RESUMEN
OBJECTIVE: Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. MATERIAL AND METHODS: We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. RESULTS: A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). CONCLUSION: High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis.
Asunto(s)
Genes de la Neurofibromatosis 2/fisiología , Mutación/genética , Neoplasias/diagnóstico , Neurofibromatosis 2/genética , Neurofibromatosis 2/metabolismo , Adulto , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Neurofibromatosis 2/diagnóstico , Patología MolecularRESUMEN
The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes , Técnicas de Transferencia de Gen , ARN Guía de Kinetoplastida/genética , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Células Clonales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Endonucleasas/metabolismo , Exones , Marcación de Gen/métodos , Genoma , Células HEK293 , Humanos , Intrones , Ratones , Células 3T3 NIH , Mutación Puntual , ARN Guía de Kinetoplastida/metabolismo , Reparación del ADN por Recombinación , Transcripción GenéticaRESUMEN
AIMS: Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features. METHODS: We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes. RESULTS: Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes. CONCLUSIONS: Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies.
Asunto(s)
Análisis Mutacional de ADN , Genes Relacionados con las Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Hepáticas/genética , Melanoma/genética , Mutación Missense , Neoplasias de la Úvea/genética , Humanos , Neoplasias Hepáticas/secundario , Melanoma/secundario , Proteínas de Neoplasias/genética , Estudios Retrospectivos , Neoplasias de la Úvea/patologíaRESUMEN
Constitutional dominant loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisted of multiple café-au-lait macules, axillary freckling, learning disabilities and macrocephaly. SPRED1 is a negative regulator of the RAS MAPK pathway and can interact with neurofibromin, the NF1 gene product. Individuals with NF1 have a higher risk of haematological malignancies. SPRED1 is highly expressed in haematopoietic cells and negatively regulates haematopoiesis. SPRED1 seemed to be a good candidate for leukaemia predisposition or transformation. We performed SPRED1 mutation screening and expression status in 230 paediatric lymphoblastic and acute myeloblastic leukaemias (AMLs). We found a loss-of-function frameshift SPRED1 mutation in a patient with Legius syndrome. In this patient, the leukaemia blasts karyotype showed a SPRED1 loss of heterozygosity, confirming SPRED1 as a tumour suppressor. Our observation confirmed that acute leukaemias are rare complications of the Legius syndrome. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority of AMLs at diagnosis compared with normal or paired complete remission bone marrows. SPRED1 decreased expression correlated with genetic features of AML. Our study reveals a new mechanism which contributes to deregulate RAS MAPK pathway in the vast majority of paediatric AMLs.