Duplication of 7q34 in pediatric low-grade astrocytomas detected by high-density single-nucleotide polymorphism-based genotype arrays results in a novel BRAF fusion gene

Abstract

In the present study, DNA from 28 pediatric low-grade astrocytomas was analyzed using Illumina HumanHap550K single-nucleotide polymorphism oligonucleotide arrays. A novel duplication in chromosome band 7q34 was identified in 17 of 22 juvenile pilocytic astrocytomas and three of six fibrillary astrocytomas. The 7q34 duplication spans 2.6 Mb of genomic sequence and contains approximately 20 genes, including two candidate tumor genes, HIPK2 and BRAF. There were no abnormalities in HIPK2, and analysis of two mutation hot-spots in BRAF revealed a V600E mutation in only one tumor without the duplication. Fluorescence in situ hybridization confirmed the 7q34 copy number change and was suggestive of a tandem duplication. Reverse transcription polymerase chain reaction-based sequencing revealed a fusion product between KIAA1549 and BRAF. The predicted fusion product includes the BRAF kinase domain and lacks the auto-inhibitory N-terminus. Western blot analysis revealed phosphorylated mitogen-activated protein kinase (MAPK) protein in tumors with the duplication, consistent with BRAF-induced activation of the pathway. Further studies are required to determine the role of this fusion gene in downstream MAPK signaling and its role in development of pediatric low-grade astrocytomas.

Figure 1

Illumina BeadStudio results for chromosome 7. A. In case 02‐112 there is a split in the B‐allele frequency (top) and increase in the log R ratio (bottom) at 7q34 consistent with a duplication. B. In case 99‐173 the log R ratio (bottom) is elevated along the entire chromosome with a peak in the 7q34 region (top), consistent with a whole chromosome 7 copy number gain and an additional duplication in the 7q34 region.

Figure 2

Representative fluorescence in situ hybridization (FISH) results. A. Interphase FISH for case 02‐112. Two duplicated signals for the 7q34 BAC clone for LUC7L2 (red) are most consistent with a pair of tandem duplications. The chromosome 7 centromere (green) probe was present in two copies. B. Metaphase spread from case 01‐065. The two green signals mark the centromeric regions of chromosome 7. The LUC7L2 probe is present in single copies on both chromosomes 7, as well as a supernumerary marker with amplification of the 7q34 region.

Figure 3

Western blot analysis with a BRAF antibody demonstrated variable protein expression in tumors with and without the 7q34 duplication. Fifty micrograms of tumor lysate was probed with anti‐BRAF (A), anti‐ACTIVE MAPK (B) and anti‐total MAPK (C). Lanes (4) 06‐219, (5) 01‐080, (6) 99‐146, and (8) 04‐098 were from tumors with the 7q34 duplication. Lanes 1 (00‐309), 3 (05‐040) and 7 (05‐255) were from tumors without the duplication. Lane 2 (01‐122) was the only tumor with a V600E BRAF mutation.

Figure 4

7q34 duplication is the result of a structural rearrangement. A. 7q34 duplication is the result of a tandem duplication. B. Proposed KIAA1549‐BRAF fusion gene. Shown is a ∼1 kb polymerase chain reaction product with sequence spanning KIAA1549 exon 15 to BRAF exons 9 through 15.