RESUMO
Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Defeitos Congênitos da Glicosilação , Humanos , Glicosilação , Proteínas Adaptadoras de Transporte Vesicular/genética , Fibroblastos/metabolismo , Defeitos Congênitos da Glicosilação/genética , FenótipoRESUMO
Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.
Assuntos
Hidrolases Anidrido Ácido/deficiência , Deficiência Intelectual/patologia , Microcefalia/patologia , Hipotonia Muscular/patologia , Mutação , Transtornos do Neurodesenvolvimento/patologia , Monoéster Fosfórico Hidrolases/genética , Alelos , Animais , Pré-Escolar , Feminino , Humanos , Lactente , Deficiência Intelectual/etiologia , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Microcefalia/etiologia , Microcefalia/metabolismo , Hipotonia Muscular/etiologia , Hipotonia Muscular/metabolismo , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo , Linhagem , FenótipoRESUMO
The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. We show that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. We suggest that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division.
Assuntos
Divisão Celular Assimétrica/fisiologia , Polaridade Celular/fisiologia , Proteínas de Membrana/genética , Microcefalia/virologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Animais , Divisão Celular , Drosophila melanogaster/metabolismo , Humanos , Mutação , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Neurônios/citologia , Zika virusRESUMO
DNA rearrangements resulting in human genome structural variants (SVs) are caused by diverse mutational mechanisms. We used long- and short-read sequencing technologies to investigate end products of de novo chromosome 17p11.2 rearrangements and query the molecular mechanisms underlying both recurrent and non-recurrent events. Evidence for an increased rate of clustered single-nucleotide variant (SNV) mutation in cis with non-recurrent rearrangements was found. Indel and SNV formation are associated with both copy-number gains and losses of 17p11.2, occur up to â¼1 Mb away from the breakpoint junctions, and favor C > G transversion substitutions; results suggest that single-stranded DNA is formed during the genesis of the SV and provide compelling support for a microhomology-mediated break-induced replication (MMBIR) mechanism for SV formation. Our data show an additional mutational burden of MMBIR consisting of hypermutation confined to the locus and manifesting as SNVs and indels predominantly within genes.
Assuntos
Cromossomos Humanos Par 17 , Mutação , Anormalidades Múltiplas/genética , Pontos de Quebra do Cromossomo , Transtornos Cromossômicos/genética , Duplicação Cromossômica/genética , Variações do Número de Cópias de DNA , Reparo do DNA/genética , Replicação do DNA , Rearranjo Gênico , Genoma Humano , Variação Estrutural do Genoma , Humanos , Mutação INDEL , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Análise de Sequência de DNA/métodos , Síndrome de Smith-Magenis/genéticaRESUMO
De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.
Assuntos
Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Doenças Genéticas Inatas/embriologia , Doenças Genéticas Inatas/genética , Instabilidade Genômica , Mutação , Pontos de Quebra do Cromossomo , Duplicação Cromossômica , Replicação do DNA , Desenvolvimento Embrionário , Feminino , Gametogênese , Humanos , MasculinoRESUMO
ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane protein implicated in mitochondrial dynamics, nucleoid organization, protein translation, cell growth, and cholesterol metabolism. We identified a recurrent de novo ATAD3A c.1582C>T (p.Arg528Trp) variant by whole-exome sequencing (WES) in five unrelated individuals with a core phenotype of global developmental delay, hypotonia, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. We also describe two families with biallelic variants in ATAD3A, including a homozygous variant in two siblings, and biallelic ATAD3A deletions mediated by nonallelic homologous recombination (NAHR) between ATAD3A and gene family members ATAD3B and ATAD3C. Tissue-specific overexpression of borR534W, the Drosophila mutation homologous to the human c.1582C>T (p.Arg528Trp) variant, resulted in a dramatic decrease in mitochondrial content, aberrant mitochondrial morphology, and increased autophagy. Homozygous null bor larvae showed a significant decrease of mitochondria, while overexpression of borWT resulted in larger, elongated mitochondria. Finally, fibroblasts of an affected individual exhibited increased mitophagy. We conclude that the p.Arg528Trp variant functions through a dominant-negative mechanism that results in small mitochondria that trigger mitophagy, resulting in a reduction in mitochondrial content. ATAD3A variation represents an additional link between mitochondrial dynamics and recognizable neurological syndromes, as seen with MFN2, OPA1, DNM1L, and STAT2 mutations.
