The ND Protein Sequence Homologies and Three-Dimensional Structure
Analysis of the ND protein defined two characteristic domains: a signal peptide of 24 residues at the N-terminus, suggesting that this protein is secreted, and a cysteine-rich C-terminal domain.40,53 Database searches have identified homologies of this cysteine-rich domain with mucins, the Drosophila slit protein, von Willebrand factor, and the growth factor-binding proteins Cef-10 and Cyr61.40,43 Mucin proteins represent highly glycosylated extracellular proteins present in mucosal fluids and are primarily responsible for their gel-like character.54 The homology extends from the first cysteine residue of the ND protein at position 39 to the very C-terminal end (see Fig. 239-2). Transient expression of in vitro-mutated mucin molecules in COS-7 cells revealed a function of this Cys-rich C-terminal domain in dimerization of the protein.55
In Drosophila, the slit protein is expressed in a subset of glial cells along the midline of the developing central nervous system.56 Down-regulation of slit leads to disruption of the developing midline cells and the commissural axon pathway. Slit is thought to be an extracellular protein involved in interactions between midline glial cells, their extracellular environment, and the commissural axons crossing the midline.
The von Willebrand protein is a coagulation factor, and its C-terminal Cys-rich domain, which displays homology with the ND polypeptide, is responsible for dimerization.57
The growth factor-binding protein Cyr61 was shown recently to trigger migration of endothelial cells, angiogenesis, and tumor growth.58 Cef-10, another protein showing homology with the C-terminal Cys-rich domain of the ND protein, can be considered a chicken orthologue of Cyr61 (89 percent protein sequence identity and 92 percent similarity).59 Cyr61 belongs to a family of connective tissue growth factors that show an overall identity of 30 to 38 percent with insulin-like growth factor-binding proteins (IGFBPs) at the N-terminus.60 The protein induces chemotactic endothelial cell migration through the αvβ3-integrin-mediated pathway, and in the rat, it was shown that Cyr61 is capable of inducing corneal neovascularization in vivo.58
Transfection of COS-7 cells with different constructs encoding the ND protein, as well as mutant alleles, has provided a first clue to its biochemical function.61 These studies showed that the ND protein is secreted and forms dimers and oligomers that are associated with the extracellular matrix. Crosslinking experiments revealed that the dimers constitute an important structural unit of the oligomers. Oligomerization, but not dimerization, was abolished by replacing the cysteine residue at position 95 by an alanine. Two additional mutations, introduced by site-directed mutagenesis (V60E and R121Q), show a decrease in the amount of extracellular protein compared with the intracellular level, inversely to the wild-type construct. These data suggest that these altered proteins are aberrantly secreted. Decreased extracellular protein levels and additional structural alterations in the mutant protein may be responsible for the disease phenotype.
Molecular modeling of the ND gene product predicts a tertiary structure very similar to that of transforming growth factor β and put it into a family of growth factors containing a cysteine-knot motif.53 The computer analysis also predicts the formation of dimers, which is in good agreement with experimental data obtained from transient expression of the ND protein, the submaxillary mucin, and their experimentally altered isoforms.55,61
It is noteworthy in this context that female as well as male offspring of ND gene mutation carriers can develop peripheral inner retinal vascular abnormalities reminiscent of regressed ROP. Similar alterations were found in carrier females, but offspring of affected males did not show peripheral retinopathy.62 These observations suggest a role of the protein in vasculogenesis rather than neuroectodermal development. Mintz-Hittner et al.62 speculated that the mutated ND protein may have a transplacental effect on normal vascularization of the inner retina.
Cloning of the Mouse Orthologue and Generation of an Animal Model for ND
The human cDNA was used to isolate the homologous transcript of the mouse from a brain cDNA library. The gene was shown to be X-linked in the mouse and to consist of three exons.63,64 It encodes a polypeptide of 131 amino acids, and sequence comparison between mouse and human proteins revealed 94 percent identity. Tissue-specific expression was analyzed by RNA in situ hybridization. High expression levels were found in the inner nuclear layer and the ganglion cell layer of the retina, in Purkinje cells of the cerebellum, and in the mitral and sensory cell layers of the olfactory epithelia 2 weeks after birth.63 In contrast, no transcript was detected in the ear, gonads, kidney, liver, lung, spleen, gut, and tongue of 4- to 6-month-old mice.
