Abstract
We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs. The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes. An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions. Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.
Highlights
We previously found a dominant mutation, retinal white spots (Rwhs), causing white spots on the retina accompanied by retinal folds
Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes
In nanophthalmos eye length is reduced by 30% or more, and is usually associated with other ocular features, notably a thickened choroid and sclera, as well as a high incidence of glaucoma, corneal defects, vascular defects, and a range of retinal features including retinitis pigmentosa, retinoschisis, retinal detachments, and retinal folds
Summary
The Rwhs mutation was identified to be a missense mutation in Tmem[98] by genetic mapping and sequencing. Clinical examinations were performed as previously described.[37] Fundus imaging was carried out as described elsewhere.[38] Mice carrying a targeted knockout-first conditional-ready allele of Tmem[98], Tmem98tm1a(EUCOMM)Wtsi (hereafter Tmem98tm1a) were obtained from the Sanger Institute.[39] Tmem98tm1a/þ mice were crossed with mice expressing Cre in the germline to convert this ‘‘knockout-first’’ allele to the reporter knockout allele Tmem98tm1b(EUCOMM)Wtsi (hereafter Tmem98tm1b). In this allele the DNA between the loxP sites in the targeting cassette, which includes the neoselection gene and the critical exon 4 of Tmem[98], is deleted. To create the Tmem98H196P allele the CRISPR design site http://www.crispr.mit.edu (in the public domain) was used to design guides, and the selected guide oligos ex7_Guide[1] and ex7_Guide[2] (Supplementary Table S1) were annealed and cloned into the Bbs I site of the SpCas[9] and chimeric guide
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