Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
Nature ; 605(7910): 503-508, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35545669

RESUMO

Mutations in the germline generates all evolutionary genetic variation and is a cause of genetic disease. Parental age is the primary determinant of the number of new germline mutations in an individual's genome1,2. Here we analysed the genome-wide sequences of 21,879 families with rare genetic diseases and identified 12 individuals with a hypermutated genome with between two and seven times more de novo single-nucleotide variants than expected. In most families (9 out of 12), the excess mutations came from the father. Two families had genetic drivers of germline hypermutation, with fathers carrying damaging genetic variation in DNA-repair genes. For five of the families, paternal exposure to chemotherapeutic agents before conception was probably a key driver of hypermutation. Our results suggest that the germline is well protected from mutagenic effects, hypermutation is rare, the number of excess mutations is relatively modest and most individuals with a hypermutated genome will not have a genetic disease.


Assuntos
Doenças Genéticas Inatas , Células Germinativas , Mutação em Linhagem Germinativa , Fatores Etários , Doenças Genéticas Inatas/genética , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Mutagênese/genética , Mutação , Pais , Polimorfismo de Nucleotídeo Único
2.
Hum Mol Genet ; 33(17): 1506-1523, 2024 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-38776958

RESUMO

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.


Assuntos
Mutação , Transtornos do Neurodesenvolvimento , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Apoptose/genética , Masculino , Feminino , Ubiquitina/metabolismo , Ubiquitina/genética , Células HEK293
3.
Hum Mol Genet ; 30(7): 595-602, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33772547

RESUMO

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.


Assuntos
Transtornos Cromossômicos/genética , Fenda Labial/genética , Fissura Palatina/genética , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genética , Animais , Proliferação de Células/genética , Deleção Cromossômica , Transtornos Cromossômicos/metabolismo , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/metabolismo , Fenda Labial/embriologia , Fenda Labial/metabolismo , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Proteínas Repressoras/deficiência , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
4.
Clin Genet ; 104(2): 186-197, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37165752

RESUMO

POU3F3 variants cause developmental delay, behavioral problems, hypotonia and dysmorphic features. We investigated the phenotypic and genetic landscape, and genotype-phenotype correlations in individuals with POU3F3-related disorders. We recruited unpublished individuals with POU3F3 variants through international collaborations and obtained updated clinical data on previously published individuals. Trio exome sequencing or single exome sequencing followed by segregation analysis were performed in the novel cohort. Functional effects of missense variants were investigated with 3D protein modeling. We included 28 individuals (5 previously published) from 26 families carrying POU3F3 variants; 23 de novo and one inherited from an affected parent. Median age at study inclusion was 7.4 years. All had developmental delay mainly affecting speech, behavioral difficulties, psychiatric comorbidities and dysmorphisms. Additional features included gastrointestinal comorbidities, hearing loss, ophthalmological anomalies, epilepsy, sleep disturbances and joint hypermobility. Autism, hearing and eye comorbidities, dysmorphisms were more common in individuals with truncating variants, whereas epilepsy was only associated with missense variants. In silico structural modeling predicted that all (likely) pathogenic variants destabilize the DNA-binding region of POU3F3. Our study refined the phenotypic and genetic landscape of POU3F3-related disorders, it reports the functional properties of the identified pathogenic variants, and delineates some genotype-phenotype correlations.


Assuntos
Transtorno Autístico , Epilepsia , Deficiência Intelectual , Humanos , Criança , Deficiência Intelectual/genética , Transtorno Autístico/genética , Fenótipo , Epilepsia/genética , Mutação de Sentido Incorreto/genética , Deficiências do Desenvolvimento/genética , Fatores do Domínio POU/genética
5.
J Med Genet ; 59(8): 737-747, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34716235

RESUMO

BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.


Assuntos
Anormalidades Múltiplas , Ciliopatias , Anormalidades do Olho , Doenças Renais Císticas , Anormalidades Múltiplas/genética , Ciliopatias/diagnóstico , Ciliopatias/genética , Ciliopatias/patologia , Anormalidades do Olho/genética , Humanos , Doenças Renais Císticas/genética , Fenótipo , Medicina Estatal
6.
Brain ; 144(5): 1422-1434, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33970200

RESUMO

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.


