RESUMO
Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.
Assuntos
Acidose Láctica/genética , Encefalopatias/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Ligação ao GTP/genética , Processamento de Proteína Pós-Traducional , Acidose Láctica/fisiopatologia , Sequência de Aminoácidos , Encéfalo/patologia , Encefalopatias/fisiopatologia , Cardiomiopatia Hipertrófica/fisiopatologia , Linhagem Celular , Criança , Pré-Escolar , Consanguinidade , Feminino , Fibroblastos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Biossíntese de Proteínas , Interferência de RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Alinhamento de SequênciaRESUMO
PURPOSE: Our goal was to design a customized microarray, arrEYE, for high-resolution copy number variant (CNV) analysis of known and candidate genes for inherited retinal dystrophy (iRD) and retina-expressed noncoding RNAs (ncRNAs). METHODS: arrEYE contains probes for the full genomic region of 106 known iRD genes, including those implicated in retinitis pigmentosa (RP) (the most frequent iRD), cone-rod dystrophies, macular dystrophies, and an additional 60 candidate iRD genes and 196 ncRNAs. Eight CNVs in iRD genes identified by other techniques were used as positive controls. The test cohort consisted of 57 patients with autosomal dominant, X-linked, or simplex RP. RESULTS: In an RP patient, a novel heterozygous deletion of exons 7 and 8 of the HGSNAT gene was identified: c.634-408_820+338delinsAGAATATG, p.(Glu212Glyfs*2). A known variant was found on the second allele: c.1843G>A, p.(Ala615Thr). Furthermore, we expanded the allelic spectrum of USH2A and RCBTB1 with novel CNVs. CONCLUSION: The arrEYE platform revealed subtle single-exon to larger CNVs in iRD genes that could be characterized at the nucleotide level, facilitated by the high resolution of the platform. We report the first CNV in HGSNAT that, combined with another mutation, leads to RP, further supporting its recently identified role in nonsyndromic iRD.Genet Med 19 4, 457-466.
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Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Distrofias Retinianas/genética , Acetiltransferases/genética , Proteínas da Matriz Extracelular/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , RNA não Traduzido/genética , Deleção de SequênciaRESUMO
OBJECTIVES: To implement non-invasive prenatal testing (NIPT) for fetal aneuploidies with semiconductor sequencing in an academic cytogenomic laboratory and to evaluate the first 15-month experience on clinical samples. METHODS: We validated a NIPT protocol for cell-free fetal DNA sequencing from maternal plasma for the detection of trisomy 13, 18 and 21 on a semiconductor sequencing instrument. Fetal DNA fraction calculation for all samples and several quality parameters were implemented in the workflow. One thousand eighty-one clinical NIPT samples were analysed, following the described protocol. RESULTS: Non-invasive prenatal testing was successfully implemented and validated on 201 normal and 74 aneuploid samples. From 1081 clinical samples, 17 samples showed an abnormal result: 14 trisomy 21 samples, one trisomy 18 and one trisomy 16 were detected. Also a maternal copy number variation on chromosome 13 was observed, which could potentially lead to a false positive trisomy 13 result. One sex discordant result was reported, possibly attributable to a vanishing twin. Moreover, our combined fetal fraction calculation enabled a more reliable risk estimate for trisomy 13, 18 and 21. CONCLUSIONS: Non-invasive prenatal testing for trisomy 21, 18 and 13 has a very high specificity and sensitivity. Because of several biological phenomena, diagnostic invasive confirmation of abnormal results remains required. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Assuntos
Transtornos Cromossômicos/diagnóstico , DNA/sangue , Diagnóstico Pré-Natal/métodos , Semicondutores , Análise de Sequência de DNA/métodos , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , DNA/análise , Variações do Número de Cópias de DNA , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Reações Falso-Positivas , Feminino , Testes Genéticos/métodos , Humanos , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trissomia/diagnóstico , Trissomia/genética , Síndrome da Trissomia do Cromossomo 13 , Síndrome da Trissomía do Cromossomo 18RESUMO
Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms--such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)--were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.
Assuntos
Blefarofimose , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Fatores de Transcrição Forkhead , Recombinação Homóloga/genética , Menopausa Precoce , Anormalidades da Pele , Alelos , Blefarofimose/etiologia , Blefarofimose/genética , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genoma Humano , Humanos , Menopausa Precoce/genética , Estrutura Terciária de Proteína , Deleção de Sequência , Anormalidades da Pele/etiologia , Anormalidades da Pele/genética , Moldes GenéticosRESUMO
A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl-tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood, whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In-gel activity staining after blue native-polyacrylamide gel electrophoresis confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole-exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3' splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV-transformed lymphoblasts, a specific decrease in the amount of charged mt-tRNA(Asn) was demonstrated as compared with controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease-associated aaRS2.
