New insights in the molecular pathogenesis of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1; OMIM 162200) is one of the most common autosomal dominant genetic disorders, affecting approximately 1 in 3500 individuals worldwide. The most common clinical manifestations are pigmentary abnormalities together with the development of benign peripheral nerve sheath tumors or neurofibromas. In addition, NF1 patients are prone to a pleiotropy of other phenotypic features. Determining when and in what cell type the crucial molecular defect actually occurs has proven to be surprisingly difficult for many NF1-related symptoms. Impeding factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types within affected tissues, and the NF1 heterozygous state of all cells in the body. The major goal of this thesis was to gain insight into the developmental concepts underlying various NF1-related symptoms by exploiting improved somatic mutation detection strategies on a diverse spectrum of cellular entities

Identification and characterization of neurofibromatosis type 1 (NF1) gene mutations

La neurofibromatose 1 (NF 1) affecte un individu sur 3000 et se caractérise par l'extrême variabilité des symptôme cliniques. Le gène de NF/, situé sur le chromosome l 7q 11.2, contient 350 kb et 60 exons. Le taux de mutation est de 1 x 104/gamète/génération. Environ 50% des mutations sont d'origine familiale et 50% sporadique. Nous avons identifié et caractérisé une mutation qui cause l'exclusion des exons 11 et l 2a, six délétions et un polymorphisme HinclI et aussi déterminé le déséquilibre de liaison dans la population québécoise. Une mutation originale a été identifiée grâce au test de tronquation des protéines; elle serait la cause de l'exclusion d'exons. Le séquençage de la région génomique entre les introns 1 Oc et 12a a permis d'identifier le changement d'un G en A en position 2056+ l dans le site d' épissage 5' de l 'exon 12a. Ceci engendre l'excision des deux exons l l et 12a sans changement du cadre de lecture et sans que la quantité des ARN messagers en soit affectée. Des essais d'épissage in vivo et in vitro nous ont permis de démontrer pour la première fois que le mutation naturelle supprime la définition des exons. Nos résultats montrent aussi que la séquence encodée par les exons 11 et l 2a est essentielle pour l'activité de la neurofibromine. L 'haplotypage de 19 familles a permis de détecter la perte d'hétérozygosité (LOH) grâce aux 4 microsatellites et aux RFLPs RsaI et EcoRI. Six délétions (2 familles:76 l 0 et 74 73, et 4 patients: 178, 184, 236 et 23 7) ont été identifiées et caractérisées par la LOH et des buvardages de type Southern. La délétion dans la famille 7610 est d'origine maternelle et seul restent les exons l-4b. La délétion dans la famille 7473 est aussi d'origine maternelle et les exons de là 5 sont délétés, la délétion s'arrêtant quelque part avant l' intron 26. Avec le patient 1 78 la délétion commence entre les exons 23-2 et 27b pour se terminer après la région 3' de NFJ. La délétion dans le patient 184 commence au 5' du gène et se termine entre les exons 27b-29. La délétion dans le patient 236 commence entre les exons 14-18 et se termine après l'extrémité 3' de NFJ. Avec le patient 237, la délétion commence entre les exons 38-45 et se termine après la région 3' du gène. Ces délétions sont distribuées au hasard dans le gène NF 1.Abstract: Neurofibromatosis type 1 (NF1) afflicts 1 in 3,000 individuals and is characterized with variable clinical presentations. The NF1 gene spans 350 kb on chromosome 17q11.2 with 60 exons. The gene exhibits high mutation rate of 1 × 104/gamete/generation and approximately 50% are sporadic new mutations. In this study, a splice site mutation and 6 gross deletions have been identified and characterized. Also, a HincII polymorphism was detected and the linkage disequilibrium was investigated. Using a protein truncation assay, we have identified a exon skipping mutation. The mutation, which consists of a G to A transition at position +1 (2056+1) of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. The mutation inactivates the 5' splice site of exon 12a, prevents exon definition and leads to the skipping of both exons 11 and 12a. These results document the first example of a natural mutation that inactivates exon definition, and suggest that the 11-12a region of NF1 plays an important role in the activity of neurofibromin. Six gross deletions (families 7610 and 7473, patients 178, 184, 236 and 236) have been identified. The breakpoint of the deletion is located between exons 5 and 26. The deletions are unique and different from those reported previously. The deletions are not associated with unusual clinical features. The HincII polymorphism is located between exons 1 and 4a as defined by probe GE2-400 bp. Three alleles are detected: A1 (3.1 and 3.3 kb), A2 (2.4 and 4.0 kb), A3 (1.3 and 5.1 kb). The detected heterozygote forms are A1-A2, or A1-A3 and the homozygote forms A1-A1 or A2-A2. Analysis haplotypes of the 19 NF1 families indicates that the four intragenic polymorphic microsatellites are strongly linked with the NF1 disease. However, no linkage disequilibrium and founder effect was observed in this Québec population collection. In conclusion, the study shows that the analysis of NF1 gene mutation is complex. No particular founder mutation has been observed in the Québec population."--Résumé abrégé par UM

SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that the

SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints

Background: Genomic disorders are caused by copy number changes that may exhibit recurrent breakpoints processed by nonallelic homologous recombination. However, region-specific disease-associated copy number changes have also been observed which exhibit non-recurrent breakpoints. The mechanisms underlying these non-recurrent copy number changes have not yet been fully elucidated. Results: We analyze large NF1 deletions with non-recurrent breakpoints as a model to investigate the full spectrum of causative mechanisms, and observe that they are mediated by various DNA double strand break repair mechanisms, as well as aberrant replication. Further, two of the 17 NF1 deletions with non-recurrent breakpoints, identified in unrelated patients, occur in association with the concomitant insertion of SINE/variable number of tandem repeats/Alu (SVA) retrotransposons at the deletion breakpoints. The respective breakpoints are refractory to analysis by standard breakpoint-spanning PCRs and are only identified by means of optimized PCR protocols designed to amplify across GC-rich sequences. The SVA elements are integrated within SUZ12P intron 8 in both patients, and were mediated by target-primed reverse transcription of SVA mRNA intermediates derived from retrotranspositionally active source elements. Both SVA insertions occurred during early postzygotic development and are uniquely associated with large deletions of 1 Mb and 867 kb, respectively, at the insertion sites. Conclusions: Since active SVA elements are abundant in the human genome and the retrotranspositional activity of many SVA source elements is high, SVA insertion-associated large genomic deletions encompassing many hundreds of kilobases could constitute a novel and as yet under-appreciated mechanism underlying large-scale copy number changes in the human genome

Characterisation of Mutations in the Gene for Neurofibromatosis Type 1

Neurofibromatosis-1 is an autosomal dominant disorder with a prevalence of approximately 1 in 3000 individuals. Its manifestations involve tissues derived from the neural crest and include mainly cafe au lait spots, neurofibromas and Lisch nodules. The gene for NF-1 was identified in 1990 and found to encode a protein, neurofibromin, with sequence similarity to a family of GTPase activating proteins (GAP). The region of homology is called the NF-1 GAP related domain (NF-1 GRD). Its expression has been shown to complement yeast strains deficient in the yeast GAP homologues IRA1 and IRA2 and to interact with human ras proteins and accelerate the conversion of active GTP bound ras to inactive GDP bound ras. Neurofibromin is also known to associate with cytoplasmic microtubules and the connection between ras mediated signal transduction and the cytoskeleton suggests that neurofibromin may play multiple roles in the regulation of cell division. Neurofibromatosis type-1 is caused by mutations in the NF-1 gene. Mutation analysis in NF-1 is complicated due to the large size of the gene, which extends for over 300 kb on chromosome 17 and is made up of more than 49 exons, due to its high mutation rate and due to the presence of NF-1 pseudogenes and homologous sequences. The present study involved the characterisation of germline and somatic mutations within the NF-1 gene. Characterisation of germline mutations was carried out in 25 randomly selected, unrelated patients with neurofibromatosis type-1 from Scotland and included both inherited and sporadic cases. Characterisation of somatic mutations was carried out in tumours unrelated to NF-1, to assess the tumour suppressor function of the NF-1 gene. The strategy for germline mutation analysis involved initial amplification of the NF-1 coding sequence by the polymerase chain reaction (PCR) using both DNA and RNA as templates. In order to do so, PCR primers were designed to amplify 78% the NF-1 coding sequence. Primers for amplification of selected individual exons from genomic DNA were also designed. After initial amplification by PCR, the products were electrophoresed on agarose gels in order to check for any abnormal alterations in size. If no alteration was identified, the segments amplified using DNA as a template i.e. individual exons of the NF-1 gene were analysed using single stranded conformational analysis and chemical cleavage analysis. The segments amplified using RNA as a template were larger in size (0.4-1 kb) and thus were directly subjected to chemical cleavage analysis, to precisely locate the presence of small alterations or point mutations within the NF-1 gene. Any mismatch detected by chemical cleavage was then fully characterised using direct sequencing by the dideoxy chain termination method, using single stranded DNA generated by asymmetric PCR amplification. Larger rearrangements within the NF-1 gene were screened by Southern blotting of genomic DNA. Using the above strategy, 17 positive screening results were detected on analysis of 78% of the coding sequence in 25 patients. Of these, 13 have been characterised by direct sequencing. The mutations include three splice site errors responsible for exon skipping, two other gross abnormalities of the NF-1 mRNA resulting from a partial deletion in exon 16 and the complete deletion of exon 18, two insertions, a nonsense mutation, two missense mutations, three silent mutations and a novel intragenic polymorphism in intron 41. The mutation 3113+1G to A affected the splice donor site of intron 18 and resulted in the skipping of exon 18 from the NF-1 mRNA. This loss of exon 18 did not cause a shift in the reading frame and is predicted to result in a loss of 41 amino acids from the protein product. The 41 amino acids lost include two cysteine residues at positions 1016 and 1036, whose loss may lead to altered conformation / stability of neurofibromin. This mutation was identified in a familial case of NF-1. 5749+2T to G is a splice site mutation that affects the invariant GT dinucleotide of the splice donor site of intron 30 and was identified in a sporadic case of NF-1. This mutation resulted in the skipping of exon 30 and a shift in the translational reading frame. This is predicted to result in a truncation of the protein product due to the translation of a single altered amino acid before the termination at a premature stop codon at position 1851. 1721+3A to G is a splice site mutation at position +3 of the donor site in intron 11 and was identified in a familial case of NF-1. This transition resulted in an error of splicing, leading to skipping of exon 11 from the NF-1 mRNA. This is predicted to cause a shift in the translational reading frame, resulting in the formation of 12 altered amino acids and the creation of a premature stop codon at position 560. This would result in the synthesis of a protein of 559 amino acids instead of the normal 2818 amino acids, which would lack the NF-1 GAP related domain. (Abstract shortened by ProQuest.)

