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Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages.

ABSTRACT: Mutations in membrane frizzled-related protein (MFRP) are associated with nanophthalmia, hyperopia, foveoschisis, irregular patches of RPE atrophy, and optic disc drusen in humans. Mouse mfrp mutants show retinal degeneration but no change in eye size or refractive state. The goal of this work was to generate zebrafish mutants to investigate the loss of Mfrp on eye size and refractive state, and to characterize other phenotypes observed.Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methods were used to generate multiple frameshift mutations in zebrafish mfrp causing premature translational stops in Mfrp. Spectral-domain optical coherence tomography (SD-OCT) was used to measure eye metrics and refractive state, and immunohistochemistry was used to study adult eyes. Gene expression levels were measured using quantitative PCR.Zebrafish Mfrp was shown to localize to apical and basal regions of RPE cells, as well as the ciliary marginal zone. Loss of Mfrp in mutant zebrafish was verified histologically. Zebrafish eyes that were mfrp mutant showed reduced axial length causing hyperopia, RPE folding, and macrophages were observed subretinally. Visual acuity was reduced in mfrp mutant animals.Mutation of zebrafish mfrp results in hyperopia with subretinal macrophage infiltration, phenocopying aspects of human and mouse Mfrp deficiency. These mutant zebrafish will be useful in studying the onset and progression of Mfrp-related nanophthalmia, the cues that initiate the recruitment of macrophages, and the mechanisms of Mfrp function.

SUBMITTER: Collery RF

PROVIDER: S-EPMC5215506 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages.

Collery Ross F RF Volberding Peter J PJ Bostrom Jonathan R JR Link Brian A BA Besharse Joseph C JC

Investigative ophthalmology & visual science 20161201 15

<h4>Purpose</h4>Mutations in membrane frizzled-related protein (MFRP) are associated with nanophthalmia, hyperopia, foveoschisis, irregular patches of RPE atrophy, and optic disc drusen in humans. Mouse mfrp mutants show retinal degeneration but no change in eye size or refractive state. The goal of this work was to generate zebrafish mutants to investigate the loss of Mfrp on eye size and refractive state, and to characterize other phenotypes observed.<h4>Methods</h4>Clustered regularly intersp ...[more]

PMID: 28002843

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Project description:The membrane-type frizzled-related protein (MFRP) gene is selectively expressed in the retinal pigment epithelium and ciliary body, and mutations of this gene cause nanophthalmos. The MFRP gene may not be essential for retinal function but has been hypothesized to play a role in ocular axial length regulation. The involvement of the MFRP gene in moderate to high hyperopic, isolated microphthalmic/anophthalmic, and high myopic patients was tested in two phases: a mutation screening/sequence variant discovery phase and a genetic association study phase.Eleven hyperopic, ten microphthalmic/anophthalmic, and seven non-syndromic high-grade myopic patients of varying ages and 11 control subjects participated in the mutation screening phase. Sixteen primer pairs were designed to amplify the 13 exons of the MFRP gene including intron/exon boundaries. Polymerase chain reactions were performed, and amplified products were sequenced using standard techniques. Normal and affected individual DNA sequences were compared alongside the known reference sequence (UCSC genome browser) for the MFRP gene. The genetic association study included 146 multiplex non-syndromic high-grade myopia families. Seventeen intragenic and flanking single nucleotide polymorphisms (SNPs) were chosen for the MFRP gene and genotyped in the large data set using the Taqman allelic discrimination assay. The family-based association Pedigree Disequilibrium Test (PDT) and GenoPDT were performed.The average spherical refractive error of the hyperopic patient cohort was +4.21 diopters (D; range +2.00 to +9.25 D) and of the myopic patient cohort was -12.36 D (range -8.25 to -14.50 D). A total of 16 SNPs were identified by direct sequencing. No significant association was determined between the 16 MFRP gene SNPs and the moderate to high hyperopia, microphthalmia/anophthalmia affection status, and high myopia. Family based association analysis did not reveal any association between the 17 SNPs genotyped in the larger family data set for any refractive error type.Sequence variants of the MFRP gene do not appear to be associated with either the less severe forms of hyperopia, extreme forms of limited eye growth and development, or high myopia. These results indicate that the MFRP gene may not play a role in regulating ocular axial length in these phenotypes.

Dataset Information

Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages.

Publications

Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages.

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