Tatevossian, R.G.; (2010) Molecular genetic analysis of paediatric low-grade astrocytoma. Doctoral thesis , UCL (University College London).

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Abstract

The thesis describes the molecular genetic analysis of fifty low-grade paediatric astrocytomas. DNA copy number changes were investigated in paediatric low-grade astrocytomas (WHO grade I and II), using array comparative genomic hybridisation and Affymetrix 250K and 6.0 SNP arrays. A discrete region of DNA copy number gain was identified at chromosome 7q34, primarily although not exclusively in pilocytic astrocytomas of the cerebellum. Further analysis of this region, by PCR and sequencing, demonstrated the presence of gene fusions between KIAA1549 and BRAF. Five KIAA1549- BRAF fusion variants were subsequently identified. A further gene fusion between SRGAP3 and RAF1 was also found in a single tumour with DNA copy number gain at chromosome 3p25. The fusion genes lacked the auto-inhibitory domains of BRAF and RAF1. These were replaced in-frame by N-terminal segments of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. Sequencing confirmed the presence of activating mutations in KRAS and BRAF in three tumours where gene fusions were not identified. Both gene fusions and activating mutations were shown to cause activation of the ERK/MAP kinase pathway by Western blotting. Further sequencing was performed of CDKN2A, PTEN and IDH1/2 to assess the frequency of abnormalities in paediatric low-grade astrocytoma. These genes have previously been found to contain aberrations within adult high-grade astrocytomas. To date, no significant aberrations have been identified in the paediatric astrocytoma samples examined. This confirms previous findings in adult and paediatric astrocytomas, which appear to show distinct molecular changes depending on patient age. Gene fusions or activating mutations were identified in 100% pilocytic astrocytomas studied and were also found in 28% of grade II astrocytomas. These findings highlight the importance of the ERK/MAPK pathway both in the development of paediatric low-grade astrocytomas and as a possible therapeutic target. Gene fusions may provide a means of molecular classification for pilocytic astrocytomas in the future.

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