MATERIALS AND METHODS

Families

This study comprises 92 ARRP families including four families with retinitis punctata albescens (50 consanguineous and 42 non-consanguineous pedigrees). Most of the families were examined ophthalmologically at the Hospital de la Santa Creu i Sant Pau in Barcelona or at the Fundación Jiménez Díaz in Madrid. The clinical diagnosis of RP was based on ophthalmological examination including measurements of visual acuity, ophthalmoscopy, dark adaptation, ocular tension by air tonometer, perimetry, and electroretinogram amplitudes according to ISCEV protocols.¹⁸

METHODS

Blood samples were obtained after informed consent was given and genomic DNA was extracted from leucocytes of peripheral blood from each patient.¹⁹ We used single strand conformation analysis (SSCP) to screen all exonic sequences (including intron-exon boundaries) containing the open reading frame of the RGR, CRBP1, and CRB1 genes. Primers corresponding to the complete coding sequence of the RGR and the CRBP1 genes were designed according to GenBank entries NT_033890 and NT_005832 to yield PCR products in the range 200-350 bp. The 28 primer sets described by Hollander et al^20,21 were used to amplify the complete coding region including two promoter regions of the CRB1 gene. PCR amplification was performed in a final volume of 25 μl containing MgCl₂ (1-2 mmol/l depending on the fragment amplified), 200 μmol/l of each dNTPs, 0.2 μmol/l of each primer, 0.5 units of Taq DNA polymerase (Ecogen) in the recommended buffer, and 100 ng of genomic DNA. The thermocycling conditions were 94°C for six minutes, followed by 30 cycles at 94°C for 30 seconds, from 53-63°C depending on the fragment for 30 seconds, and 72°C for two minutes, followed by a 10 minute final extension step at 72°C.

After amplification, mutation analysis was carried out using SSCP under two different conditions combining acrylamide concentration, running temperatures, and voltage. Fragments that showed abnormal patterns of migration by SSCP were analysed on an automated sequencer (ABI Prism 310, Applied Biosystems).

On detection of a sole mutant allele, the patient sample was subjected to direct sequence analysis of the remaining exons and of the promoter regions of the corresponding gene.

RESULTS

All deleterious mutations and genetic variants were assigned a nucleotide number starting at the first translated base of the RGR and CRBP1 genes according to the GenBank entries U14910 and NM_002899, respectively. The corresponding accession numbers for the CRB1 gene were AY043324 or AY043325 for isoforms I or II, respectively. Deletions were names in accordance with the HUGO recommendations.

RPE retinal G protein coupled receptor (RGR)

A PCR-SSCP strategy was used to screen each of the seven exons of the RGR gene in the 92 unrelated patients under study. SSCP band shifts were detected in DNA encompassing exons 1, 4, and 6. Sequencing of these fragments allowed the identification of six single base pair substitutions (table 2). Four of these single nucleotide changes were C>T transitions, three of which corresponded to previously described frequent polymorphisms.²² The remaining C>T substitution causing the missense change Ser241Phe was present in nine unrelated ARRP patients (eight carriers and one homozygote). Family studies in these nine ARRP families indicated that all the affected RP patients were carriers of the Ser241Phe in the four non-consanguineous pedigrees, whereas cosegregation with the disease phenotype could be excluded in the five consanguineous families. In the control group (190 chromosomes), we found five alleles with Ser241Phe. A new silent variation (Lys205Lys) was identified in a carrier state in the two affected brothers of an ARRP family. A patient showing a punctata albescens phenotype was a carrier of an A>G transition in intron 6 (IVS6+5 A>G). Segregation analysis in the patient’s family showed no cosegregation with the disease phenotype. This change was not observed in 190 control chromosomes.