Assuntos
Adenosina Trifosfatases/genética , Alelos , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mutação , Doenças do Sistema Nervoso/genética , ATPases Associadas a Diversas Atividades Celulares , Adulto , Animais , Axônios/patologia , Cardiomiopatias/genética , Criança , Pré-Escolar , Variações do Número de Cópias de DNA/genética , Deficiências do Desenvolvimento/genética , Drosophila melanogaster/genética , Feminino , Fibroblastos , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Hipotonia Muscular/genética , Músculos/patologia , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Neurônios/patologia , Atrofia Óptica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Síndrome , Adulto JovemRESUMO
Robinow syndrome is a rare congenital disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features. Recent reports have identified, in individuals with dominant Robinow syndrome, a specific type of variant characterized by being uniformly located in the penultimate exon of DVL1 and resulting in a -1 frameshift allele with a premature termination codon that escapes nonsense-mediated decay. Here, we studied a cohort of individuals who had been clinically diagnosed with Robinow syndrome but who had not received a molecular diagnosis from variant studies of DVL1, WNT5A, and ROR2. Because of the uniform location of frameshift variants in DVL1-mediated Robinow syndrome and the functional redundancy of DVL1, DVL2, and DVL3, we elected to pursue direct Sanger sequencing of the penultimate exon of DVL1 and its paralogs DVL2 and DVL3 to search for potential disease-associated variants. Remarkably, targeted sequencing identified five unrelated individuals harboring heterozygous, de novo frameshift variants in DVL3, including two splice acceptor mutations and three 1 bp deletions. Similar to the variants observed in DVL1-mediated Robinow syndrome, all variants in DVL3 result in a -1 frameshift, indicating that these highly specific alterations might be a common cause of dominant Robinow syndrome. Here, we review the current knowledge of these peculiar variant alleles in DVL1- and DVL3-mediated Robinow syndrome and further elucidate the phenotypic features present in subjects with DVL1 and DVL3 frameshift mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Anormalidades Craniofaciais/genética , Nanismo/genética , Éxons , Mutação da Fase de Leitura , Deformidades Congênitas dos Membros/genética , Fosfoproteínas/genética , Anormalidades Urogenitais/genética , Alelos , Sequência de Bases , Códon sem Sentido , Anormalidades Craniofaciais/diagnóstico , Proteínas Desgrenhadas , Nanismo/diagnóstico , Feminino , Variação Genética , Humanos , Deformidades Congênitas dos Membros/diagnóstico , Masculino , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Análise de Sequência de DNA , Deleção de Sequência , Anormalidades Urogenitais/diagnóstico , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
PURPOSE: Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of genetic disorders of the peripheral nervous system. Copy-number variants (CNVs) contribute significantly to CMT, as duplication of PMP22 underlies the majority of CMT1 cases. We hypothesized that CNVs and/or single-nucleotide variants (SNVs) might exist in patients with CMT with an unknown molecular genetic etiology. METHODS: Two hundred patients with CMT, negative for both SNV mutations in several CMT genes and for CNVs involving PMP22, were screened for CNVs by high-resolution oligonucleotide array comparative genomic hybridization. Whole-exome sequencing was conducted on individuals with rare, potentially pathogenic CNVs. RESULTS: Putatively causative CNVs were identified in five subjects (~2.5%); four of the five map to known neuropathy genes. Breakpoint sequencing revealed Alu-Alu-mediated junctions as a predominant contributor. Exome sequencing identified MFN2 SNVs in two of the individuals. CONCLUSION: Neuropathy-associated CNV outside of the PMP22 locus is rare in CMT. Nevertheless, there is potential clinical utility in testing for CNVs and exome sequencing in CMT cases negative for the CMT1A duplication. These findings suggest that complex phenotypes including neuropathy can potentially be caused by a combination of SNVs and CNVs affecting more than one disease-associated locus and contributing to a mutational burden.Genet Med 18 5, 443-451.
