Rhodopsin mutations in a Scottish retinitis pigmentosa population, including a novel splice site mutation in intron four

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1 British Journl ofophthlmology 14; 78: Medicl Genetics, Deprtment of Moleculr nd Cell Biology, University of Aberdeen Medicl School, Foresterhili, Aberdeen AB 2ZD C Bell A Osborne N E Hites Deprtment of Phrmceuticl Sciences, University of Strthclyde, George Street, Glsgow GI 1XW C A Converse Tennent Institute of Ophthlmology, University of Glsgow, Western Infirmry, Church Street, Glsgow Gll 6NT H M Hmmer Correspondence to: Christine Bell. Accepted for publiction 1 August 14 Rhodopsin muttions in Scottish retinitis pigmentos popultion, including novel splice site muttion in intron four Christine Bell, Crolyn A Converse, Hrold M Hmmer, Aileen Osborne, Nev E Hites Abstrct Retinitis pigmentos (RP) is the nme given to group of disorders, both cliniclly nd geneticlly heterogeneous, tht primrily ffect the photoreceptor function of the eye. Muttions in the genes encoding for rhodopsin, RDS-peripherin, or the subunit of the cgmp phosphodiesterse enzyme cn be responsible for the phenotype. In this study the rhodopsin gene hs been screened for muttions in pnel of RP individuls nd five different sequence chnges hve been detected to dte in three dominntly inherited nd two unclssified fmilies. One of these, bse substitution in the 3'UTR, hs not yet been confirmed s disese specific, while three missense substitutions hve previously been reported nd re likely to be responsible for the phenotype. The fifth chnge, bse substitution t the intron 4 cceptor splice site, represents novel muttion nd is ssumed to be the custive muttion. (BrJ Ophthlmol 14; 78: 33-38) The term retinitis pigmentos (RP) describes group of retinl degenertions tht ffect the photoreceptor function of the eye nd re heterogeneous in their genetic nd clinicl presenttion. The disorder my be inherited in n utosoml dominnt, utosoml recessive, or X linked recessive mnner, or occur s n isolted cse with no previous fmily history. Within these subgroups the clinicl picture my differ; from mild impirment to severe visul hndicp in combintion with n erly or lte ge of onset. The symptoms include night blindness, commonly occurring erly in the disese, followed by progressive loss of peripherl vision nd chrcteristic chnges in the fundus. The disorder hs been clculted to ffect pproximtely one in 3500 of the popultion' nd thus constitutes mjor cuse of visul impirment. Recent dvnces in moleculr genetics hve led to the identifiction of severl genes tht re responsible for the phenotype. The first of these followed the demonstrtion of linkge of the RP gene in lrge Irish fmily with the mrker CRI-C17 on 3q.2 Dryj nd collegues soon reported muttion in the rhodopsin gene, lso on 3q, in 17 utosoml dominnt RP ptients.3 Further studies hve reveled over 40 different muttions in this gene cn be responsible for the disorder in dominnt RP fmilies.4 A muttion in the rhodopsin gene hs lso been shown to ccount for the phenotype in n individul with utosoml recessive RP.5 Non-llelic genetic heterogeneity hs been shown in these two subtypes by the demonstrtion tht muttion in the peripherin-rds gene my lso result in dominnt RP,67 nd tht muttions in the gene which codes for the B subunit of the rod phosphodiesterse enzyme my result in utosoml recessive RP.8 Other linkge studies in severl dominnt fmilies indicte tht the respective RP genes re locted on chromosome 7p, 7q, 8q, nd 1q.-12 There is lso evidence supporting the involvement of the ROM-l gene on chromosome 1 lq'3 in dominnt RP fmily. Considering tht t lest two genes re responsible for the X linked form of the disese,'4 it is cler tht muttion in one of numerous genes is cpble of producing the clssic RP phenotype. In ddition to cusing RP, muttions in these genes my result in other retinl degenertions, including mculr degenertion, congenitl sttionry nightblindness, nd retinitis punctt lbescens. 15-'7 We hve collected DNA from members of 30 RP fmilies nd from 23 isolted cses of retinl degenertions, nd hve screened these ptients using single strnded conformtion polymorphism (SSCP) nlysis nd direct sequencing to identify the custive gene. Here we present the results from the study of the rhodopsin gene. Ptients nd methods Smples were collected from ptients with RP, who presented to the RP clinic in Tennent Institute, Western Infirmry s nd when referred by their generl prctitioners nd ophthlmologists. Alterntively, ptients were recruited when referred to the genetic clinic in Aberdeen Royl Hospitls by their ophthlmologists. The smples comprised pnel of RP ptients representing 30 fmilies nd 23 isolted cses from geogrphicl re covering the west nd north est of Scotlnd. Of the fmilies, 12 hve been confirmed s being dominnt due to the observtion of mle to mle inheritnce, three re considered to be recessive fmilies, nd 15 re designted unclssified. These unclssified fmilies hve ffected members in severl genertions but no observed mle to mle inheritnce. Genomic DNA ws extrcted from 20 ml whole blood using method bsed on tht described by Kunkel.'8 Five different sequence chnges were detected, three of which were in designted dominnt fmilies. The fourth nd fifth chnges occur in unclssified fmilies (Fig 1). The first sequence chnge hs been reported' nd occurs in dominnt fmily from the Tyside/Grmpin region (referred to s drp3 in Lester et l20) with diffuse form of the Br J Ophthlmol: first published s /bjo on 1 December 14. Downloded from on 24 Jnury 201 by guest. Protected by copyright.

