Developmental basis of nanophthalmos: MFRP Is required for both prenatal ocular growth and postnatal emmetropization

Abstract

Background: Nanophthalmos is a genetic disorder characterized by very small, hyperopic eyes that are without gross structural defects. Recessive nanophthalmos is caused by severe mutations in the MFRP gene, which encodes a Frizzled-related transmembrane protein that is selectively expressed in the retinal pigment epithelium (RPE) and ciliary body.

Results: For two MFRP -/- adults, we have obtained records of refraction that begin in early childhood. At the age of 6 months, one patient's eyes already had a refractive error of +12.25 D, and over the next 20 years this slowly increased to +17.50 D. Adults homozygous for null mutations in MFRP have eyes with axial lengths shorter than those of normal newborns. Furthermore, the unusually high curvature of their corneas is consistent with eyes that had been smaller than normal during late fetal development. MFRP protein was first detected at 14 weeks of gestation, when it was restricted to the posterior pole RPE. By 20 weeks gestation, MFRP expression had spread laterally, and was found throughout the RPE. MFRP protein was detected in both posterior and lateral RPE of the adult eye.

Conclusions: Embryonic function of the MFRP gene appears necessary for the eye to reach its full size at birth. Its onset of expression in the RPE during mid-gestation suggests that MFRP does not participate in early formation of the optic cup, and is consistent with a role in later growth and development of the eye. Patients without MFRP gene function exhibit no correction of refractive error during childhood, which suggests that this gene is essential for emmetropization, a complex process by which vision regulates axial growth of the eye.

Figure 1. Structural features of hyperopic and myopic eyes

Diagrams of A. nanophthalmic, B. normal and C. myopic eyes are shown in horizontal section. The nanophthalmic eye is a composite of biometric features of MFRP −/− homozygotes (10) and histological data reviewed earlier (6,7,8). Relative biometric and histological features of eyes from emmetropes and high axial myopes (6) were the basis of B. and C.

Figure 2. Biometry of MFRP-null homozygotes and heterozygotes

Biometric measurements of Amish-Mennonites ages 18–64 with the 1143insC frameshift, a null mutation of MFRP, compared with the Caucasian population described by Lyhne (18). Solid arrows: null homozygotes (−/−). Open-headed arrows: null heterozygotes(−/+). The −/h heterozygote (only in panel A) is a compound heterozygote for the 1143insC allele and HM-1, a presumed hypomorphic allele of MFRP. A. Spherical equivalent refractive error, determined with cycloplegia, and averaged of both eyes. B. Axial length (corneal surface to retina), by ultrasound. C. Corneal radius of curvature, R, in mm. The conversion to K-value (diopters of corneal refractive power) is: K = 337.5/R. D. Anterior chamber depth, from endothelium to lens surface (ultrasound). E. Axial lens thickness (ultrasound).

Figure 3. Adult MFRP null-homozygote have eyes shorter than those of normal newborns

Distributions of individual ocular axial lengths, normalized to percent of total and assigned to 0.5 mm wide bins. Open circles: 7 eyes of adults homozygous for null mutation MFRP^1143insC. Caucasians, aged 20 to 32. (10). Solid squares: 160 eyes of normal Caucasian newborns 1 to 5 days (from Larsen et al. ref. 18). A 2-tailed Student’s t-test gives a P-value of 1.1 × 10−⁵, which allows us to confidently exclude the null hypothesis that these populations are identical. Solid circles: 228 normal Caucasian adult eyes (from Lyhne et al. ref. 17).

Figure 4. Developmental changes in refractive error in the absence of MFRP

Spherical equivalent refractive error was obtained for two siblings featured in Figs 2 and 3. Each is homozygous for the 1143insC null allele of MFRP. Red curve: patient #5 (ref. 10 ), better eye; Blue curve: Patient #7 (ref. 10 ), average of both eyes. Points without circles: lensometry values from the child’s glasses, collected by the mother. The earliest set was prescribed at 6 months. Circled points: retinoscopic cycloplegic refraction from our study or from medical records. Green curve: average refractive error in the general population (ref. 30).

Figure 5. MFRP protein in the adult RPE

A. Neural retina, detached from RPE. Ros-arrow: Rod outer segments. B. Retinal pigment epithelium, detached from neural retina. Blue BCIP-alkaline phosphatase reaction product. Br-arrow: Bruch’s membrane. C. Control, without primary antibody. RPE: Faint golden color in RPE of control remains after bleach. Bar: 20 μm. A–C are at the same magnification.

Figure 6. MFRP protein in the embryonic eye at 7 and 20 weeks

Immunocytochemical localization of MFRP in retinal sections as in Fig. 5. A. 7-week neural retina (Ret), with RPE attached. No signal visible in RPE, which retains a faint golden color after bleaching of melanin. Underlying mesenchyme (Mes) shows no signal. B, C: 20-week neural retina and RPE+choroid (Chor), respectively. s: space between neural retina and RPE, resulting from post-mortem detachment. nos: Nascent rudiments of photoreceptor outer segments, showing signal. D, E: Adult retina, neural retina and RPE, respectively, in region of detachment. Signal appears associated with extracellular matrix surrounding the distal portion of photoreceptor outer segments (OS). F: Control section of detached adult RPE+choroid, without primary antibody. G: Region in which neural retina and RPE remained attached, same retinal section shown in panels D,E. Bar: 20 μm. A–G are at the same magnification.

Figure 7. Expression of MFRP in the human eye at 14 weeks

Prior to sectioning, the dissected optic cup fragment extended from one margin of the optic cup, and contained the posterior pole. Sections included retina, RPE, developing choriocapillaris, and sclera. A. Hematoxylin and eosin staining. A gradient of retinal development is visible from the margin to the posterior pole, with the inner plexiform layer visible in lateral and posterior retina. The RPE is heavily pigmented, and detached at both edges of the specimen. Gcl: ganglion cell layer. Nbl: neuroblastic layer. RPE: retinal pigment epithelium. B. MFRP protein. Signal is intense in the posterior pole sector of RPE, intermittently visible in the lateral RPE, and absent in anterior and marginal RPE (insets 1, 2, 3). All observed MFRP expression is located towards the apical side of the RPE.