Mutations in the CRB1 Gene Cause Leber Congenital Amaurosis

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: EDWIN M. STONE, MD, PHD Mutations in the CRB1 Gene Cause Leber Congenital Amaurosis

Andrew J. Lotery, MD, FRCOphth; Samuel G. Jacobson, MD, PhD; Gerald A. Fishman, MD; Richard G. Weleber, MD; Anne B. Fulton, MD; P. Namperumalsamy, MD; Elise He´on, MD; Alex V. Levin, MD; Sandeep Grover, MD; Justin R. Rosenow, BS; Kelly K. Kopp, BS; Val C. Sheffield, MD, PhD; Edwin M. Stone, MD, PhD

Objectives: To test the hypothesis that mutations in the amino acid–altering sequence variations in the CRB1 gene CRB1 gene cause Leber congenital amaurosis (LCA) and, (P=.003). if so, to describe the ocular phenotype of patients with LCA who harbor CRB1 sequence variations. Conclusions: In our cohort of patients with LCA, coding sequence variations were observed in the CRB1 gene Patients: One hundred ninety probands with a clinical more frequently than in any of the other 5 known LCA- diagnosis of LCA were selected from a cohort of 233 pro- associated genes. Likely disease-causing sequence varia- bands ascertained in 5 different countries. The remain- tions have now been identified in 64 (28%) of 233 sub- ing 43 probands (18%) were excluded because they har- jects in this cohort. bored sequence variations in previously identified LCA genes. Clinical Relevance: Molecular diagnosis can confirm and clarify the diagnosis in an increasing fraction of pa- Methods: One hundred ninety unrelated individuals with tients with LCA. As genotype data accumulate, clinical LCA were screened for coding sequence mutations in the phenotypes associated with specific mutations may be es- CRB1 gene with single-strand conformation polymor- tablished. This will facilitate the counseling of patients phism analysis followed by automated DNA sequencing. regarding their visual prognosis and the likelihood of associated systemic anomalies. Results: Twenty-one of the 190 probands (9% of the total cohort of 233) and 2 (1.4%) of 140 controls harbored Arch Ophthalmol. 2001;119:415-420

EBER CONGENITAL amauro- most often neurological, are observed in sis (LCA) is a term used to a small number of individuals. The visual refer to a group of inherited outcome can vary widely. Some children retinal disorders character- with LCA maintain measurable acuity for ized by severe, bilateral vi- decades, while others are completely and sualL impairment in infancy. The pupil- permanently blind in infancy.1-3 From the University of Iowa lary responses are sluggish, nystagmus is College of Medicine, Iowa City frequently present, and the electroretino- For editorial comment (Drs Lotery, Sheffield, and graphic responses are markedly attenu- see page 426 Stone, Mr Rosenow, and ated. The eyes of affected children ap- Ms Kopp); the Scheie Eye These disparate clinical findings are Institute, Philadelphia, Pa pear grossly normal, with clear media, pink (Dr Jacobson); the University of optic discs, and completely attached reti- in part owing to the genetic heterogene- Illinois Eye and Ear Infirmary, nas. The fundus can initially appear nor- ity of this disorder. To date, mutations in Chicago (Drs Fishman and mal, although many patients exhibit a de- 5 genes have been reported to cause a sub- Grover); the Casey Eye gree of vascular attenuation when first set of LCA. These include GUCY2D, en- Institute, Portland, Ore examined. Pigmentary abnormalities, rang- coding retinal guanylate cyclase4; RPE65, (Dr Weleber); Children’s ing from white dots to nummular dark pig- encoding a retinal pigment epithelium– Hospital, Boston, Mass ment clumps and even bone spicule–like specific 65 KD5,6; CRX, encoding the cone- (Dr Fulton); Aravind Eye changes, can be seen in some patients. rod homeobox-containing gene7; TULP1, Hospital, Madurai, India Coloboma-like lesions in the macula are encoding the Tubby-like protein 18; and (Dr Namperumalsamy); the a less common finding. High refractive er- AIPL1, encoding aryl-hydrocarbon inter- Eye Research Institute of 9 Canada (Dr He´on), and rors are sometimes present, and older pa- acting protein–like 1. The discovery of The Hospital for Sick tients may develop keratoconus, presum- these genes has stimulated the search for Children (Dr Levin), ably from the chronic trauma of the novel therapies for this currently untreat- Toronto, Ontario. oculodigital reflex. Systemic disorders, able disease.10

