RESUMO
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
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.
Assuntos
Aberrações Cromossômicas , Reparo do DNA , Deficiências do Desenvolvimento/genética , Neoplasias/genética , Sequência de Bases , Criança , Pré-Escolar , Quebra Cromossômica , Hibridização Genômica Comparativa , Replicação do DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Dados de Sequência MolecularRESUMO
BACKGROUND: Complement-mediated thrombotic microangiopathy (CM-TMA), also called atypical hemolytic uremic syndrome (aHUS), is a difficult-to-diagnose rare disease that carries severe morbidity and mortality. Anti-C5 monoclonal antibodies (aC5-mab) are standard treatments, but large studies and long-term data are scarce. Here, we report our single institution experience to augment the knowledge of CM-TMA treated with aC5-mab therapy. METHODS: We aimed to assess the short and long-term effects of aC5-mab in patients diagnosed with CM-TMA treated outside of a clinical trial. This was a retrospective study. We included all patients diagnosed with CM-TMA and treated with aC5-mab at our institution. There were no exclusion criteria. Endpoints included complete TMA response (CR) defined as normalization of hematological parameters and ≥25% improvement in serum creatinine (Cr) from baseline in patients with renal disease, relapse defined as losing the previously achieved CR, morbidity, adverse events, and survival. RESULTS: We found 28 patients with CM-TMA treated with aC5-mab. The median age was 50 years. Baseline laboratories: platelet counts 93 × 109 /L, hemoglobin 8.6 g/dL, lactate dehydrogenase 1326 U/L, serum Cr 4.7 mg/dL, and estimated glomerular filtration rate 19 mL/min. One individual was on renal replacement therapy (RRT) and 10 initiated RRT within 5 days of the first dose of aC5-mab. Genetic variants associated with CM-TMA included mutations in C3, CFB, CFH, CFHR1/3, CFI, and MCP. The mean duration of hospitalization was 24 days. The median time to initiation of aC5-mab was 10 days. Sixteen subjects received RRT. At the time of hospital discharge, 27 were alive, 14 remained on RRT, and 4 had a CR. At 6 months, 23 patients were alive, 18 continued aC5-mab, 8 remained on RRT, and 9 had a CR. At the last follow-up visit past 6 months, 20 were alive, 14 continued aC5-mab, 5 remained on RRT, 12 had a CR, and 1 was lost to follow-up. CONCLUSIONS: Our study provides real-world experience and insight into the long-term outcomes of CM-TMA treated with aC5-mab. Our findings validate that CM-TMA is an aggressive disease with significant morbidity and mortality, and confirm that aC5-mab is a relatively effective therapy for CM-TMA. Our study adds practical, real-world experience to the literature, but future research remains imperative.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Proteínas Inativadoras do Complemento , Microangiopatias Trombóticas , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/tratamento farmacológico , Microangiopatias Trombóticas/etiologia , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/genética , Proteínas do Sistema ComplementoRESUMO
Kidney transplantation is the most successful kidney replacement therapy available, resulting in improved recipient survival and societal cost savings. Yet, nearly 70 years after the first successful kidney transplant, there are still numerous barriers and untapped opportunities that constrain the access to transplant. The literature describing these barriers is extensive, but the practices and processes to solve them are less clear. Solutions must be multidisciplinary and be the product of strong partnerships among patients, their networks, health care providers, and transplant programs. Transparency in the referral, evaluation, and listing process as well as organ selection are paramount to build such partnerships. Providing early culturally congruent and patient-centered education as well as maximizing the use of local resources to facilitate the transplant work up should be prioritized. Every opportunity to facilitate pre-emptive kidney transplantation and living donation must be taken. Promoting the use of telemedicine and kidney paired donation as standards of care can positively impact the work up completion and maximize the chances of a living donor kidney transplant.
