The genomic landscape of familial glioma

Abstract

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Fig. 1.. Study analysis pipeline.

Variants were classified as CNVs and smaller events. SNVs and indels were selected according to rarity or presence using the ClinVar database. The resulting variants were then divided into four groups: highly deleterious variants, intronic cryptic splicing variants, variants in TFBSs, and known glioma-associated GWAS SNPs (see Methods). Affected genes were stratified on the basis of whether they were known or novel CPGs. Genes with identified variants are shown below each category. Discovered genes were then validated in a second cohort (bold), and a subset of genes was examined via in vivo screening (underlined). B, billion; SNV, single-nucleotide variant; MAF, minor allele frequency in gnomAD overall; K, thousand; P/LP, pathogenic or likely pathogenic; LoF, loss of function; TFBS, transcription factor binding site; GWAS, genome-wide association study; CNV, copy number variant; CPG, cancer predisposition gene.

Fig. 2.. Proportion of the glioma exploratory cohort (green) or control cohort (blue) with variants in seven cancer genes and overall.

P values were calculated with Fisher’s exact test and Bonferroni correction.

Fig. 3.. CNVs identified in the glioma exploratory cohort.

The relative coverage of HERC2 (A), ATM (B), CHEK2 (C), DMBT1 (D), and POT1 (E) is shown in green; the average coverage is shown by a dashed black line. Exons (blue lines) and introns (thin lines) are shown below the coverage plots.

Fig. 4.. Schematic of variants identified in HERC2.

Copy gain (blue bar) and variants (arrows: red, orange, and gray indicate CADD PH scores of 35, ≥30 but <35, and frameshift mutations, respectively) are shown relative to the regulator of chromosome condensation protein domains (green bar). Approximately 99% of all variants identified by WGS were noncoding; however, the functional consequences and MAF distributions of these variants are not as well understood as those of coding variants. To avoid numerous spurious associations, we restricted our analysis to deep-intronic (i.e., >5 bp from the exon) variants that were predicted to cause cryptic splicing and variants in the immediate upstream region of genes that overlapped a TFBS. We identified 16 deep-intronic variants in LoF-intolerant and novel CPGs that were predicted to cause cryptic splicing in our exploratory cohort (see table S5). Identical variants in two genes, NCAM1 and SMG6, were identified in the validation cohort.

Fig. 5.. In vivo functional screening identified regulators of glioma tumorigenesis.

(A) Schematic of barcoded screening, where the 3xCr glioma system was combined with 72 barcoded gRNAs and co-electroporated into the embryonic cortex. (B) Next-generation sequencing to determine barcode amplification. The barcode for each gRNA (red) and the input signal (black) are shown (n = 3 tumors). Data are indicated as the mean and SEM. (C) Kaplan-Meier curves from individual validation studies (n = 20, px330 control, median survival 104 days; n = 20, ΔZC3H7B, median survival 76.5 days; n = 20, ΔDMBT1, median survival 78 days; n = 20, ΔHP1BP3, median survival 86.5 days). (D) BrdU staining of end-stage tumors; quantification for each group is derived from five different tumors. *P < 0.05, **P < 0.01, and ***P < 0.005. Scale bar, 50 μm.

Fig. 6.. ZC3H7B, DMBT1, and HP1BP3 are key regulators of glioma tumorigenesis–related gene populations.

(A) Venn diagram of DEGs affected by ZC3H7B-LOF, DMBT1-LOF, and HP1BP3-LOF. (B) GO analysis of the common DEGs. (C) Heatmap of GO-related genes from the RNA-seq results of 3xCr [control (C)], ZC3H7B-LOF (Z), DMBT1-LOF (D), and HP1BP3-LOF (H). (D and E) VEGF and FGF2 staining from a postnatal day 60 tumor. DAPI, 4′,6-diamidino-2-phenylindole. (F) Intensities of VEGF and FGF2 staining determined by ImageJ software. The quantification of each group is derived from three different tumors. *P < 0.05, **P < 0.01, and ***P < 0.005. Scale bars, 50 μm. a.u., arbitrary units.