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 also cause some cases of LCA. Indeed, 3 of the known LCA- associated genes—RPE65, CRX, and TULP1—are each SUBJECTS, MATERIALS, known to cause some cases of RP.11-14 AND METHODS CRB1 is a recently discovered gene that is respon- sible for a distinctive form of autosomal recessive RP re- ferred to as RP12.15 Clinically, RP12 exhibits the un- Informed consent was obtained from all study pa- usual feature of preservation of the periarteriolar retinal tients or their legal guardians. Two hundred thirty- pigment epithelium. Some individuals affected with RP12 three probands with LCA were ascertained from the first experience visual loss in childhood and some have United States (146), Canada (43), India (41), Israel 16 (3), and Switzerland (1). Forty-three of these pa- hyperopic refractive errors. The purpose of this study tients were known from a previous study2 to harbor was to determine whether mutations in CRB1 might cause a likely disease-causing sequence variation in a pre- LCA in some individuals. viously described LCA gene. These patients were excluded from the present study because the likeli- RESULTS hood of finding additional disease-associated mutations was judged to be too low to warrant fur- ther consumption of their often irreplaceable DNA MOLECULAR RESULTS samples. The DNA was extracted from peripheral blood using a previously described protocol.17 One Thirty instances of 20 different amino acid–altering se- hundred ninety probands were screened for muta- quence variations were observed in this study (Table 1). tions in the coding sequence of the CRB1 gene with Twenty-eight of these changes were found among 21 LCA single-strand conformation polymorphism analysis. probands while, only 2 were observed in the heterozy- Ninety-four control subjects from Iowa and 46 con- gous state in 2 control individuals (P=.003). Six LCA pro- trol subjects from India were screened in an identi- bands were each found to harbor 2 amino acid–altering cal fashion. The primer sequences used for the single- changes (presumably on different alleles). One proband strand conformation polymorphism screening have was homozygous (for Cys948Tyr) and another was ho- 15 been previously described with the exception that, mozygous for Cys480Gly. The other 4 probands were com- for exon 2, we used the following primer sequences: pound heterozygotes. Fifteen of the probands exhibited forward, GCAGCACAAAGGTCACAAG and re- verse, TCCTGATGGCAAATACCTCC. The poly- 1 heterozygous amino acid–altering change (Table 1). merase chain reaction (PCR) amplification prod- A total of 5 synonymous codon changes were ob- ucts were denatured for 3 minutes at 94°C and then served—each unique, 4 present in single LCA probands electrophoresed on 6% polyacrylamide, 5% glycerol and 1 in an Indian control. These were significantly more gels at 25 W for approximately 3 hours. The gels were likely to be found in an LCA proband with a single amino then stained with silver nitrate.18,19 The PCR prod- acid–changing CRB1 variation (2/15) than in LCA pro- ucts from samples with aberrant electrophoretic pat- bands without an amino acid–altering CRB1change terns were then sequenced bidirectionally with fluo- (2/169; P=.03) or in controls (1/140; P=.03). rescent dideoxynucleotides on an automated DNA Four different intronic single nucleotide polymor- sequencer (ABI model 377; PE Applied Biosystems, phisms (SNPs) were found among the study partici- Foster City, Calif). Clinical records of 19 probands and 1 affected sibling with amino acid–changing se- pants (Table 1), but these were not significantly skewed quence variations in the CRB1 gene were available toward LCA probands or controls (either singly or as a for review, and the resulting phenotypic informa- group). The most common intronic SNP was located 12 tion was tabulated. bases upstream from the start of exon 2 and was identi- cally distributed (thymine, 40%; adenine, 60%) among the alleles of LCA probands and control individuals. A single instance of a 4–base pair deletion in intron 2 was The fraction of LCA resulting from these 5 genes var- observed in a single LCA proband. ies widely. TULP1 has been associated with the LCA phe- The most common amino acid–changing variant that notype in only a single family from the Dominican Re- we observed in the LCA cohort (Cys948Tyr) has been public.8 The contribution of the other genes to the previously observed in patients with RP12.15 The other worldwide prevalence of LCA has been reported to be 17 amino acid–changing variants have not been previ- CRX, 2.8%; GUCY2D, 6.3%; RPE65, 6.8%2; and AILP1, ously reported. 7%.11 Thus, the molecular cause for most cases of LCA is still unknown. CLINICAL RESULTS Retinitis pigmentosa (RP) is a term used to refer to another clinically and genetically heterogeneous group of Clinical records were available for 18 of the LCA pro- retinal degenerations that are closely related to LCA. In fact, bands with amino acid–altering sequence variations and the distinction between LCA and RP is largely based on age 1 affected sibling (Table 2). Nystagmus was noted in of onset of the visual dysfunction. Patients whose condi- 18 of 19 patients, with “roving eye movements” re- tions are diagnosed when they are younger than 1 year are corded for the remaining patient. The visual acuity ranged likely to be classified as having LCA, while those older than from 20/40 in 1 eye of 1 patient, to light perception, with 1 year who develop photoreceptor degeneration are more a median of 20/250. A refraction was recorded in 37 likely to be diagnosed as having RP. The similarity of these eyes of 19 patients, and the average spherical equiva- conditions suggests that genes known to cause RP might lent was +4.9 diopters (D) (range, −10.00 to +9.00 D).

