Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing.

Physiological double-stranded breaks (DSBs) are a major source of genomic instability. Here, we present a protocol for mapping physiological DSBs by in-suspension break labeling in situ and sequencing (sBLISS) in a single-nucleotide resolution. We describe steps for cell fixation, labeling of DSBs, DNA isolation followed by in vitro transcription (IVT), reverse transcription, and library preparation. sBLISS provides a map of DSBs over the genome and can be used to study the role of different factors in DSB formation. For complete details on the use and execution of this protocol, please refer to Hidmi et al.1.

Neurological Disorders Associated with WWOX Germline Mutations-A Comprehensive Overview.

The transcriptional regulator WW domain-containing oxidoreductase (WWOX) is a key player in a number of cellular and biological processes including tumor suppression. Recent evidence has emerged associating WWOX with non-cancer disorders. Patients harboring pathogenic germline bi-allelic WWOX variants have been described with the rare devastating neurological syndromes autosomal recessive spinocerebellar ataxia 12 (SCAR12) (6 patients) and WWOX-related epileptic encephalopathy (DEE28 or WOREE syndrome) (56 patients). Individuals with these syndromes present with a highly heterogenous clinical spectrum, the most common clinical symptoms being severe epileptic encephalopathy and profound global developmental delay. Knowledge of the underlying pathophysiology of these syndromes, the range of variants of the WWOX gene and its genotype-phenotype correlations is limited, hampering therapeutic efforts. Therefore, there is a critical need to identify and consolidate all the reported variants in WWOX to distinguish between disease-causing alleles and their associated severity, and benign variants, with the aim of improving diagnosis and increasing therapeutic efforts. Here, we provide a comprehensive review of the literature on WWOX, and analyze the pathogenic variants from published and unpublished reports by collecting entries from the ClinVar, DECIPHER, VarSome, and PubMed databases to generate the largest dataset of WWOX pathogenic variants. We estimate the correlation between variant type and patient phenotype, and delineate the impact of each variant, and used GnomAD to cross reference these variants found in the general population. From these searches, we generated the largest published cohort of WWOX individuals. We conclude with a discussion on potential personalized medicine approaches to tackle the devastating disorders associated with WWOX mutations.