Assuntos
Aspartato-tRNA Ligase/genética , Mutação de Sentido Incorreto , Adulto , Aspartato-tRNA Ligase/metabolismo , Sequência de Bases , Células Cultivadas , Consanguinidade , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , Doenças Musculares/genética , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNARESUMO
PURPOSE: Autosomal recessive retinal dystrophies are clinically and genetically heterogeneous, which hampers molecular diagnosis. We evaluated identity-by-descent-guided Sanger sequencing or whole-exome sequencing in 26 families with nonsyndromic (19) or syndromic (7) autosomal recessive retinal dystrophies to identify disease-causing mutations. METHODS: Patients underwent genome-wide identity-by-descent mapping followed by Sanger sequencing (16) or whole-exome sequencing (10). Whole-exome sequencing data were filtered against identity-by-descent regions and known retinal dystrophy genes. The medical history was reviewed in mutation-positive families. RESULTS: We identified mutations in 14 known retinal dystrophy genes in 20/26 (77%) families: ABCA4, CERKL, CLN3, CNNM4, C2orf71, IQCB1, LRAT, MERTK, NMNAT1, PCDH15, PDE6B, RDH12, RPGRIP1, and USH2A. Whole-exome sequencing in single individuals revealed mutations in either the largest or smaller identity-by-descent regions, and a compound heterozygous genotype in NMNAT1. Moreover, a novel deletion was found in PCDH15. In addition, we identified mutations in CLN3, CNNM4, and IQCB1 in patients initially diagnosed with nonsyndromic retinal dystrophies. CONCLUSION: Our study emphasized that identity-by-descent-guided mutation analysis and/or whole-exome sequencing are powerful tools for the molecular diagnosis of retinal dystrophy. Our approach uncovered unusual molecular findings and unmasked syndromic retinal dystrophies, guiding future medical management. Finally, elucidating ABCA4, LRAT, and MERTK mutations offers potential gene-specific therapeutic perspectives.
Assuntos
Consanguinidade , Análise Mutacional de DNA , Exoma , Mutação , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Adolescente , Proteínas Relacionadas a Caderinas , Caderinas/genética , Criança , Pré-Escolar , Feminino , Genes Recessivos , Estudo de Associação Genômica Ampla , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Oftalmoscópios , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Dente/patologiaRESUMO
PURPOSE: Radial ray deficiencies are characterized by unilateral or bilateral absence of varying portions of the radius and thumb. Both isolated and syndromic forms have been described, and although for some of the syndromes the causal gene has been identified, many patients remain without a genetic diagnosis. METHODS: In this study, a cohort of 54 patients with radial ray deficiencies was screened for genomic aberrations by molecular karyotyping. RESULTS: In 8 of 54 cases, an aberration was detected. Two unrelated patients inherited a 1q21.1 microduplication from a healthy parent, whereas in a third patient, a 16p13.11 microduplication was identified. Two other interesting microdeletions were detected: a 10q24.3 deletion at the split hand-foot malformation (SHFM3) locus and a 7p22.1 deletion including the RAC1 gene. CONCLUSION: The finding of these microduplications may just be coincidental or, alternatively, they may illustrate the broad phenotypic spectrum of these microduplications. Duplications in the 10q24.3 region result in split hand-foot malformations, and our observation indicates that deletions may cause radial ray defects. Finally, a candidate gene for radial ray deficiencies was detected in the 7p22.1 deletion. RAC1 plays an important role in the canonical Wnt pathway and conditional RAC1 knockout mice exhibit truncated-limb defects.