2 34 Bell, Converse, Hmmer, Osborne, Hites F-6246 Figure I Pedigrees of ptients with muttions identified in the rhodopsin gene. Individuls testedfor the presence of the prticulr muttion re indicted (*). Arrows refer to individuls indicted in the text. Htched symbols represent individuls in which the RP dignosis hs not been confirmed. Figure 2 SSCP shift (A) nd sequence (B) ofthe muttion (cg>tg in codon 17) detected in fmily B The mobility shift is indicted by the rrows (lne number indicted beside rrow) in lne I in A, nd the corresponding sequence in B(i). Control sequence is shown in B(ii). disese. Onset of symptoms (nightblindness) usully ppered in lte childhood with preservtion of centrl vision into the third or fourth decde. Clinicl fetures include typicl field losses, trophic retinl pigment epithelil chnges with chrcteristic bone spicule pigmenttion. Electroretinogrphic (ERG) testing revels grossly reduced rod nd cone function, with flt responses hving been recorded in one fmily member t ge 12 yers (rrowed in Fig 1). This individul hd vision of 6/18 in both left nd right eyes nd mrked constriction of visul fields (pproximtely 10 degrees) in ech eye. The second nd third sequence chnges were identified in two dominnt fmilies (M-611 nd K-6056) who come from the Strthclyde region nd hve typicl RP symptoms including constricted fields, ple discs, nd ttenuted rteries. Both fmilies hve nightblindness s the onset symptom. Limited detiled clinicl informtion is vilble for fmily K The individul (48 yers old) indicted by n rrow in fmily M-611 presented to the RP clinic 7 yers go with 20 yer history of nightblindness, which hd incresed in severity in the pst 5 yers. At tht time he hd good centrl vision in ech eye (6/6 N5) nd erly retinl nd visul field chnges. ERG testing reveled norml photopic signl, but mrkedly A B(i) 3, bnorml scotopic signl. Recent exmintions confirm the retention of centrl vision, with progression of fundus chnges nd very mrked constriction of visul fields (pproximtely 15 degrees in ech eye). His photopic ERG is now reduced to pproximtely 60% of norml nd scotopic signls to below 10% of norml. The fourth chnge ws identified in two ffected members of n unclssified fmily (B-6183) tht ppers to suffer mild form of sectorl RP with lter ge of onset. The individul indicted by the rrow in fmily B-6183, presented 6 yers go (ge 58) t generl ophthlmic clinic fter referrl from her generl prctitioner, complining of hzy vision in both eyes. She ws subsequently referred to the RP clinic, where exmintion reveled vision of 6/ nd 6/12 in the right nd left eyes, respectively. Fundus findings showed typicl re of sector RP ffecting the inferior fundus, prticulrly long the inferotemporl vessels nd visul field chnges corresponded with this in tht there ws mrked loss of vision in the upper field in ech eye. Her photopic ERG ws recorded t 43% nd 37% of norml, while her scotopic ERG ws 41% nd 2% of norml in right nd left eyes respectively. Her ffected dughter (sterisked) shows erly but similr fetures ffecting the lower fundus. t --A g Gly 18 - c: Thr 17 > Met 17 C )Al 16 A C G T\ 5/ It Y-rf b B(i ) 3' 4- - \xgly 18,,_ c )~~Thr 17 A C G T \ g Al 16 Br J Ophthlmol: first published s /bjo on 1 December 14. Downloded from on 24 Jnury 201 by guest. Protected by copyright.

3 Rhodopsin muttions in Scottish retinitis pigmentos popultion, including novel splice site muttion in intronfour 35 A B(i) 3' B(ii) F.~- 3' 1 -_ -i i fii Alit.. 5 twf Figure 3 SSCP shift (A) nd sequence (B) ofthe muttion (cgg>tgg in codon 135) detected in fmily F The mobility shift is indicted by the rrows (lne number indicted beside rrow) in lne I in A, nd the corresponding sequence in B(i). Control sequence is shown in B(ii). Figure 4 SSCP shifts (A) nd sequences (B,C) ofthe muttions detected in the fmilies K-6056 (g> trnsition t position 52) nd M-611 (g> in intron 4 cceptor splice site). The mobility shifts re indicted by the rrows (with lne numbers indicted beside rrows) in lnes I (K-6056) nd 2 (M-611) in A, nd the corresponding sequences in B(i) nd C(i) respectively. Control sequence t these points re lso shown (B(ii) nd C(ii)). A C G T Arg 135 > Trp 135 c>t Glu 134 The fifth sequence chnge ws observed in one individul representing smll unclssified fmily (F-6246). This mn ws first exmined 3 yers go (ge 25) when he ttended generl clinic with divergent squint. On noting signs of the disese, he ws then referred to the RP clinic. Only on close questioning did he dmit to hving some visul field defect nd difficulty in drk conditions. His visul cuities were t tht time, 6/ N5 right nd 6/18 N14 left. He hs the clssic fundus ppernce of RP with mrkedly constricted fields to less thn 10 degrees in ech eye. ERG testing reveled flt photopic responses nd flt left scotopic, with smll response (10% of norml) in right scotopic. He is lso mrkedly hypermetropic with moderte degree of stigmtism. Both unclssified fmilies hve origins in the Strthclyde region. Sequence chnges were detected using SSCP nlysis2' followed by direct sequencing.22 SSCP primers were designed to mplify the whole coding region of the rhodopsin gene nd A ON : c C(i) -p.u.;-. AC... c X T. T 5 g Arg 135 Giu 134 bp of the intron/exon junctions nd mplifiction products were electrophoresed in 5% nondenturing crylmide gels (with 5% glycerol t both 5 C nd 20 C). Smples which displyed mobility shift were mplified gin using primers upstrem of ech sense nd ntisense primer, enbling the whole region under SSCP nlysis to be sequenced. Results SSCP shifts were detected in exons 1, 2, 3, nd 5 ofthe rhodopsin gene in five distinct fmilies. No mobility shifts were identified in the isolted cses. Sequencing reveled the shift in exon 1 (Fig 2A) to originte from the heterozygous bse substitution ACG to ATG (threonine to methionine) in codon 17 of the rhodopsin gene. The sequence chnge is shown in Figure 2B next to the norml sequence. This muttion ws observed in two ffected individuls in two genertions of this fmily (B-6183), but ws bsent from n unffected member. In ddition, 3' C *.. g A CG T n L 3 0 C 5, t Phe 313 t exon 5...g. intron 4 A C G T 5 B(ii) CO., ; ACi 3, 3' /7 I I t- c g C t \ t \C t Phe 313 A CG T 51 g> exon 5 intron Br J Ophthlmol: first published s /bjo on 1 December 14. Downloded from on 24 Jnury 201 by guest. Protected by copyright.

4 36 Bell, Converse, Hmmer, Osborne, Hites the muttion hs been observed in other fmilies with drp2324 nd is therefore believed to be responsible for the phenotype. The shift in exon 2 ws observed in one individul, belonging to the fmily F The SSCP shift is shown in Figure 3A nd sequencing reveled heterozygous bse substitution in codon 135: CGG (rginine) to TGG (tryptophn). The chnge is shown in Figure 3B. Although this sequence chnge hs only been identified in one individul in n unclssified fmily, it is likely to be the responsible muttion, s it hs previously been reported in dominnt RP.24 The shift in exon 3 ws observed in n ffected member of the fmily designted drp3 nd found to be due to bse substitution in codon 178 (TAC (tyrosine) to TGC (cysteine) results not shown). This muttion bolishes Rsl restriction site nd restriction nlysis shows the segregtion of the muttion with the phenotype s reported previously." Two different mobility shifts were observed ner exon 5 of the rhodopsin gene. The two shifts re shown in Figure 4A. The shift shown in lne 1 ws due to heterozygous G to A substitution t position 52 in the 3' untrnslted region (3'UTR) of the gene. This muttion ws detected in n ffected individul of fmily K-6056 nd is shown in Figure 4B. The shift in lne 2 ws due to heterozygous G to A (AG to AA) substitution t the 3' cceptor site of intron 4 nd ws observed in three ffected fmily members of fmily M-611. The sequence is shown in Figure 4C. Norml sequence t these points re shown longside s comprison. Discussion RHODOPSIN MUTATIONS IN RP The muttions identified in exons 1, 2, nd 3 hve ll previously been identified in utosoml dominnt cses of retinitis pigmentos2125 nd therefore, lthough the exon 2 muttion in fmily F-6246 hs only been identified in one individul, it is likely to be the custive muttion in tht fmily. Similrly, the muttions identified in exons 1 nd 3 re considered responsible for the phenotype in the respective fmilies (B-6183 nd drp3). The two sequence chnges in exon 5 hve not been reported previously. The first of these, G to A substitution t position 52 (fmily K-6056) hs not been confirmed in ny other ffected fmily members owing to the limited vilbility of dditionl smples, nd it is therefore conceivble tht the chnge is merely neutrl polymorphism. We hve screened pnel of 88 control smples (178 lleles) for the presence of this sequence chnge nd hve found the chnge to be bsent in this smple popultion. However, until it cn be shown to segregte with RP in fmily it is best considered (rre) polymorphism. Attempts re under wy to locte dditionl fmily members to determine the nture of the chnge. The novel splice site muttion ws observed in three ffected siblings nd bsent in fourth unffected sibling of dominnt fmily" M-611). Although this muttion hs not been confirmed through two genertions it is likely tht it is the disese specific muttion in the fmily. The sequence chnge destroys the conserved 3' splice cceptor site (AG) which is essentil for ccurte splicing of mrna. Muttions within these splice sites re known to result in bnorml splicing nd commonly result in disese. This hs been observed in choroideremi nd gyrte trophy EFFECT OF MUTATION ON RHODOPSIN MOLECULE Rhodopsin is the mjor protein of the rod outer segments nd functions s the light bsorbing protein, inititing cscde of rections tht medite dim vision. However, the mechnisms by which these nd previously identified muttions in the rhodopsin gene result in disese re uncler. Sung nd collegues hve studied severl mutnt rhodopsin protein species using trnsfected cells nd hve designted two mutnt clsses: the first of these, known s clss I, groups together those proteins which resemble wild type rhodopsin in yield, regenerbility with the chromophore nd subcellulr loclistion (the plsm membrne (PM)), while clss II mutnts refers to the proteins which ccumulte to significntly lower levels thn the wild type, regenerte the chromophore inefficiently nd re retined predominntly in the endoplsmic reticulum (ER) (clss II). Clss IIb proteins re similr to clss II except in their cellulr loclistion, which is distributed roughly between the ER nd PM.28 Abnorml proteins tht fil to fold correctly or ssemble into the required complexes hve been shown to be retined in the ER, in mnner similr to those described s clss II mutnts, nd re degrded therein.2 Tht three of the muttions discussed here (codons 17, 135, nd 178) hve been ssigned to the clss II type28 nd thus retined in the ER is not surprising ifwe consider the likely effect ofech sequence chnge. The first muttion, Thr 17 Met, destroys the three residue recognition sequence (Asn'5-Al'6- Thr'7) tht is required for glycosyltion t Asn15 nd the subsequent prevention of glycosyltion presumbly results in n bnormlly folded protein. The close proximity ofthe second muttion (Tyr 178 Cys) to the conserved Cys'87, which is involved in forming the disulphide bridge with Cys"', nd crucil in the tertiry structure ofrhodopsin, my be lso be considered deleterious to correct folding. The muttion Arg 135 Trp occurs in one of the trnsmembrne segments, which contin severl invrint glycine nd proline residues nd re thought to be importnt in forming the three dimensionl structure of the protein, especilly in forming pocket for the invrint Lys"*. The substitution of this mino cid my gin ffect the proper folding of the protein. The effect of the remining sequence chnge is s yet uncler. Muttions in splice sites commonly led to the use of cryptic sites, or exon skipping, which result in the production of n ltered protein, either with severl residues or lrge portions.of the gene deleted. Alterntively, the intron my be retined in the processed trnscript nd if the trnscript is stble then the intron sequence will be trnslted. We re currently Br J Ophthlmol: first published s /bjo on 1 December 14. Downloded from on 24 Jnury 201 by guest. Protected by copyright.

