Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A)

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1 H j H Q M oleculr Vision 2005; 11: < t" Received 29 November 2004 I Accepted 18 Jnury 2005 I Published 19 Februry 2005 Autosoml dominnt cone dystrophy cused by novel muttion in the GCAP1 gene (GUCA1A) Li Jing,1,2,3 Brdley J. K tz,1,2 Z henglin Yng,1,2,4 Yu Zho,1,2 N thn Fulkner,1,2 Jinbin H u,1,2,4 Jennifer B ird,1 W olfgng Behr,1,3,5 Donnell J. C reel,1 K ng Z hng1,2,5 Deprtments o f 1Ophthlmology nd Visul Sciences, 3Biology, nd 5Neurobiology nd Antomy, nd the 2Progrm in Humn Moleculr Biology nd Genetics, University o f Uth Helth Sciences Center, Slt Lke City, UT; 4Sichun Provincil Medicl Acdemy & Sichun Provincil People's Hospitl, Sichun, People's Republic of Chin P u r p o s e : To describe the clinicl fetures nd genetic nlysis of fmily with n utosoml dominnt cone dystrophy (dcd). M eth o d s : Selected members of fmily with n utosoml dominnt cone dystrophy underwent ophthlmic evlution. Blood smples were obtined, genomic DNA ws isolted, nd genomic frgments were mplified by PCR. Linkge to locus D6S1017 ws estblished. DHPLC muttionl nlysis nd direct sequencing were used to identify muttion in GUCA1A, the gene encoding the gunylte cyclse ctivting protein 1 (GCAP1). R e s u lts : Of 24 individuls who re t risk of the disese in five genertion fmily, 11 members were ffected. Clinicl presenttions included photophobi, color vision defects, centrl cuity loss, nd legl blindness with dvnced ge. The disese phenotype ws observed in the second nd third decdes of life nd segregted in n utosoml dominnt fshion. An electroretinogrm performed on one probnd reveled profoundly subnorml nd prolonged photopic nd flicker responses, but preserved scotopic ERGs, consistent with cone dystrophy. Muttionl nlysis nd direct sequencing reveled C451T trnsition in GUCA1A, corresponding to novel L151F muttion in GCAP1. Like the E155G muttion, this muttion occurs in the EF4 hnd domin, region of GCAP1 criticl in conferring clcium sensitivity to the protein. The leucine t this position is highly conserved mong vertebrte gunylte cyclse ctivting proteins. C o n c lu s io n s : A novel L151F missense muttion in the EF4 high ffinity C2+ binding site of GCAP1 is linked to dcd in lrge pedigree. The cone dystrophy in this fmily shres clinicl nd electrophysiologic chrcteristics with other previously described dcd cused by muttions in GUCA1A. The cone dystrophies re phenotypiclly heterogeneous group of hereditry retinl degenertions chrcterized by progressive dysfunction of the photopic (cone medited) system, presenting with hemerlopi (dy blindness), loss of color vision, reduced centrl visul cuity, nd preserved peripherl vision [1]. Cone dystrophies my be contrsted to rod dystrophies, such s retinitis pigmentos, which re chrcterized by bnormlities in scotopic (rod medited) functions, mnifesting s night blindness, preserved centrl visul cuity erly in the disese process, nd constricted peripherl vision. Erly in the disese process, the fundus ppernce of ptient with cone dystrophy my be norml. The disese is then dignosed bsed on chrcteristic chnges in the electroretinogrm. Lter in the disese process, the retinl pigment epithelium my tke on grnulr ppernce tht my progress to centrl trophy. Like retinis pigmentos (RP), the cone dystrophies re geneticlly heterogeneous nd my present s spordic, utosoml dominnt, utosoml recessive, or X-linked recessive trit (RetNet). Identified genes linked to utosoml dominnt Correspondence to: Dr. Kng Zhng, Eccles Institute of Humn Genetics, 15 North 2030 Est, Building 533, Room 3060A, University of Uth Helth Science Center, Slt Lke City, UT, 84132; Phone: (801) ; FAX: (801) ; emil: kng.zhng@hmbg.uth.edu 143 cone dystrophies include GUCY2D, encoding photoreceptor gunylte cyclse 1 (retgc-1, or GC1) nd GUCA1A, encoding the photoreceptor specific C2+ binding protein termed gunylte cyclse ctivting protein 1 (GCAP1). GC1 nd GCAP1 re key components in the phototrnsduction cscde in rod nd cone photoreceptors. In the drk dpted stte, photoreceptors hve high levels of cgmp nd this molecule holds photoreceptor plsm membrne ction chnnels open. Exposure to light initites the phototrnsduction cscde in photoreceptors, resulting in the hydrolysis of cgmp nd closure of the cgmp-gted ction chnnels [2-4]. The concentrtion of free intrcellulr C2+ decreses s consequence of continued ctivity of the N+/C2+-K+ exchnger (NCKX) exchnger lso locted in the plsm membrne, n event tht in turn ctivtes gunylte cyclses [4]. The stimultion of the gunylte cyclses by decresed levels of clcium occurs indirectly vi GCAPs [5-7]. The GCAP-stimulted GC ctivity eventully returns cgmp to levels tht re sufficient to reopen the cgmp-gted ction chnnels, estblishing the drk dpted stte. The GCAPs re neuron specific clcium binding proteins (NCBPs), subgroup of the lrge clmodulin supergene fmily with four EF hnd [8] motifs [9]. NCBPs include recoverins, nother set of photoreceptor specific C2+ binding proteins, frequenins, hippoclcins nd hippoclcin-like proteins, neu-

2 ronl C2+ sensors nd mny others [10]. Unlike clmodulin nd other NCBPs, the GCAPs ctivte their trget proteins, the gunylte cyclses, when the concentrtion of free intrcellulr C2+ is low [11], while other NCBPs ctivte their trgets when the concentrtion of free C2+ is high. Up to eight GCAPs hve been identified in vertebrtes [12], but in humn retin there re only three (GCAP1, GCAP2, nd GCAP3). The mouse ppers to express only GCAP1 nd GCAP2, but not GCAP3 [11,13]. The humn nd mouse GCAP1 nd GCAP2 genes reside in til-to-til rry on chromosome 6p21.1 nd chromosome 17, respectively, nd shre fourexon/three-intron rrngement [14,15]. GCAP3 hs the sme exon/intron rrngement, but hs been loclized to 3q13.1 [11]. Thus, it ppers tht these three GCAP genes rose s result of the dupliction nd trnsloction of common ncestrl gene. Four different muttions linked to retin disese hve been previously described in GCAP1 (Y99C, P50L, E155G, nd I143NT) [16-20]. All re missense muttions, one (I143NT) hs n insertion of n dditionl mino cid residue. Loss of C2+ sensitivity is ssocited with the Y99C, E155G, nd I143NT muttions. Common to these is tht the muttion ffects one of the three functionl EF hnd motifs in GCAP1 (EF3 or EF4). The biochemicl effect of the muttions consists of the inbility of the mutnt GCAPs to inhibit photoreceptor GC in the drk, when C2+ is elevted [21,22]. The result is tht cgmp levels re elevted in mutnt photoreceptors, nd lrger number of ction chnnels remin open, which eventully leds to elevted C2+ concentrtion nd to photoreceptor cell deth [23]. In this mnuscript, we describe the clinicl fetures of nd the genetic muttions in fmily with n utosoml dominnt cone dystrophy crrying novel missense muttion in the GUCA1 gene. M ETHODS Ptients: This study ws pproved by the Institutionl Review Bord of the University of Uth Hospitls nd Clinics nd ll subjects provided informed consent prior to prticiption. Some subjects underwent complete ophthlmologic ex- T b le 1. O l i g o n u c l e o t i d e p r im e r s u s e d f o r m p lif ic t io n n d SEQUENCING OF EXONS 1-4 OF THE HUMAN G U C A 1 A (G C A P 1 ) GENE T b l e 2. A c c e ssio n n u m b ers o r r e f e r e n c e s fo r G C A P s e quences used in the lignm ent of F igure F igure 3B Nme Species Source bgcap1 Bos turus AAB31698 hgcap1 Homo spiens NP_ mgcap1 