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LCA Alleles Control Alleles Variation DNA Sequence Change (n = 380) (n = 280) Nonconservative, charge/size/polarity (patient No.) 1 bp del codon 37 T 1 0 2 bp ins codons 86-87 (15)† GT 1 0 5 bp del codons 143-144 GATTC 1 0 7 bp del codons 204-207 (3)† AAATAGG 2 0 Thr289Met ACG→ATG 1 0 Cys480Arg (3)† TGT→CGT 1 0 Arg764Cys (9)† CGT→TGT 2 0 Arg769Gln CGC→CAG 0 1 Ͼ100 bp poly A ins codon 812-813 AAAA. . . 1 0 4 bp del codons 850-851 GGCT 1 0 1 bp ins codon 871 (5)† T 1 0 Gly1205Arg GGA→AGA 1 0 Asn1317His AAC→CAC 1 0 Cys1332Stop TGC→TGA 1 0 Conservative Phe144Val (2)† TTC→GTC 1 0 Cys383Tyr (5)† TGT→TAT 1 0 Cys480Gly† TGT→GGT 2 0 Cys681Tyr (7)† TGT→TAT 1 0 Cys948Tyr (9)(15)(16,16)† TGT→TAT 7 0 Arg1331His CGC→CAC 0 1 Synonymous codon changes Leu470Leu (2)† CTG→CTA 1 0 Thr476Thr ACC→ACT 1 0 Asn549Asn (7)† AAT→AAC 1 0 Pro941Pro CCG→CCA 0 1 Asn1057Asn AAC→AAT 1 0 Intronic variations T12bp5Јto exon 2 153 73 A12bp5Јto exon 2 227 111 4 bp del 48 bp 5Ј to exon 3 TGCT 1 0 C→T35bp3Јto exon 5 21 G→T54bp5Јto exon 6 20

*LCA indicates Leber congenital amaurosis; bp, base pair; del, deletion at; and ins, insertion at. †Eight patients (including patients 2, 3, 5, 7, 9, 15, and 16 from Table 2) each harbored 2 rare CRB1 sequence variants, presumably on different alleles. These notes indicate which specific mutations were present in each of these patients (eg, individual 16 was homozygous for Cys948Tyr).

