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.

Novel variants causing megalencephalic leukodystrophy in Sudanese families.

Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare form of leukodystrophy characterized by macrocephaly, epilepsy, spasticity, and slow mental deterioration. Genetic studies of MLC are lacking from many parts of the world, especially in Sub-Saharan Africa. Genomic DNA was extracted for 67 leukodystrophic patients from 43 Sudanese families. Mutations were screened using the NGS panel testing 139 leukodystrophies and leukoencephalopathies causing genes (NextSeq500 Illumina). Five homozygous MLC1 variants were discovered in seven patients from five distinct families, including three consanguineous families from the same region of Sudan. Three variants were missense (c.971 T > G, p.Ile324Ser; c.344 T > C, p.Phe115Ser; and c.881 C > T, p.Pro294Leu), one duplication (c.831_838dupATATCTGT, p.Ser280Tyrfs*8), and one synonymous/splicing-site mutation (c.762 C > T, p.Ser254). The segregation pattern was consistent with autosomal recessive inheritance. The clinical presentation and brain MRI of the seven affected patients were consistent with the diagnosis of MLC1. Due to the high frequency of distinct MLC1 mutations found in our leukodystrophic Sudanese families, we analyzed the coding sequence of MLC1 gene in 124 individuals from the Sudanese genome project in comparison with the 1000-genome project. We found that Sudan has the highest proportion of deleterious variants in MLC1 gene compared with other populations from the 1000-genome project.