Downregulation of MGMT expression by targeted editing of DNA methylation enhances temozolomide sensitivity in glioblastoma.

Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.

Lost in translation: the pitfalls of Ensembl gene annotations between human genome assemblies and their impact on diagnostics.

BACKGROUND: Gene models based on GRCh37 human genome assembly are preferred by many international projects over other updated assemblies (GRCh38 and T2T). Discrepant genes (DGs), those recognized as protein coding in the new but not the old assembly, are ignored by several genomic resources and discarded by variant prioritization tools relying on information based on GRCh37. METHODS: We curated a set of Ensembl genes with discrepant annotations between GRCh37 and GRCh38, additionally matching their RefSeq transcripts. Furthermore, we examined their clinical and phenotypic relevance. RESULTS: A total of 337 genes were reclassified as 'protein-coding' in GRCh38 but not in GRCh37, with 194 having a discrepant HGNC gene symbol. Many remain missing from the current known RefSeq gene models (N = 73). We found many clinically relevant genes in this group of neglected genes, and we anticipate that many more will be found relevant in the future. Important additional annotations such as evolutionary constraint metrics are also not calculated for these genes, further relegating them into oblivion. CONCLUSION: For discrepant genes, the inaccurate label of 'non-protein-coding' has relevant ramifications on clinical genetics. Accurate collation of these genes allows for manual curation in clinically relevant scenarios.

Age, absolute CD4 count, and CD4 percentage in relation to HPV infection and the stage of cervical disease in HIV-1-positive women.

A subgroup of women who are co-infected with human immunodeficiency virus type 1 (HIV-1) and human papillomavirus (HPV), progress rapidly to cervical disease. We characterized HPV genotypes within cervical tumor biopsies, assessed the relationships of cervical disease stage with age, HIV-1 status, absolute CD4 count, and CD4 percentage, and identified the predictive power of these variables for cervical disease stage in a cohort of South African women.We recruited 181 women who were histologically diagnosed with cervical disease; 87 were HIV-1-positive and 94 were HIV-1-seronegative. Colposcopy-directed tumor biopsies were confirmed by histology and used for genomic DNA extraction. The Roche Linear Array HPV genotyping test was used for HPV genotyping. Peripheral whole blood was used for HIV-1 rapid testing. Fully automated FC500MPL/CellMek with PanLeucogate (PLG) was used to determine absolute CD4 count, CD4 percentage, and CD45 count. Chi-squared test, a logistic regression model, parametric Pearson correlation, and ROC curves were used for statistical analyses. We used the Benjamini-Horchberg test to control for false discovery rate (FDR, q-value). All tests were significant when both P and q were <.05.Age was a significant predictor for invasive cervical cancer (ICC) in both HIV-1-seronegative (P < .0001, q < 0.0001) and HIV-1-positive women (P = .0003, q = 0.0003). Sixty eight percent (59/87) of HIV-1-positive women with different stages of cervical disease presented with a CD4 percentage equal or less than 28%, and a median absolute CD4 count of 400 cells/µl (IQR 300-500 cells/µl). Of the HIV-1-positive women, 75% (30/40) with ICC, possessed ≤28% CD4 cells vs 25% (10/40) who possessed >28% CD4 cells (both P < .001, q < 0.001). Furthermore, 70% (28/40) of women with ICC possessed CD4 count >350 compared to 30% (12/40) who possessed CD4 count ≤ 350 (both P < .001, q < 0.001).Age is an independent predictor for ICC. In turn, development of ICC in HIV-1-positive women is independent of the host CD4 cells and associates with low CD4 percentage regardless of absolute CD4 count that falls within the normal range. Thus, using CD4 percentage may add a better prognostic indicator of cervical disease stage than absolute CD4 count alone.

Challenges imposed by minor reference alleles on the identification and reporting of clinical variants from exome data.

BACKGROUND: The conventional variant calling of pathogenic alleles in exome and genome sequencing requires the presence of the non-pathogenic alleles as genome references. This hinders the correct identification of variants with minor and/or pathogenic reference alleles warranting additional approaches for variant calling. RESULTS: More than 26,000 Exome Aggregation Consortium (ExAC) variants have a minor reference allele including variants with known ClinVar disease alleles. For instance, in a number of variants related to clotting disorders, the phenotype-associated allele is a human genome reference allele (rs6025, rs6003, rs1799983, and rs2227564 using the assembly hg19). We highlighted how the current variant calling standards miss homozygous reference disease variants in these sites and provided a bioinformatic panel that can be used to screen these variants using commonly available variant callers. We present exome sequencing results from an individual with venous thrombosis to emphasize how pathogenic alleles in clinically relevant variants escape variant calling while non-pathogenic alleles are detected. CONCLUSIONS: This article highlights the importance of specialized variant calling strategies in clinical variants with minor reference alleles especially in the context of personal genomes and exomes. We provide here a simple strategy to screen potential disease-causing variants when present in homozygous reference state.

Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways.

The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions-deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions-deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins.