Full ophthalmic examination and fundus photography were performed on seven family members and one spouse, the proband’s father (Fig. 1) . The family and 150 control individuals were white. All patients gave informed consent to participate in this study, which adhered to the tenets of the Declaration of Helsinki and was approved by internal institutional review (QUB 74/03). Electrophysiology was performed in three of the second-generation individuals and optical coherence scanning tomography (OCT) was performed on a subset of the family. 

DNA was extracted from a blood sample provided under consent. Sixteen amplicons spanning specific exons of several candidate genes associated with retinal degeneration were analyzed (Table 1) . In addition, 14 primer pairs (Table 2 ; Invitrogen Life Technologies, Paisley, UK) were used to amplify all 12 exons of the CRB1 gene and their flanking donor and acceptor splice sites, the 5′ noncoding promoter region and part of the 3′ untranslated region. In 150 control individuals only exon 2 of the CRB1 gene was amplified. Each amplicon was generated by polymerase chain reaction (PCR; Platinum PCR SuperMix; Invitrogen-Life Technologies) after initial denaturation at 94°C for 1 minute and 30 cycles of 94°C, 10 seconds; 50°C, 10 seconds; and 72°C, 20 seconds; and one cycle of 72°C, 7 minutes. PCR amplicons were column purified (Promega, Madison, WI) and subjected to DNA sequence analysis by dye termination chemistry (Big Dye Sequencing; Applied Biosystems, Warrington, UK). Homology modeling of the fourth EGF-like calcium-binding domain of CRB1 was performed (Swiss-Model, ver. 36.0003; http://www.expasy.org/spdbv/ provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland). ^{20 21}  

A 28-year-old man (proband) was referred for evaluation of a pigmentary retinopathy (Fig. 2) . A diagnosis of PPCRA with optic disc drusen was made. The patient reported no symptoms on presentation; however, he subsequently suffered a serious decline in visual function over the next 3 years. 

Clinical examination revealed that the proband’s father was unaffected, but although his mother and two siblings were asymptomatic, they showed clinical signs of early PPCRA. The proband’s mother (II:2) was aged 62 at the time of examination. She showed moderate chorioretinal atrophy in the inferior quadrant associated with paravenous pigmentary changes in the far periphery. The woman, who was ill, was examined at home and is now deceased. It was therefore difficult to capture the pigmentary changes in the far periphery with the handheld camera. However, moderate chorioretinal atrophic changes in the inferior quadrant were recorded (Figs. 3 A 3B) . Individual II:5, a woman, was severely affected with very poor visual acuity. Although the changes in this 59-year-old individual were panfundoscopic, the more severe chorioretinal atrophy and pigmentation were again located in the inferior quadrant (Figs. 3C 3D) . Individual II:6, aged 56, was found to have no evidence of any atrophy or pigmentation and was deemed unaffected. Individual III:2, a 36-year-old woman, showed very early chorioretinal atrophy in the inferior quadrant but, as yet no pigmentary changes, and had normal electrophysiology. Individual III:4 (a 27-year-old man) showed more obvious chorioretinal atrophy inferiorly, with some very early pigmentary changes (Fig. 4)and had subnormal rod ERGs in both eyes. 

OCT scanning in this family indicated that retinal thickness and lamination were within normal limits (Fig. 5A) . An OCT scan taken through an area of bony spicule pigmentation indicated that the pigment lies in the superficial neuroretina (Figs. 5B 5C) . 

A heterozygous c.619G→A single nucleotide variation in CRB1 (GenBank accession number AY043325) resulting in an amino acid change from valine to methionine was detected at codon 162 within the fourth EGF-like domain. Of the seven family members examined, the Val162Met variation was found to cosegregate with six affected family members and was not found in one unaffected family member (Fig. 1) , giving a lod score of 1.8. This nucleotide variation was not detected in 150 control individuals analyzed. 

Homology modeling of the fourth EGF-like domain was performed (Swiss-Model, ver. 36.0003; http://www.expasy.org/spdbv/), ^{20 21} based on structural data for the calcium binding EGF-like domains of coagulation factor VII, which has a high degree of primary sequence identity (Fig. 6A) . The Val162Met variation is adjacent to the fourth highly conserved cysteine and is a semiconservative substitution, with valine being replaced with the larger, nonaliphatic methionine residue. Deduction of any structural effect is challenging because Val162 is not conserved, but that amino acid residue is involved in hydrogen bonding between the antiparallel β-strands of the major β-sheet; in CRB1 the backbone amide/carbonyl of Val162 hydrogen bonds with those of Phe173 (Fig. 6B) . A possibility is that changing the side-chain would result in sufficient perturbation of the backbone that the domain-stabilizing hydrogen bond does not form or is weakened. 