Table 2. Clinical Features*

Study No. Variation Age, y Visual Acuity, OD Refraction, OD Visual Acuity, OS Refraction OS ERG 1 5 bp del 143-144 4 20/80 +7.50 + 3.00 ϫ 90 20/80 +7.50 + 2.00 ϫ 90 ↓ 2 Phe144Val, Leu470Leu 6 20/200 +5.75 + 1.25 ϫ 095 20/400 +6.00 + 0.75 ϫ 080 ↓ 3 7 bp del 204-207, Cys480Arg 46 LP +4.00 + 1.00 ϫ 10 LP +5.75 . . . 4 Thr298Met 1 20/3700 +1.50 20/3700 +2.00 ↓ 5 Cys383Tyr, 1 bp ins T 871 35 CF +6.00 + 0.50 ϫ 090 HM +4.25 + 1.25 ϫ 090 ND 6 7 bp del 612-619 5 20/200 +6.75 + 1.00 ϫ 090 20/400 +6.25 + 1.00 ϫ 084 ↓ 7 Asn549Asn, Cys681Tyr 61 CF +6.50 CF +8.50 . . . 8 Arg764Cys 9 20/200 +2.75 + 0.75 ϫ 121 20/200 +2.25 + 2.25 ϫ 090 ND 9 Cys948Tyr, Arg764Cys 10 20/200 +6.00 + 1.00 ϫ 55 20/100 +7.25 + 1.75 ϫ 155 . . . 10 4 bp del 850-851 21 20/40 −0.50 + 1.00 ϫ 80 20/400 −0.25 + 0.25 ϫ 60 ND 11 Cys948Tyr 4 20/200 +7.25 + 0.75 ϫ 180 20/200 +7.50 . . . 12 Cys948Tyr 15 20/250 +7.00 + 0.75 ϫ 47 20/200 +7.00 + 1.25 ϫ 100 ND 13 Cys948Tyr 35 CF +8.00 HM +7.50 . . . 14 Cys948Tyr 20 20/400 . . . 20/400 +6.50 + 2.00 ϫ 70 ... 15 Cys948Tyr, 2 bp ins codon 86-87 19 CF +8.50 20/400 +6.00 ND 16 Cys948Tyr, Cys948Tyr 0.6 F&FL +6.50 F&FL +6.50 ↓ 17 Gly1205Arg 9 20/50 +3.75 + 1.50 ϫ 100 LP +2.50 + 1.00 ϫ 180 ND 18 Asn1317His 17 20/200 −10.00 + 1.00 ϫ 90 20/250 −9.50 + 1.00 ϫ 90 ↓ 19 Cys1332Stop 10 LP +4.50 LP +4.50 . . .

*ERG indicates electroretinogram; bp, base pair; del, deletion at; ↓, reduced in amplitude; LP, light perception; ellipses, not recorded; ins, insertion at; CF, counting fingers; HM, hand movements; ND, nondetectable; and F&FL, fixes and follows light.

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 Figure 1. Midperipheral fundus of patient 13 at age 19 years. The visual Figure 4. Posterior pole of the right eye of patient 12 at age 11 years. The acuity at this visit was counting fingers. Small white spots are admixed with visual acuity on that visit was 20/300. The optic nerve head appears fairly nummular pigment clumps at the level of the retinal pigment epithelium. normal, but the fovea is not well developed. The most striking finding is a zonal atrophy or hypoplasia of the choriocapillaris and retinal pigment epithelium temporally and inferiorly, which largely spares the macula.

