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. 2020 Feb 5;8(1):8.
doi: 10.1186/s40478-020-0882-4.

H3F3A Mutant Allele Specific Imbalance in an Aggressive Subtype of Diffuse Midline Glioma, H3 K27M-mutant

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H3F3A Mutant Allele Specific Imbalance in an Aggressive Subtype of Diffuse Midline Glioma, H3 K27M-mutant

Sachi Maeda et al. Acta Neuropathol Commun. .
Free PMC article

Abstract

Diffuse midline glioma, H3 K27M-mutant is a lethal brain tumor located in the thalamus, brain stem, or spinal cord. H3 K27M encoded by the mutation of a histone H3 gene such as H3F3A plays a pivotal role in the tumorigenesis of this type of glioma. Although several studies have revealed comprehensive genetic and epigenetic profiling, the prognostic factors of these tumors have not been identified to date. In various cancers, oncogenic driver genes have been found to exhibit characteristic copy number alterations termed mutant allele specific imbalance (MASI). Here, we showed that several diffuse midline glioma, H3 K27M-mutant exhibited high variant allele frequency (VAF) of the mutated H3F3A gene using droplet digital polymerase chain reaction (ddPCR) assays. Whole-genome sequencing (WGS) revealed that these cases had various copy number alterations that affected the mutant and/or wild-type alleles of the H3F3A gene. We also found that these MASI cases showed a significantly higher Ki-67 index and poorer survival compared with those in the lower VAF cases (P < 0.05). Our results indicated that the MASI of the H3F3A K27M mutation was associated with the aggressive phenotype of the diffuse midline glioma, H3 K27M-mutant via upregulation of the H3 K27M mutant protein, resulting in downregulation of H3K27me3 modification.

Keywords: Diffuse midline glioma; Droplet digital polymerase chain reaction; H3 K27M-mutant; Mutant allele specific imbalance; Whole-genome sequencing.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Genetic analyses of H3F3A gene mutation using ddPCR and Sanger sequencing. a Results of ddPCR using various mixture DNA consisting of H3F3A K27M and wild-type H3F3A. Upper dot plots indicate wild-type H3F3A DNA dots (green) and lower dot plots indicate H3F3A K27M DNA dots (blue). The x-axis is the number of droplets including PCR products. The y-axis shows the intensity of HEX and FAM. The pink line shows the threshold (HEX: 1700, FAM: 2400) b Correlation of VAFs calculated by ddPCR with expected VAF. The x-axis is the expected VAF and the y-axis is VAF calculated by ddPCR (P < 0.01, r2 = 0.99). c 2D cluster plot of droplet fluorescence of H3F3A K27M and wild-type H3F3A dots in more than 50% VAF of H3F3A K27M case (case 12). FAM positive and HEX negative droplets (blue) include H3F3A K27M. HEX positive and FAM negative droplets (green) include wild-type H3F3A. VAF of H3F3A K27M was calculated as (blue dot numbers/blue dot numbers + green dot numbers). d Sanger sequencing of lower than 50% VAF case (left; 13.1% of VAF; case 8) or more than 50% VAF case (right; 79.9% of VAF; case 12). Hot spots of H3F3A K27M mutation are indicated by the red arrows
Fig. 2
Fig. 2
Flowchart for the identification of the most appropriate chromosomal structure model. Using the total copy number of chromosome 1 obtained by WGS, tumor content of the tumor specimen (H3 K27M positive cells/DAPI positive cells in IHC), BAF of common SNPs obtained by WGS, and VAF of H3F3A K27M obtained by ddPCR, we identified the most consistent chromosomal structure model. Then, we calculated the tumor content with VAF of H3F3A K27M in the most appropriate model and compared the calculated tumor content with that of the tumor specimen obtained by IHC for validation
Fig. 3
Fig. 3
MASI of H3F3A K27M mutation determined with whole-genome sequencing. a Total copy number analyses of chromosome 1 on which H3F3A gene located (upper) and B allele frequency (BAF) of common SNPs (lower) with whole-genome sequencing (WGS) of four more than 50% VAF cases (cases 5, 15, 10, and 12). The red line shows the H3F3A gene region. The x-axis shows the gene location and the y-axis shows the copy number. Gray arrows indicate the H3F3A gene region and PRKACB gene region. Red and blue bars indicate gain and loss, respectively (upper). Higher or lower values between the moving average of the mismatch ratio of SNPs and (1-moving average of mismatch ratio of SNPs) are shown as a yellow or green dot, respectively (lower). b Hematoxylin-Eosin staining of tumor specimen (case 5, 15, 10, and 12; bars: 200um). In each case, the tumor content was calculated as H3 K27M positive cell numbers/DAPI positive cell numbers. c Expected chromosomal structure models of chromosome 1 in cases 5, 15, 10, and 12. The red line indicates the H3F3A K27M mutation. d FISH analysis of chromosome 1p and 1q in MASI cases (case 5; left and case 10; right). Red arrow heads indicated 1p36 signals (red) and white arrow heads indicated 1q25 signals (green) in DAPI-stained nuclei
Fig. 4
Fig. 4
Aggressive phenotype in cases exhibiting MASI of H3F3A K27M. a Immunofluorescence staining of H3 K27M (red) and histone H4 (green) as an internal control of MASI case (case 5; bars, 30 μm) and lower VAF case (case 6). b Bar graph of relative fluorescence intensity of H3 K27M to those of histone H4 in lower VAF and MASI cases (n = 6 and n = 4, respectively). Error bars indicate the standard deviation (SD) (**P < 0.05). (c) Bar graph indicates the frequency of H3K27me3 positive cells among histone H4 positive cells in lower VAF and MASI cases (n = 6 and n = 4, respectively). Error bars indicate the SD (***P < 0.01). d Immunofluorescence staining of Ki-67 (green), H3 K27M (red), and DAPI (blue) using the tumor specimen of the MASI case (case 5; bars, 20 μm) and lower VAF case (case 4). e Bar graphs indicating the frequency of Ki-67 positive cells among H3 K27M positive cells in lower VAF cases (n = 6, blue) and MASI cases (n = 4, red). Error bars indicate the SD (**P < 0.05). f Kaplan-Meier survival curves illustrating the progression-free survival (PFS) of MASI cases (n = 4) and lower VAF cases (n = 11; P = 0.03). g Kaplan-Meier survival curves illustrating the overall survival (OS) of MASI cases (n = 4) and lower VAF cases (n = 11; P = 0.01)

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