Subtle symmetrical chorioretinal atrophy in the inferior quadrant is the earliest clinical sign of PPCRA detectable within this family. This geographic pattern correlates with the clinical spotting of the inferior nasal quadrant of the fundus of rd8 mice, which have a point mutation in the Crb1 gene. ²² Paravenous pigmentation occurs initially in the far periphery but, as the disease progresses, moves more centrally with the chorioretinal atrophy becoming more widespread, with involvement of the nasal, then the temporal and finally the upper quadrant. Both males and females have variable expressivity. Males are more likely to exhibit a severe phenotype, whereas females may remain virtually asymptomatic, even in later years. The reason for the differential expression of the disease process remains unexplained; however, it is consistent with a mutation in CRB1, ^{5 9 10 23} in which mutations have been reported to cause a wide spectrum of phenotypes. ^{12 13 14 24} The specific CRB1 mutation reported affects the phenotype observed, with loss of function more likely to result in LCA and residual function associated with RP. ²⁴ In addition, the same mutations can lead to different phenotypes, with inconsistent characteristics such as PPRPE and Coats’-like exudative vasculopathy. ^{13 14 24} Therefore other genetic modifiers and environmental factors may influence the effect of CRB1 mutations. 

The earliest reported cases of PPCRA suggested an inflammatory cause because one affected individual was diagnosed with tuberculous spondylitis, ² a child with congenital syphilis, ²⁵ another with rubeola retinopathy, ²⁶ and another with ocular inflammation with cystoid macular edema. ⁷ In accordance with the previous reports relating to the hereditary etiology of this disease, ^{3 5 9 23 27} we found no evidence of an inflammatory or infectious origin within this family. The autosomal dominant mode of inheritance observed in this family is consistent with most of the information available from previous reports of PPCRA. ^{9 10} Our experience supports suggestions that sporadic cases may in fact be revealed to be familial, if asymptomatic relatives are examined. ^{3 6}  

The CRB1 gene is composed of 12 exons and encodes a transmembrane protein with 19 epidermal growth factor (EGF)-like domains, three laminin A globular-like domains, and a 37-amino-acid cytoplasmic tail. It is expressed in human retina and brain, with evidence of alternative splicing at its 3′ end and is thought to encode four different isoforms, two of which are found in human retina. ²⁸ Crumbs, the Drosophila orthologue of CRB1, forms part of one of the many protein complexes along the plasma membrane that participate in cell-to-cell contact and has been shown to be a key regulator of epithelial apical–basal polarity. ²⁹ The function of the intracellular domain of Crumbs and CRB1 in localizing the phototransduction complex to the apical membranes of photoreceptors appears to be conserved from Drosophila to humans. ^{28 30 31} The extracellular domain of Crumbs is essential to suppress light-induced photoreceptor degeneration. ³² In mice, Crb1, the murine orthologue of CRB1 is located apical to the zonula adherens in photoreceptors and is necessary for the integrity of the external limiting membrane. ^{22 33} A report of abnormal lamination and increased retinal thickness in patients with certain CRB1 mutations ¹⁸ suggests a role in laminar development. This function does not appear to be affected by the Val162Met mutation, because retinal thicknesses measured by OCT were within the normal range. 

All the CRB1 mutations previously reported to be associated with retinal degeneration in humans have also been localized to the extracellular portion of the protein, leading to amino acid substitutions, frame shifts, or premature stop codons (with the exception of one documented mutation at codon 1354 within the transmembrane domain, reported in an individual with RP with Coats’-like exudative vasculopathy, in conjunction with two additional mutations found within the extracellular domain). ^{12 13 14 15 16} Among these mutations, Cys948Tyr is the most common, having been associated with LCA, RP, RP with Coats’-like exudative vasculopathy and RP with PPRPE. A family has been reported in which affected individuals homozygous for the Cys948Tyr mutation experience a severe LCA phenotype. ¹⁴ This observation is in contrast to a compound heterozygous family member with the Cys948Tyr mutation and a nonsynonymous Ile1100Thr mutation, also within the CRB1 gene, resulting in an early onset RP phenotype without PPRPE. It was concluded that the homozygous mutation results in a complete loss of function of the CRB1 gene, whereas heterozygosity is still likely to result in partial function. den Hollander et al. ¹² concurred with these findings, reporting homozygous Cys948Tyr to be a severe mutation likely to lead to LCA. When found heterozygously in combination with a splice-site mutation that was unlikely to completely inactivate the protein, the less severe RP with PPRPE resulted. Cys948Tyr occurs in what appears to be a truncated EGF-like domain (the 14th EGF-like domain). Although no high-resolution structural data are available, the amino acid sequence up to the fourth cysteine conforms to the EGF consensus sequence. In the typical EGF structure, this cysteine forms a disulfide bond with the second highly conserved cysteine (Cys933), and a nonsynonymous substitution would therefore be expected to disrupt the native fold of the domain. 

The Val162Met mutation within CRB1 occurs in the fourth EGF-like domain. EGF-like domains are widely distributed in nature and are independently folding modules found in many transmembrane and extracellular proteins. ³⁴ Sequence similarity among EGF-like domains is low at approximately 30%, they are defined largely on the basis of the spacing between the cysteines, ³⁵ and they typically consist of six cysteine residues that form disulfide bonds in a 1-3, 2-4, 5-6 arrangement. A subset of EGF-like domains contains a consensus associated with calcium binding. The residues that define this consensus comprise those that directly bind the calcium atom (through both side chain and backbone carbonyl H-bonding), those involved in interdomain packing interactions, and those for which clear functions have not yet been determined. ³⁶ The main structural feature of all EGF-like domains (both calcium-binding and non–calcium-binding) is a central two-stranded β-sheet that is located in the region of the sequence containing the 1-3 and 2-4 disulfide bonds. The region separated by the 5-6 disulfide bond forms a β-strand with hairpin turns at either end. The 19 EGF domains comprise ∼50% of CRB1 but contain ∼70% of the nontruncating mutations, half of which directly substitute one of the six signature cysteine amino acid residues that disulfide bond to stabilize the global fold of the domain. Other mutations introduce cysteine residues that could incorrectly form disulfide bonds. The remaining mutations have no clear structural effect, and could mediate their disease-causing phenotype through disruption of intradomain, interdomain, or protein–protein interactions. 

Human fibrillin-1 is a gene composed of similar calcium-binding EGF domains and demonstrates similar mutation frequency ratios within the EGF-like domains, emphasizing the functional importance of these domains. ^{37 38} A relatively conservative Val1128Ile mutation similar to CRB1 Val162Met occurs at the corresponding domain location within a calcium-binding EGF-like domain of fibrillin 1 and is associated with Marfan syndrome. ³⁹ A variant of Marfan syndrome is also caused by a conservative mutation at G1127S, ⁴⁰ corresponding to codon 161 within CRB1, a site previously reported to be associated with RP through a conservative alanine-to-valine substitution. ¹² Both mutations within each gene have been associated with disease, and all mutations are nominally conservative, although each substituted residue is larger than its native counterpart. Smallridge et al. ³⁷ reports that the G1127S is likely to impair folding of the calcium-binding EGF-like domain, possibly because of the exchange of the glycine for a less flexible residue at the start of the major β-hairpin. 

The mechanisms whereby mutations in CRB1 cause retinal degeneration are unclear. Crb1 is required for maintenance of the adherens junctions between photoreceptor and Müller glia cells. ³³ Complete loss of Crb1 results in retinal disorganization and dystrophy in mice, and null mutations in CRB1 result in LCA in humans. ¹⁴ EGF-like domains mediate protein interactions, and the severity of the phenotype associated with mutations within them presumably reflects the importance of the specific interactions affected. Although previously reported mutations within CRB1 are recessive, dominant mutations within EGF-like domains of other proteins have been reported to cause retinal disease, for example EFEMP1 Arg345Trp (dominant drusen) ⁴¹ and HEMICENTIN-1 Gln5345Arg (ARMD1 phenotype). ⁴² It has been proposed that mutations within the calcium-binding EGF-like domains of fibrillin-1 exert a dominant negative effect by disrupting the function of the multicomponent 10- to 12-nm microfibrils of which fibrillin-1 is a major constituent. ⁴³ It is possible that the fourth CRB1 EGF-like calcium-binding domain is involved in the assembly of an extracellular scaffold structure and that the Val162Met mutation disrupts the organization of this network. Further investigation is necessary to confirm whether the mutational effect is local to the domain or is implicated in a potential site of protein–protein interaction. However, the extensive variation in phenotypic presentation within this family suggests that multiple interactions are likely to be involved. 

 

The authors thank the family involved in this investigation for their patience and cooperation and Gerry Mahon, Justin O’Neill, Vittorio Silvestri, Anne Hughes, and Ronald Hunter for assistance with the research.