Assuntos
Deformidades Congênitas das Extremidades Superiores/genética , Duplicação Cromossômica , Cromossomos Humanos Par 10 , Cromossomos Humanos Par 7 , Hibridização Genômica Comparativa , Proteínas F-Box/genética , Feminino , Humanos , Masculino , Radiografia , Deformidades Congênitas das Extremidades Superiores/diagnóstico , Deformidades Congênitas das Extremidades Superiores/diagnóstico por imagem , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
To increase the throughput, lower the cost, and save scarce test reagents, laboratories can pool patient samples before SARS-CoV-2 RT-qPCR testing. While different sample pooling methods have been proposed and effectively implemented in some laboratories, no systematic and large-scale evaluations exist using real-life quantitative data gathered throughout the different epidemiological stages. Here, we use anonymous data from 9673 positive cases to model, simulate and compare 1D and 2D pooling strategies. We show that the optimal choice of pooling method and pool size is an intricate decision with a testing population-dependent efficiency-sensitivity trade-off and present an online tool to provide the reader with custom real-time 1D pooling strategy recommendations.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19 , Teste para COVID-19 , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
PURPOSE: Despite different treatment options for patients with refractory epilepsy such as epilepsy surgery and neurostimulation, many patients still have seizures and/or drug-related cerebral and systemic side effects. Local intracerebral delivery of antiepileptic compounds may represent a novel strategy with specific advantages such as the option of higher local doses and reduced side effects. In this study we evaluate the antiepileptic effect of local delivery of adenosine in the kainic acid rat model, a validated model for temporal lobe epilepsy. METHODS: Fifteen rats, in which intraperitoneal kainic acid injection had induced spontaneous seizures, were implanted with a combination of depth electrodes and a cannula in both hippocampi. Cannulas were connected to osmotic minipumps to allow continuous hippocampal delivery. Rats were freely moving and permanently monitored by video-EEG (electroencephalography). Seizures were scored during 2 weeks of local hippocampal delivery of saline (baseline), followed by 2 weeks of local adenosine (6 mg/ml) (n = 10) or saline (n = 5) delivery (0.23 µl/h) (treatment). In 7 of 10 adenosine-treated rats, saline was also delivered during a washout period. RESULTS: During the treatment period a mean daily seizure frequency reduction of 33% compared to the baseline rate was found in adenosine-treated rats (p < 0.01). Four rats had a seizure frequency reduction of at least 50%. Both nonconvulsive and convulsive seizures significantly decreased during the treatment period. In the saline-control group, mean daily seizure frequency increased with 35% during the treatment period. CONCLUSIONS: This study demonstrates the antiseizure effect of continuous adenosine delivery in the hippocampi in rats with spontaneous seizures.
Assuntos
Adenosina/farmacologia , Anticonvulsivantes/farmacologia , Epilepsia do Lobo Temporal/prevenção & controle , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Adenosina/administração & dosagem , Animais , Anticonvulsivantes/administração & dosagem , Cateterismo/métodos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Eletrodos Implantados , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/métodos , Eletroencefalografia/estatística & dados numéricos , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/fisiopatologia , Lateralidade Funcional/fisiologia , Humanos , Injeções Intraperitoneais , Ácido Caínico , Masculino , Ratos , Ratos Sprague-Dawley , Técnicas EstereotáxicasRESUMO
CONTEXT.: In routine clinical practice, tumor tissue is stored in formalin-fixed, paraffin-embedded blocks. However, the use of formalin-fixed, paraffin-embedded tissue for genome analysis is challenged by poorer DNA quality and quantity. Although several studies have reported genome-wide massive parallel sequencing applied on formalin-fixed, paraffin-embedded samples for mutation analysis, copy number analysis is not yet commonly performed. OBJECTIVE.: To evaluate the use of formalin-fixed, paraffin-embedded tissue for copy number alteration detection using shallow whole-genome sequencing, more generally referred to as copy number variation sequencing. DESIGN.: We selected samples from 21 patients, covering a range of different tumor entities. The performance of copy number detection was compared across 3 setups: array comparative genomic hybridization in combination with fresh material; copy number variation sequencing on fresh material; and copy number variation sequencing on formalin-fixed, paraffin-embedded material. RESULTS.: Very similar copy number profiles between paired samples were obtained. Although formalin-fixed, paraffin-embedded profiles often displayed more noise, detected copy numbers seemed equally reliable if the tumor fraction was at least 20%. CONCLUSIONS.: Copy number variation sequencing of formalin-fixed, paraffin-embedded material represents a trustworthy method. It is very likely that copy number variation sequencing of routinely obtained biopsy material will become important for individual patient care and research. Moreover, the basic technology needed for copy number variation sequencing is present in most molecular diagnostics laboratories.
RESUMO
OBJECTIVE: To report the clinical, radiologic, biochemical, and molecular characteristics in a 46-year-old participant with adult-onset Leigh syndrome (LS), followed by parkinsonism. METHODS: Case description with diagnostic workup included blood and CSF analysis, skeletal muscle investigations, blue native polyacrylamide gel electrophoresis, whole exome sequencing targeting nuclear genes involved in mitochondrial transcription and translation, cerebral MRI, 123I-FP-CIT brain single-photon emission computed tomography (SPECT), and C-11 raclopride positron emission tomography (PET). RESULTS: The participant was found to have a defect in the oxidative phosphorylation caused by a c.626C>T mutation in the gene coding for mitochondrial methionyl-tRNA formyltransferase (MTFMT), which is a pathogenic mutation affecting intramitochondrial protein translation. The proband had a normal concentration of lactate in blood and no abnormal microscopic findings in skeletal muscle. Cerebral MRI showed bilateral lesions in the striatum, mesencephalon, pons, and medial thalamus. Lactate concentration in CSF was increased. FP-CIT SPECT and C-11 raclopride PET demonstrated a defect in the dopaminergic system. CONCLUSIONS: We report on a case with adult-onset LS related to a MTFMT mutation. Two years after the onset of symptoms of LS, the proband developed a parkinson-like disease. The c.626C>T mutation is the most common pathogenic mutation found in 22 patients reported earlier in the literature with a defect in MTFMT. The age of the previously reported cases varied between 14 months and 24 years. Our report expands the phenotypical spectrum of MTFMT-related neurologic disease and provides clinical evidence for involvement of MTFMT in extrapyramidal syndromes.