Mus musculus NP_ rgcap1 Rttus norvegicus cgcap1 Gllus gllus P7988C fgcap1 Rn pipiens O73761 zgcap1 Dnio reric AAK95947 fugcap1 Fugu rubripes CAD silugcap1 orygcap1 Xenopus (Silurn) tropiclis Oryzis ltipes AL BAB83093 bgcap2 Bos turus L43001 hgcap2 Homo spiens mgcap2 Mus musculus NP_ sbgcap2 Morone sxtilis unpublished dt cgcap2 Gllus gllus P79881 fgcap2 Rn pipiens O73762 silugcap2 orygcap2 Xenopus (Silurn) tropiclis Oryzis ltipes AL BAB83094 fugcap2 Fugu rubripes CAD zgcap2 Dnio rerio AAK95948 hgcap3 Homo spiens AAD19944 E x o n P r i m e r s e q u e n c e zgcap3 Dnio rerio AAK95949 G C A P1_1F GCA P1_1R G C A P1_2F GCA P1_2R G C A P1_3F GCA P1_3R G C A P1_4F GCAP1 4R 5'-G G CCTG TCCATCTCAGACGT-3' 5'-CCCCAGCTGGTCAGGCTTCCA G-3' 5'-GCCTGAGGCTGGAGTGAGCG-3' 5'-C TAACCCTGGGCTCTCA GTTCC-3' 5'-CCTGAGATAGGATAAGGATGG-3' 5'-ACCCCA CATCCATGG TGA CC-3' 5'-CTGGACTGCAGAAATGAACACCCTC-3' 5'-GGCGAGCTAAGCCTCTGAGTTC-3' zgcap4 Dnio rerio AY zgcap5 Dnio rerio AY zgcap7 Dnio rerio AY fgcip Rn pipiens O fugugcip Fugu rubripes [12] Up to eight GCAP genes hve been identified in vertebrtes (mmmls, fishes, nd mphibi). One sequence (sbgcap2, from M. Primers mrked with F re forwrd (sense) primers, while those mrked with R re reverse (ntisense) primers. Amplicons generted with F/R pirs were directly sequenced to identify muttions in from genomic contigs s described in [12], is not vilble from sxtiolis) is unpublished, nd one sequence (fugugcip), derived the GCAP1 gene. Genbnk. 144

3 Figure 1. Identifiction of the GUCA1A muttion in n Americn Pedigree. A: Pedigree of the study fmily. Individuls re identified by pedigree number. Squres indicte mles, circles indicte femles, slshed symbols indicte decesed, solid symbols indicte ffected individuls, open symbols indicte unffected individuls, +/+ indictes two copies of wild type GUCA1A, M/+ indictes one copy of wild type GUCA1A, one copy of mutnt GUCA1A. The probnd nlyzed in this study is identified by n rrow. B: Electroretinogrm (ERG) obtined from individul IV:1. Trces obtined from the study subject re shown in the right column. Trces from norml subject re shown in the left column for comprison. The scotopic ERGs obtined with blue, red, nd white flshes re nerly norml (top three rows). The photopic ERG is non-recordble consistent with lck of cone function. 30 Hz flicker following is ttenuted, but still recordble. These results re consistent with dignosis of cone dystrophy nd re similr to electrophysiologic findings described in subjects with Y99C nd E155G muttions in GUCA1A. C: Comprison of DNA sequences of the ffected individul IV: 1 (top) to norml control (bottom). The probnd DNA reveled C-to-T trnsition (boxed) t the beginning of in exon 4 of GUCA1A, resulting in leucine to phenyllnine chnge (L151F) in GCAP1 (boxed). This muttion segregted with the disese phenotype nd ws not found in 200 norml controls. 145

4 mintion including visully cuity mesurements nd fundus exmintions. Other subjects were interviewed by telephone. Blood smples were obtined by venipuncture. Ptients were dignosed with cone dystrophy if they showed the clssic trid of photophobi, decresed color vision, nd decresed visul cuity. Electroretinogrphy: One ptient underwent electroretinogrphy. Electroretinogrms (ERGs) were recorded using stndrd electrophysiologic methods (D. Creel, Clinicl Electrophysiology, Webvision). The subject ws drk dpted for 30 min. Reference nd ground electrodes were ech ttched to n erlobe. Using n indirect hedlmp with severl Wrtten 26 red filters simulting mobile drk room sfe light, Burin-Lwwill speculum contct lenses ws inserted to record ERGs from the corne. Responses were obtined using Nicolet gnzfeld bowl, mplifier, nd computer. Responses