Assuntos
Acessibilidade aos Serviços de Saúde , Falência Renal Crônica , Transplante de Rim , Obtenção de Tecidos e Órgãos , Humanos , Obtenção de Tecidos e Órgãos/métodos , Falência Renal Crônica/cirurgia , Doadores Vivos/provisão & distribuição , Listas de EsperaRESUMO
Lumbar punctures (LP) are routinely used to administer intrathecal chemotherapy for children and adults with hematologic malignancies. The current guidelines suggest a platelet threshold of ≥ 50 × 109/L prior to LP for intrathecal chemotherapy (ITC). This can be challenging in patients with hematological malignancies who are thrombocytopenic. We conducted a retrospective chart review of 900 LPs for ITC and compared adverse events in patients with a platelet count of ≥ 50 × 109/L and < 50 × 109/L. Cohort 1 included 682 LPs (75.8%) with a pre-procedure platelet count ≥ 50 × 109/L, and cohort 2 included 218 LPs (24.2%) with a pre-procedure platelet count < 50 × 109/L. Cohort 2 was further subdivided into pre-procedure platelet counts of 41 × 109/L-49 × 109/L (n = 43), 31 × 109/L-40 × 109/L (n = 77), 21 × 109/L-30 × 109/L (n = 84), and 11 × 109/L-20 × 109/L (n = 14). Among 900 LP procedures, a pre-procedure platelet count < 50 × 109/L did not demonstrate a higher rate of post-procedure adverse events (6.5% vs 6.8%, p = 0.8237). When cohort 2 was further stratified, the cohort with a pre-procedure platelet count of 21 × 109/L-30 × 109/L had the highest percentage of complications from LP (9.5%) and the highest rates of traumatic taps with observed LP RBC count > 200 (35.7%, p = 0.0015). The rate of red blood cells (RBC) in the CSF was significantly higher in the group with platelets < 50 × 109/L with observed LP RBC count ≥ 200 (31.2% vs 20.5%, p = 0.0016), ≥ 500 (27.1% vs 14.6%, p < 0.0001), and ≥ 1000 (23% vs 11.6%, p < 0.0001). No instances of epidural hematomas were seen. We found no significant difference in bleeding complications between patients undergoing LPs for ITC with a platelet count above or below 50 × 109/L.
Assuntos
Neoplasias Hematológicas , Trombocitopenia , Criança , Adulto , Humanos , Punção Espinal/efeitos adversos , Estudos Retrospectivos , Trombocitopenia/etiologia , Lipopolissacarídeos , Transfusão de Plaquetas , Neoplasias Hematológicas/complicaçõesRESUMO
Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels by counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insights into endosomal trafficking, illuminate the cooperativity between an ubiquitin ligase and a deubiquitinating enzyme, and establish a role for USP7 in human neurodevelopmental disease.
Assuntos
Transtorno do Espectro Autista/enzimologia , Endossomos/metabolismo , Deficiência Intelectual/enzimologia , Proteínas dos Microfilamentos/metabolismo , Ubiquitina Tiolesterase/fisiologia , Adolescente , Transtorno do Espectro Autista/genética , Criança , Pré-Escolar , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Feminino , Células HCT116 , Haploinsuficiência , Humanos , Hipotálamo/metabolismo , Deficiência Intelectual/genética , Masculino , Neurônios/enzimologia , Proteínas Nucleares/metabolismo , Transporte Proteico , Proteólise , Deleção de Sequência , Peptidase 7 Específica de Ubiquitina , UbiquitinaçãoRESUMO
To describe an experience using a protocol using de novo belatacept (DNB) based maintenance immunosuppression in the setting of lymphocyte depletion. A retrospective, observational study was performed on 37 kidney transplant recipients treated with the DNB protocol, which was defined as belatacept initiated within 7 days after a kidney transplant with steroids and mycophenolate with anti-thymocyte globulin (ATG) induction without concomitant calcineurin inhibitors (CNIs). Patients who received a deceased donor kidney meeting one or more of the following criteria: anticipated cold ischemia time (CIT) greater than 24 h, donation after cardiac death, donor acute kidney injury, and a Kidney Donor Profile Index (KDPI) >85% during the study period were included. Patient survival at 1 year was 97.3% and graft survival was 94.6%. Delayed graft function (DGF) occurred in 40.54% of the patients. Two patients experienced a Banff 1B acute cellular rejection. BK viremia was detected in 32.4% of patients. The mean estimated glomerular filtration rate (eGFR) calculated with the use of modification of diet in renal disease (MDRD) equation at 1 year in the study group was 54.7 ml/min/1.73 m2 . We believe that utilization of the DNB protocol, which allows early CNI avoidance, may decrease organ discard rates.
