Page 44 - Molecular features of low-grade developmental brain tumours

2
Gene
Exon variant (KIAA1549:BRAF)
Forward Primer (5’->3’)
Reverse primer (5’->3’)
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CHAPTER 2
further purified using QIAamp DNA mini Kit (Qiagen). PCR amplification for the entire extent of exon 15 of BRAF including codon 600 was performed as previously described using primers TCATAATGCTTGCTCTGATAGGA and GGCCAAAAATTTAATCAGTGGA26. Purified PCR products were sequenced by the Sanger method using the Big Dye Terminator Cycle Sequencing Kit (PerkinElmer Biosystems, Foster City, CA, USA).
KIAA1549–BRAF gene fusion
Six SEGA tissue samples were tested for KIAA1549-BRAF fusions in a diagnostic setting. Total RNA was extracted from frozen tissue samples using miRNeasy mini kit (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed into cDNA, followed by PCR using primer sets corresponding to different KIAA1549-BRAF fusion genes and the PBGD and B2M reference genes (Table 2). PCR products were analyzed on a 2% agarose gel. Pilocytic astrocytoma tissue containing the KIAA1549-BRAF fusions was used as a positive control. Additionally, tonsil tissue known to lack the KIAA1549-BRAF fusion genes was used as a negative control.
Table 2. Primer sequences for detection of KIAA1549:BRAF fusion genes.
KIAA1549-BRAF fusion KIAA1549-BRAF fusion KIAA1549-BRAF fusion KIAA1549-BRAF fusion KIAA1549-BRAF fusion
Ex16:Ex9
Ex15:Ex9
Ex16:Ex11
Ex18:Ex10
CTACAGCCCAGCCCAGAC GTGAGCCAGGTAATGAGGCAG CCACAACTCAGCCTACATCGG GTGAGCCAGGTAATGAGGCAG
AGACGGCCAACAATCCCTGC GTCCCACTGTAATCTGCCC GAGGGATCTACTCGGAGGAG GTGAGCCAGGTAATGAGGCAG
Ex19:Ex9 PBGD -
GAAGCGGGGCGAAGAGAG
CTGGTAACGGCAATGCGGCT AGCATTCAGACTTGTTTCAG
GTGAGCCAGGTAATGAGGCAG
GCAGATGGCTCCGATGGTGA GATGCTGCTTAGATGTCTCG
B2M -
TSC1/TSC2 mutation and LOH analysis of SEGAs
In 3 cases (fresh frozen samples), targeted MPS was performed using a HaloPlex custom capture array as described previously 38. In the other 31 cases (24 FFPE and 7 fresh frozen samples), targeted MPS was performed using a customized gene bait set (Agilent platform) designed in the Kwiatkowski lab that covers the entire TSC1 and TSC2 genes including 10 kb upstream and downstream and all coding exons and introns. This bait set also covered all coding exons and adjacent introns of BRAF. MPS was performed according to the following methods. Briefly, DNA was subjected to fragmentation using Covaris sonication to an average size of 250bp. The fragmented DNA was purified using Agencourt AMPure XP beads and