Figure 2. Midperiphery of patient 9 at age 10 years. The visual acuity at this visit was 20/200 OD. Numerous small white spots are admixed with fairly typical bone spicule–like pigmentation. Figure 5. Posterior pole of the right eye of patient 4 at age 1 year. The visual acuity in this eye was 20/3700 (with Teller acuity cards). The retinal vasculature is attenuated, and a large area of atrophy or hypoplasia of the retinal pigment epithelium and choriocapillaris is present in the macula. This finding has often been called a macular coloboma.

Figure 3. Posterior pole of patient 10 at age 21 years. The visual acuity in this eye at this visit was 20/400 OS. The disc appears healthy, but there is mild vascular attenuation. There are diffuse fine white spots throughout the fundus. There is an oval area of retinal pigment epithelium (RPE) pigment disruption centered on the fovea and a few clumps of bone spicule–like Figure 6. Posterior pole of patient 7 at age 61 years. The visual acuity at this intraretinal pigment as well. There is a circular area of atrophy or hypoplasia visit was counting fingers. The optic disc is obscured by an area of vitreous of the RPE and choriocapillaris along the superotemporal arcade at the opacity. There is widespread pigment disruption of the retinal pigment margin of the photograph. epithelium (RPE) in addition to some overlying bone spicule–like changes in the retina. There is a circular area of atrophy or hypoplasia of the RPE and choriocapillaris just temporal to the normal location of the fovea. Electroretinogram (ERG) data were available for 12 patients. In all cases the full-field ERG was markedly re- and Figure 3) were specifically mentioned in the notes duced, and in 7 of 12 patients it was nondetectable. De- of 5 of 14 patients. Large zones of retinal pigment epi- tailed retinal notes were available for 14 patients. thelium atrophy were present in 1 patient (Figure 4), Nummular pigment clumps were seen in 9 of 14 pa- and coloboma-like lesions of the macula were present in tients (Figure 1), and white spots (Figure 1, Figure 2, 3 others (Figure 5 and Figure 6). Keratoconus was