Assuntos
Soro Antilinfocitário , Inibidores de Calcineurina , Abatacepte/efeitos adversos , Aloenxertos , Soro Antilinfocitário/efeitos adversos , Inibidores de Calcineurina/efeitos adversos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Terapia de Imunossupressão , Imunossupressores/efeitos adversos , Rim , Estudos Retrospectivos , EsteroidesRESUMO
COVID-19 has been associated with acute kidney injury and published reports of native kidney biopsies have reported diverse pathologies. Case series directed specifically to kidney allograft biopsy findings in the setting of COVID-19 are lacking. We evaluated 18 kidney transplant recipients who were infected with SARS-CoV-2 and underwent allograft biopsy. Patients had a median age of 55 years, six were female, and five were Black. Fifteen patients developed COVID-19 pneumonia, of which five required mechanical ventilation. Notably, five of 11 (45%) biopsies obtained within 1 month of positive SARS-CoV-2 PCR showed acute rejection (four with arteritis, three of which were not associated with reduced immunosuppression). The remaining six biopsies revealed podocytopathy (n = 2, collapsing glomerulopathy and lupus podocytopathy), acute tubular injury (n = 2), infarction (n = 1), and transplant glomerulopathy (n = 1). Biopsies performed >1 month after positive SARS-CoV-2 PCR revealed collapsing glomerulopathy (n = 1), acute tubular injury (n = 1), and nonspecific histologic findings (n = 5). No direct viral infection of the kidney allograft was detected by immunohistochemistry, in situ hybridization, or electron microscopy. On follow-up, two patients died and most patients showed persistent allograft dysfunction. In conclusion, we demonstrate diverse causes of kidney allograft dysfunction after COVID-19, the most common being acute rejection with arteritis.
Assuntos
Injúria Renal Aguda , COVID-19 , Aloenxertos , Biópsia , Feminino , Rejeição de Enxerto/etiologia , Humanos , Rim , Pessoa de Meia-Idade , SARS-CoV-2RESUMO
To further assess the scale and level of parental somatic mosaicism, we queried the CMA database at Baylor Genetics. We selected 50 unrelated families where clinically relevant apparent de novo CNV-deletions were found in the affected probands. Parental blood samples screening using deletion junction-specific PCR revealed four parents with somatic mosaicism. Droplet digital PCR (ddPCR), qPCR, and amplicon-based next-generation sequencing (NGS) were applied to validate these findings. Using ddPCR levels of mosaicism ranged from undetectable to 18.5%. Amplicon-based NGS and qPCR for the father with undetectable mosaicism was able to detect mosaicism at 0.39%. In one mother, ddPCR analysis revealed 15.6%, 10.6%, 8.2%, and undetectable levels of mosaicism in her blood, buccal cells, saliva, and urine samples, respectively. Our data suggest that more sensitive and precise methods, e.g. CNV junction-specific LR-PCR, ddPCR, or qPCR may allow for a more refined assessment of the potential disease recurrence risk for an identified variant.
Assuntos
Variações do Número de Cópias de DNA , Mosaicismo , Reação em Cadeia da Polimerase , Técnicas de Laboratório Clínico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Padrões de Herança , Masculino , Análise de Sequência de DNARESUMO
Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.
Assuntos
Transtornos do Neurodesenvolvimento/genética , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Animais , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Deleção de Genes , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Polimorfismo de Nucleotídeo Único , Peixe-Zebra/genéticaRESUMO
PURPOSE: Copy-number analysis to detect disease-causing losses and gains across the genome is recommended for the evaluation of individuals with neurodevelopmental disorders and/or multiple congenital anomalies, as well as for fetuses with ultrasound abnormalities. In the decade that this analysis has been in widespread clinical use, tremendous strides have been made in understanding the effects of copy-number variants (CNVs) in both affected individuals and the general population. However, continued broad implementation of array and next-generation sequencing-based technologies will expand the types of CNVs encountered in the clinical setting, as well as our understanding of their impact on human health. METHODS: To assist clinical laboratories in the classification and reporting of CNVs, irrespective of the technology used to identify them, the American College of Medical Genetics and Genomics has developed the following professional standards in collaboration with the National Institutes of Health (NIH)-funded Clinical Genome Resource (ClinGen) project. RESULTS: This update introduces a quantitative, evidence-based scoring framework; encourages the implementation of the five-tier classification system widely used in sequence variant classification; and recommends "uncoupling" the evidence-based classification of a variant from its potential implications for a particular individual. CONCLUSION: These professional standards will guide the evaluation of constitutional CNVs and encourage consistency and transparency across clinical laboratories.