were differentilly mplified (bnd pss Hz), verged, nd stored. Amplitude bnd pss sensitivity ws one millivolt. ERGs were recorded using single scotopiclly blnced dim blue nd red flshes, nd bright white flshes. Ptients were then light dpted with bckground illumintion of 3.18x104 cd/m2 for 10 min nd photopic ERGs were recorded using 30 Hz flicker following nd the sme bright white flsh. Genetic linkge nd muttion screening: Genomic DNA ws extrcted from blood smples using Qigen DNA isoltion kit ccording to the m nufcturer s specifictions (QIAGEN, Vlenci, CA). Linkge to microstellite mrker D6S1017, linked to the GCAP1 locus ws ssessed using estblished methods [24]. Ech of the four exons of GCAP1 were then mplified by PCR using flnking intron specific primers (Tble 1) nd screened for muttions by denturing high performnce liquid chromtogrphy (DHPLC; WAVE System, Trnsgenomic, Omh, NE). Sequence ltertions were identified by direct sequencing with CEQ Dye Termintor Cycle Sequencing Kit on Beckmn-Coulter CEQ 8000 Genetic Anlysis System, ccording to the mnufcturer s instructions nd using estblished methods [25,26]. Fundus photogrphy: Fundus photogrphs nd fluorescein ngiogrphy were performed using TOPCON digitl fundus cmer ccording to the mnufcturer s specifiction (TOPCON Americ Corportion, Plesnton, CA). Sequence lignments: The lignments of FstA versions of GCAP mino cid sequences were generted by Clustl W (version 1.82). The GenBnk ccession numbers for the mino cid sequences re in Tble 2. RESULTS Clinicl evlution o f the fmily with dcord: The pedigree consisted of 30 living members of five genertion fmily (Figure 1A) in which the disese ws inherited in n utosoml dominnt pttern. Clinicl chrcteristics of the fmily (Tble 3) included photophobi, color vision defects nd centrl cuity loss. Most subjects within the fmily noted symptoms in the second nd third decdes of life. Visul cuity rnged from younger individuls with norml or nerly norml visul function to older individuls with legl blindness. Representtive fundus photos from two individuls from this pedigree re presented in Figure 2. The probnd IV:1 t ge 36 is mildly ffected. Fundus exmintions reveled only subtle pigmentry chnges in the mcul (Figure 2A,B). However, the fundus fluorescent ngiogrm of this individul (Figure 2C,D) reveled more mrked trophic chnges in the mcul thn cn be seen on fundus exmintion. The mother of the probnd t ge 64 is more severely ffected nd hs geogrphic trophy of the retin nd retinl pigment epithelium (Figure Tble 3. Clinicl chrcteristics of individuls from study fmily Age t Current cuity Age t time of Current onset of correct Individul Gender ge OD OS symptoms dignosis II:3 F <20/200 <20/200 3 F 88 <20/200 <20/ III:2 F 64 20/400 20/ * III:5 F 60 <20/300 <20/ III:7 M 64 20/200 20/ III:9 M 52 20/400 20/ LO I I M 38 20/40 20/50 24 M 24 20/40 20/ M 33 20/40 20/ F 36 20/400 20/ M 39 20/30 20/ V:4 F 8 20/20 20/20 ** 6 > I I I I I 1 4 O In the Age t time of correct dignosis column, the two individuls hving single sterisk were originlly dignosed with juvenile mculr dystrophy. In the Age t onset of symptoms column, the symptoms include photophobi, decresed color vision, nd decresed visul cuity. In this sme column, the double sterisk indictes tht this individul is symptomtic, most likely due to her youth, though she hs inherited n L151F muttion. 146 * 42

