METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma
Yang Pan, Akiko Suga, Itaru Kimura, Chojiro Kimura, Yuriko Minegishi, Mao Nakayama, Kazutoshi Yoshitake, Daisuke Iejima, Naoko Minematsu, Megumi Yamamoto, Fumihiko Mabuchi, Mitsuko Takamoto, Yukihiro Shiga, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Toru Nakazawa, Takeshi Iwata
Yang Pan, Akiko Suga, Itaru Kimura, Chojiro Kimura, Yuriko Minegishi, Mao Nakayama, Kazutoshi Yoshitake, Daisuke Iejima, Naoko Minematsu, Megumi Yamamoto, Fumihiko Mabuchi, Mitsuko Takamoto, Yukihiro Shiga, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Toru Nakazawa, Takeshi Iwata
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Research Article Ophthalmology

METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma

Abstract

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23–knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/– and Mettl23–/–) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB–mediated TNF-α and IL-1β feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

Authors

Yang Pan, Akiko Suga, Itaru Kimura, Chojiro Kimura, Yuriko Minegishi, Mao Nakayama, Kazutoshi Yoshitake, Daisuke Iejima, Naoko Minematsu, Megumi Yamamoto, Fumihiko Mabuchi, Mitsuko Takamoto, Yukihiro Shiga, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Toru Nakazawa, Takeshi Iwata

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Figure 2

The METTL23 c.A83G mutation leads to splicing in transfected HEK293T cells and iPSCs.

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The METTL23 c.A83G mutation leads to splicing in transfected HEK293T cel...
(A) The splicing construct minigene was generated by incorporating the genomic region of the METTL23 gene into the pSpliceExpress vector via XhoI and XbaI restriction sites. Vector exons are depicted as black boxes, and the METTL23 exon 2 is shown as a gray box. The locations of the mutations are marked by arrowheads (red: c.A83G; yellow: c.84+60delAT). (B) Gel electrophoresis of RT-PCR products from transfected HEK293T cells. The primers are indicated by arrows in A. EV, empty vector; PBS, cells transfected with PBS only; PCR-n, PCR negative control. WT and mutant transcript contents were determined by Sanger sequencing and are depicted to the right of the gel image. (C) Graphic display of iPSC preparation. Collected samples are marked with a red box in Figure 1A. Scale bar: 200 μm. (D) Characterization of iPSCs. A83G-iPSC colonies (glaucoma a and b) were derived from lymphocytes from patients with NTG. There was no difference between iPSC controls (normal a/b) and A83G-iPSCs (glaucoma a/b) during induction and cultivation. Scale bar: 200 μm. (E) Gel electrophoresis of RT-PCR products from iPSCs. TA-cloning analysis following Sanger sequencing identified 4 splicing variants in A83G-iPSCs (glaucoma a/b). (F) Schematic representation of the splicing variants. (G) Predicted protein structures of the splicing variants (red: p.E28G). The 2 predicted proteins lack motif 1 and motif post 1, suggesting that the mutant alleles are functionally null. C-term, C-terminal; N-term, N-terminal.