Evaluation of the melting temperature of TaqMan probes as a genotyping method for IDH1, IDH2, and H3F3A in pediatric astrocytomas

Abstract

Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-Ta-qMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

The Transcriptomic Landscape of Pediatric Astrocytoma

“…However, these mutations are not present in the pediatric population [ 7 , 19 ]. Accordingly, we did not detect the typical mutations in IDH1/2 and H3F3A genes in grade II astrocytoma [ 20 ]. We then evaluated the transcriptional changes in grade II astrocytoma with various locations compared to matching tissues and locations ( Supplementary Figure S3 ) and found 84 and 6 up-and downregulated genes, respectively ( Figure 4 a and Supplementary Table S1 ).…”

“…Purified DNA was used to perform Sanger sequencing of driver mutations within IDH1/2 and H3F3A as recommended by the WHO [ 4 ]. In a previous study, we reported the genotypic and histopathologic/molecular classification of the samples used in this work [ 20 ] ( Supplementary Table S1 ). Purified RNA with an acceptable RIN value of 5.5 to 7.6 was used to perform sequencing libraries with the TruSeq Stranded total RNA with Illumina Ribo-Zero Plus rRNA depletion kit (Illumina, Inc. San Diego, CA 92122, USA).…”

“…In a previous study, we genotyped the status of IDH1/2 and H3FA3 in the samples used for our RNA sequencing assay [ 20 ]. For genotyping of paraffin-embedded grade IV astrocytoma, DNA from five different grade IV astrocytoma and two healthy cerebrums was extracted.…”

“…DNA concentration and purity were read with a spectrophotometer (Genova Nano micro-volume spectrophotometer). DNA from each of the grade IV astrocytoma biopsies and healthy control cerebrum was used for IDH1 / IDH2 and H3K27M genotyping as previously reported [ 20 ].…”