Integration of microarray analysis into the clinical diagnosis of hematological malignancies: How much can we improve cytogenetic testing?

PurposeTo evaluate the clinical utility, diagnostic yield and rationale of integrating microarray analysis in the clinical diagnosis of hematological malignancies in comparison with classical chromosome karyotyping/fluorescence in situ hybridization (FISH).MethodsG-banded chromosome analysis, FISH and microarray studies using customized CGH and CGH+SNP designs were performed on 27 samples from patients with hematological malignancies. A comprehensive comparison of the results obtained by three methods was conducted to evaluate benefits and limitations of these techniques for clinical diagnosis.ResultsOverall, 89.7% of chromosomal abnormalities identified by karyotyping/FISH studies were also detectable by microarray. Among 183 acquired copy number alterations (CNAs) identified by microarray, 94 were additional findings revealed in 14 cases (52%), and at least 30% of CNAs were in genomic regions of diagnostic/prognostic significance. Approximately 30% of novel alterations detected by microarray were >20 Mb in size. Balanced abnormalities were not detected by microarray; however, of the 19 apparently "balanced" rearrangements, 55% (6/11) of recurrent and 13% (1/8) of non-recurrent translocations had alterations at the breakpoints discovered by microarray.ConclusionMicroarray technology enables accurate, cost-effective and time-efficient whole-genome analysis at a resolution significantly higher than that of conventional karyotyping and FISH. Array-CGH showed advantage in identification of cryptic imbalances and detection of clonal aberrations in population of non-dividing cancer cells and samples with poor chromosome morphology. The integration of microarray analysis into the cytogenetic diagnosis of hematologic malignancies has the potential to improve patient management by providing clinicians with additional disease specific and potentially clinically actionable genomic alterations

Molecular characterization of neurofibromatosis type 1–associated malignant peripheral nerve sheath tumours and functional identification of genes involved in their pathogenesis

