Am. J. Hum. Genet. 63:1307–1315, 1998 A Range of Clinical Phenotypes Associated with Mutations in CRX, a Photoreceptor Transcription-Factor Gene Melanie M. Sohocki,1 Lori S. Sullivan,1,2 Helen A. Mintz-Hittner,2 David Birch,3 John R. Heckenlively,4 Carol L. Freund,5 Roderick R. McInnes,5 and Stephen P. Daiger1,2 1Human Genetics Center, School of Public Health, and 2Department of Ophthalmology and Visual Science, The University of Texas Health Science Center, Houston; 3Retina Foundation of the Southwest, Dallas; 4Jules Stein Eye Institute, University of California, Los Angeles; and 5Program in Developmental Biology, Research Institute, Hospital for Sick Children, Toronto Summary is the diagnosis of some of these diseases difficult because of overlapping phenotypes, but mutations within a single Mutations in the retinal-expressed gene CRX (cone-rod gene may cause very different phenotypes. For example, homeobox gene) have been associated with dominant different mutations in one gene, peripherin/RDS, have cone-rod dystrophy and with de novo Leber congenital been associated with several forms of retinal degenera- amaurosis. However, CRX is a transcription factor for tion, such as cone-rod dystrophy, cone degeneration, and several retinal genes, including the opsins and the gene retinitis pigmentosa, affecting both the macula and the for interphotoreceptor retinoid binding protein. Because panretinal structures (Weleber et al. 1993; Wells et al. loss of CRX function could alter the expression of a 1993; Keen et al. 1994; Nakazawa et al. 1994, 1996). number of other retinal proteins, we screened for mu- Recently, mutations within the photoreceptor-ex- tations in the CRX gene in probands with a range of pressed gene CRX (cone-rod homeobox gene; MIM degenerative retinal diseases. Of the 294 unrelated in- 120970) have been reported to cause dominant cone- dividuals screened, we identified four CRX mutations rod dystrophy at the CORD2 locus (Freund et al. 1997; in families with clinical diagnoses of autosomal domi- Swain et al. 1997); in addition, de novo CRX mutations nant cone-rod dystrophy, late-onset dominant retinitis have been found in isolated cases of Leber congenital pigmentosa, or dominant congenital Leber amaurosis amaurosis (Freund et al. 1998). CRX belongs to the (early-onset retinitis pigmentosa), and we identified four orthodenticle homeobox (OTX) family of homeobox additional benign sequence variants. These findings im- genes, members of which are involved in the develop- ply that CRX mutations may be associated with a wide ment of anterior head structures and other embryonic range of clinical phenotypes, including congenital retinal features (Finkelstein and Boncinelli 1994). The three ex- dystrophy (Leber) and progressive diseases such as cone- ons of CRX encode a 299–amino acid polypeptide, rod dystrophy or retinitis pigmentosa, with a wide range which shows a high degree of sequence similarity to of onset. OTX and OTX-related homeodomain proteins: the CRX homeobox sequence has up to 88% identity with the homeobox sequences of other members of the OTX gene family. In addition to the homeodomain, CRX and the other OTX-related genes share two highly conserved Introduction peptide sequences, a 13–amino acid WSP motif and a 12–amino acid OTX tail (Furukawa et al. 1997; Swain Inherited retinal diseases are exceptionally heterogene- et al. 1997; Freund et al. 1998). CRX is also expressed ous, both genetically and phenotypically. A total of 96 in the pineal gland and is a transcription factor for pin- genes causing inherited retinal diseases have been eal-specific genes (Li et al. 1998). mapped to chromosomal sites in man, but less than half CRX binds specifically to conserved sequences up- have been cloned (RetNet; Daiger et al. 1998). Not only stream of several photoreceptor-specific genes, including the opsins, and CRX activates transcription of inter- Received April 14, 1998; accepted for publication September 12, photoreceptor retinoid binding protein, arrestin, and b- 1998; electronically published October 16, 1998. phosphodiesterase (Chen et al. 1997; Furukawa et al. Address for correspondence and reprints: Dr. Stephen P. Daiger, 1997). Because CRX appears to affect many retinal Human Genetics Center, The University of Texas Health Science genes, mutations within the CRX gene may be associated Center, P.O. Box 20334, Houston, TX 77225-0334. E-mail: sdaiger with different retinal-disease phenotypes. @utsph.sph.uth.tmc.edu ᭧ 1998 by The American Society of Human Genetics. All rights reserved. We screened the CRX gene in a large cohort of patients 0002-9297/98/6305-0008$02.00 with a wide range of clinical diagnoses (table 1). We 1307 1308 Am. J. Hum. Genet. 