Homozygosity mapping was performed in the Italian consanguineous family with autosomal recessive RP to identify the genetic defect causing the disease. Two affected individuals (II-3 and II-4) were genotyped with 262,000 SNPs. Two shared homozygous regions were identified in these individuals: a 6-Mb region on chromosome 20 containing 520 homozygous SNPs and a 40-Mb region on chromosome 10 spanning 3780 SNPs. Haplotype analysis in all family members excluded the involvement of the chromosome 20 region, since one of the affected individuals (II-1) was not homozygous for alleles in this interval. The haplotypes at the locus on chromosome 10 completely segregated with the disease in this family. Two recombination events were observed, which reduced the critical interval to 9 Mb between SNPs rs2460551 and rs7898315 ().
Figure 1 Molecular genetic analysis in a consanguineous family with autosomal recessive RP. (A) Pedigree of the family; the parents were first cousins and had no history of visual loss. All four affected siblings shared a 9-Mb homozygous region on chromosome 10, (more ...)
This interval contains 57 annotated genes. All genes in the region were evaluated as candidates for disease by assessment of their expression pattern, function, and the phenotype of available targeted knockout mice. Twenty-three genes are not expressed in the eye, 29 genes are expressed in the eye but also broadly in other tissues, and 4 genes (CHAT
, and SLC18A3
) are expressed in the eye and only a small number of other tissues. RBP3
was considered to be the best candidate gene in the region because it is the only gene that is highly and exclusively expressed in the retina. In addition, the photoreceptor degeneration observed in transgenic Irbp−/−
correlates with the disease in this family. Chat−/−
mice have severe neuromuscular deficits26–28
and were therefore excluded as candidate genes. Although it is ubiquitously expressed, the oxoglutarate dehydrogenase-like (OGDHL
) gene was considered to be a functional candidate gene for RP, since oxoglutarate dehydrogenase is the enzyme that catalyzes the conversion of 2-oxoglutarate to succinyl-coA and CO2
within the Krebs cycle. This is the step after the IDH3 reaction, which was recently found to be associated with RP.29
Sequence analysis of the GDF10 gene in an affected individual did not reveal any sequence changes. The individual was homozygous for the “A” allele of a previously documented SNP in the OGDHL gene (rs7090775; c.657G>A; p.Gln219Gln). The individual was apparently heterozygous at three SNPs in the FRMPD2 gene: c.2620A>G, Ser874Gly (SNP rs1346694); c.2889T>C, Ser963Ser (SNP rs2579678); and c.2902A>G; Ile968Val. The heterozygous state of the changes identified in the FRMPD2 gene is inconsistent with the homozygosity of the entire region in the patients. It is explained because this gene is located in a portion of the chromosome known to have undergone a segmental duplication and the primers used to amplify the gene most likely coamplify two or more regions of nearly identical sequence. None of the changes found were deemed pathogenic as most are known SNPs and the Ile968Val change was also found in a normal control.
Sequence analysis of the RBP3
gene identified a homozygous missense mutation (c.3238G>A, p.Asp1080Asn) in exon 2 in all affected individuals (). The p.Asp1080Asn mutation was not observed in 762 unrelated probands with autosomal recessive or isolate RP, including 179 Italian probands, nor was it found among 94 unaffected control subjects of mixed North American ethnicity or 116 Italian control subjects without known photoreceptor disease. Asp1080 is located in the fourth IRBP repeat module, and the homologous residue is completely identical among all four IRBP modules of vertebrate species (). The IRBP protein sequence and structure show similarity to a family of enzymes called C-terminal-processing proteases, which are part of the photosynthetic system in bacteria, algae, and plants.13,30–32
The homologous aspartic acid is also identical among C-terminal processing proteases from these distant species, demonstrating that it is highly conserved during evolution (). IRBP has been found to have structural homology with the enoyl Co-A hydratase/isomerase superfamily by using three-dimensional search programs, but the sequence identity was reported to be low.23
It is, therefore, not surprising that Asp1080 is not conserved among that family.
