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The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH. These tumors, designated TERTpWT-IDHWT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTpWT-IDHWT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDHWT-TERTSV), and an ALT-positive subgroup (IDHWT-ALT) with mutations in ATRX or SMARCAL1., Glioblastoma can be classified based on IDH and TERT promoter mutations, but ~20% of glioblastoma do not have these mutations (TERTpWT-IDHWT glioblastoma). Here, the authors present a genetic landscape of TERTpWT-IDHWT glioblastoma, identifying a telomerase-positive subgroup driven by TERT-structural rearrangements and an ALT-positive subgroup with mutations in ATRX or SMARCAL1.

Precise genetic counseling and prenatal diagnosis are often hindered by incomplete penetrance of risk variance and complex patterns of inheritance. Here, we performed a clinical and genetic study of a five-generation Pakistani family with a history of multiple cases of childhood brain tumors. Six affected individuals died of brain tumors at very early ages and three were confirmed as having a homozygous mutation in exon 6 of the PMS2 gene (c.543delT). Fifteen members of the family were identified as heterozygous carriers of this mutation with a lack of cancer incidence. Both clinical manifestations and genetic test results of brain tumor patients in the family support the diagnosis of constitutional mismatch repair deficiency (CMMRD) syndrome, a condition in which individuals carry homozygous germline mutations in mismatch repair machinery genes with an early onset of malignancies such as glioma. This information was used to guide prenatal diagnosis with genetic testing on chorionic villus samples for the family. This is the first report of prenatal genetic diagnosis of hereditary brain tumor.

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor in adults. Mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH1/2) can classify ~80% of GBMs into molecular subgroups with distinct clinical courses. These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, approximately 20% of GBMs lack alterations in TERTp and IDH1/2. These tumors, designated TERTp(WT)-IDH(WT) or triple-negative glioblastomas (as they also lack 1p19q co-deletion) do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we performed whole-exome, whole-genome, and RNA-sequencing on a cohort of triple-negative GBMs to define their genetic landscape. We discovered recurrent inactivating mutations in SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) in 21% (8/39) of triple-negative GBMs. Using telomere maintenance characterization assays, we show that SMARCAL1-mutant cases exhibited the ALT phenotype. Using CRISPR/Cas9 gene editing in GBM cell lines, we found that inducing loss of SMARCAL1 generates features of ALT. Rescue of expression of SMARCAL1 in SMARCAL1-null cell lines markedly suppressed ALT features and was dependent on the enzyme helicase activity. Furthermore, using break-apart FISH and whole genome sequencing, we identified recurrent rearrangements upstream of TERT in ~50% of triple-negative GBMs. These TERT-rearranged tumors exhibited elevated levels of TERT mRNA expression. This represents a novel mechanism of telomerase activation in GBMs lacking the well-known TERT promoter hotspot mutations. Finally, we identify recurrent BRAF V600E mutations in younger patients with GBM. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDH(WT)-TERT(SV), ~50%), and an ALT-positive subgroup (IDH(WT)-ALT, ~40%) with mutations in ATRX or SMARCAL1. We also establish SMARCAL1 inactivating

Background Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase ( TERT ) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. Methods TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. Results TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. Conclusions TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets.

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor in adults. Mutations in the TERT promoter (TERTp) and isocitrate dehydrogenase 1 or 2 (IDH1/2) can classify ~80% of GBMs into molecular subgroups with distinct clinical courses. These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX-mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, approximately 20% of GBMs lack alterations in TERTp and IDH1/2. These tumors, designated TERTp(WT)-IDH(WT) or triple-negative glioblastomas (as they also lack 1p19q co-deletion) do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we performed whole-exome, whole-genome, and RNA-sequencing on a cohort of triple-negative GBMs to define their genetic landscape. We discovered recurrent inactivating mutations in SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) in 21% (8/39) of triple-negative GBMs. Using telomere maintenance characterization assays, we show that SMARCAL1-mutant cases exhibited the ALT phenotype. Using CRISPR/Cas9 gene editing in GBM cell lines, we found that inducing loss of SMARCAL1 generates features of ALT. Rescue of expression of SMARCAL1 in SMARCAL1-null cell lines markedly suppressed ALT features and was dependent on the enzyme helicase activity. Furthermore, using break-apart FISH and whole genome sequencing, we identified recurrent rearrangements upstream of TERT in ~50% of triple-negative GBMs. These TERT-rearranged tumors exhibited elevated levels of TERT mRNA expression. This represents a novel mechanism of telomerase activation in GBMs lacking the well-known TERT promoter hotspot mutations. Finally, we identify recurrent BRAF V600E mutations in younger patients with GBM. Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT-structural rearrangements (IDH(WT)-TERT(SV), ~50%), and an ALT-positive subgroup (IDH(WT)-ALT, ~40%) with mutations in ATRX or SMARCAL1. We also establish SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT in cancer.

The Kolmogorov-Arnold stochasticity parameter technique is applied for the first time to the study of cancer genome sequencing, to reveal mutations. Using data generated by next generation sequencing technologies, we have analyzed the exome sequences of brain tumor patients with matched tumor and normal blood. We show that mutations contained in sequencing data can be revealed using this technique thus providing a new methodology for determining subsequences of given length containing mutations i.e. its value differs from those of subsequences without mutations. A potential application for this technique involves simplifying the procedure of finding segments with mutations, speeding up genomic research, and accelerating its implementation in clinical diagnostic. Moreover, the prediction of a mutation associated to a family of frequent mutations in numerous types of cancers based purely on the value of the Kolmogorov function, indicates that this applied marker may recognize genomic sequences that are in extremely low abundance and can be used in revealing new types of mutations., To appear in Royal Society Open Science, 12 pages, 2 figures