RESUMO
Gene discovery using massively parallel sequencing has focused on phenotypes diagnosed postnatally such as well-characterized syndromes or intellectual disability, but is rarely reported for fetal disorders. We used family-based whole-exome sequencing in order to identify causal variants for a recurrent pattern of an undescribed lethal fetal congenital anomaly syndrome. The clinical signs included intrauterine growth restriction (IUGR), severe microcephaly, renal cystic dysplasia/agenesis and complex brain and genitourinary malformations. The phenotype was compatible with a ciliopathy, but not diagnostic of any known condition. We hypothesized biallelic disruption of a gene leading to a defect related to the primary cilium. We identified novel autosomal recessive truncating mutations in KIF14 that segregated with the phenotype. Mice with autosomal recessive mutations in the same gene have recently been shown to have a strikingly similar phenotype. Genotype-phenotype correlations indicate that the function of KIF14 in cell division and cytokinesis can be linked to a role in primary cilia, supported by previous cellular and model organism studies of proteins that interact with KIF14. We describe the first human phenotype, a novel lethal ciliary disorder, associated with biallelic inactivating mutations in KIF14. KIF14 may also be considered a candidate gene for allelic viable ciliary and/or microcephaly phenotypes.
Assuntos
Anormalidades Múltiplas/genética , Transtornos da Motilidade Ciliar/genética , Predisposição Genética para Doença/genética , Cinesinas/genética , Proteínas Oncogênicas/genética , Fenótipo , Anormalidades Múltiplas/patologia , Sequência de Bases , Transtornos da Motilidade Ciliar/patologia , Exoma/genética , Genes Recessivos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Mutação/genéticaRESUMO
We studied the potential of systemically administered aminoglycosides as a therapy for retinal degeneration resulting from premature termination codon (PTC) mutations. Aminoglycosides were systemically delivered to two rodent models of retinal degeneration: a transgenic rat model of dominant disease caused by a PTC in rhodopsin (S334ter); and a mouse model of recessive disease (rd12) caused by a PTC in the retinoid isomerase Rpe65. Initial luciferase reporter assays were undertaken to measure the efficiency of gentamicin-induced read-through in vitro. These experiments indicated that gentamicin treatment induced on average a 5.3% extra read-through of the S334ter PTC in vitro, but did not affect the rd12 PTC. Beginning at postnatal day 5, animals received daily subcutaneous injections of gentamicin or geneticin at a range of doses. The effect of the treatment on retinal degeneration was examined by histopathology and electroretinography (ERG). Systemic treatment with aminoglycoside significantly increased the number of surviving photoreceptors in the S334ter rat model over several weeks of treatment, but was not effective in slowing the retinal degeneration in the rd12 mouse model. Similarly, ERG recordings indicated better preservation of retinal function in the treated S334ter rats, but no difference was observed in the rd12 mice. Daily subcutaneous injection of 12.5mug/g gentamicin was the only regimen that inhibited retinal degeneration without apparent adverse systemic side effects. Reduced effectiveness beyond postnatal day 50 correlated with reduced ocular penetration of drug as seen in gentamicin-Texas red (GTTR) conjugation experiments. We conclude that, in the rat model, an approximately 5% reduction of abnormal truncated protein is sufficient to enhance photoreceptor survival. Such a change in truncated protein is consistent with beneficial effects seen when aminoglycosides has been used in other, non-ocular animal models. In the rd12 mouse, lack of efficacy was seen despite this particular PTC being theoretically more sensitive to aminoglycoside modification. We conclude that aminoglycoside read-through of PTCs in vitro and in vivo cannot be predicted just from genomic context. Because there is considerable genetic heterogeneity amongst retinal degenerations, pharmacologic therapies that are not gene-specific have significant appeal. Our findings suggest that if adverse issues such as systemic toxicity and limited ocular penetration can be overcome, small molecule therapeutics, such as aminoglycosides, which target classes of mutation could hold considerable potential as therapies for retinal disease.
Assuntos
Aminoglicosídeos/uso terapêutico , Retinose Pigmentar/prevenção & controle , Aminoglicosídeos/farmacocinética , Animais , Proteínas de Transporte/genética , Códon sem Sentido , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Eletrorretinografia/efeitos dos fármacos , Proteínas do Olho/genética , Corantes Fluorescentes , Gentamicinas/farmacocinética , Gentamicinas/uso terapêutico , Camundongos , Camundongos Mutantes , Ratos , Ratos Transgênicos , Retina/metabolismo , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Rodopsina/genética , Xantenos , cis-trans-IsomerasesRESUMO
Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.