Assuntos
Oxigenases/genética , Paraplegia Espástica Hereditária/genética , Animais , Feminino , Humanos , Masculino , Camundongos , Mutação , Linhagem , Ratos , Peixe-Zebra
7.
Am J Hum Genet ; 100(1): 75-90, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28041643

RESUMO

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.


Assuntos
Análise Mutacional de DNA , Variação Genética/genética , Genoma Humano/genética , Doenças Retinianas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Sequência de Bases , Coroideremia/genética , Etnicidade/genética , Exoma/genética , Feminino , Genes Recessivos/genética , Humanos , Íntrons/genética , Masculino , Mutação , Doenças Raras/genética
8.
Clin Genet ; 95(6): 693-703, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30859559

RESUMO

Noonan syndrome (NS) is characterised by distinctive facial features, heart defects, variable degrees of intellectual disability and other phenotypic manifestations. Although the mode of inheritance is typically dominant, recent studies indicate LZTR1 may be associated with both dominant and recessive forms. Seeking to describe the phenotypic characteristics of LZTR1-associated NS, we searched for likely pathogenic variants using two approaches. First, scrutiny of exomes from 9624 patients recruited by the Deciphering Developmental Disorders (DDDs) study uncovered six dominantly-acting mutations (p.R97L; p.Y136C; p.Y136H, p.N145I, p.S244C; p.G248R) of which five arose de novo, and three patients with compound-heterozygous variants (p.R210*/p.V579M; p.R210*/p.D531N; c.1149+1G>T/p.R688C). One patient also had biallelic loss-of-function mutations in NEB, consistent with a composite phenotype. After removing this complex case, analysis of human phenotype ontology terms indicated significant phenotypic similarities (P = 0.0005), supporting a causal role for LZTR1. Second, targeted sequencing of eight unsolved NS-like cases identified biallelic LZTR1 variants in three further subjects (p.W469*/p.Y749C, p.W437*/c.-38T>A and p.A461D/p.I462T). Our study strengthens the association of LZTR1 with NS, with de novo mutations clustering around the KT1-4 domains. Although LZTR1 variants explain ~0.1% of cases across the DDD cohort, the gene is a relatively common cause of unsolved NS cases where recessive inheritance is suspected.


Assuntos
Exoma , Síndrome de Noonan/genética , Fatores de Transcrição/genética , Adolescente , Alelos , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Ontologia Genética , Genes Dominantes , Genes Recessivos , Heterozigoto , Humanos , Lactente , Masculino , Mutação , Síndrome de Noonan/fisiopatologia , Linhagem , Fenótipo
9.
Prenat Diagn ; 38(1): 33-43, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29096039

RESUMO

OBJECTIVE: Rare genetic disorders resulting in prenatal or neonatal death are genetically heterogeneous, but testing is often limited by the availability of fetal DNA, leaving couples without a potential prenatal test for future pregnancies. We describe our novel strategy of exome sequencing parental DNA samples to diagnose recessive monogenic disorders in an audit of the first 50 couples referred. METHOD: Exome sequencing was carried out in a consecutive series of 50 couples who had 1 or more pregnancies affected with a lethal or prenatal-onset disorder. In all cases, there was insufficient DNA for exome sequencing of the affected fetus. Heterozygous rare variants (MAF < 0.001) in the same gene in both parents were selected for analysis. Likely, disease-causing variants were tested in fetal DNA to confirm co-segregation. RESULTS: Parental exome analysis identified heterozygous pathogenic (or likely pathogenic) variants in 24 different genes in 26/50 couples (52%). Where 2 or more fetuses were affected, a genetic diagnosis was obtained in 18/29 cases (62%). In most cases, the clinical features were typical of the disorder, but in others, they result from a hypomorphic variant or represent the most severe form of a variable phenotypic spectrum. CONCLUSION: We conclude that exome sequencing of parental samples is a powerful strategy with high clinical utility for the genetic diagnosis of lethal or prenatal-onset recessive disorders. © 2017 The Authors Prenatal Diagnosis published by John Wiley & Sons Ltd.