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 present in 2 siblings, and periarteriolar preservation of sometimes quite different—phenotype. These include (1) the retinal pigment epithelium was seen in a different pair the RDS gene, which was initially found to cause RP but of siblings. was later discovered to be associated with pattern dystrophy and other maculopathies20-22; (2) rhodopsin and COMMENT PDEB, which were initially associated with RP but later found to be associated with stationary night blind- Leber congenital amaurosis is a severe, genetically het- ness23,24; (3) CRX and GUCY2D, which each cause LCA erogeneous, autosomal recessive retinal dystrophy. In- and cone-rod dystrophy4,7,25,26; and (4) RPE65, which cluding CRB1, mutations in 6 genes have now been shown causes LCA and RP.5,12 to cause the LCA phenotype. In our complete cohort of This lack of perfect correspondence between clinical 233 LCA probands, amino acid–changing CRB1 vari- and molecular nomenclature systems is not a serious ob- ants were found in 21 (9%), making CRB1 the most com- stacle to accurate communication as long as one makes it monly mutated gene in this group of patients. However, clear which system is being used in a specific situation. Both most patients with LCA in our cohort do not harbor se- systems serve a useful purpose. Infants with severely im- quence variations in any of these 6 genes. This is in part paired vision, nystagmus, no striking ophthalmoscopic ab- because other LCA genes undoubtedly exist, and in part normalities, hyperopia, and a nonrecordable ERG will con- because some mutations will be missed by PCR-based as- tinue to be given the clinical diagnosis of LCA, and it remains says of the coding sequences such as the ones employed an important goal to ultimately identify all the genes that in this study. Such assays will not detect deletions in- are capable of causing this phenotype. In contrast, scien- volving the primer sites or mutations in the promoters. tists studying the biological roles of individual genes like Indeed, even among the 21 patients with amino acid– CRB1 will continue to be interested in identifying all the changing CRB1 variants in this study, we were unable to different phenotypes that can be caused by variations in detect any CRB1 variations in 15 (36%) of the alleles. Fur- that particular gene. In the present study, the frame of ref- ther extensive investigation of these 15 alleles is war- erence was the clinical diagnosis of LCA, and the clini- ranted to try to identify additional regions of the gene cally relevant finding is that 9% of a carefully defined LCA that will be fruitful to reexamine in the entire cohort. cohort harbor mutations in the CRB1 gene. The distribution of the synonymous codon variants Less than 5 years ago, no patients with LCA could observed in this study is interesting. Although one would be molecularly diagnosed. With the addition of CRB1 to not usually expect such changes to contribute to an al- the panel of LCA genes, we now have a greater than 25% tered phenotype (because the predicted protein sequence chance of detecting a disease-causing mutation in a new is not changed), the significant distribution among pa- patient with LCA. Molecular diagnosis is important for tients with LCA vs controls (as well as among patients with providing accurate counseling about recurrence risk to LCA with CRB1 mutations vs those without mutations) sug- affected families, as well as for the identification of spe- gests that these alleles may also be disease causing, per- cific molecular subsets of patients for future studies of haps by altering the stability or processing of the CRB1 mes- novel interventions. senger RNA. Functional assays will be necessary to support this hypothesis. Evaluation of a common SNP found within Accepted for publication January 5, 2001. intron 1 does not show a disease association, which indi- This study was supported in part by National Institutes cates that our mutation screening assay is unlikely to be of Health, Bethesda, Md, grants EY10539 and EY05627; the missing a common CRB1 mutation that would account for Foundation Fighting Blindness, Hunt Valley, Md; the Grous- a large portion of the as yet undetected mutant alleles. beck Family Foundation, Stanford, Calif; the Carver Endow- The biochemical function of CRB1 is currently un- ment for Molecular Ophthalmology, Muscatine, Iowa; the known. Comparison of CRB1 with the homologous Dro- Daniel Matzkin Research Fund; the Grant Healthcare Foun- sophila melanogaster crumbs protein (CRB) suggests that dation; and an unrestricted grant from Research to Prevent it may be involved in neuronal development of the retina.15 Blindness Inc, New York, NY. Dr Lotery is a recipient of a Re- This hypothesis would be consistent with the early on- search to Prevent Blindness Career Development Award. set of retinal dysfunction seen in the patients in this study. The authors thank Creig Hoyt, MD; James Jan, MD; By- Although the number of patients with LCA in whom we ron Lam, MD; Ronald Carr, MD; John Heckenlively, MD; found CRB1 coding sequence variations is small, we ob- William Scott, MD; Douglas Fredrick, MD; Ehud Zamir, MD; served 2 fairly constant phenotypic features: the pres- SaulMerin,MD;FrancisMunier,MD;ArleneDrack,MD;Terry ence of moderate to high hyperopia and the relatively early Schwartz,MD;JeanBennett,MD;AlessandroIannaccone,MD; appearance of white spots and nummular pigment clumps. Maria Musarella, MD; and Benedetto Falsini, MD, for shar- The preservation of the periarteriolar retinal pigment epi- ing their patients with us for this study. The authors also thank thelium phenotype (characteristic of RP1215) was seen LuanStreb,BA;ChristineTaylor,BS;HeidiHaines,MS;Louisa in 2 of our patients. This is a good example of an increas- Affatigato, BS; and Gretel Beck, BA, for their excellent tech- ingly common situation in which an established, clini- nical assistance and Jessica Emmons, BA; Elaine De Castro, cally based nomenclature system fails to correspond neatly BS; and Leigh Gardner, BA, for clinical coordination. with molecular reality at the DNA level. There are now Corresponding author and reprints: Edwin M. Stone, numerous examples in the field of retinal degeneration MD, PhD, Department of Ophthalmology and Visual Sci- research in which a gene that is initially shown to cause ences, The University of Iowa College of Medicine, 200 disease in patients with one clinical phenotype is later Hawkins Dr, Iowa City, IA 52242 (e-mail: edwin-stone@ found to cause disease in patients with another— uiowa.edu).

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