RESUMO
Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems (ALK, Th-MYCN, Dbh-MYCN and Lin28b). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.
RESUMO
Purpose: Neuroblastoma (NB) is a heterogeneous disease characterized by distinct clinical features and by the presence of typical copy-number alterations (CNAs). Given the strong association of these CNA profiles with prognosis, analysis of the CNA profile at diagnosis is mandatory. Therefore, we tested whether the analysis of circulating cell-free DNA (cfDNA) present in plasma samples of patients with NB could offer a valuable alternative to primary tumor DNA for CNA profiling.Experimental Design: In 37 patients with NB, cfDNA analysis using shallow whole genome sequencing (sWGS) was compared with arrayCGH analysis of primary tumor tissue.Results: Comparison of CNA profiles on cfDNA showed highly concordant patterns, particularly in high-stage patients. Numerical chromosome imbalances as well as large and focal structural aberrations including MYCN and LIN28B amplification and ATRX deletion could be readily detected with sWGS using a low input of cfDNA.Conclusions: In conclusion, sWGS analysis on cfDNA offers a cost-effective, noninvasive, rapid, robust and sensitive alternative for tumor DNA copy-number profiling in most patients with NB. Clin Cancer Res; 23(20); 6305-14. ©2017 AACR.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , Variações do Número de Cópias de DNA , Neuroblastoma/genética , Sequenciamento Completo do Genoma , Pré-Escolar , Hibridização Genômica Comparativa , Genes myc , Humanos , Lactente , Biópsia Líquida , Metástase Neoplásica , Estadiamento de Neoplasias , Neuroblastoma/diagnósticoRESUMO
Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.
Assuntos
Genoma Humano , Linhagem Celular , Cromossomos Humanos/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Dosagem de Genes , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
OBJECTIVE: To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. DESIGN: Study of diagnostic accuracy. SETTING: University hospital. PATIENT(S): Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. INTERVENTION(S): Trophectoderm biopsy was performed on 47 blastocysts. MAIN OUTCOME MEASURE(S): In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. RESULT(S): In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a â¼ 4.5 Mb deletion/duplication. CONCLUSION(S): This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.
Assuntos
Blastocisto/patologia , Transtornos Cromossômicos/genética , Cromossomos Humanos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Implantação/métodos , Translocação Genética , Adulto , Biópsia , Transtornos Cromossômicos/patologia , Hibridização Genômica Comparativa , Técnicas de Cultura Embrionária , Feminino , Marcadores Genéticos , Hospitais Universitários , Humanos , Masculino , Valor Preditivo dos Testes , Injeções de Esperma IntracitoplásmicasRESUMO
Structural genomic variations play an important role in human disease and phenotypic diversity. With the rise of high-throughput sequencing tools, mate-pair/paired-end/single-read sequencing has become an important technique for the detection and exploration of structural variation. Several analysis tools exist to handle different parts and aspects of such sequencing based structural variation analyses pipelines. A comprehensive analysis platform to handle all steps, from processing the sequencing data, to the discovery and visualization of structural variants, is missing. The ViVar platform is built to handle the discovery of structural variants, from Depth Of Coverage analysis, aberrant read pair clustering to split read analysis. ViVar provides you with powerful visualization options, enables easy reporting of results and better usability and data management. The platform facilitates the processing, analysis and visualization, of structural variation based on massive parallel sequencing data, enabling the rapid identification of disease loci or genes. ViVar allows you to scale your analysis with your work load over multiple (cloud) servers, has user access control to keep your data safe and is easy expandable as analysis techniques advance. URL: https://www.cmgg.be/vivar/
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Biologia Computacional/métodos , Variação Estrutural do Genoma , Genoma Humano , Humanos , Internet , CariótipoRESUMO
Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14-18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.
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Anormalidades Múltiplas/genética , Aberrações Cromossômicas , Sequenciamento de Nucleotídeos em Larga Escala , Deficiência Intelectual/genética , Anormalidades Múltiplas/diagnóstico , Bandeamento Cromossômico , Duplicação Cromossômica , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 21 , Hibridização Genômica Comparativa , Biologia Computacional , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Cariótipo , Masculino , Recombinação GenéticaRESUMO
Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.