63:1307–1315, 1998 Table 1 tained and tested are indicated by “DNA” to the upper Clinical Diagnoses of Individuals Screened for CRX Mutations, in right of the symbol. This Study No. of Unrelated DNA Isolation Clinical Diagnosis Probands Tested DNA was isolated from peripheral blood by use of Retinitis pigmentosa, autosomal the Puregene DNA extraction kit (Gentra), in accor- dominant 164 dance with the manufacturer’s instructions. Retinitis pigmentosa, autosomal recessive 22 Retinitis pigmentosa, isolated 63 Genotyping Cone-rod dystrophy or degeneration, Primer pairs for the chromosome 19 linkage markers autosomal dominant 24 Rod-cone dystrophy, autosomal were obtained from Research Genetics. The forward- 32 dominant 6 strand primer was end labeled with P-ATP and poly- Cone degeneration, autosomal dominant 6 nucleotide kinase (Promega). Amplified DNA was di- Bardet-Biedl syndrome, autosomal luted 2:3 (vol:vol) with a solution of 95% formamide, recessive 3 20 mM EDTA, 0.05% bromophenyl blue, and 0.05% Leber congenital amaurosis, autosomal dominant 1 xylene cyanol, and fragments were separated on 6% Leber congenital amaurosis, isolated 1 denaturing acrylamide gels (Promega). Chorioretinitis, isolated 2 Usher syndrome, autosomal recessive 1 SSCP Analysis Optic atrophy, autosomal dominant 1 Genomic DNA samples from patients were screened by SSCP, by use of five sets of primers (table 2). All primers were synthesized by a commercial source (Geno- report a new CRX mutation associated with autosomal sys Biotechnologies). PCR was performed with Ampli- dominant cone-rod dystrophy in one family and the Taq polymerase (Perkin Elmer). Products were radio- E80A mutation reported by Freund et al. (1997) in a labeled by incorporation of 1 mCi 32P-dCTP. After an second, unrelated family with cone-rod dystrophy. The initial denaturation step for 5 min at 95ЊC, PCR was present study also demonstrates association between conducted for 30 cycles, each with a denaturation step CRX mutations and a family with late-onset, autosomal of 30 s at 95ЊC. Annealing was for 30 s at 64ЊC for dominant retinitis pigmentosa or mild, atypical, cone- fragments 1, 2, 3a, and 3b. Annealing for fragment 3c rod dystrophy and a family with severe congenital cone- was for 30 s at 60ЊC. Extension for all fragments was rod dystrophy with the clinical description “dominant for 30 s at 72ЊC, with a final elongation step for 10 min Leber congenital amaurosis” (Heckenlively 1988). Clin- at 72ЊC. The amplified products for exons 1, 2, and 3a ical summaries of the diseases in these families are pre- were analyzed as described elsewhere by Freund et al. sented, to emphasize the broad impact of CRX muta- (1997). In this study, the 3b fragment of Freund et al. tions. (1997) was amplified in two smaller independent and overlapping fragments, 3b and 3c. The amplification Subjects, Material, and Methods products of exon 3c were digested with EcoRI (Strata- gene), yielding fragments of 209 bp and 132 bp for SSCP Subjects analysis. For SSCP, PCR products were denatured and sepa- Subjects were ascertained at one of the following sites: rated on a 0.6# MDE gel (FMC Bioproducts), either at (1) the Anderson Vision Research Center, Retina Foun- room temperature or at 4ЊC. The gel was prepared in dation of the Southwest, Dallas (108 unrelated pro- 0.6# Tris-borate EDTA buffer and was subjected to bands); (2) the Jules Stein Eye Institute, UCLA School autoradiography after electrophoresis. of Medicine, Los Angeles (167 unrelated probands); or (3) the Hermann Eye Center, Department of Ophthal- Sequence Analysis mology, The University of Texas Health Science Center, Houston (19 unrelated probands, including the large In general, for sequence analysis, the fragment show- cone-rod dystrophy family from Texas). Informed con- ing an SSCP variant was amplified, by PCR, from the sent was obtained from all subjects. For each case, clin- stock DNA for the patient. The fragment then was ical evaluation was by at least one of the coauthors and, treated with shrimp alkaline phosphatase (Amersham) for several cases, by two coauthors jointly. Families in and exonuclease I (Amersham), followed by sequencing which CRX mutations were found are shown in figures with the AmpliCycle sequencing kit (Perkin Elmer) and 1–4; individuals from whom DNA samples were ob- a primer end labeled with 33P-ATP. The PCR sequencing Sohocki et al.: Mutations in the CRX Gene 1309 Figure 1 Family UTAD148, cone-rod dystrophy, autosomal dominant. Left, Pedigree. Blackened symbols represent affected individuals. A question mark (?) designates an individual of unknown phenotype. “DNA” to the upper right of the symbol indicates that a DNA sample was tested. Right, Sequence of exon 2, showing the ArC substitution in one allele (indicated by arrow), at nucleotide 238. protocol consisted of a 2-min denaturation step at 95ЊC, Results followed by 30 cycles of 30 s at 95ЊC and 30 s at 60ЊC each. The sequence was separated on 6% Long Ranger Of the 294 probands from unrelated families screened (FMC) denaturing acrylamide gels. for CRX mutation (table 1), we identified four disease- For insertion or deletion mutations detected by PCR- associated mutations in families with inherited retinop- product sequencing, a second amplification of the frag- athies and an additional four benign variants (table 3).