Fig. 4: Functional consequence of GGGCC repeat expansions in NAXE.

a Experimental principles of NET-CAGE. The nuclei were isolated in the presence of α-amanitin, which maintains the nascently transcribed mRNAs. The nascent transcripts were enriched and processed using 5’ cap trapping technology to capture the full-length transcripts for subsequent sequencing (cap analysis of gene expression (CAGE)). b NET-CAGE of the proband fibroblasts compared to the controls (NHDF, normal neonatal human dermal fibroblast; Pt2659, patient with splicing and missense variants in NAXE) showing markedly suppressed nascent transcripts in the promoter region of NAXE (arrowhead). Note the comparable level of signals at GPATCH4 (arrow), which is reciprocally transcribed. c CpG hypermethylation (methylation indicated in red) was detected within the NAXE promoter region by analysis of long-read sequencing data in the proband as well as in the mother, whereas not in Pt2659 and NHDF. Reads were aligned to the reference genome (GRCh38). d Left panel, long-reads mapped against a sequence with expanded repeat showing hypermethylation in the GGGCC repeat per se as well as in the region downstream of the repeat stretch. The bidirectional arrow at the bottom denotes the repeat stretch. Right panel, magnified view of the dotted rectangle area in the left panel. Red and light blue lines below the right panel show the sites of normal length repeat in the controls (Pt2659 and NHDF) and the promoter region downstream of the repeat stretch, respectively. e Diagram of the suggested pathologic mechanism. GGGCC repeat expansion causes hypermethylation and suppressed transcription.