e Controles , Elementos Facilitadores Genéticos , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Haplótipos , Doença de Hirschsprung/etnologia , Doença de Hirschsprung/fisiopatologia , Humanos , Masculino , Mutação , Penetrância , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Proteínas Proto-Oncogênicas c-ret/metabolismo , Fatores de Transcrição SOXE/metabolismo , Fatores Sexuais , Ativação Transcricional , População Branca
10.
EMBO J ; 29(10): 1637-51, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-20360680

RESUMO

Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Neurônios/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Diferenciação Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Rim/metabolismo , Cinesinas/química , Modelos Biológicos , Neurônios/metabolismo , Peixe-Zebra
11.
Gene Expr Patterns ; 9(4): 231-7, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19272335

RESUMO

SCG10 (Superior Cervical Ganglia 10, STMN2) is a member of the stathmin family of proteins. Stathmins regulate microtubule dynamics by inhibiting polymerization and promoting their depolymerization. SCG10 is believed to be a neuronal-specific stathmin that is enriched in the growth cones of developing neurons and plays a role in regulating neurite outgrowth. In all species examined so far, SCG10 is expressed in both the CNS and PNS. We have cloned two zebrafish SCG10 homologues and have determined the temporal and spatial expression pattern of both of these genes by RT-PCR and in situ hybridization. RT-PCR shows that both transcripts are expressed maternally and zygotically through at least 5 days. In situ hybridization analysis reveals that both SCG10 orthologues have dynamic, spatial expression patterns that are nearly identical to each other. Initially, these orthologues are expressed in discrete areas of the forebrain, midbrain, and hindbrain, as well as in the anterior and posterior lateral line ganglia and transiently in the spinal cord Rohon-Beard neurons. From 48hpf onwards, the level of expression of both genes increases and becomes mainly restricted to the anterior CNS (the forebrain region, retina, optic tectum, and hindbrain), and to the cranial ganglia. From 72 to 96hpf, SCG10 genes are also expressed in the developing neurons in the gut and in the surrounding intestinal mesenchyme. Our results provide a starting point for future studies that will investigate the in vivo function of SCG10 orthologues in zebrafish neural development.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Estatmina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Hibridização In Situ , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Masculino , Mesoderma/metabolismo , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificação
12.
Trends Genet ; 22(11): 627-36, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16979782

RESUMO

The receptor tyrosine kinase RET is expressed in cell lineages derived from the neural crest and has a key role in regulating cell proliferation, migration, differentiation and survival during embryogenesis. Germline and somatic mutations in RET that produce constitutively activated receptors cause the cancer syndrome multiple endocrine neoplasia type 2 and several endocrine and neural-crest-derived tumors, whereas mutations resulting in nonfunctional RET or lower expression of RET are found in individuals affected with Hirschsprung disease. This review focuses on the genetics and molecular mechanisms underlying the different inherited human neural-crest-related disorders in which RET dysfunction has a crucial role and discusses RET as a potential therapeutic target.


Assuntos
Doença de Hirschsprung/genética , Neoplasia Endócrina Múltipla Tipo 2a/genética , Polimorfismo Genético , Proteínas Proto-Oncogênicas c-ret/fisiologia , Animais , Ativação Enzimática , Haplótipos , Doença de Hirschsprung/metabolismo , Humanos , Camundongos , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-ret/genética , Transdução de Sinais
13.
Am J Hum Genet ; 77(1): 120-6, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15883926

RESUMO

We identified, by homozygosity mapping, a novel locus on 10q21.3-q22.1 for Goldberg-Shprintzen syndrome (GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development.


Assuntos
Códon sem Sentido , Sistema Nervoso Entérico/anormalidades , Malformações do Sistema Nervoso/genética , Anormalidades Múltiplas , Sequência de Bases , Cromossomos Humanos Par 10 , Consanguinidade , Feminino , Doença de Hirschsprung/genética , Humanos , Deficiência Intelectual/genética , Masculino , Proteínas do Tecido Nervoso , Linhagem , Síndrome
14.
Am J Hum Genet ; 76(5): 850-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15759212