5 Figure 2. Fundus photogrphs of ffected subjects from the study fmily. A: Individul IV:1, ge 36, with visul cuities of 20/40 in the right eye nd 20/50 in the left eye. Color fundus photogrph of the right eye. Subtle pigmentry chnges re evident in the mcul. B: Color fundus photogrph of the left eye show similr chnges. C: Fundus fluorescein ngiogrm of the right eye. Tken t 5:42 min fter injection of sodium fluorescein, this photogrphic method revels more mrked chnges in the retin tht re not immeditely evident in the color photogrphs. Ares of brightness within the mcul (rrows) re referred to s window defects nd represent res of retinl nd retinl pigment epithelium trophy. D: Fundus fluorescein ngiogrm of the left eye tken t 6:22 min fter injection shows similr chnges. E,F: Color fundus photogrphs from the more severely ffected mother of the subject depicted in Figure 2. Individul III:2, ge 64, hs visul cuities of 20/400 in the right eye nd 20/400 in the left eye. E: Color fundus photogrph of the right eye revels trophy of the mcul. F: Color fundus photogrphs of the left eye revel similr chnges. The peripherl retin looks norml. 147

6 2E,F). The dim blue scotopic ERG response (mostly rod response) of the probnd is essentilly norml. The dim red scotopic ERG predominntly reflects rod ctivity, but lso reflects some cone ctivity. Thus the norml subject s dim red ERG hs n -wve, n erly, smll b-wve lbeled bx, nd oscilltory potentils on the scending limb of the b-wve. The erly -wve, bx-wve, nd oscilltory potentils reflect cone ctivity, nd these responses re bsent in the probnd s ERG. The scotopic bright white ERG is minimlly bnorml with n ttenuted b-wve mplitude nd slow implicit time. The 30 Hz flicker response nd the photopic white ERG were profoundly subnorml nd prolonged. Electrophysiologiclly, the mnifesttions of the L151F phenotype closely resemble tht of the Y99C, E155G, nd I143NT muttions where photopic responses nd flickers were non-recordble [16,19,20]. Rod responses nd mximl drk dpted single white flsh responses were only mildly subnorml in mplitude. Genetic nlysis: An initil genotype nlysis with mrker D6S1017 yielded LOD score of 3.3 t 0=0.00, consistent with linkge to GUCA1A. Subsequently, DHPLC muttion screening nd direct sequencing identified C451T trnsition resulting in novel L151F chnge in the GCAP1 mino cid sequences in ll ffected individuls (Figure 1C). This muttion segregted with the disese phenotype nd ws not found in 200 norml controls. Like the E155G [16] nd the I143NT [20] muttion, the L151F muttion occurs within the fourth EF-hnd domin of GCAP1. d i s c u s s i o n Progressive cone dystrophies re inherited in n utosoml recessive, utosoml dominnt, or X-linked fshion, nd re cused by heterogeneous set of genes (Tble 4) [1]. These genes include phototrnsduction genes like GNAT2, which encodes the cone trnsducin subunit, the CNGA3 nd CNGB3 genes, which encode subunits of the cgmp-gted ction chnnel, nd the genes GUC2D nd GUC1A, which encode GC1 nd GCAP1. Also contined in this group re trnscription fctors (CRX), genes involved in the retinoid cycle (ABCA4, RDH5), nd genes involved in protein trnsport through the cilium (RPGR nd RPGRIP). A number of loci re known for which the corresponding gene hs yet to be identified (RetNet). We hve described the clinicl fetures of twelve subjects in five genertion fmily with n utosoml dominnt cone dystrophy (Tble 3). This disese ws found to be cused by novel muttion in GUCA1A ffecting the EF4 high ffinity C2+ binding site of GCAP1. The phenotype of ffected fmily members crrying the L151F muttion is cliniclly very similr to three of the four previously described muttions in GUCA1A. Initil symptoms of reduced centrl cuity nd loss of color vision becme pprent in the second nd third decdes. It ppers tht ll ffected individuls eventully progress to legl blindness, with visul cuities between 20/ 200 nd 20/400. Funduscopic chnges were initilly subtle, but progressed to centrl trophy over time. The L151F phenotype resembles the Y99C muttion in severity, but the ge of onset of clinicl symptoms is somewht erlier in ptients with the L151F muttion. EF hnds represent high ffinity C2+ binding sites nd re responsible for the clcium sensitivity of the GCAP/GC system. Not surprisingly, muttions within these domins were shown to lter clcium sensitivity [16,21,22]. Some of the mino cids within the domin contin oxygen in their side chins tht fcilitte clcium coordintion [27,28]. Although the L151 residue does not directly prticipte in clcium binding, the L151F muttion ffects the structure of the EF-hnd T ble 4. Genetic loci ssocited with progressive cone dystrophies Inheritnce Locus Symbol OMIM Gene Reference AR 1p GNAT2 [33] AR 1p22.1 CORD ABCA4 [34] AR 1q12-q2 4 CORD [35] AR 2q CNGA3 [36] AD 6p21.1 COD GUCA1A [19] AD 6q13 CORD RIMS1 [37] AD 6q RCD1 [38] AR 8q CNGB3 [39] AR 12q RDH5 [40] AD 17p13.1 CORD GUCY2D [41] 18q21.1-q21.3 CORD [42] AD 19q13.3 CORD CRX [43] XR Xp11.4 CORDX RPGR [25] XR Xp11.4-q13.1 CORDX [44] XR Xq2 7 CORDX [45] The tble lists the inheritnce mode (AR represents utosoml recessive; AD represents utosoml dominnt; XR represents X-linked recessive), the chromosoml locliztion ( Locus ), the disese nomenclture from RetNet, the Online Mendelin Inheritnce in Mn (OMIM) ccession number, gene symbol, nd reference. 148

7 LO P H X c o P Q CO Q Q O O i O < PL 3 g tq E- < pl 3 g < & tq U X U co u H < tq H m o i Q C H E s s H PL s Em E l tq P l tq P l tq P l > i co < S H E H CO U O P- Em El Em El s P-i p. S!z X X H X U H E H > Q Pi! S i-= O P (J (J (J u S S!Z (J (J (J u Q Q Q Q >H >H >H >- J J J Em Em Em S I - Em El!z < x Em El H X El >h >h >h >- g S H O I I < tq < p X P l ft < X P l CO p. i i < tq m cu!z m ix (J (J QC QC Q Q ft i m n i s s >H m m ix h oc e- i-q q 1-q h= co CC H S i - Em Cl > Q > H H C q cc cc S Em Em El H H El H H Cl J CO J H H Cl (J (J U Q (J (J U i!z M > I Q I I CC CC q Em Em El > S 2 CO S S!z H H E Em Em El H H C H H H Cl E-i E-i E-i E S S S E H < H C Q H O H I I!Zi S H P l!z CC CC (J (S i fcfce ie if C fc f C rt ix ix ix ix ix ix ix c r H H E h Eh H H H H H H H P l Q Q Q Q C _ D C _ D ; i n i X pz pz pz pz pz pz pz pi Q M Q Q Q Q Q C (J (J (J (S i (J (J (J (S Q Q Q Q >H >H >H >- J J J h= Em Em Em El >h >h >h >- S 2 > < m QC QC!Zi O m o i i co S I - < s > co i i co m p. H!Z Q S O S J < X H S Em > H S H CO QC > ( j ( j u H H Pl H P l Q!Z J J H O m p M > QC i-= H i-= > i -!Zi I d! I P I E I I i-= I I U I O I O CO C CO > H P l > H CO H U CO < U!Zi I I i - < QC > < co d! M > S < d! QC QC U QC QC O I I < E I s s co u co u P-I p-!z S I s J h= s s > i -!Z i E J J Q d! J i - P-I p-!z 2 s J h=!z QCQC!Zi QC QC QC O Q co P P- co & < C Em H Q J CO J t Pl CO J O!Z J J d Em O t QCQC > H d : Q d! Q CO P P p P P P P > I H CO H CO i-= J J J O I U Q O H!Z!Zi P P- x U m E- < co H d : Q d! X X X H U E i i < QX co H d l oc < J h= >h >- u < s m ix >h >- QC QC O CO M > U!Z (J U P P- > E- S i-= s X X X x cy > w u t s i-= E- CC x Q d : m > H H Q O J J!z H d : I I Q d! o co X X X CJ < Q X X < P- CO QC d! QC U O!Z H X S i-= (j co < E- P-i < S > i i i S!z J > P-l Cl QC d! H H CO < >h O!z!Z!z > d i co pq tq &: V V V i i CO CO <!Z dq S CC O u Em 2 O d S U i - P IX (J E- l l i 1 co I d : QC E- i-= s i-= 2 E- P Cl d O f< I J >- I I i - d i co u i i > QC X Q!Z U E- > t Q d! H I I I X M X H H < S > CO i co QC >h x < X X X V X m X X < O Pi! d! < H m >h CO O Q U >h >!Zi i - CO C > t f M > CC X J CO S CO VV QC!Zi d! Em >- E- cc x (j (j u tq d: VV H J > J i_q X V V V H Cu P- CO E-i E X X X q x x p-i P-i P C P;q P- Q X m m ix < d! h h c CO i i (j (j u co pq [ S i - i i Em Em d M M Ci! M M Ci! J i_q X CO E E-i E-i E J M M Ci! (J (J U Q d! M M Ci! U!Z Q CO < > H C < P- CO p- (J (J U i!zi ;z > i Q Q QC Q tq d i Em d- i i J i_q X