1000 Reports JOVS, April 1999, Vol. 40, No. 5 6. Bunt-Milam AH, SaariJC. Immunocytochemical localization of two from bovine retina and retinal pigment epithelium. / Biol Chem. retinoid-binding proteins in vertebrate retina. / Cell Biol. 1983;97: 1982;257:13329-13333. 703-712. 11. Saari JC, Bredberg D, Noy N. Control of substrate flow at a branch 7. Bok D. The retinal pigment epithelium: a versatile partner in in the visual cycle. Biochemistry. 1994;33:3106-3112. vision./ Cell Sci. 1993;17:189-195. 12. Morimura H, Berson EL, Dryja TP. Recessive mutations in the 8. Bernstein PS, Law WC, Rando RR. Isomerization of all-trans-retin- RLBP1 gene encoding cellular retinaldehyde-binding protein in a oids to 11-czs-retinoids in vitro. Proc Natl Acacl Sci USA. 1987;84: form of retinitis punctata albescens. Invest Ophthalmol Vis Sci. 1849-1853. 1999;40:1000-1004. 9. Deigner PS, Law WC, Canada FJ, Rando RR. Membranes as the 13. Broman KW, Murray JC, Sheffield VC, White RL, Weber JL. Compre- energy source in the endergonic transformation of vitamin A to hensive human genetic maps: individual and sex-specific variation in 11-cfe-retinol. Science. 1989;244:968-971. recombination. 1998 http://www.marshmed.org/genetics/. 10. SaariJC, Bredberg L, Garwin GG. Identification of the endogenous 14. Hudson T, Stein L, Gerety S, et al. An STS-based map of the human retinoids associated with three cellular retinoid-binding proteins genome. Science. 1995;270:1945-1954. Recessive Mutations in the missense: one was a frameshift, and one affected a canon- ical splice donor site. RLBP1 Gene Encoding Cellular CONCLUSIONS. Recessive mutations in the RLBP1 gene are Retinaldehyde-Binding Protein an uncommon cause of retinal degeneration in humans. The phenotype produced by RLBP1 mutations seems to in a Form of Retinitis be a form of retinitis punctata albescens. (Invest Ophthal- Punctata Albescens mol Vis Sci. 1999;40:1000 -1004) 1 2 Hiroyuki Morimura, Eliot L Berson, and he first step in vision occurs when a photon converts an 1 1 Thaddeus P. Dryja ' T 1 l-cis retinal chromophore, which is covalently linked to rod or cone opsin, to the all-trans isomer.1 In mammals, this PURPOSE. TO determine the frequency and spectrum of reaction takes place in the outer segments of the photorecep- mutations in the RLBP1 gene encoding cellular retinalde- tor cells of the retina. The aH-trans chromophore subsequently hyde-binding protein (CRALBP) in patients with heredi- leaves rod or cone opsin and travels as all-fraws-retinol to a 2 tary retinal degeneration. neighboring retinal pigment epithelial cell, where it is con- verted through a series of intermediates back to 11-m-retinal- METHODS. The single-strand conformation polymorphism dehyde. The protein CRALBP seems to play a role in this (SSCP) technique and a direct genomic sequencing tech- pathway. CRALBP is present in the retinal pigment epithelium nique were used to screen the coding exons of this gene and the Miiller cells of the retina.3 The protein forms com- (exons 2-8) for mutations in 324 unrelated patients with plexes with regenerated 11-a's-retinaldehyde and its immediate recessive or isolate retinitis pigmentosa, retinitis punctata precursor 11-czs-retinol.4'5 Still uncertain is whether these com- albescens, Leber congenital amaurosis, or a related dis- plexes are essential intermediates in this pathway or whether ease. Variant DNA fragments revealed by SSCP analysis CRALBP has another physiological role. were subsequently sequenced. Selected alleles that altered The requirement for CRALBP in the retina was highlighted the coding region or intron splice sites were evaluated by a recent report of the missense mutation Argl50Gln in the further through segregation analysis in the families of the RLBP1 gene (chromosome 15q26)6 in a family from India with index cases. a recessive retinal degeneration that was termed retinitis pig- mentosa.7 The mutation was homozygous in the affected mem- RESULTS. Four novel mutations were identified in this gene bers but not in the unaffected members of an 11-member among three unrelated patients with recessively inherited sibship that was the product of a first-cousin marriage. Evi- retinitis punctata albescens. Two of the mutations were dence for the pathogenicity of this mutation came from the observations that the mutant protein in vitro was less water soluble than normal and that it did not bind to 11-m-retinal- From the 'Ocular Molecular Genetics Institute and the 2Berman- dehyde.7 In the present report, we provide additional evi- Guncl Laboratory for the Study of Retinal Degenerations, Harvard Med- dence, through the identification of mutations in patients of ical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. European ancestry, that recessive defects in the human RLBP1 gene are pathogenic. Furthermore, the clinical findings from Supported by Grants EY08683 and EY00169 from the National Eye 8 Institute, National Institutes of Health, Bethesda, Maryland; the Foun- our cases and those in the report by Burstedt et al in this issue dation Fighting