Assuntos
Doença de Charcot-Marie-Tooth/genética , GTP Fosfo-Hidrolases/genética , Proteínas Mitocondriais/genética , Proteínas da Mielina/genética , Polineuropatias/genética , Adulto , Idade de Início , Doença de Charcot-Marie-Tooth/fisiopatologia , Pré-Escolar , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Exoma/genética , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Proteína P0 da Mielina/genética , Condução Nervosa/genética , Polimorfismo de Nucleotídeo Único/genética , Polineuropatias/fisiopatologiaRESUMO
Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases-about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual's susceptibility to acquiring disease-associated alleles.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Genoma Humano , Doenças Renais Císticas/congênito , Proteínas de Membrana/genética , Alelos , Animais , Hibridização Genômica Comparativa , Proteínas do Citoesqueleto , Dosagem de Genes , Rearranjo Gênico , Variação Estrutural do Genoma , Haplótipos , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , PrimatasRESUMO
Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in â¼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Exoma , Carga Genética , Doenças do Sistema Nervoso Periférico/genética , Fenótipo , Animais , Feminino , Variação Genética , Proteínas de Choque Térmico HSP40/genética , Humanos , Masculino , Mutação , Proteína P2 de Mielina/genética , Linhagem , Penetrância , Serina C-Palmitoiltransferase/genética , Supressão Genética , Peixe-ZebraRESUMO
BACKGROUND: The antecedents that contribute to health disparities in maternal child health populations begin before birth and extend into the early prenatal and gestational growth periods. Mothers and infants living in rural poverty in particular are at considerable risk for problems associated with reproductive health, including pregnancy complications and premature births. OBJECTIVE: The aim of this manuscript is thus two-fold, to describe the epidemiologic makeup of the community and the intervention model of the Community Caring Collaborative. METHODS: Innovative models of early-integrated care for high-risk mothers and children are showing promise for long-term outcomes. They foster environments that enable mothers to trust health systems while maintaining a workforce of high functioning health workers who understand the mechanisms that underpin maternal and child health disparities. The Community Caring Collaborative in Washington County, Maine developed one such model that has made inroads in bridging such gaps. This manuscript explicates a case study of how the Community Caring Collaborative came into being and why it established the Bridging model of comprehensive care. The focus of this manuscript is thus two-fold, the community and the intervention model. RESULTS: The "bridging model" develops trust-based relationships between high-risk mothers with the health system and its multiple resources. Community members with advanced training provide the support and care linkages that are critical for family success. CONCLUSIONS: Innovative models of collaborative care impact the health of vulnerable mothers and their children working toward a marked decrease in health related disparities.
Assuntos
Redes Comunitárias/organização & administração , Mães , Síndrome de Abstinência Neonatal , Saúde da População Rural , População Rural , Apoio Social , Feminino , Acessibilidade aos Serviços de Saúde , Disparidades em Assistência à Saúde , Humanos , Recém-Nascido , Terapia Intensiva Neonatal , Maine , Modelos Organizacionais , Síndrome de Abstinência Neonatal/terapia , Pobreza , Serviço SocialRESUMO
CONTEXT: Pituitary stalk interruption syndrome (PSIS) is a rare, congenital anomaly of the pituitary gland characterized by pituitary gland insufficiency, thin or discontinuous pituitary stalk, anterior pituitary hypoplasia, and ectopic positioning of the posterior pituitary gland (neurohypophysis). The clinical presentation of patients with PSIS varies from isolated growth hormone (GH) deficiency to combined pituitary insufficiency and accompanying extrapituitary findings. Mutations in HESX1, LHX4, OTX2, SOX3, and PROKR2 have been associated with PSIS in less than 5% of cases; thus, the underlying genetic etiology for the vast majority of cases remains to be determined. OBJECTIVE: We applied whole-exome sequencing (WES) to a consanguineous family with two affected siblings who have pituitary gland insufficiency and radiographic findings of hypoplastic (thin) pituitary gland, empty sella, ectopic neurohypophysis, and interrupted pitiutary stalk-characteristic clinical diagnostic findings of PSIS. DESIGN AND PARTICIPANTS: WES was applied to two affected and one unaffected siblings. RESULTS: WES of two affected and one unaffected sibling revealed a unique homozygous missense mutation in GPR161, which encodes the orphan G protein-coupled receptor 161, a protein responsible for transducing extracellular signals across the plasma membrane into the cell. CONCLUSION: Mutations of GPR161 may be implicated as a potential novel cause of PSIS.
Assuntos
Hipopituitarismo/genética , Hipófise/anormalidades , Receptores Acoplados a Proteínas G/genética , Adolescente , Pré-Escolar , Exoma , Feminino , Estudo de Associação Genômica Ampla , Humanos , MutaçãoRESUMO
Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.
Assuntos
Adenosina Trifosfatases/genética , Elementos Alu/genética , Proteínas de Transporte de Cátions/genética , Variações do Número de Cópias de DNA/genética , Paraplegia Espástica Hereditária/genética , Sequência de Bases , Linhagem Celular Transformada , Genótipo , Humanos , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , Deleção de Sequência , EspastinaRESUMO
CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.