Assuntos
Anormalidades Congênitas/genética , Sequenciamento do Exoma , Doenças Genéticas Inatas/diagnóstico , Pais , Diagnóstico Pré-Natal/métodos , Feminino , Genes Recessivos , Humanos , Masculino , Gravidez
11.
Eur J Hum Genet ; 2024 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-39448799

RESUMO

An increasing number of individuals with intellectual developmental disorder (IDD) and heterozygous variants in BCL11A are identified, yet our knowledge of manifestations and mutational spectrum is lacking. To address this, we performed detailed analysis of 42 individuals with BCL11A-related IDD (BCL11A-IDD, a.k.a. Dias-Logan syndrome) ascertained through an international collaborative network, and reviewed 35 additional previously reported patients. Analysis of 77 affected individuals identified 60 unique disease-causing variants (30 frameshift, 7 missense, 6 splice-site, 17 stop-gain) and 8 unique BCL11A microdeletions. We define the most prevalent features of BCL11A-IDD: IDD, postnatal-onset microcephaly, hypotonia, behavioral abnormalities, autism spectrum disorder, and persistence of fetal hemoglobin (HbF), and identify autonomic dysregulation as new feature. BCL11A-IDD is distinguished from 2p16 microdeletion syndrome, which has a higher incidence of congenital anomalies. Our results underscore BCL11A as an important transcription factor in human hindbrain development, identifying a previously underrecognized phenotype of a small brainstem with a reduced pons/medulla ratio. Genotype-phenotype correlation revealed an isoform-dependent trend in severity of truncating variants: those affecting all isoforms are associated with higher frequency of hypotonia, and those affecting the long (BCL11A-L) and extra-long (-XL) isoforms, sparing the short (-S), are associated with higher frequency of postnatal microcephaly. With the largest international cohort to date, this study highlights persistence of fetal hemoglobin as a consistent biomarker and hindbrain abnormalities as a common feature. It contributes significantly to our understanding of BCL11A-IDD through an extensive unbiased multi-center assessment, providing valuable insights for diagnosis, management and counselling, and into BCL11A's role in brain development.

12.
Eur J Med Genet ; 66(4): 104714, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36724812

RESUMO

INTRODUCTION: Recurrent chromosome 16p13.11 microduplication has been characterised in the literature as a cause of developmental delay, learning difficulties and behavioural abnormalities. It is a neurosusceptibility locus and has incomplete penetrance and variable expression. Other clinical features, such as cardiac abnormalities have also been reported. The duplicated region contains the MYH11 gene, which encodes the protein myosin-11 and is a component of the myosin heavy chain in smooth muscle. Recent literature has suggested 16p13.11 microduplication as one of the possible risk factors for thoracic aortic aneurysms and dissection (TAAD). Therefore, we studied the detailed phenotype of cases of chromosome 16p13.11 microduplication from seven centres in the United Kingdom (UK) to expand the phenotype, focusing on the cardiac abnormalities. METHODS: All individuals with a chromosome 16p13.11 microduplication seen in Clinical Genetics prior to June 2017 in 6 centres (prior to 2018 in the seventh centre) were identified through the regional genetics laboratory databases. A Microsoft Excel® proforma was created and clinical data was collected retrospectively from clinical genetics databases from the seven genetics services in the UK. The data was collated and analysed collectively. RESULTS: The majority of the individuals presented with (72%) developmental delay and (62%) behavioural abnormalities, in keeping with the published literature. 27% had some dysmorphic features, 14% had visual impairment and 8% had congenital cardiac abnormalities. Echocardiograms were performed in 50% of patients, and only 3.8% patients had aortic dilatation and no one had aortic dissection. 9.7% of patients were found to have a second genetic/chromosomal diagnosis, especially where there were additional phenotypic features. CONCLUSION: 16p13.11 microduplication is a neurosusceptibility locus and is associated with variable expression. It may be helpful to refer children with 16p13.11 microduplication for a cardiac review for congenital cardiac abnormalities and also for ophthalmological assessment. Further prospective studies with cardiac assessments are recommended in this cohort of patients to determine whether ongoing aortic surveillance is indicated. Guidelines about the frequency of surveillance are indicated, especially in individuals with normal cardiac findings. We also highlight the importance of considering a second diagnosis if the phenotype is inconsistent with that reported.


