Missense Mutations in the Human Nanophthalmos GeneTMEM98Cause Retinal Defects in the Mouse

Abstract

Citation: Cross SH, Mckie L, Keighren M, et al. Missense mutations in the human nanophthalmos gene TMEM98 cause retinal defects in the mouse. Invest Ophthalmol Vis Sci. 2019;60:2875-2887. https://doi.org/ 10.1167/iovs.18-25954PURPOSE. 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.METHODS. 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.RESULTS. 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. CONCLUSIONS.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.

Dopachrome tautomerase variants in patients with oculocutaneous albinism

“…To create the Dct 118 A/A, Dct 183 G/G and Dct −/− alleles the CRISPR design site https://chopchop.cbu.uib.no/ (in the public domain) was used to design guides with default settings (g1 for Dct 118 A/A and g2 for Dct 183 G/G and Dct −/−, Supplementary Materials). Genetic engineering of CRISPR-Cas9 lines with each of the Cys substitutions was performed as already described 12 and detailed in Supplementary Materials and Methods.…”

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification

“…In the eye it is expressed during development in the RPE, and to a lesser extent in the ganglion cell layer (Fig 1A). In the adult eye it is expressed in the RPE, ciliary body and the iris (S1 Fig) [15].…”

“…At embryonic day (E)16.5-E17.5 the eyes of the majority (5/6) knockout mice have an elongated shape when compared to wild-type and heterozygous knockout eyes ( Fig 1B), suggesting that loss of Tmem98 impacts control of eye size. As complete knockout of Tmem98 is perinatal lethal [15] to examine the phenotype of adult eyes in the absence of Tmem98 we generated mice where Tmem98 is knocked-out only in the eye. We converted the "knockout-first" allele Tmem98 tm1a to the conditional allele Tmem98 tm1c as described in Materials and Methods.…”

“…We also tested two missense mutants of Tmem98, I135T and A193P, to ascertain if the ability of TMEM98 to inhibit MYRF self-cleavage was compromised by these mutations in cotransfection experiments (S8 Fig). The I135T missense mutation of Tmem98 causes folds in the retinal layers which is accompanied by a retinal white spotting phenotype when heterozygous and is lethal when homozygous [15] and the A193P missense mutation is associated with dominant nanophthalmos in humans [6] and causes a recessive retinal phenotype in the mouse that is similar to the dominant phenotype caused by I135T [15]. Neither of these missense mutations affected the ability of TMEM98 to inhibit MYRF self-cleavage to any noticeable degree suggesting that the mutant phenotypes caused by these missense mutations are probably not due to a change in the interaction between TMEM98 and MYRF (S7 Fig).…”

“…A different missense mutation in Tmem98, I135T, was found to cause a similar dominant retinal phenotype but was homozygous lethal [15]. The eyes of mice that are heterozygous for a knockout allele for Tmem98 are normal showing that in the mouse haploinsufficiency for Tmem98 does not cause an eye phenotype [15]. When pathogenic mutations in other genes causing nanophthalmos in humans have been studied in mice it has also been found that significant changes in eye size do not result, rather the predominant phenotype is retinal defects in line with what we found with Tmem98 [16][17][18][19][20][21][22][23][24][25][26][27].…”