The wild-type ND gene was inactivated in mouse embryonic stem (ES) cells by homologous recombination of a targeting construct replacing the coding portion of exon 2 by a neomycin cassette, which removes 56 amino acid residues from the N-terminus of the ND protein (Fig. 239-3 A). Similar mutations, e.g., exon 2 deletions, have been observed in ND patients.40,43 ES cells carrying the inactivated gene copy were injected into blastocysts, and embryos were transferred to pseudopregnant carrier mothers. This gave rise to chimeric male offspring that transmitted the mutation through their germ line. Heterozygous female offspring of these males were mated with wild-type males, which resulted in hemizygous mutant mice. Macroscopically, eyes were normally developed, and ocular atrophy was not observed until age 2 years and 3 months (W Berger, unpublished results, 1999). Hemizygous mutant males as well as carrier females were analyzed by ophthalmologic, morphologic, and electrophysiologic means.
Partial replacement of exon 2 of the mouse Nd gene by gene targeting (A) and histologic findings in wild-type (B) and mutant retinas (C,D). (A) The targeting construct replaces the protein coding part of exon 2 with a neomycin cassette yielding a truncated protein that lacks the 56 N-terminal amino acids. (B) Eosin-stained 5-μm section of a wild-type mouse retina. The different layers are indicated on the right: vitreous body (VB), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segments (OS), and the retina pigment epithelium (RPE). (C) Mildly affected area from a retina of a mutant mouse. The picture shows vascularized material within the posterior vitreous and conspicuous dysgenesis of ganglion cell nuclei. Some of these cells seem to migrate in the inner plexiform layer. The distal retinal layers slightly undulate but otherwise are inconspicuous. (D) A severely affected region from the retina of a mutant animal. In the outer retinal layers, OPL and the outer segments of the photoreceptor cells have disappeared, whereas vitreal changes and ganglion cell disorganization are similar to the more mildly affected segment of the retina.
Histologic and Electrophysiologic Characterization of the Retina in Mutant Mice
Initial ophthalmologic examination in mutant animals was performed by slit-lamp biomicroscopy 3 weeks after birth.63 Precipitate-like structures were observed in the vitreous body that were absent in wild-type animals. Histologic data were obtained from mouse eyes enucleated between 4 and 26 weeks after birth. The most prominent finding was the presence of blood vessels in the posterior vitreal chamber surrounded by fibrous tissue in all eyes examined.63,65 Another conspicuous abnormality was a general disorganization of the retinal ganglion cell layer in hemizygous mutant animals (see Fig. 239-3 C,D). Some of the nuclei from the ganglion cell layer seemed to migrate into the inner plexiform layer, whereas the ganglion cell layer was found to be completely disorganized. Changes involving the outer and inner nuclear layers as well as the outer plexiform layer and outer segments of the photoreceptor cells showed a patchy distribution. In severely affected areas, the outer plexiform layer and the outer segments of the photoreceptors disappeared, and hyperpigmentation of the retinal pigment epithelium was seen. Otherwise, the retina was normally differentiated, and there was no evidence of progression of the disease in older animals. In mutant mice, alterations in the vitreous are considerably milder than in patients with ND, but the retinal histology bears striking similarities, in particular the dysgenesis of the ganglion cell layer and focal changes in the outer nuclear and photoreceptor layers.8,9
In ND mice, the vascular system of the retina also was examined.65 At postnatal day 9 of normal mouse development, hyaloid vessels become atrophic, and the inner retinal vessels start to sprout. At this developmental stage, inactivation of the ND gene does not lead to dramatic qualitative and quantitative changes. In contrast, vascular changes were observed at postnatal day 14. The outer layers of the neuroretina (OPL, outer plexiform layer; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer) show an almost complete lack of blood vessels except for the main branches of the retinal artery. In contrast, the number of blood vessels in the ganglion and nerve fiber layers is increased, possibly due to neovascularization processes or remnants of hyaloid vessels incorporated in the inner limiting membrane. In mutant animals older than 20 days, fenestration in retinal vessels occurs. Moreover, penetration of vessels through the inner limiting membrane, as well as persistence of hyaloid vessels, has been observed in the vitreous of gene-targeted mice older than 20 days. Most prominent morphologic changes were found in retinal cell layers, which showed elevated expression of the ND gene 14 days after birth.63