[cat] El tumor maligne de la beina dels nervis perifèrics (MPNST) és un sarcoma de teixit tou del sistema nerviós perifèric. Al voltant del 50% dels MPNSTs es desenvolupen en el context de la neurofibromatosi de tipus 1 (NF1) a partir de neurofibromes plexiformes (pNFs) preexistents. Mentre que els pNFs no mostren grans alteracions genòmiques, els MPNSTs presenten un genoma hiperploide amb diverses alteracions somàtiques en el nombre de còpies (SCNAs), que són recurrents. En aquesta tesi s'hipotetiza que les SCNAs tenen un impacte en l'expressió gènica de manera regional, i que aquesta informació transcriptòmica pot ser emprada per trobar gens implicats en la patogènesi dels MPNSTs. L'objectiu principal d'aquesta tesi ha estat la caracterització del genoma dels MPNSTs i la utilització de la informació genòmica regional per a la identificació de gens i mecanismes moleculars implicats en la patogènesi dels MPNSTs associats a la NF1. En la caracterització genòmica es va desenvolupar un assaig de qPCR per a la detecció de delecions constitucionals i somàtiques del gen NF1. Un segon assaig de qPCR va ser dissenyat per a la detecció de SCNAs en MPNSTs. Els SNP arrays d'un grup de MPNSTs van confirmar el genoma hiperploide i les SCNAs recurrents d'aquest tipus de tumors. A partir de dades de microarrays d'expressió d'un altre grup de MPNSTs es van identificar regions del genoma amb una abundància significativa de gens sobre- o infraexpressats, coneguts com a desequilibris transcripcionals (Tls). Es va trobar una associació global dels Tls amb les SCNAs, sobretot dels Tls de sobreexpressió amb guanys genòmics. Experiments de RNAi i assajos de genètica funcional en línies cel-lulars derivades de MPNSTs semblaven indicar que els Tls, tot i incloure algun gen "driver" de la tumorigènesi dels MPNSTs, capturarien principalment expressió de tipus "passenger". La utilització d'informació exclusiva proporcionada pels Tls va facilitar la identificació de gens candidats i possibles mecanismes moleculars implicats en la patogènesi dels MPNSTs, com ara la borealina i el "cromosomal passenger complex", i algunes kinesines mitòtiques, com K1F11, K1F15 i KIF23. A més, es va trobar la kinesina K1F11 com a nova potencial diana terapèutica per al tractament dels MPNSTs.[eng] Malignant peripheral nerve sheath tumour (MPNST) is a soft tissue sarcoma of the peripheral nervous system. Around 50% of MPNSTs develop in the context of the hereditary cancer neurofibromatosis type 1 (NF1), normally arising from pre-existing benign plexiform neurofibromas (pNFs). While pNFs do not show gross genomic alterations, MPNSTs present a hyperploid genome with recurrent somatic copy number alterations (SCNAs). We hypothesized that these SCNAs have an impact on gene expression in a regional manner, which can be informative when studying MPNST pathogenesis. This regional transcriptomic information could be employed for finding genes involved in the pathogenesis of MPNSTs, including some potential therapeutic targets. The main objective of this thesis project was to characterize the MPNST genome and use regional genomic information by the integration of DNA copy number and gene expression in order to identify genes and molecular mechanisms involved in the pathogenesis of MPNSTs arising in the context of NF1. Copy number analysis was performed by using SNP array and two DNA-based qPCR assays. A probe-based qPCR assay was developed for an accurate detection of constitutional and somatic deletions in the NF1 gene. A second qPCR assay was designed to detect SCNAs in MPNSTs, where genomic repetitive sequences were found to improve the normalization of copy number data. The analysis of the genome of a set of MPNSTs by SNP array confirmed a hyperploid genome with recurrent SCNAs for these tumours. ln addition, expression microarray data from a different set of tumours was used to identify regions of the MPNST genome with a significant abundance of over- or underexpressed genes, known as transcriptional imbalances (Tls). These Tls were found to be globally associated with the identified recurrent SCNAs, especially for Tls of overexpression with genomic gains. Tls of underexpression were found to be associated with genomic losses and significatively enriched in genes that were hypermethylated in MPNSTs compared to benign pNFs. All together these results indicated a remarkable impact of regional genomic copy number alterations (and probably also a regional epigenomic status) on the expression of the genes contained in these regions. RNAi experiments and functional genetic approaches used in MPNST cell lines seemed to indicate that Tls, despite probably including one or few drivers of MPNST pathogenesis, would be mainly capturing passenger gene expression. T1 information, together with previous biological knowledge of cancer genes in other tumour types, was used to search drivers of MPNST pathogenesis encompassed within Tls. Furthermore, the exclusive information provided by Tls was used to select genes with an opposed differential expression to the T1 overall expression, which facilitated the identification of candidate genes and molecular mechanisms for MPNST pathogenesis: borealin and the chromosomal passenger complex seemed to be involved in MPNST pathogenesis, and some mitotic kinesins, such as K1F11, K1F15 and KIF23, were also proposed as important players. ln addition, K1F11 was found to be a novel potential therapeutic target for the treatment of MPNSTs