Assuntos
Duplicação Cromossômica , Cromossomos Humanos Par 11 , Humanos , Estudos Prospectivos , Estudos Retrospectivos , Fenótipo
13.
Eur J Hum Genet ; 30(1): 95-100, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34645992

RESUMO

White-Sutton syndrome (WHSUS) is a neurodevelopmental disorder caused by heterozygous loss-of-function variants in POGZ. Through the Deciphering Developmental Disorders study and clinical testing, we identified 12 individuals from 10 families with pathogenic or likely pathogenic variants in POGZ (eight de novo and two inherited). Most individuals had delayed development and/or intellectual disability. We analyzed the clinical findings in our series and combined it with data from 89 previously reported individuals. The results demonstrate WHSUS is associated with variable developmental delay or intellectual disability, increased risk of obesity, visual defects, craniofacial dysmorphism, sensorineural hearing loss, feeding problems, seizures, and structural brain malformations. Our series includes further individuals with rod-cone dystrophy, cleft lip and palate, congenital diaphragmatic hernia, and duplicated renal drainage system, suggesting these are rare complications of WHSUS. In addition, we describe an individual with a novel, de novo missense variant in POGZ and features of WHSUS. Our work further delineates the phenotypic spectrum of WHSUS highlighting the variable severity of this disorder and the observation of familial pathogenic POGZ variants.


Assuntos
Anormalidades Múltiplas/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Fenótipo , Transposases/genética , Anormalidades Múltiplas/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Feminino , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Masculino , Mutação de Sentido Incorreto , Linhagem , Síndrome
14.
Genome Med ; 14(1): 62, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35698242

RESUMO

BACKGROUND: Genomics enables individualized diagnosis and treatment, but large challenges remain to functionally interpret rare variants. To date, only one causative variant has been described for KCNK9 imprinting syndrome (KIS). The genotypic and phenotypic spectrum of KIS has yet to be described and the precise mechanism of disease fully understood. METHODS: This study discovers mechanisms underlying KCNK9 imprinting syndrome (KIS) by describing 15 novel KCNK9 alterations from 47 KIS-affected individuals. We use clinical genetics and computer-assisted facial phenotyping to describe the phenotypic spectrum of KIS. We then interrogate the functional effects of the variants in the encoded TASK3 channel using sequence-based analysis, 3D molecular mechanic and dynamic protein modeling, and in vitro electrophysiological and functional methodologies. RESULTS: We describe the broader genetic and phenotypic variability for KIS in a cohort of individuals identifying an additional mutational hotspot at p.Arg131 and demonstrating the common features of this neurodevelopmental disorder to include motor and speech delay, intellectual disability, early feeding difficulties, muscular hypotonia, behavioral abnormalities, and dysmorphic features. The computational protein modeling and in vitro electrophysiological studies discover variability of the impact of KCNK9 variants on TASK3 channel function identifying variants causing gain and others causing loss of conductance. The most consistent functional impact of KCNK9 genetic variants, however, was altered channel regulation. CONCLUSIONS: This study extends our understanding of KIS mechanisms demonstrating its complex etiology including gain and loss of channel function and consistent loss of channel regulation. These data are rapidly applicable to diagnostic strategies, as KIS is not identifiable from clinical features alone and thus should be molecularly diagnosed. Furthermore, our data suggests unique therapeutic strategies may be needed to address the specific functional consequences of KCNK9 variation on channel function and regulation.


Assuntos
Deficiência Intelectual , Canais de Potássio de Domínios Poros em Tandem , Genótipo , Humanos , Deficiência Intelectual/genética , Hipotonia Muscular , Mutação , Fenótipo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo
15.
BMC Cell Biol ; 12: 22, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21599933