Cellular retinol binding protein 1 (CRBP1)

The mutational screening of the four exons and their flanking regions of the CRBP1 gene in 92 unrelated patients allowed the identification of two single base pair substitutions in the amplimer containing exon 1 (table 2). These single nucleotide changes were C>T transitions in the 5′ UTR region (position −37 and position −134) and correspond to two new polymorphisms with a minor allele frequency of 6% and 10%, respectively.

Crumbs homologue 1 (CRB1)

A total of 19 germline sequence variants were observed in this study, including seven pathogenic mutations, eight rare sequence variants, and four intronic polymorphisms (table 3). Seven mutations meet the criteria of pathogenicity, namely, absence in controls and segregation with the disease within the family. Six out of seven are novel: the non-conservative change (Ile>Thr) located in two different CRB1 codons (205 and 1100, respectively); two in frame deletions causing the loss of serine (cd 749) and leucine (cd 962) residues; an amino acid changing variant Cys891Gly and one out of frame deletion caused by the insertion of a guanine between codons 160-161 creating a stop codon in position 168. We also observed the previously reported Cys948Tyr mutation. Fig 1 shows the pedigrees of the ARRP families in which these mutations were identified.

In addition to deleterious mutations, we detected eight rare sequence variants that include three synonymous codon changes (Leu470Leu, Asn549Asn, and Asn1057Asn), two conservative amino acid changes (Arg769His and Arg 1331His), two non-conservative amino acid changes (Thr289Met and Gln679Glu), and a G to A substitution at position −268 of the 5′ UTR region (table 3). Family studies in all these eight substitutions excluded cosegregation with the disease phenotype. We identified four intronic variants (IVS1-12 T/A, IVS2+42 T/A, IVS4-53 T/G, and IVS4-64 T/G), all of them with a >1% frequency in the general population, which were regarded as polymorphisms.

DISCUSSION

RGR is a seven transmembrane domain receptor, a close relative of rhodopsin, found in the support cells for the photoreceptors, the RPE, and the Müller glia. Unlike rhodopsin, the RGR protein is coupled to all-trans-retinal that is isomerised to 11-cis-retinal upon light exposure.²³ The essential role of RGR in the process of vision was reinforced when (1) RP associated mutations in the RGR gene were described by Morimura et al²² and (2) the phenotype of mice with targeted disruption of Rgr was described.²⁴ Two mutations, a number of other changes less likely to be pathogenic, and four frequent polymorphisms were found in the mutational screening of the RGR gene performed by Morimura et al,²² which included a large group of patients with photoreceptor degeneration. In our group of Spanish ARRP patients, we found three of the previously described polymorphisms with similar allelic frequencies, two variations (IVS6+5 A>G and Lys205Lys) and the Ser241Phe substitution (table 2). Morimura et al²² found the latter change heterozygously in two cases of recessive RP, two simplex cases, and one of 95 unrelated normal controls; one simplex case was homozygous Ser241Phe. We found the same substitution in nine unrelated ARRP patients (eight carriers and one homozygote). The pathogenic significance of this variant is difficult to assess because although there is a higher frequency of this variant in ARRP patients (10/182 alleles) compared with the frequency in control population (5/190 alleles) (p=0.0005), no cosegregation exists between the variant Ser241Phe and the disease in the Spanish consanguineous families.

CRBP1 is the carrier protein involved in the intracellular transport of retinol. Analysis of the visual cycle in CRBP1 knockout mice suggests that the binding protein participates in a process that drives diffusion of all-transretinol from photoreceptor cells to RPE, perhaps delivering vitamin A to lecithin-retinol acyltransferase for esterification. This alleged involvement of the CRBP1 gene in the visual process and the lack of mutational studies of this gene in patients affected by retinal degenerations prompted us to screen ARRP patients for pathological mutations in the four exons and flanking sequences of the CRBP1 gene. We detected no disease causing mutation in our set of families but two new single nucleotide polymorphisms were found both in the 5′ UTR region of the gene (table 2). These polymorphisms were observed in controls as well as in unaffected family members.

