Loss-of-function Mutations in FREM2 Undermine the Morphogenesis of Eye

Abstract

Purpose : Cryptophthalmos is a rare congenital disorder characterized by ocular dysplasia with eyelid malformation. Complete cryptophthalmos is characterized by complete fusion of the eyelids. However, the genetic basis and pathogenesis of isolated bilateral cryptophthalmos are poorly understood.

Methods : In the present study, we characterized the clinical manifestations of three patients with isolated bilateral cryptophthalmos by ocular B-scan ultrasound, MRI and exploratory surgery. Whole exome sequencing and genetic analysis revealed that these patients shared a same missense mutation c.6499C>T in the FREM2 gene, while each individual had an additional nonsense mutation in the same gene. Then, we used CRISPR/Cas9 to generate mice carrying compound heterozygous mutations Frem2^R725X/R2156W. Hematoxylin-eosin staining was used for morphology examinations. A set of different expressed genes which may contribute secondarily to the phenotypes were identified by RNA-seq from the fetal Frem2 mutant mice.

Results : We found a transparent anterior cyst underneath the subcutaneous tissue in Patient #1. His iris and suspensory ligament of the lens were absent, besides a lens-like transparent spherical body was observed in the posterior cyst. Whole gourd-shaped cysts within the eye sockets were also identified in Frem2^R725X/R2156W mice with abnormal eyelids. All the dysplastic eyes exhibited structural malformation in the anterior segment, including disorganized or absent cornea, complete iris synechia, iris hypoplasia, an absent ciliary body and an absent pupil. What's more, 8/11 mice exhibited a single kidney and 4/11 mice exhibited syndactyly. The lower expression of Ecm2, Col6a1, Lor, Sprr1a, Cryge, and the 3 genes Cblc, Esp8, Agr2 participating in the EGFR signaling pathway, were identified by RNA-seq and verified by real-time quantitative PCR in Frem2^R725X/R2156W mice.

Conclusions : Our findings extend the mutation spectrum of FREM2, and provide insight into the opportunities for prenatal diagnosis of isolated cryptophthalmos. Furthermore, our work highlights the importance of FREM2 protein during eyeball development, establish a suitable animal model for studying epithelial reopening during eyelid development and serves as a valuable reference for further mechanistic studies on the pathogenesis of isolated cryptophthalmos.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Probands showing bilateral complete cryptophthalmos.

 

Phenotypes of Frem2^R725X/R2156W mice.