RESUMO

BACKGROUND: GGAs (Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog. RESULTS: Here we have quantified protein expression in Drosophila and show that there is >3-fold higher expression of GGA in male flies relative to female flies. In female flies the majority of GGA expression is in the head. In male flies GGA is not only expressed at high levels in the head but there is a gender specific increased expression which is due to the abundant expression of GGA in the testes. Using a highly specific antibody we have localised endogenous GGA protein in testes squashes, and visualised it in somatic and germ line cells. We show that GGA is expressed during multiple stages of sperm development, and co-stains with a marker of the trans-Golgi Network. This is most striking at the acroblast of early spermatids. In spite of the high expression of GGA in testes, knocking down its expression by >95% using transgenic RNAi fly lines did not affect male fertility. Therefore spermatogenesis in the male flies appears to progress normally with <5% GGA, most likely because alternative adaptors may be able to substitute partially or completely for the function of GGA. We also identify 'cueball' as a novel cargo for GGA, and mutants of cueball have been shown to have a male sterility phenotype. CONCLUSION: In Drosophila we have uncovered a potential role for GGA in the testes of male flies. The gender specific higher expression of GGA, its specific enrichment in testes and its localisation to developing spermatocytes and at the acroblast of spermatids supports a role for GGA function in Drosophila spermatogenesis, even though spermatogenesis still occurs when GGA expression is depleted to <5% of control.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Espermatogênese/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Fertilidade , Masculino , Fenótipo , Interferência de RNA , Testículo/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
16.
Bioconjug Chem ; 22(4): 709-16, 2011 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-21395337

RESUMO

Optimal PET imaging of tumors with radiolabeled engineered antibodies requires, among other parameters, matching blood clearance and tumor uptake with the half-life of the engineered antibody. Although diabodies have favorable molecular sizes (50 kDa) for rapid blood clearance (t(1/2) = 30-60 min) and are bivalent, thereby increasing tumor uptake, they exhibit substantial kidney uptake as their major route of clearance, which is especially evident when they are labeled with the PET isotope (64)Cu (t(1/2) = 12 h). To overcome this drawback, diabodies may be conjugated to PEG, a modification that increases the apparent molecular size of the diabody and reduces kidney uptake without adversely affecting tumor uptake or the tumor to blood ratio. We show here that site-specific attachment of monodispersed PEGn of increasing molecular size (n = 12, 24, and 48) can uniformly increase the apparent molecular size of the PEG-diabody conjugate, decrease kidney uptake, and increase tumor uptake, the latter due to the increased residence time of the conjugate in the blood. Since the monodispersed PEGs were preconjugated to the chelator DOTA, the conjugates were able to bind radiometals such as (111)In and (64)Cu that can be used for SPECT and PET imaging, respectively. To allow conjugation of the DOTA-PEG to the diabody, the DOTA-PEG incorporated a terminal cysteine conjugated to a vinyl sulfone moiety. In order to control the conjugation chemistry, we have engineered a surface thiolated diabody that incorporates two cysteines per monomer (four per diabody). The thiolated diabody was expressed and purified from bacterial fermentation and only needs to be reduced prior to conjugation to the DOTA-PEGn-Cys-VS. This novel imaging agent (a diabody with DOTA-PEG48-Cys-VS attached to introduced thiols) gave up to 80%ID/g of tumor uptake with a tumor to blood ratio (T/B) of 8 at 24 h when radiolabeled with (111)In and 37.9% ID/g of tumor uptake (T/B = 8) at 44 h when radiolabeled with (64)Cu in PET imaging in an animal model. Tumor uptake was significantly improved from the 50% ID/g at 24 h observed with diabodies that were pegylated on surface lysine residues. Importantly, there was no loss of immunoreactivity of the site-specific Cys-conjugated diabody to its antigen (TAG-72) compared to the parent, unconjugated diabody. We propose that thiolated diabodies conjugated to DOTAylated monodisperse PEGs have the potential for superior SPECT and PET imaging in a clinical setting.


Assuntos
Compostos Heterocíclicos com 1 Anel , Rim/metabolismo , Neoplasias/metabolismo , Polietilenoglicóis , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Radioisótopos de Cobre/química , Radioisótopos de Cobre/farmacocinética , Feminino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Rim/diagnóstico por imagem , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Neoplasias/diagnóstico por imagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual
17.
Biochem J ; 412(2): 191-209, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18466116

