Abstract
BackgroundWe had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors. This has the advantage that DNA fingerprinting identifies the genetic alterations in a manner not biased for locus.MethodsIn this study we used RAPD-PCR to identify novel genomic alterations in the astrocytic tumors of WHO grade II (Low Grade Diffuse Astrocytoma) and WHO Grade IV (Glioblastoma Multiforme). Loss of heterozygosity (LOH) of the altered region was studied by microsatellite and Single Nucleotide Polymorphism (SNP) markers. Expression study of the gene identified at the altered locus was done by semi-quantitative reverse-transcriptase-PCR (RT-PCR).ResultsBands consistently altered in the RAPD profile of tumor DNA in a significant proportion of tumors were identified. One such 500 bp band, that was absent in the RAPD profile of 33% (4/12) of the grade II astrocytic tumors, was selected for further study. Its sequence corresponded with a region of FAT, a putative tumor suppressor gene initially identified in Drosophila. Fifty percent of a set of 40 tumors, both grade II and IV, were shown to have Loss of Heterozygosity (LOH) at this locus by microsatellite (intragenic) and by SNP markers. Semi-quantitative RT-PCR showed low FAT mRNA levels in a major subset of tumors.ConclusionThese results point to a role of the FAT in astrocytic tumorigenesis and demonstrate the use of RAPD analysis in identifying specific alterations in astrocytic tumors.
Highlights
We had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors
Grades II to IV follow the same lineage of progression and Grade II (Low-grade Diffuse Astrocytomas, Low grade diffuse astrocytoma (DA)) have an inherent tendency to progress to grade III (Anaplastic Astrocytoma, Anaplastic astrocytoma (AA)) and grade IV (Glioblastoma Multiforme, GBM)[2,3]
RAPD-fingerprinting of human astrocytic tumors and characterization RAPD-PCR was done on 23 astrocytic tumors (12 Grade II and 11 Grade IV) along with the corresponding normal leucocyte DNA with 5 RAPD primers mentioned (Table 1)
Summary
We had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors. This has the advantage that DNA fingerprinting identifies the genetic alterations in a manner not biased for locus. WHO has classified astrocytic tumors into 4 grades [1]. The median survival time of Grade II and IV tumors is 6 years and 1 year respectively [4], and over the years, not much has changed regarding their outcome. Malignant transformation of glial cells is a complex process[5] that is still incompletely understood [6,7]
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