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ABSTRACTRNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

Abstract Background Phenotypically discordant monozygotic twins (PDMZTs) offer a unique opportunity to study post‐zygotic genetic variation and provide insights into the linkage between genotype and phenotype. We report a comprehensive analysis of a pair of PDMZTs. Methods Dysmorphic features and delayed neuro‐motor development were observed in the proband, whereas her twin sister was phenotypically normal. Four tissues (blood, skin, hair follicles, and buccal mucosa) from both twins were studied using four complementary methods, including whole‐exome sequencing, karyotyping, array CGH, and SNP array. Results In the proband, tetrasomy 18p affecting all studied tissues except for blood was identified. Karyotyping of fibroblasts revealed isochromosome 18p [i(18p)] in all metaphases. The corresponding analysis of the phenotypically normal sister surprisingly revealed low‐level mosaicism (5.4%) for i(18p) in fibroblasts. Conclusion We emphasize that when mosaicism is suspected, multiple tissues should be studied and we highlight the usefulness of non‐invasive sampling of hair follicles and buccal mucosa as a convenient source of non‐mesoderm‐derived DNA, which complements the analysis of mesoderm using blood. Moreover, low‐level mosaic tetrasomy 18p is well tolerated and such low‐level mosaicism, readily detected by karyotyping, can be missed by other methods. Finally, mosaicism for low‐level tetrasomy 18p might be more common in the general population than it is currently recognized, due to detection limitations.