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Title: Characterisation of mutations in the gene for neurofibromatosis type 1
Author: Purandare, Smita Madhav
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1994
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available