RESUMO

Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative disease caused by a CAG trinucleotide repeat expansion encoding an abnormally long polyglutamine tract in the huntingtin protein. Much has been learnt since the mutation was identified in 1993. We review the functions of wild-type huntingtin. Mutant huntingtin may cause toxicity via a range of different mechanisms. The primary consequence of the mutation is to confer a toxic gain of function on the mutant protein and this may be modified by certain normal activities that are impaired by the mutation. It is likely that the toxicity of mutant huntingtin is revealed after a series of cleavage events leading to the production of N-terminal huntingtin fragment(s) containing the expanded polyglutamine tract. Although aggregation of the mutant protein is a hallmark of the disease, the role of aggregation is complex and the arguments for protective roles of inclusions are discussed. Mutant huntingtin may mediate some of its toxicity in the nucleus by perturbing specific transcriptional pathways. HD may also inhibit mitochondrial function and proteasome activity. Importantly, not all of the effects of mutant huntingtin may be cell-autonomous, and it is possible that abnormalities in neighbouring neurons and glia may also have an impact on connected cells. It is likely that there is still much to learn about mutant huntingtin toxicity, and important insights have already come and may still come from chemical and genetic screens. Importantly, basic biological studies in HD have led to numerous potential therapeutic strategies.


Assuntos
Doença de Huntington , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/patologia , Doença de Huntington/terapia , Mitocôndrias/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Ubiquitina/metabolismo
18.
Expert Rev Mol Med ; 5(20): 1-21, 2003 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-14585171

RESUMO

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HD gene. The expanded repeats are translated into an abnormally long polyglutamine tract close to the N-terminus of the HD gene product ('huntingtin'). Studies in humans and mouse models suggest that the mutation is associated with a deleterious gain-of-function. Several studies have suggested that the large huntingtin protein is cleaved to produce a shorter N-terminal fragment containing the polyglutamine expansion, and that the polyglutamine expansion causes the protein fragment to misfold and form aggregates (inclusions) in the nuclei and processes of neurons. It is likely that neurotoxicity is caused by the misfolded protein in its soluble form, and/or in aggregates, and/or in the process of aggregation. A wide range of potential mechanisms for neurotoxicity have been proposed, including caspase activation, dysregulation of transcriptional pathways, increased production of reactive oxygen species, and inhibition of proteasome activity. In this review we consider the current status of research in the field and possible mechanisms whereby the HD mutation might result in neurodegeneration.


Assuntos
Encéfalo/patologia , Doença de Huntington/genética , Doença de Huntington/patologia , Degeneração Neural/patologia , Agregação Celular , Morte Celular , Cromossomos Humanos Par 4 , Corpo Estriado/patologia , Humanos , Doença de Huntington/terapia , Mutação , Neurônios/patologia , Peptídeos/genética , Repetições de Trinucleotídeos
19.
Neurosci Lett ; 330(3): 270-4, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12270644

RESUMO

Huntington's disease (HD) is caused by expansions of more than 35 CAG repeats in the HD gene. These repeats are translated into a long polyglutamine tract that confers a deleterious gain-of-function on the mutant protein. Intraneuronal inclusions comprising mutant huntingtin are found in HD patient brains. Here we show that the bacterial chaperonin GroEL can reduce aggregation of mutant huntingtin in COS-7 cells and requires GroES for efficient activity, analogous to what has been described in bacteria. The reduction in aggregation of mutant huntingtin by GroEL/GroES was associated with protection against polyglutamine-induced cell death.


Assuntos
Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Doença de Huntington/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Animais , Western Blotting , Células COS , Morte Celular , Chaperonina 10/genética , Chaperonina 60/genética , Doença de Huntington/patologia , Imuno-Histoquímica , Modelos Biológicos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Transfecção
20.
Curr Neurol Neurosci Rep ; 6(5): 437-46, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16928355

RESUMO

This review focuses on malformations of the central nervous system that have a genetic etiology. One can view each malformation as giving us unique details on a map entitled "how to make a human brain." The gene(s) that cause each malformation are being identified, allowing discovery of their specific role in neurodevelopment, and defining a "road" on the map. The malformation is then the developmental consequence of "taking a wrong turn." Assimilation of complementary data from other species with human malformation phenotype and genotype is revealing just how wonderful and complex the neurodevelopment map is. Here we highlight recent research on brain malformations and how this is illuminating the map of normal human brain formation.


Assuntos
Encéfalo/fisiologia , Desenvolvimento Humano/fisiologia , Malformações do Sistema Nervoso/genética , Humanos , Malformações do Sistema Nervoso/classificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA