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Retinitis pigmentosa (RP) is the termapplied to a clinically and genetically

heterogeneous group of retinal degenera-tions that primarily affects the rod pho-toreceptors and has a prevalence ofapproximately 1 in 3,000. RP is character-ized by progressive loss of vision, initiallymanifesting as night blindness and reduc-tion in the peripheral visual field, and laterinvolving loss of central vision. It may beinherited as an autosomal dominant,autosomal recessive, digenic or X-linkedtrait. Autosomal dominant RP (adRP)accounts for 20−25% of all cases (forreview, see ref. 1). There are nine mappedadRP loci, but mutations causing adRPhave so far been identified in only twogenes: RHO (encoding rhodopsin) andRDS (http://www.sph.uth.tmc.edu/Retnet/disease.htm).

We determined the disease locus andcausative gene in a large adRP pedigree(RP251) by full-genome linkage analysisand candidate gene screening. Significantexclusion was obtained for all knownadRP loci. Linkage was obtained betweenadRP and markers at 14q11, with a maxi-mum lod score of 5.72 (θ=0.00) for themarker D14S64.

D14S64 resides in a cosmid containingthe NRL gene2. NRL was considered a can-

didate because it encodes a basic motif-leucine zipper (bZIP) DNA-binding pro-tein that is highly and specificallyexpressed in adult retina3,4. We screenedthe three exons of NRL for mutations inaffected members of RP251 by heterodu-plex analysis and direct sequencing5. Allaffected individuals were found to have aT→A change at nt 1,942, resulting in a ser-ine (Ser) to threonine (Thr) substitution atcodon 50 of the NRL protein (Fig. 1a). Noother sequence change was observed.Because this nucleotide change abolishesan HphI site, the amplified NRL exon 2product was digested with HphI to confirmthe T→A sequence change in all affectedmembers of the RP251 family, and itsabsence in their unaffected siblings(Fig. 1b). Complete digestion by HphI wasobserved in 250 unrelated control samples,indicating that the T→A nucleotidechange was not present.

NRL has been shown to upregulate theactivity of the RHO promoter6,7. Similarto other bZIP transcription factors, NRLis a modular protein with two distinctdomains3. A transactivation (TA) domainrich in proline, serine and threonineresidues is present in the first half of theprotein, encoded by exon 2. The DNA-binding (DB) domain at the carboxy ter-minus (encoded by exon 3) contains aleucine zipper motif for dimerization, pre-ceded by a stretch of basic amino acidsthat are involved in DNA binding. TheSer50 residue is located in one of twohighly conserved regions of the TAdomain (located at residues 3−27 and 41−54, respectively) of NRL, and is also pre-

sent in other members of the Maf familyof proteins that contain a TA domain.

While Nrl transcripts are detected in allpost-mitotic neurons and the lens duringmouse embryonic development, theirexpression is restricted to retinal cells inthe adult4. On the basis of this develop-mental expression pattern and a demon-strated lack of sequence variation in thecoding region2, it is predicted that a nullmutation in NRL might be lethal. Aminoacid substitutions in the TA domain mayalter the activity, specificity or ability ofNRL to interact with other transcriptionfactors. Mutations in the cone-rodhomeobox gene (CRX), encoding a home-odomain protein that functions synergis-tically8 with NRL in regulating RHOpromoter activity, have been shown tocause autosomal dominant cone-rod dys-trophy9,10 (CORD2) and recessive Lebercongenital amaurosis11.

To assess the effect of the S50T mutationon the ability of NRL to transactivate theRHO promoter, either alone or in combi-nation with CRX, we performed transienttransfection experiments in CV-1 and 293cell lines6−8. Using an expression constructgenerated by cloning the NRL cDNA in thepED mammalian expression vector12

(a derivative of pMT3; ref. 6), we saw a sta-tistically significant increase in the transac-tivation of the RHO promoter in CV-1cells with the mutant NRLS50T proteincompared with wild-type NRL (Fig. 2). Inthe presence of CRX, however, NRLS50T

demonstrated enhanced synergistic trans-activation of the RHO promoter at rela-tively low levels of the expression construct(Fig. 2). With CRX, the concentration ofpED-NRLS50T required for half-maximaltransactivation synergy was almost 90%less than that of pED-NRL. The degree ofsynergy achieved in the presence of satu-rating amounts of NRL, however, wasidentical with both normal and mutant

A mutation in NRL is associatedwith autosomal dominantretinitis pigmentosa

Fig. 1 Identification of NRL mutation. a, Sequence of the mutated NRL allele demonstrating a T→A change in the forward sequence at nt 1,942 (codon 50), indi-cated by ‘N’. A normal sequence is shown below for comparison. Forward and reverse primers used were as described2. b, Restriction analysis of the amplifiedNRL exon 2 in generation III of family RP251, demonstrating the abolition of the HphI site caused by the T→A change. HphI cleaves the normal exon 2 productinto two fragments of 65 bp and 205 bp. The presence of an undigested band of 270 bp, indicating heterozygosity of the substituted allele, is observed in allseven affected subjects (lanes 2, 4−6, 8, 10 and 12).

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NRL proteins. Similar results wereobtained in independent experimentsusing the human 293 cell line (data notshown). Levels of protein expression werenot ascertained.

Although it is difficult to extrapolatethe in vivo significance from cell culturestudies, our data suggest that NRLS50T

may result in altered (probablyincreased) transcription of RHO, andpossibly of other photoreceptor genes, invivo. Rhodopsin is the major structuralprotein of rod outer segments, compris-ing over 90% of the total protein content.RHO mutations are responsible forapproximately 25% of all cases of adRP(ref. 13). In animal models both overex-pression and underexpression of rho-dopsin have been shown to causephotoreceptor cell death14,15, and thismay be the mechanism by which NRLS50T

elicits retinal degeneration.NRL is only the third gene in which an

adRP-causing mutation has beendetected. The evidence in support ofS50T being the disease-causing mutationin the RP251 family are: (i) linkagebetween adRP and D14S64, the closestgenetic marker to NRL; (ii) retina-spe-cific expression of NRL; (iii) lack of T→Asequence change in 250 normal controls;(iv) conservation of the NRL coding

region, as revealed by sequencing of 53independent retinal dystrophy patients2

and negative heteroduplex screening ofNRL in an additional 200 individuals(data not shown); (v) conservation ofSer50 in all Maf proteins containing thetransactivation domain; and (vi)enhanced transactivation of RHO pro-moter activity by NRLS50T, particularlywhen mutant NRL acts synergisticallywith CRX.

AcknowledgementsWe thank family members for their participation.D.A.R.B. and A.M.P. are supported by the MedicalResearch Council of the U.K. (grant no.G9301094) and Q.-L.W. is a recipient of a KnightsTemplar Foundation fellowship. This research wassupported by grants from the National Institutes ofHealth (EY11115, EY09769), the FoundationFighting Blindness, Research to Prevent Blindness,The Rebecca P. Moon, Charles M. Moon Jr and DrP. Thomas Manchester Research Fund, and theMrs Harry J. Duffey AMD Research Fund. A.S. isa recipient of the Lew R. Wasserman Merit Awardand D.J.Z. a Career Development Award, bothfrom Research to Prevent Blindness.

David A.R. Bessant1,2*, Annette M. Payne1*,Kenneth P. Mitton3*, Qing-Liang Wang5,Prabodha K. Swain3, Catherine Plant2,Alan C. Bird2, Donald J. Zack5,6,7,

Fig. 2 Effect of the S50T mutation on NRL-medi-ated transactivation of RHO promoter activity inCV1 cells. Different concentrations of pED-NRL andpED-NRLS50T expression constructs (0.003−0.3 µg)were cotransfected with pBR130-luc (RHO pro-moter/luciferase reporter, 0.3 µg; refs 6−8) withand without pCDNA-bCRX (0.3 µg; ref. 7) as indi-cated. Luciferase activity was normalized for trans-fection efficiency with that of β-galactosidase. Theexperiment was performed three times to ensurereproducibility. Fold activation in relative lightunits (luciferase/β-galactosidase) was calculatedover the pED vector in presence of pBR130-lucreporter construct10 (=1 fold). The luciferase activ-ity in the presence of CRX expression constructalone is indicated by the dotted line. An increasedtransactivation of the RHO promoter was observedwith NRLS50T compared with NRL. The synergistictransactivation of NRLS50T with CRX was enhancedover that of NRL+CRX. t-test, P<*0.05, **0.01,***0.001; bars show s.d.

NRLS50T + CRXNRL + CRXNRLS50T

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Anand Swaroop3,5

& Shomi S. Bhattacharya1

*These authors contributed equally to this work.1Department of Molecular Genetics, Institute ofOphthalmology, University College London,and 2Moorfields Eye Hospital, London, UK.Departments of 3Ophthalmology and 4HumanGenetics, W.K. Kellogg Eye Centre, University ofMichigan, Ann Arbor, Michigan, USA.Departments of 5Ophthalmology,6Neuroscience, and 7Molecular Biology andGenetics, The Johns Hopkins University Schoolof Medicine, Baltimore, Maryland, USA.Correspondence should be addressed to S.S.B.(e-mail: sbhatach@hgmp.mrc.ac.uk) or A.S. (e-mail: swaroop@umich.edu).

1. Bird, A.C. Am. J. Ophthalmol. 119, 543–562 (1995).2. Farjo, Q. et al. Genomics 45, 395–401 (1997).3. Swaroop, A. et al. Proc. Natl Acad. Sci. USA 89,

266–270 (1992).4. Liu, Q., Ji X., Breitman, M.L., Hitchcock, P.F. &

Swaroop, A. Oncogene 12, 207–211 (1996).5. Keen, J., Lester, D., Inglehearn, C.F., Curtis, A. &

Bhattacharya, S.S. Trends Genet. 7, 5 (1991).6. Rehemtulla, A. et al. Proc. Natl Acad. Sci. USA 93,

191–195 (1996).7. Kumar, R. et al. J. Biol. Chem. 271, 29612–29618

(1996).8. Chen, S.M. et al. Neuron 19, 1017–1030 (1997).9. Freund, C.L. et al. Cell 91, 543–553 (1997).10. Swain, P.K. et al. Neuron 19, 1329–1336 (1997).11. Freund, C.L. et al. Nature Genet. 18, 311–312 (1998).12. Kaufman, R.J. in Gene Amplification in Mammalian

Cells A Comprehensive Guide (ed. Kellems, R.E.)315−343 (Marcel Dekker, New York, 1992).

13. Inglehearn, C.F. et al. Hum. Mol. Genet. 1, 41–45(1992).

14. Olsson, J. et al. Neuron 9, 815–830 (1992).15. Humphries, M.M. et al. Nature Genet. 15, 216–219

(1997).

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