Assuntos
Neuropilina-1/genética , Neuropilina-1/metabolismo , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias da Próstata/tratamento farmacológico , Regulação para Cima , Antagonistas de Androgênios/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Análise de SobrevidaRESUMO
Congenital colobomata of the eye are important causes of childhood visual impairment and blindness. Ocular coloboma can be seen in isolation and in an impressive number of multisystem syndromes, where the eye phenotype is often seen in association with severe neurological or craniofacial anomalies or other systemic developmental defects. Several studies have shown that, in addition to inheritance, environmental influences may be causative factors. Through work to identify genes underlying inherited coloboma, significant inroads are being made into understanding the molecular events controlling closure of the optic fissure. In general, severity of disease can be linked to the temporal expression of the gene, but this is modified by factors such as tissue specificity of gene expression and genetic redundancy.
Assuntos
Coloboma/genética , Animais , Anoftalmia/genética , Coloboma/embriologia , Coloboma/etiologia , Meio Ambiente , Olho/embriologia , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica , Aconselhamento Genético , Proteínas Hedgehog , Proteínas de Homeodomínio/fisiologia , Humanos , Microftalmia/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas Repressoras/fisiologia , Transativadores/fisiologiaRESUMO
OBJECTIVE: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. DESIGN: Molecular genetic analysis. PARTICIPANTS: Ten unrelated families and 17 young drusen patients. MAIN OUTCOME MEASURES: Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. RESULTS: Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. CONCLUSIONS: EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.
Assuntos
Proteínas da Matriz Extracelular/genética , Genes Dominantes , Polimorfismo de Nucleotídeo Único , Drusas Retinianas/genética , Adulto , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 6 , Estudos de Coortes , Feminino , Ligação Genética , Variação Genética , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Radiografia , Drusas Retinianas/diagnóstico por imagemRESUMO
BACKGROUND: Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. RESULTS: The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb). The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. CONCLUSIONS: Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.
Assuntos
Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Componentes do Gene , Humanos , Melanócitos/química , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Distribuição Tecidual , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab27 de Ligação ao GTPRESUMO
Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Morte Celular/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Técnicas de Silenciamento de Genes , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Nr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis were identified.
Assuntos
Encéfalo/anormalidades , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Retina/anormalidades , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genoma , Humanos , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos , Fenótipo , Retina/embriologia , Retina/metabolismoAssuntos
Proteínas do Olho/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Sequências Reguladoras de Ácido Nucleico/genética , Retinopatia da Prematuridade/genética , Alelos , Pré-Escolar , Análise Mutacional de DNA , Doenças em Gêmeos/genética , Mecanismo Genético de Compensação de Dose , Éxons/genética , Feminino , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Masculino , Regiões Promotoras Genéticas/genética , Sítios de Splice de RNA/genética , Retinopatia da Prematuridade/fisiopatologia , Gêmeos/genéticaAssuntos
Fundo de Olho , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Adulto , Humanos , Masculino , Síndrome , Verteporfina , Transtornos da Visão/tratamento farmacológico , Transtornos da Visão/etiologiaAssuntos
Neovascularização de Coroide/complicações , Degeneração Retiniana/complicações , Adulto , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/fisiopatologia , Feminino , Fundo de Olho , Humanos , Degeneração Retiniana/fisiopatologia , Esteroides/uso terapêutico , Acuidade VisualRESUMO
We have identified a highly polymorphic intragenic marker composed of two dinucleotide repeats in an intron of the human interferon regulatory factor (IRF3) gene. This polymorphic marker has allowed us to map IRF3 to the boundary of 19q13.3-13.4 between the polymorphic markers D19S604 and D19S206 close to KLK1. An intron is present in a homologous position in the mouse Irf3 gene, but lacks the dinucleotide repeats present in the human intron. Syntenic conservation between this region of 19q13.3-13.4 maps Irf3 to either mouse chromosome 7 or 17.
Assuntos
Cromossomos Humanos Par 19 , Proteínas de Ligação a DNA/genética , Polimorfismo Genético , Fatores de Transcrição/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Frequência do Gene , Marcadores Genéticos , Humanos , Fator Regulador 3 de Interferon , Dados de Sequência MolecularRESUMO
In this study, we report the identification and characterisation of a novel carbonic anhydrase related-protein. We have determined that the full length coding sequence of an anonymous expressed sequenced tag, D19S799E, encodes a novel carbonic anhydrase related-protein (CARP-2) that is 328 amino acids in length. This peptide exhibits between 23.1-28.8% amino acid identity with the seven active human carbonic anhydrase (CA) isozymes. Four substitutions of key amino acids in the catalytic domain of CAs (equivalent to His94Arg, His96Leu, His119Gln, and Thr199Ser) are likely to render CARP-2 inactive as a carbonic anhydrase. Northern blot analysis of 23 human tissues indicates that CARP2 is expressed abundantly in the brain with moderate expression also present in spinal cord and thyroid. D19S799E (and thus CARP2) has previously been localised close to the polymorphic marker D19S412 and the genes DBP and FUT1/FUT2 on 19q13. 3.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Cromossomos Humanos Par 19/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência de Bases , Anidrases Carbônicas/biossíntese , Mapeamento Cromossômico , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Especificidade de Órgãos/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Syndromic Hirschsprung disease has been associated with mutations in ZFHX1B, a Smad-interacting transcriptional repressor protein. Tissue in situ hybridization has demonstrated strong expression of ZFHX1B in the developing eye, suggesting that some mutations in this gene may cause visual loss. However, none of the reported mutations have been associated with an ocular phenotype. We describe a patient with Down syndrome and Hirschsprung disease with high myopia and ocular coloboma affecting the iris and retina. In addition to trisomy 21, a novel, de novo heterozygous A to G transition in exon 8 of the ZFHX1B gene was identified, which results in a R953G amino acid substitution. This abnormality was not seen in a screen of 200 chromosomes from ethnically matched, normal controls. The arginine residue at position 953 is an extremely conserved amino acid throughout evolution. This is the first report associating Hirschsprung disease and severe eye defects with a specific genetic mutation and is the first report of a mutation in ZFHX1B causing a developmental ocular anomaly.
Assuntos
Anormalidades Múltiplas/genética , Síndrome de Down/patologia , Oftalmopatias/patologia , Doença de Hirschsprung/patologia , Proteínas de Homeodomínio/genética , Mutação de Sentido Incorreto , Proteínas Repressoras/genética , Anormalidades Múltiplas/patologia , Sequência de Bases , Criança , Coloboma/patologia , DNA/química , DNA/genética , Análise Mutacional de DNA , Feminino , Humanos , Cariotipagem , Masculino , Miopia/patologia , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
OBJECTIVE: Little is known of the cytopathology of photoreceptors in human inherited retinal dystrophies that initially affect the central retina, including the macula. The current study sought to determine the cytologic features of dysfunctional cone and rod photoreceptors, as well as the pattern of degeneration of the cells in representative cases of central retinal dystrophy. STUDY DESIGN: Comparative human tissue study. MATERIALS: Four human donor eyes with the following forms of central retinal dystrophy: cone-rod dystrophy (CRD), central areolar choroidal dystrophy, Bardet-Biedl syndrome, and cone dystrophy-cerebellar ataxia. The cytologic features of retinal photoreceptors in these eyes were compared with those in an eye with retinitis pigmentosa and six normal human eyes. METHODS AND OUTCOME MEASURES: Immunocytochemistry and electron microscopy were used to evaluate the retinal histopathology in the donor eyes. RESULTS: Cone numbers were decreased in the case of CRD, particularly in the central and far peripheral retina, and both cone and rod outer segments were slightly shortened. Occasional degenerate cones had dense cytoplasm and pyknotic nuclei dislocated sclerad to the external-limiting membrane. The most prominent alteration in this retina was marked enlargement and distortion of the cone photoreceptor pedicles, which contained reduced numbers of synaptic vesicles. The retina with central areolar choroidal dystrophy contained a few cones with similarly abnormal synapses. However, comparable cone synapse abnormalities were not observed in the cases of Bardet-Biedl syndrome, cone dystrophy-cerebellar ataxia, retinitis pigmentosa, or in the normal retinas. CONCLUSIONS: The functional consequences of the cone synapse abnormalities in CRD are not known but may correlate with the electroretinographic abnormalities documented in some cases of CRD. To our knowledge, comparable synapse changes have not been noted in either rods or cones in other forms of retinal dystrophy, including retinitis pigmentosa, suggesting that different cytopathologic mechanisms may be involved.
Assuntos
Células Fotorreceptoras de Vertebrados/ultraestrutura , Degeneração Retiniana/patologia , Sinapses/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Ataxia Cerebelar/patologia , Doenças da Coroide/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Síndrome de Laurence-Moon/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus, spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus, and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex.
Assuntos
Mapeamento de Sequências Contíguas/métodos , Degeneração Retiniana/genética , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 2/genética , Feminino , Marcadores Genéticos , Genótipo , Humanos , Escore Lod , Masculino , Linhagem , Recombinação Genética , Sitios de Sequências RotuladasRESUMO
We report the identification and characterisation of a novel human orphan G-protein-coupled receptor (GPR) which maps to chromosome 2p16. We have determined the full-length coding sequence and genomic structure of a gene corresponding to the anonymous expressed sequenced tag, WI-31133. This gene encodes a novel protein that is 540 amino acids in length. Protein sequence analysis predicts the presence of seven transmembrane domains, a characteristic feature of GPRs. In situ hybridisation to human retina and Northern blot analysis of human retinal pigment epithelium (RPE) showed localisation of this transcript to the RPE and cells surrounding retinal arterioles. In contrast, the transcript was localised to the photoreceptor inner segments and the outer plexiform layer in mouse sections. Northern blot analysis demonstrated a 7 kb transcript highly expressed in the brain. No mutations were identified during a screen of patients suffering from Doyne's honeycomb retinal dystrophy (DHRD), an inherited retinal degeneration which maps to chromosome 2p16.
Assuntos
Cromossomos Humanos Par 2 , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Etiquetas de Sequências Expressas , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Polimorfismo Genético , Doenças Retinianas/genética , Vasos Retinianos/metabolismo , Distribuição TecidualRESUMO
Cone--rod homeobox (CRX), a paired-like homeobox transcription factor, plays a major role in photoreceptor development and maintenance of the retina. Fifteen different mutations in the CRX gene have been identified as a cause of blinding retinal dystrophy. As a step towards characterizing the underlying pathophysiology of disease, temporal and spatial gene expression patterns during human and mouse eye development were investigated for CRX and for downstream retinally expressed genes, postulated to be transactivated by CRX. We found that human CRX was expressed at 10.5 weeks post-conception (p.c.). This was significantly later than observed in mouse development. Immunocytochemistry in human retina showed that CRX protein was not detected until >4 weeks later at 15 weeks p.c., implying that it would be unable to transactivate PDEB, IRBP and arrestin, which were all expressed before 15 weeks. These data therefore eliminate CRX as the major transcriptional activator of these three genes from a wide group of retinal genes that can be transactivated by CRX in vitro. Additionally, PDEB was expressed 2 weeks before CRX whereas murine Pdeb was expressed after Crx, highlighting a potential difference for the role of PDEB in human eye development. Previous data had shown CRX expression in the adult human retina to be photoreceptor-specific; however, we demonstrate that this gene is also expressed in the inner nuclear layer (INL) of the human and mouse retina by in situ hybridization and immunocytochemistry. INL localization of murine Crx was confirmed in rd/rd,cl mice, as in this mouse model the photoreceptors are absent. We have found important differences in the temporal expression of this gene in human and mouse retina, although spatial expression of the CRX gene appears to be conserved. In addition, downstream targets of CRX in vitro might not represent in vivo function during development. These data support concerns about the extent to which we can extrapolate from rodent models regarding embryonic development and disease pathophysiology.
Assuntos
Olho/embriologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Transativadores/genética , Transativadores/metabolismo , Animais , Sítios de Ligação , Western Blotting , DNA Complementar/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Fatores de Tempo , Distribuição Tecidual , Ativação TranscricionalRESUMO
OBJECTIVE: To describe the clinical features of autosomal dominant cone-rod retinal dystrophy (CRD) in a British family mapping to chromosome 17p12-p13 (CORD6), with a heterozygous mutation (Glu837Asp/ Arg838Ser) of GUCY2D. DESIGN: A prospective, clinical family survey. PATIENTS: Ten affected members of a family with autosomal dominant CRD. METHODS: Full clinical examinations were undertaken. Selected affected family members underwent electrophysiologic evaluation, scotopic static perimetry, dark adaptometry, and color vision assessment. MAIN OUTCOME MEASURES: Clinical appearance and electroretinographic responses. RESULTS: Typical clinical and electroretinographic features of childhood-onset CRD were recorded. In addition, moderate myopia and pendular nystagmus were seen in affected individuals. Color vision assessment in the youngest affected individual showed no color discrimination on a tritan axis, but retention of significant red-green discrimination. Electronegative electroretinogram responses were seen on electrophysiology in the only young family member examined. CONCLUSIONS: The phenotype associated with GUCY2D CRD is clinically distinct from that associated with other dominant CRD loci. Unusual electroretinographic responses may indicate that this mutation of GUCY2D is associated with early defects in photoreceptor synaptic transmission to second-order neurons.
Assuntos
Cromossomos Humanos Par 17/genética , Genes Dominantes/genética , Ligação Genética , Guanilato Ciclase/genética , Células Fotorreceptoras de Vertebrados/enzimologia , Mutação Puntual , Degeneração Retiniana/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Mapeamento Cromossômico , Defeitos da Visão Cromática/diagnóstico , Eletrorretinografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miopia/diagnóstico , Nistagmo Patológico/diagnóstico , Linhagem , Células Fotorreceptoras de Vertebrados/patologia , Estudos Prospectivos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/enzimologia , Visão Ocular/genética , Acuidade VisualRESUMO
Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors.