Rett syndrome (RTT) is a rare X-linked dominant neurodevelopmental disorder caused by pathogenic variants in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a methyl-CpG-binding protein (MeCP2) that acts as a repressor of gene expression, crucial in neurons. Dysfunction of MeCP2 due to its pathogenic variants explains the clinical features of RTT. Here, we performed histological and RNA analyses on a post-mortem brain sample from an RTT patient carrying the p.Arg106Trp missense mutation. This patient is part of a cohort of 56 genetically and clinically characterized RTT patients, for whom we provide an overview of the mutation landscape. In the RTT brain specimen, RT-PCR analysis detected preferential transcription of the mutated mRNA. X-inactivation studies revealed a skewed X-chromosome inactivation ratio (95:5), supporting the transcriptional findings. We also mapped the MECP2 transcript in control human brain regions (temporal cortex and cerebellum) using the RNAscope assay, confirming its high expression. This study reports the MECP2 transcript representation in a post-mortem RTT brain and, for the first time, the in situ MECP2 transcript localization in a human control brain, offering insights into how specific MECP2 mutations may differentially impact neuronal functions. We suggest these findings are crucial for developing RNA-based therapies for Rett syndrome.

NF-κB-activating protein (NKAP) gene maps to Xq24 and encodes a nuclear protein involved in NF-κB transcription factor activation. Missense variants in NKAP have previously been reported in males with X-linked intellectual disability. Here, we identified a recurrent, novel frameshift variant, c.172del (p.His58Ilefs74*), by targeted X-chromosome sequencing (X-TRS) in seven females from a Sardinian family showing fully skewed X-chromosome inactivation (XCI). To investigate the functional effect of the mutation, we knocked out (KO) and knocked down (KD) NKAP in healthy donor fibroblasts and mouse embryonic stem cells, respectively. We performed RNA sequencing, immunofluorescence, and Western blot analysis on fibroblasts derived from the index subject. We demonstrated that the NKAP frameshift variant leads to monoallelic wild-type (WT) transcription and does not directly affect XCI, indicating secondary rather than primary XCI skewing. NKAP-edited human fibroblasts underwent cell death, leading to the positive selection of WT cells. The NKAP correctly localized to the nucleus but showed reduced expression. We also showed that XIST expression was not affected by this NKAP mutation, further excluding a primary XCI event. Our findings demonstrate that this novel NKAP frameshift variant causes early cell death and post-mitotic negative selection of mutant cells, resulting in a fully secondary XCI. Female carriers are healthy and fertile, whereas affected males are likely nonviable. In conclusion, our results provide insights into both the role of NKAP in cells and novel clinical implications for human fertility.