Blindness, Hunt Valley, Maryland; and the Massachu- suggest that the phenotype can be distinguished from typical setts Lions Eye Research Fund, Northborough; and by private dona- retinitis pigmentosa. tions to the Taylor Smith Laboratory and the Ocular Molecular Genetics Institute. TPD is a Senior Scientific Investigator of Research to Prevent Blindness, New York, New York. METHODS Submitted for publication October 16, 1998; accepted December 11, 1998. Ascertainment of Patients Proprietary interest category: N. Reprint requests: Thaddeus P. Dryja, MD, Massachusetts Eye and This study, which involved human subjects, conformed to the Ear Infirmary, 243 Charles Street, Boston, MA 02114. tenants of the Declaration of Helsinki. The index patients in Downloaded from iovs.arvojournals.org on 09/25/2021 IOVS, April 1999, Vol. 40, No. 5 Reports 1001 this study had a diagnosis of retinal degeneration or malfunc- containing 20 mM Tris-HCl (pH 8.4 or 8.6), 0.25 to 1.5 mM tion made through ophthalmologic examination including MgCl2, 50 mM KC1, 0.02 mM deoxyadenosine triphosphate electroretinography (ERG). Most patients resided in the United (dATP), 0.02 mM deoxythymidine triphosphate (dTTP), 0.02 States or Canada. mM deoxyguanosine triphosphate (dGTP), and 0.002 mM de- Patients with autosomal recessive retinitis pigmentosa had oxycytidine triphosphate (dCTP), 0.6 mCi [a-32P] deoxycyti- unaffected parents and at least one affected sibling or were the dine triphosphate (3000 Ci/millimole), 0.1 mg/ml bovine se- offspring of a consanguineous mating. If all affected siblings rum albumin, 0% or 10% dimethyl sulfoxide, and 0.25 units of with retinitis pigmentosa in a sibship were males, the possibil- Taq polymerase. The pH, Mg2+ concentration, and presence ity of X-linked disease was evaluated through an ophthalmo- or absence of 10% dimethyl sulfoxide were tailored to each logic examination of the mother, including an ERG to search primer pair to yield optimal amplification. After initial denatur- for the carrier state of X-linked retinitis pigmentosa; families ation (94°C for 5 minutes), 35 cycles of PCR amplification were with X-linked retinitis pigmentosa were excluded from this performed. Each cycle consisted of denaturation (94°C for 30 study. Some isolate cases were included. They had no affected seconds), primer annealing (54°C for 30 seconds), and exten- relatives and were not the offspring of a consanguineous mat- sion (71°C for 30 seconds). The final extension was at 71°C for ing. Atypical retinitis pigmentosa was diagnosed in these pa- 5 minutes. The amplified DNA fragments were heat denatured, tients if they had some combination of the following features: and the single-stranded fragments were separated, each reduced but unusually large ERGs for their age, an unusual through two sets of 6% polyacrylamide gels, one set with and pigmentary pattern, or no abnormality of dark adaptation one without 10% glycerol. Gels were run at 8 W to 12 W for 5 threshold after 45 minutes of dark adaptation; all were isolate to 20 hours before drying and autoradiography. cases. Variant bands were evaluated by sequencing of corre- Patients with retinitis punctata albescens had ERG findings sponding PCR-amplified DNA segments using a cycle-sequenc- indicating a generalized retinal degeneration, elevated dark ing protocol in a radiolabeled terminator cycle sequencing kit adaptation threshold after 45 minutes of dark adaptation, and (Thermo Sequenase; Amersham Life Science, Cleveland, OH). fundi that showed attenuated arterioles and subretinal yellow Sequence variations expected to affect protein sequence or or yellow-white deposits as a predominant feature. Some also expression were evaluated further by recruiting the relatives of had intraretinal pigment deposits. the index patient to participate in a study to determine Patients with Leber congenital amaurosis had absent or whether the variant allele cosegregated with the disease. For severely diminished vision within the first year of life and this purpose, leukocyte DNA samples from the relatives were markedly diminished ERGs; all had unaffected parents. Patients analyzed by SSCP or direct genomic sequencing for the pres- with fundus albipunctatus had normal retinal vessels, diffuse ence of the variant sequence. subretinal white deposits, elevated dark adaptation thresholds, and reduced ERGs after 45 minutes of dark adaptation that became normal after 6 to 12 hours of dark adaptation. RESULTS Patients with other forms of stationary night blindness had fundi without the stigmata of retinitis pigmentosa, elevated Based on the previously reported 5' untranslated, 3' untrans- dark-adaptation thresholds, reduced rod ERGs, and cone ERGs lated, and flanking intron sequences for the human RLBPJ 6 that were normal when corrected for age and refractive error. gene, we developed assays using the SSCP technique to Control subjects had no known blood relative with hereditary screen for mutations in the entire coding sequence and the retinal degeneration and no symptoms of retinal malfunction.