The recent work of two groups of investigators^20,21,25,26 has identified a number of mutations in the CRB1 gene in patients affected by (1) a form of autosomal recessive RP (RP12), characterised by a preserved para-arteriolar retinal pigment epithelium (PPRPE) and by a severe loss of vision at age <20 years, (2) Leber congenital amaurosis, (3) RP with Coats-like exudative vasculopathy, and (4) a severe form of RP with common features. Table 4 summarises the reported mutations in the CRB1 gene.

Bearing in mind these findings, we undertook the study of the CRB1 gene in our set of ARRP families. Overall, seven pathogenic mutations were detected, six of which are reported for the first time. In addition, a number of rare sequence variants and intronic polymorphisms were identified.

The insertion of a G residue between nucleotides 478-481 was found in a homozygous state in the affected patient of a consanguineous family (M-717 in fig 1). This sequence alteration generates a stop signal in codon 168. All the remaining asymptomatic members of this family were heterozygous carriers of this mutation or homozygous for the wild type allele. Clinical findings of the affected patient (table 5) show an RP pattern with an early onset and a severe loss of vision under the age of 20. Given that the PPRPE phenotype can only be identified in the early/middle stages of the disease, the present fundus examination of this patient with advanced RP does not allow us to exclude a previous typical RP12 pattern.

A homozygous point mutation (G>A nucleotide 2843) causing the substitution Cys948Tyr was found in two RP sisters of family M-69 (fig 1). The clinical findings in these patients were consistent with a diagnosis of LCA (table 5). This substitution was also present in another branch of this family where the affected RP subject carried a non-conservative change Ile1100Thr in addition to Cys948Tyr. Clinical data of this patient (table 5) indicate an early onset of typical RP with macular symptoms. An extensive search for PPRPE signs yielded a negative result. The Cys948Tyr mutation was also identified in the two patients from family M-641 (fig 1), who also had a deletion of a serine residue in position 749. Ophthalmological examination of both affected sibs showed an RP12 phenotype with PPRPE, night blindness, and loss of visual field before the age of 10 years. Nystagmus and hyperopia were also observed as described in cases with an RP12 phenotype. Since the cysteine residue in position 948 is involved in the formation of disulphide bridges in the 14th EGF-like domain of CRB1, the correct folding of this domain may be impaired by changes affecting this position. The mutation Cys948Tyr, which is the CRB1 mutation that is most frequently found, has been described in patients with Leber congenital amaurosis (LCA), with RP characterised by a preserved para-arteriolar retinal pigment epithelium, and in patients who had RP with Coats-like exudative vasculopathy. The three homozygous Cys948Tyr patients described to date were diagnosed with LCA. This clinical phenotype has also been associated with the presence of Cys948Tyr in combination with a frameshift mutation, with a missense mutation, and in patients in whom only the mutated allele Cys948Tyr has been found.^20,21,26 The identification, in the present work, of two LCA patients (M-69 family) who are homozygous Cys948Tyr reinforces the view that Cys948Tyr is a mutation that leads to a severe phenotype when present homozygously, resulting in the complete loss of function of CRB1.²¹

The inheritance of Cys948Tyr in combination with Ile1100Thr (family M-69) is associated with an early onset RP phenotype without PPRPE whereas the coinheritance of Cys948Tyr and 749delSer (family M-641) manifests as an RP12 with PPRPE. These cases may have residual CRB1 function as postulated by den Hollander et al,²¹ who identified four PPRPE patients who carried Cys948Tyr in combination with other missense mutations.

A new point mutation causing the substitution of the cysteine residue in position 891 by a glycine was found in compound heterozygosity with a non-conservative change (Ile1100Thr) in the proband of family B-102. Ophthalmological examination of this patient (table 5) showed a typical RP with an early onset and a rapid progression of the disease. Cys891 is a conserved residue located in the 13th EGF-like domain of CRB1 and its substitution, impairing the formation of disulphide bridges, probably causes domain misfolding with secondary deleterious effects on the global conformation of the protein. The mutation Ile1100Thr that cosegregates in this family affects the same isoleucine residue found by Hollander et al²¹ to be mutated (Ile1100Arg) in a LCA proband.

A deletion of three nucleotides leading to the 962 del Leu of the laminin 3 motif was found, in a heterozygous state, in the proband of family B-15. Sequence analysis of the entire CRB1 ORF did not show any additional mutation. The disease in this patient may be the result of an additional missing mutant CRB1 allele (a point mutation in the non-coding regions of the CRB1 gene or a large genomic rearrangement that would not be detected with the methodology used). Clinical findings in this patient (table 5) meet all the standard definitions of retinitis pigmentosa except for the fundus appearance. He had night blindness from an early age, with a progressive visual field loss and a non-recordable ERG. The most striking finding in the fundus of this patient is the extensive atrophy of the retinal pigment epithelium and choroid. Choroid atrophy is widespread leaving areas of bare sclera, although other areas are still spared. There is little dispersion in pigment scattered all across the fundus. The pigment does not tend to assume the spicular configuration of deposits in typical RP. The disc is not pale but the retinal arteries are thin. This choroideremia (CHM)-like fundus prompted us to analyse the segregation of three intragenic markers of the REP-1 gene (two SNPs located in exons 5 and 9 and a VNTR in exon 14) in the members of family B-15. Linkage between these informative markers and the disease phenotype can be excluded (data not shown). Thus, the diagnosis of a CHM associated with REP-1 abnormalities can be rejected.

A novel non-conservative missense mutation, Ile205Thr, was identified in the affected brothers of family M-489 (fig 1). The Ile205Thr mutation changes an apolar residue conserved through evolution by a polar amino acid, in the 5th epidermal growth factor (EGF)-like domain of the CRB1 protein. The mutational analysis of the entire coding region and promoter sequences of the CRB1 gene failed to detect any additional mutation. The ophthalmological investigations of these affected members in family M-489 showed an LCA pattern defined by congenital blindness, nystagmus, extinguished ERG, and the presence of Franceschetti sign in both patients (table 5).

Six out of the seven rare sequence variants identified in this study have been previously reported in Leber congenital amaurosis patients.²⁶ Arg769His and Arg1331His were identified by Lotery et al²⁶ in control chromosomes and we were able to exclude cosegregation with the disease phenotype in our families. The lack of cosegregation has also been shown in the three synonymous codon changes (Leu470Leu, Asn549Asn, and Asn1057Asn) previously reported in LCA probands, indicating that these substitutions are not pathogenic. Two non-conservative amino acid changes (Thr289Met and Gln679Glu) were identified in Spanish ARRP patients. Thr289Met was initially considered to be related to LCA by Lotery et al,²⁶ who found this change in an affected proband. Nevertheless, we regard this change as non-pathogenic in line with the family studies that indicate a lack of cosegregation in the Spanish ARRP family (data not shown). A similar conclusion can be drawn for Gln679Glu, a previously unreported sequence variant identified in the present study.

Four intronic single nucleotide polymorphisms were found among the study participants. The most common change, located 12 bp upstream from the start of exon 2, has been previously reported to be identically distributed among the alleles of LCA probands and controls.²⁶ Similar results were obtained in our Spanish population. Analysis of the three new variants indicates that they represent polymorphisms in the human population, suggesting no particular relationship to retinal degeneration.

In conclusion, the data reported here provide (1) strong evidence against a direct involvement of the RGR and CRBP1 genes in our set of Spanish autosomal recessive retinitis pigmentosa families and (2) data to establish the implication of the CRB1 gene in the development of different types of retinal degeneration. The wide range of phenotypes associated with CRB1 mutations underlines how the relationship between pathogenic mutations and disease phenotype is becoming increasingly complex. Further molecular and biochemical studies to elucidate the function of this protein will help us to define the events that result in blindness and will provide insights into the physiology of vision.