Assuntos
Variações do Número de Cópias de DNA/genética , Testes Genéticos/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Anormalidades Múltiplas/genética , Consenso , Variação Genética/genética , Genoma Humano/genética , Genômica/normas , Humanos , Mutação/genética , Estados UnidosRESUMO
RATIONALE & OBJECTIVES: Posttransplantation membranous nephropathy (MN) represents a rare complication of kidney transplantation that can be classified as recurrent or de novo. The clinical, pathologic, and immunogenetic characteristics of posttransplantation MN and the differences between de novo and recurrent MN are not well understood. STUDY DESIGN: Multicenter case series. SETTING & PARTICIPANTS: We included 77 patients from 5 North American and European medical centers with post-kidney transplantation MN (27 de novo and 50 recurrent). Patients with MN in the native kidney who received kidney allografts but did not develop recurrent MN were used as nonrecurrent controls (n = 43). To improve understanding of posttransplantation MN, we compared de novo MN with recurrent MN and then contrasted recurrent MN with nonrecurrent controls. FINDINGS: Compared with recurrent MN, de novo MN was less likely to be classified as primary MN (OR, 0.04; P < 0.001) and had more concurrent antibody-mediated rejection (OR, 12.0; P < 0.001) and inferior allograft survival (HR for allograft failure, 3.2; P = 0.007). HLA-DQ2 and HLA-DR17 antigens were more common in recipients with recurrent MN compared with those with de novo MN; however, the frequency of these recipient antigens in recurrent MN was similar to that in nonrecurrent MN controls. Among the 93 kidney transplant recipients with native kidney failure attributed to MN, older recipient age (HR per each year older, 1.03; P = 0.02), recipient HLA-A3 antigen (HR, 2.5; P = 0.003), steroid-free immunosuppressive regimens (HR, 2.84; P < 0.001), and living related allograft (HR, 1.94; P = 0.03) were predictors of MN recurrence. LIMITATIONS: Retrospective case series, limited sample size due to rarity of the disease, nonstandardized nature of data collection and biopsies. CONCLUSIONS: De novo and recurrent MN likely represent separate diseases. De novo MN is associated with humoral alloimmunity and guarded outcome. Potential predisposing factors for recurrent MN include recipients who are older, recipient HLA-A3 antigen, steroid-free immunosuppressive regimen, and living related donor kidney.
Assuntos
Glomerulonefrite Membranosa/imunologia , Antígenos HLA/análise , Transplante de Rim , Complicações Pós-Operatórias/imunologia , Adulto , Idoso , Aloenxertos/imunologia , Europa (Continente)/epidemiologia , Feminino , Glomerulonefrite Membranosa/epidemiologia , Glomerulonefrite Membranosa/etiologia , Glomerulonefrite Membranosa/cirurgia , Teste de Histocompatibilidade , Humanos , Imunossupressores , Isoanticorpos/imunologia , Isoantígenos/imunologia , Masculino , Pessoa de Meia-Idade , América do Norte/epidemiologia , Complicações Pós-Operatórias/etiologia , Receptores da Fosfolipase A2/imunologia , Recidiva , Estudos RetrospectivosRESUMO
PURPOSE: Haploinsufficiency of USP7, located at chromosome 16p13.2, has recently been reported in seven individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), autism spectrum disorder (ASD), seizures, and hypogonadism. Further, USP7 was identified to critically incorporate into the MAGEL2-USP7-TRIM27 (MUST), such that pathogenic variants in USP7 lead to altered endosomal F-actin polymerization and dysregulated protein recycling. METHODS: We report 16 newly identified individuals with heterozygous USP7 variants, identified by genome or exome sequencing or by chromosome microarray analysis. Clinical features were evaluated by review of medical records. Additional clinical information was obtained on the seven previously reported individuals to fully elucidate the phenotypic expression associated with USP7 haploinsufficiency. RESULTS: The clinical manifestations of these 23 individuals suggest a syndrome characterized by DD/ID, hypotonia, eye anomalies,feeding difficulties, GERD, behavioral anomalies, and ASD, and more specific phenotypes of speech delays including a nonverbal phenotype and abnormal brain magnetic resonance image findings including white matter changes based on neuroradiologic examination. CONCLUSION: The consistency of clinical features among all individuals presented regardless of de novo USP7 variant type supports haploinsufficiency as a mechanism for pathogenesis and refines the clinical impact faced by affected individuals and caregivers.
Assuntos
Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Transtornos do Neurodesenvolvimento/genética , Comportamento Problema , Adolescente , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Criança , Pré-Escolar , Deleção Cromossômica , Proteínas de Ligação a DNA/genética , Genoma Humano/genética , Haploinsuficiência/genética , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/fisiopatologia , Transtornos do Desenvolvimento da Linguagem/fisiopatologia , Transtornos do Neurodesenvolvimento/fisiopatologia , Proteínas Nucleares/genética , Fenótipo , Proteínas/genética , Sequenciamento do ExomaRESUMO
Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.
Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Adulto , Animais , Comportamento Animal , Transtorno Bipolar/genética , Cromossomos Humanos Par 22/genética , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Dosagem de Genes/genética , Expressão Gênica/genética , Genes Duplicados/genética , Humanos , Hipercinese/genética , Hipercinese/fisiopatologia , Potenciais Pós-Sinápticos Inibidores , Lítio/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Convulsões/genética , Ácido Valproico/farmacologia , Ácido Valproico/uso terapêuticoRESUMO
Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.
Assuntos
Aberrações Cromossômicas , Inversão Cromossômica/genética , Replicação do DNA/genética , Duplicação Gênica/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Feminino , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Masculino , Análise de Sequência de DNA , Translocação GenéticaRESUMO
Detailed characterization of chromosomal abnormalities, a common cause for congenital abnormalities and pregnancy loss, is critical for elucidating genes for human fetal development. Here, 2,186 product-of-conception samples were tested for copy-number variations (CNVs) at two clinical diagnostic centers using whole-genome sequencing and high-resolution chromosomal microarray analysis. We developed a new gene discovery approach to predict potential developmental genes and identified 275 candidate genes from CNVs detected from both datasets. Based on Mouse Genome Informatics (MGI) and Zebrafish model organism database (ZFIN), 75% of identified genes could lead to developmental defects when mutated. Genes involved in embryonic development, gene transcription, and regulation of biological processes were significantly enriched. Especially, transcription factors and gene families sharing specific protein domains predominated, which included known developmental genes such as HOX, NKX homeodomain genes, and helix-loop-helix containing HAND2, NEUROG2, and NEUROD1 as well as potential novel developmental genes. We observed that developmental genes were denser in certain chromosomal regions, enabling identification of 31 potential genomic loci with clustered genes associated with development.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Desenvolvimento Embrionário/genética , Fatores de Transcrição/genética , Animais , Transtornos Cromossômicos/patologia , Variações do Número de Cópias de DNA/genética , Feminino , Genoma Humano , Humanos , Camundongos , Análise em Microsséries , Gravidez , Peixe-Zebra/genéticaRESUMO
Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with â¼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.
Assuntos
Elementos Alu/genética , Cromossomos Humanos Par 17/genética , Variações do Número de Cópias de DNA , Alelos , Sequência de Bases , Hibridização Genômica Comparativa , Feminino , Duplicação Gênica , Rearranjo Gênico , Genoma Humano , Instabilidade Genômica , Genômica , Recombinação Homóloga , Humanos , Masculino , Dados de Sequência Molecular , Duplicações Segmentares Genômicas , Deleção de SequênciaRESUMO
Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B, and HUWE1 and deubiquitinating enzyme genes USP7 and USP9X has been reported in patients with neurodevelopmental delays. To date, only a handful of single-nucleotide variants (SNVs) and copy-number variants (CNVs) involving TRIP12, encoding a member of the HECT domain E3 ubiquitin ligases family on chromosome 2q36.3 have been reported. Using chromosomal microarray analysis and whole-exome sequencing (WES), we have identified, respectively, five deletion CNVs and four inactivating SNVs (two frameshifts, one missense, and one splicing) in TRIP12. Seven of these variants were found to be de novo; parental studies could not be completed in two families. Quantitative PCR analyses of the splicing mutation showed a dramatically decreased level of TRIP12 mRNA in the proband compared to the family controls, indicating a loss-of-function mechanism. The shared clinical features include intellectual disability with or without autistic spectrum disorders, speech delay, and facial dysmorphism. Our findings demonstrate that E3 ubiquitin ligase TRIP12 plays an important role in nervous system development and function. The nine presented pathogenic variants further document that TRIP12 haploinsufficiency causes a childhood-onset neurodevelopmental disorder. Finally, our data enable expansion of the phenotypic spectrum of ubiquitin-proteasome dependent disorders.
Assuntos
Transtorno do Espectro Autista/genética , Proteínas de Transporte/genética , Fácies , Haploinsuficiência , Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Transtorno do Espectro Autista/complicações , Criança , Pré-Escolar , Estudos de Coortes , Variações do Número de Cópias de DNA , Feminino , Humanos , Lactente , Deficiência Intelectual/complicações , Transtornos do Desenvolvimento da Linguagem/complicações , MasculinoRESUMO
New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.