RESUMO

Patients with sporadic Hirschsprung disease (HSCR) show increased allele sharing at markers in the 5' region of the RET locus, indicating the presence of a common ancestral RET mutation. In a previous study, we found a haplotype of six SNPs that was transmitted to 55.6% of our patients, whereas it was present in only 16.2% of the controls we used. Among the patients with that haplotype, 90.8% had it on both chromosomes, which led to a much higher risk of developing HSCR than when the haplotype occurred heterozygously. To more precisely define the HSCR-associated region and to identify candidate disease-associated variant(s), we sequenced the shared common haplotype region from 10 kb upstream of the RET gene through intron 1 and exon 2 (in total, 33 kb) in a patient homozygous for the common risk haplotype and in a control individual homozygous for the most common nonrisk haplotype. A comparison of these sequences revealed 86 sequence differences. Of these 86 variations, 8 proved to be in regions highly conserved among different vertebrates and within putative transcription factor binding sites. We therefore considered these as candidate disease-associated variants. Subsequent genotyping of these eight variants revealed a strong disease association for six of the eight markers. These six markers also showed the largest distortions in allele transmission. Interspecies comparison showed that only one of the six variations was located in a region also conserved in a nonmammalian species, making it the most likely candidate HSCR-associated variant.


Assuntos
Predisposição Genética para Doença , Variação Genética , Doença de Hirschsprung/genética , Proteínas Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Sequência Consenso , Sequência Conservada , Frequência do Gene , Marcadores Genéticos , Haplótipos , Humanos , Dados de Sequência Molecular , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-ret , Risco
15.
Cancer Res ; 65(5): 1729-37, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15753368

RESUMO

The RET proto-oncogene encodes a receptor tyrosine kinase whose dysfunction plays a crucial role in the development of several neural crest disorders. Distinct activating RET mutations cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Despite clear correlations between the mutations found in these cancer syndromes and their phenotypes, the molecular mechanisms connecting the mutated receptor to the different disease phenotypes are far from completely understood. Luciferase reporter assays in combination with immunoprecipitations, and Western and immunohistochemistry analyses were done in order to characterize the signaling properties of two FMTC-associated RET mutations, Y791F and S891A, respectively, both affecting the tyrosine kinase domain of the receptor. We show that these RET-FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line-derived neurotrophic factor. Moreover, we show that the dysfunctional signaling properties of these mutants, when compared with wild-type RET, involve constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, we show that STAT3 activation is mediated by a signaling pathway involving Src, JAK1, and JAK2, differing from STAT3 activation promoted by RET(C634R) which was previously found to be independent of Src and JAKs. Three-dimensional modeling of the RET catalytic domain suggested that the structural changes promoted by the respective amino acids substitutions lead to a more accessible substrate and ATP-binding monomeric conformation. Finally, immunohistochemical analysis of FMTC tumor samples support the in vitro data, because nuclear localized, Y705-phosphorylated STAT3, as well as a high degree of RET expression at the plasma membrane was observed.


Assuntos
Carcinoma Medular , Mutação/genética , Fatores de Crescimento Neural/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Neoplasias da Glândula Tireoide , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Animais , Western Blotting , Carcinoma Medular/genética , Carcinoma Medular/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Janus Quinase 1 , Janus Quinase 2 , Luciferases/metabolismo , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Proteínas Oncogênicas/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Proteínas Proto-Oncogênicas pp60(c-src) , Receptores Proteína Tirosina Quinases/genética , Fator de Transcrição STAT3 , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Transativadores/metabolismo
16.
Eur J Hum Genet ; 12(8): 604-12, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15138456

RESUMO

Hirschsprung disease (HSCR), a congenital disorder characterized by intestinal obstruction due to absence of enteric ganglia along variable lengths of the intestinal tract, occurs both in familial and sporadic cases. RET mutations have been found in approximately 50% of the families, but explains only a minority of sporadic cases. This study aims at investigating a possible role of RET in sporadic HSCR patients. Haplotypes of 13 DNA markers, within and flanking RET, have been determined for 117 sporadic HSCR patients and their parents. Strong association was observed for six markers in the 5' region of RET. The largest distortions in allele transmission were found at the same markers. One single haplotype composed of these six markers was present in 55.6% of patients versus 16.2% of controls. Odds ratios (ORs) revealed a highly increased risk of homozygotes for this haplotype to develop HSCR (OR>20). These results allowed us to conclude that RET plays a crucial role in HSCR even when no RET mutations are found. An unknown functional disease variant(s) with a dosage-dependent effect in HSCR is likely located between the promoter region and exon 2 of RET.


Assuntos
Doença de Hirschsprung/genética , Mutação/genética , Proteínas Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Adulto , Criança , Primers do DNA , Éxons/genética , Componentes do Gene , Frequência do Gene , Predisposição Genética para Doença/genética , Haplótipos/genética , Humanos , Desequilíbrio de Ligação , Repetições de Microssatélites/genética , Países Baixos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-ret
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