Figure 2 Alignment of the four IRBP repeat modules of vertebrate species, and C-terminal-processing proteases of bacteria, algae, and plants. The black highlighting indicates identical residues, the gray highlighting indicates conserved residues, and no highlighting (more ...)
Detailed clinical evaluations were performed on two of the four affected patients of this family in the Berman-Gund Laboratory (II-1 and II-3; , ). Patient II-3 reported loss of central vision and onset of nightblindness at age 32, whereas patient II-1 reported loss of central vision at age 60 and no night deficiency even at age 67. Patient II-1 had visual acuities of 20/60 and 20/80 at age 67, and patient II-3 had acuities of 20/200 in each eye at age 46. Patient II-1 had normal color vision on the Farnsworth-D-15 panel, and patient II-3 had a tritan axis of confusion on this test. The visual fields showed a marked constriction with the I-4e and a midperipheral scotoma with the V-4e test light in both patients. The visual fields were more severely diminished in patient II-3 at age 46 than in patient II-1 at age 67. Both patients showed clumped and bone spicule pigment around the periphery. Patient II-3 had waxy pallor of the optic disc and attenuated retinal vessels. Patient II-1 had normal color in each disc with a large area of atrophy just temporal to each disc and had attenuated retinal vessels. The central macula in each patient retained a normal or near-normal color. Both patients had posterior subcapsular cataracts at the first visit. Patient II-3 subsequently had cataract surgery with posterior chamber lens implants. The ERGs in both patients showed a profound loss of rod and cone function. Cone ERG amplitudes of both patients were substantially smaller than normal and could be detected only by computer averaging and narrow band-pass filtering. Cone amplitudes were smaller in patient II-3 at age 46 than in patient II-1 at age 67 (, ). The delayed cone implicit times were compatible with progressive disease.33
The other two affected siblings (patients II-2 and II-4) were examined in Italy and were reported to have fundus findings of RP by ophthalmoscopy and very reduced ERG responses that were virtually nondetectable without computer averaging. The unaffected sibling (individual II-5) had no symptoms or signs of RP. The parents had died in later life and had no visual symptoms.
Clinical Characteristics of Patients II-1 and II-3
Figure 3 Clinical characteristics of two affected siblings demonstrated variability in the severity of the disease. The older brother (II-1) was less severely affected than the younger brother was (II-3). (A) More clumped and bone spicule pigment was noted on (more ...)
Patient II-3 had reduced central retinal thickness on OCT and patient II-1 had increased macular thickness due to cystoid macular edema as possible explanations for their decreased acuities. Patient II-3 was evaluated at several time points which permitted an analysis of the progression of visual decline (). He began treatment with vitamin A palmitate, 15,000 IU/d after his second examination. The annual decline of his remaining visual field area contiguous to the center was 5.4%, and the annual decline in his remaining cone ERG amplitude was 4.4%, with both values being averages across both eyes. The rates of decline of the remaining field area and cone ERG amplitude were slower, respectively, than the average rates of 7.2% and 10% per year reported for patients with typical RP who were not receiving treatment and had been tested in the same way.19
To further examine the structural consequences of substituting Asn for the residue Asp1080, we used the x-ray crystallographic structure of the second module of Xenopus
Currently, the only known crystal structure for IRBP is that of X2IRBP. Although Asp1080 is located in the fourth module of human IRBP, this Asp is strictly conserved among all modules in all IRBPs (). Furthermore, homology modeling and biochemical studies indicate that the individual modules are remarkably conserved and represent functional units of the protein.25,34
shows a ribbon representation of the x-ray crystal structure of X2IRBP, in which an all-trans
retinol molecule (magenta) is docked into the putative binding site.25
Asp1080 in human IRBP corresponds to Asp462 in X2IRBP. Of particular interest is the presence of a nearby arginine residue, Arg464 (corresponding to Arg1082 in module 4 of human IRBP). The crystal structure indicates that Asp462 and Arg464 participate in the formation of a salt bridge. A further remarkable feature of the structure is the close proximity (~9 Å) of Asp462 with the cyclohexane ring of bound retinol (). Asp462 appears to participate in the scaffold of the retinol-binding domain.