Electroretinograms (ERGs) of hemizygous mutant mice displayed a severe loss of the b wave, leading to a negatively shaped scotopic ERG and a marked reduction in oscillatory potentials.66 The a wave was normal at low-flashlight intensities and showed a moderate loss of amplitude with brighter flashes. Scanning laser ophthalmoscopy revealed snowflake-like opacities in the posterior part of the vitreous, located predominantly at the central portions. Moreover, retinal vessels were dislocated to the vitreal chamber, and some had a bulb-like appearance. Retinal pigment epithelium alterations included pigment mottling in most parts of the retina that could be better seen in the periphery. These data suggest a more severe defect of the inner retina (containing neural and glial cells) and relatively mild symptoms in the outer retina (containing photoreceptor cells) and the pigment epithelium. In heterozygous females, ERG analyses and laser scanning ophthalmoscopy did not show significant pathologic changes.
Possible Pathogenic Mechanisms
The ND protein may have an important function in vascularization of the inner retina through sprouting of small blood vessels from the main branches of the retinal artery. This hypothesis is supported by (1) histologic findings in patients with ND and allelic disorders, (2) observations in a mouse mutant carrying an inactive ND gene, (3) angiographic findings in offspring of gene mutation carriers, and (4) homology of the ND protein to Cyr61, a growth factor associated with the extracellular matrix that induces corneal neovascularization.
Biochemical analyses involving transient expression of the ND protein in COS-7 cells suggest that it forms dimers or oligomers that are associated with the extracellular matrix when released from cells of the inner retina (possibly via integrin-mediated binding). The matrix-bound form may then trigger migration of endothelial cells and sprouting of blood vessels leading to a normally vascularized retina. When the ND gene is not expressed, vascularization cannot occur, and ganglion cells as well as other neurons start to degenerate. This degeneration leads to glial cell proliferation, seen as a massive gliosis in patients with ND. Due to these primary alterations in the neuroretina, the secondary vitreous fails to develop, and the primary vitreous (as well as hyaloid vessels) persists. This explains the vitreal pathology seen in patients. In agreement with the model proposed by Warburg,3 these changes therefore are considered to be secondary effects. Additional ocular symptoms affecting the lens, iris, and cornea may be tertiary consequences.
Alternatively, the function of the ND protein could involve the maintenance and/or differentiation of ganglion or glial cells. Further data on the biochemical characterization of the ND protein and detailed examination of the mouse model are needed to clarify the precise function of the ND gene. In particular, histologic and pathophysiologic examination of the ears and brain of ND mice may reveal alterations in these organs that would render these animals particularly suitable models for studying the molecular pathogenesis of ND. Additional clues to the function of this gene can be expected from ongoing experiments aimed at identifying additional players in the molecular pathway involving the ND gene. Characterization of these genes and their products also may provide an explanation for the clinical variability in ND that is not due to genetic variation in the ND gene itself.
Recently, another disease phenotype, designated Coats disease, was shown to be associated with a mutation in the Norrie disease gene. Coats disease is characterized by unilateral abnormal development of the retinal vasculature (retinal telangiectasis). Analysis of the retinae of 9 affected males demonstrated a somatic mutation in one of them (C96w). The same DNA sequence exchange was identified in a female with unilateral Coats disease who had a son with Norrie disease. Both mother and son carried the mutation in their germline, and it was speculated that unilateral disease in the female carrier is due to nonrandom X-inactivation in the affected eye or a somatic mutation that inactivates the second, normal allele.