H < co V V V Q M M d l P-i P- CU > pq pq pq Q Emt X V X u J E U!Z X Q t P CO QC d! CO QC Q CO H QC H < S p- Q P Pr O CO (J X!Zi E U M > O X I - d : VV!Zi E co co Em i Q X Em U CO I I >h d! > t m S d O J QC > Q S O d l Q X S s co m > H H d O X H CO X I - P;q i - U < > i - QC M E- Em U I d! I < I I CO < tq >- >h QC x < co H!Z x > < (J X X X X (J > x X E- 2 l l I l l I H s i< Q Q Q Q < O >h>m > > M l l I E- CO s X O Emt Em Em S S I I i I I E-i E I I S tq tq P P- P P!Z Q CO > d i \X X X Q E-i E-i E I I J i_i i i S pq tq Cl tq co h- E E-i Q tq Cl i-= oc d! tq d: tq i-= J i_q X X J J S H H H d l tq tq Cl tq d: tq tq d : tq d: tq d: tq tq tq tq >H O E-i E-i p- X E- Q X Q X tq co O QC QC cr QC X X X X Q x x x x x X X Q X Q Q H e- I I VV 1-q i- i u U Cl UX X X co i< u (J (J u o u (J (J (S (J (J (J (S (J (J (J (S!Z!Z CO!Z!z X X X X X X X X X X X (J CC (J CC (J (J u (J (J (S (J (J (J (S (J (J (J X X Q Q Q Q X X X X > QC I- s x x X X X X X Q Q X X X X >h >- >h >- >h >- >H>H>s m> s > i-q i_q i_q X J i_q i_q X >h>- >H >H >- Em Em Em d- Em >h >h >- i - S QC Em Em d- Emt Em Em d- Em Em Em d- Em Em Em d- >h E- H H E >- >h >h >h >- >h >h nooc'.d'.dijouoconcoconojooojoolir: ~ ~ ~ ~ ~ ~ i-q i_ i i C'-Dr-'COLO co ' I H ft ft ft HHHf<HHf<UHCgCgCgUf<CgCgCgUUUUULOCOUUCO ft ftft <<<<< i< i< h- U ftftftuftftuc_dftftftftc_duftftftc_dc_dc_dc_dc_dftftc_dftc_dftftc_d UUU-PUU UUUU UUU-H UU tt UUUrdUUMpUUUUPMUUU-HppppUUPUPUUP..C.- Q MM-I O O M I M-l O NM-I O CO N N 4-1 N Pl Pl Figure 3. The EF4 C2+ binding site nd sequence lignment of vertebrte GCAPs. A: The C2+ binding loop consisting of 12 mino cids is flnked by two -helicl regions. Residues in the loop tht coordinte the C2+ion re mrked with n sterisk. Although the 151 position mino cid (rrow) does not directly coordinte the C2+ion, it is highly conserved throughout the GCAPs (leucine or isoleucine) suggesting it is essentil for structure nd function of this loop. B: The known vertebrte gunylte cyclse ctivting proteins (vertebrte GCAP1-8) hve been ligned (only the C-terminl hlves contining EF3 nd EF 4 re shown). Residues conserved in more thn 50% of the sequences shown re red. Conservtive substitutions re blue (for origin of sequences nd ccession numbers, see Methods). 149

8 domin nd thereby ffects clcium sensitivity [29]. Review of the sequences of 30 GCAPs in 8 vertebrte species (Figure 3) reveled tht the L151 residue is highly conserved. Only the more distntly relted GCAP6-8 of pufferfish nd the gunylte cyclse-inhibitory proteins (GCIPs) crry chemiclly similr isoleucine t this position. The presence of isoleucine is not considered to interfere with C2+ binding to EF4 since it conforms with the EF hnd consensus sequence [30]. Replcement of L151 by phenyllnine is lso considered conservtive substitution since both Leu nd Phe re hydrophobic nd not much different in size [29]. However, even conservtive substitutions cn lter binding of C2+ to EF hnd loops significntly. For exmple, An E155D mutnt of GCAP 1 (replcement of n cidic Glu by nother cidic residue, Asp), drsticlly reduced C2+ sensitivity of E155D-GCAP1 [31]. We conclude tht the L151F muttion is pthogenic for the following resons. First, the muttion segregtes with disese in lrge five genertion pedigree with 30 fmily members. Second, this muttion ws not found in over 200 controls. Third, the muttion ffects the high ffinity C2+ binding site EF4, which hs been shown to be key for C2+ sensitivity of GCAP1 in vitro [31,32]. Fourth, two other muttions in EF4 hve been linked to dominnt cone dystrophy [16,20]. Fifth, the L151F muttions ws found to be pthogenic in n utosoml dominnt cone/rod dystrophy in n unrelted pedigree [29]. 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