Assuntos
Doenças do Sistema Nervoso Central/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Fosfotransferases/metabolismo , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Animais , Doenças do Sistema Nervoso Central/patologia , Cérebro/patologia , Pré-Escolar , Endorribonucleases/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos CBA , Microcefalia/genética , Doenças do Sistema Nervoso Periférico/patologia , RNA de Transferência/genética , Proteínas de Ligação a RNARESUMO
Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition.
Assuntos
Técnicas Biossensoriais/métodos , DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Linhagem Celular , Células Cultivadas , Clonagem Molecular , DNA/química , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Copy number variations (CNVs) in the human genome contribute significantly to disease. De novo CNV mutations arise via genomic rearrangements, which can occur in 'trans', i.e. via interchromosomal events, or in 'cis', i.e. via intrachromosomal events. However, what molecular mechanisms occur between chromosomes versus between or within chromatids has not been systematically investigated. We hypothesized that distinct CNV mutational mechanisms, based on their intrinsic properties, may occur in a biased intrachromosomal versus interchromosomal manner. Here, we studied 62 genomic duplications observed in association with sporadic Potocki-Lupski syndrome (PTLS), in which multiple mutational mechanisms appear to be operative. Intriguingly, more interchromosomal than intrachromosomal events were identified in recurrent PTLS duplications mediated by non-allelic homologous recombination, whereas the reciprocal distribution was found for replicative mechanisms and non-homologous end-joining, likely reflecting the differences in spacial proximity of homologous chromosomes during different mutational processes.
Assuntos
Cromossomos Humanos Par 17 , Variações do Número de Cópias de DNA , Replicação do DNA , Síndrome de Smith-Magenis/genética , Anormalidades Múltiplas , Transtornos Cromossômicos , Duplicação Cromossômica , Hibridização Genômica Comparativa , Reparo do DNA por Junção de Extremidades , Duplicação Gênica , Humanos , Linhagem , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Genomic disorders constitute a class of diseases that are associated with DNA rearrangements resulting from region-specific genome instability, that is, genome architecture incites genome instability. Nonallelic homologous recombination (NAHR) or crossing-over in meiosis between sequences that are not in allelic positions (i.e., paralogous sequences) can result in recurrent deletions or duplications causing genomic disorders. Previous studies of NAHR have focused on description of the phenomenon, but it remains unclear how NAHR occurs during meiosis and what factors determine its frequency. Here we assembled two patient cohorts with reciprocal genomic disorders; deletion associated Smith-Magenis syndrome and duplication associated Potocki-Lupski syndrome. By assessing the full spectrum of rearrangement types from the two cohorts, we find that complex rearrangements (those with more than one breakpoint) are more prevalent in copy-number gains (17.7%) than in copy-number losses (2.3%); an observation that supports a role for replicative mechanisms in complex rearrangement formation. Interestingly, for NAHR-mediated recurrent rearrangements, we show that crossover frequency is positively associated with the flanking low-copy repeat (LCR) length and inversely influenced by the inter-LCR distance. To explain this, we propose that the probability of ectopic chromosome synapsis increases with increased LCR length, and that ectopic synapsis is a necessary precursor to ectopic crossing-over.
Assuntos
Pareamento Cromossômico , Troca Genética , Recombinação Homóloga , Anormalidades Múltiplas , Alelos , Sequência de Bases , Transtornos Cromossômicos , Duplicação Cromossômica , Cromossomos Humanos Par 17/ultraestrutura , Estudos de Coortes , Hibridização Genômica Comparativa , Deleção de Genes , Dosagem de Genes , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/genética , Recombinação Genética , Síndrome de Smith-Magenis/genética , Transativadores , Fatores de Transcrição/genéticaRESUMO
We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 11 unrelated subjects. Notably, only two breakpoint junctions were generated during each rearrangement formation. All the complex rearrangement products share a common genomic organization, duplication-inverted triplication-duplication (DUP-TRP/INV-DUP), in which the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by >300 kb, a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat-mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology-driven events, via inverted repeats, and microhomologous or nonhomologous events.
Assuntos
Rearranjo Gênico , Genoma Humano , Sequências Repetitivas de Ácido Nucleico , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression.
Assuntos
Duplicação Cromossômica/genética , Dosagem de Genes , Instabilidade Genômica , Proteína de Replicação A/genética , Trissomia/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células CHO , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos Par 17/genética , Cricetinae , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mosaicismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Fase SRESUMO
Complex genomic rearrangements (CGRs) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here, we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated, we observed localization and multiple copy number changes including deletions, duplications, and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism's life cycle.