Figure 4 Molecular modeling of the Asp1080Asn mutation. (A) Structural modeling suggests that the highly conserved aspartic acid (D) and arginine (R) residues form a salt bridge in the scaffold of the retinol-binding pocket. This ribbon structure represents the (more ...)
Previous studies have called attention to conserved boxes in the primary structure of IRBP.35
The most highly conserved of these contains the Asp1080 residue, as well as several other residues including Arg1082. Substitution of asparagine (Asn) for aspartic acid (Asp) at codon 1080 would disrupt this interaction. Asp and Asn belong to different biochemical classes of amino acid residues. Asp has a carboxyl group that is negatively charged at neutral pH and is thus capable of forming a salt bridge with a positively charged arginine guanidinium group. In contrast, the Asn side chain has a neutral carboxamide group that is unable to form a salt bridge. The highly conserved Asp-Arg salt bridge would therefore be abolished by the Asp1080Asn mutation.
The entire coding region of RBP3 was additionally analyzed in DNA samples of 289 probands with autosomal recessive RP and 106 isolate patients with RP. In addition, 103 probands with autosomal dominant RP, 98 probands with CRD, 41 patients with LCA, and 438 patients with various retinal degenerations and malfunctions were analyzed. A heterozygous nonsense mutation (c.1216G>T, p.Glu406X) was identified in an isolate patient with sector RP. His unaffected brother also carried the nonsense mutation and had the same haplotype on the other allele (). The brother and carrier mother were examined and had normal fundi, ERGs, and visual fields thereby excluding RBP3 as a cause of recessive or dominant disease in this family.
Figure 5 Haplotype analysis in the family members of an isolate patient with sector RP. (A) A heterozygous nonsense mutation (c.1216G>T, p.Glu406X) was identified in the patient and in his unaffected brother, who carried the same haplotype on the other (more ...)
In addition, 23 synonymous and 40 nonsynonymous changes were identified among these 1075 probands (, ). Three variants (p.Thr279Thr, p.Leu699Leu, and p.Val884Met) were known SNPs. Fourteen variants (p.Gly272Gly, p.Pro281Pro, p.Pro308Pro, p.Arg346His, p.Arg544His, p.Ala688Val, p.Val693Met, p.Pro723Leu, p.Asp749Asp, p.Arg833Cys, p.Tyr952Tyr, p.Ser966Ser, p.His1182His, and p.Thr1194Met) were found in more than one patient with different clinical diagnoses, and therefore likely represent rare nonpathogenic variants. For nine variants (p.Ser163Pro, p.Arg267Gln, p.Val282Met, p.Arg544His, p.Val593Ala, p.Ala688Val, p.Arg747Cys, p.Asn785Lys, and p.Arg833Cys) pathogenicity was excluded because the variant did not segregate with the disease in the family. The nonsynonymous changes did not affect any of the residues that are conserved among all IRBP modules of vertebrates and C-terminal- processing proteases of bacteria, algae, and plants. All these variants were detected infrequently in only one or two patients and were all found in the heterozygous state. None of the patients carrying these changes had a second heterozygous change that could be located on the other allele except one isolate patient with RP who was heterozygous for p.Glu956Lys and p.Val1059Ile. Family members of this patient were not available, and it could therefore not be determined whether these variants were allelic or syntenic.
Synonymous Sequence Changes Detected in RBP3 in 1075 Patients
Nonsynonymous Sequence Changes Detected in RBP3 in 1075 Patients