5 Rhodopsin muttions in Scottish retinitis pigmentos popultion, including novel splice site muttion in intronfour 37 studying this muttion further using mrna isolted from peripherl blood lymphocytes of n ffected individul to determine the effect of the sequence chnge. Erly studies indicte tht the intron is retined in the trnscript (unpublished results) which subsequently results in the formtion of premture stop codon in intron four. The protein produced s result would be missing mino cids , the crboxyl terminl of the protein. This region contins severl invrint serine nd threonine residues, which re phosphorylted during the recovery phse to block the ction of rhodopsin. RHODOPSIN MUTATION AND ITS RELATION TO THE PHENOTYPE The mnner in which these muttions result in the disese phenotype is uncler. Although the muttions cn be divided into clss I/II mutnts, this does not correlte with prticulr phenotype. Within clss I nd clss II mutnts there exist both severe nd mild phenotypes28 nd this is further demonstrted in these results: the rhodopsin molecules with muttions in codons 17 nd 135/178 belong to clss II but re generlly considered to hve mild nd severe phenotypes respectively. The further subdivision of the clss II mutnts does not correlte directly with phenotype either, s Thr 17 Met (cliniclly mild) nd Tyr 178 Cys (cliniclly severe) both belong to clss II. Additionl fctors my ply role in the progression of the disese in the more severe forms with muttions in this clss. The Arg 135 Trp muttion belongs to clss IIb, where equl mounts of the protein re locted in the ER nd the PM. The phenotypic severity resulting from this muttion my be due to the presence of the mutnt protein in the outer segments, where it hs been shown to be defective in ctivting nucleotide exchnge by trnsducin,m thus preventing further mplifiction of the response. Perhps the combintion of this defect, together with competitive binding with the wild type protein re sufficient to effect severe phenotype. Clerly, further studies re required to understnd the pthogenic effect of these nd similr rhodopsin muttions in rod photoreceptors cells nd how these chnges lso ffect, in some instnces, the cone cells. Although the pthogenesis of the condition is not fully understood, knowledge of the ctul muttion my provide ophthlmologists with prognosis indictor for specific fmilies. The codon 17 muttion hs previously been reported in fmilies with regionl predilection for pigmentry chnges3 32 nd ppers to correlte with form of the disese hving modertely good prognosis, nd the severity of the disese in the fmilies with the codon 135/178 muttions lso concur with previous reports It is pprent from the bove discussion tht some unknown fctors influence the effect of the resulting mutnt protein in the rod cells nd the progression of the disese - the observtion of decresed levels of certin polyunsturted cids in the plsm of ffected individuls in fmily drp334 suggests tht gene concerned with the trnsport or synthesis of ftty cids my be linked to the progression of the disese. In ddition, light toxicity hs been suggested s secondry fctor in RP.3s Undoubtedly, s the number of custive genes increse, nd their biochemicl nture nd/ or structurl significnce re relised, the complex events of the photoreceptor cell will become clerer. Combined with n increse in knowledge of intercting fctors, definition of the pthogenic mechnisms involved in the disese process my result nd led to some form of therpeutic intervention. The uthors thnk the Wellcome Trust (No 03338/Z/1/Z/1.5), the Ntionl Retinitis Pigmentos Foundtion nd the WH Ross Foundtion (Scotlnd) for the Study of Prevention of Blindness for their support in this reserch. 1 Bunker CH, Berson EL, Bromley WC, Hyes RP, Roderick TH. Prevlence of retinitis pigmentos in Mine. AmJ Ophthlmol 184; 7: McWillim P, Frrr GJ, Kenn P, Brdley DG, Humphries MM, Shrp EM, et l. Loclistion of n ADRP gene to the long rm of chromosome 3. Genomics 18; 5: Dryj TP, McGee TL, Reichel E, Hhn LB, Cowley GS, Yndell DW, et l. A point muttion of the rhodopsin gene in one form of retinitis pigmentos. Nture 10; 343: Humphries P, Frrr GJ, Kenn P. Autosoml dominnt retinitis pigmentos: moleculr, genetic nd clinicl spects. 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