OPHTHALMIC MOLECULAR GENETICS Discordnt Phenotypes in Frternl Twins Hving n Identicl Muttion in Exon ORF15 of the RPGR Gene Sloni Wli, MD; Gerld A. Fishmn, MD; Annd Swroop, PhD; Kri E. H. Brnhm, MS; Mrtin Lindemn, COMT; Mohmmd Othmn, PhD; Richrd G. Weleer, MD Ojective: To report discordnt phenotypes, resulting from the sme muttion in exon ORF15 (GenBnk AF286472) of the retinitis pigmentos GTPse regultor gene (RPGR) (GenBnk U57629), in 2 presumed dizygotic twin rothers with X-linked retinl disese. Methods: The 2 rothers underwent complete ophthlmic exmintion tht included est-corrected visul cuity, slitlmp iomicroscopy, nd detiled fundus exmintion. isul field recording using Goldmnn kinetic perimetry nd full-field electroretinogrm were lso otined in oth ptients. Muttionl screening ws performed for RPGR ecuse of n X-linked pttern of inheritnce indicted y pedigree nlysis. Results: One rother hd phenotypic expression of cone-rod dystrophy, while the other exhiited X-linked retinitis pigmentos. A 1-nucleotide deletion ws identified in the 3 region of exon ORF15 of RPGR (ORF15 1339delA). Conclusions: An identicl muttion in RPGR-ORF15 mnifested distinct clinicl phenotypes in individuls of the sme fmily. Our dt provide strong evidence in support of dditionl modifier genes tht cn produce diverse disese phenotypes in ptients with RPGR muttions. Clinicl Relevnce: The clinicl oservtion of different retinl phenotypes in fmily with the sme muttion in exon ORF15 of RPGR implictes the potentil importnce of modifier genes for the phenotypic expression of this form of X-linked retinl disese. Arch Ophthlmol. 8;126(3):379-384 Author Affilitions: Deprtment of Ophthlmology nd isul Sciences, University of Illinois, Chicgo (Drs Wli nd Fishmn nd Mr Lindemn), Deprtments of Ophthlmology & isul Sciences nd Humn Genetics, University of Michign, Ann Aror (Drs Swroop nd Othmn nd Ms Brnhm); nd Csey Eye Institute, Oregon Helth & Science University, Portlnd (Dr Weleer). MUTATIONS IN THE RETInitis pigmentos (RP) GTPse regultor gene (RPGR) ccount for 8% to 9% of X-linked RP nd lmost 25% of mle sujects with simplex RP. 1-4 Most RPGR muttions re detected in exon ORF15, which encodes repetitive glycine nd glutmic cid rich domin of unknown function. 5 The RPGR- ORF15 isoforms re preferentilly expressed in cells with primry cili. 6-8 The high mutility of exon ORF15 seems to e relted to unusul nucleotide composition or to the repetitive nture of its sequence. This type of sequence my dopt unusul conformtions, including triplex structures, which re ssocited with reduced fidelity of repliction. 9 Exon ORF15 contins numerous potentil polymerse rrest sites, suggesting tht rrest my occur during repliction, leding to slipped-strnd mispiring events, s mny muttions involve direct repets. Ptients with muttions in the 3 region of exon ORF15 hve primrily demonstrted cone-rod dystrophies nd milder forms of RP, 2,11,12 while muttions closer to the 5 region re detected in more severe forms of X-linked RP. 1,5,13-15 Muttions in this exon hve lso een shown to cuse cone dystrophy 5,16 nd trophic mculr degenertion. 17 However, discordnt phenotypes within the sme fmily hve not een reported, to our knowledge. Herein, we descrie fmily with 2 mle presumed dizygotic twins in whom n identicl muttion in exon ORF15 resulted in 2 different phenotypes. METHODS PATIENTS AND CLINICAL ANALYSIS Informed consent ws otined from the ptients. The reserch protocol ws pproved y institutionl review ords t the University of Illinois, Chicgo, nd the University of Michign, Ann Aror. A 52-yer-old mn (ptient I-1) reported history of decresed vision in his left eye t the ge of 49 yers (Figure 1). Previous records showed tht t 14 yers of ge his visul (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 379 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.
I 1 2 1 2 3 I 3 1 2 3 4 5 6 7 8 9 11-13 14 I 2 2 2 2 1 2 3 4 5 6 7,8 9 11 12 13 14 15 16 17,18 19,21 22,23 24 25 P 1 2 3 4 5 6 7,8 2 3 2 9 11-13 14,15 16 17 Figure 1. Pedigree nlysis shows the presence of n X-linked recessive disorder in mle memers (indicted y shding). Squres indicte men; circles, women; dimond, person s sex is unknown; dot, n oligte crrier; rrow, prond; the twins, X; P, pregnncy (sex unknown). A B C Figure 2. Fundus photogrphs of ptient I-1 with cone-rod dystrophy showing n re of trophy in the mculr region nd the sence of peripherl pigmenttion. The right eye (A) nd the posterior pole (B) nd peripherl retin (C) of the left eye re shown. cuity ws / OD nd / OS. The fundus ppernce t tht time suggested erly centrl choroidl trophy. Progressive worsening of vision in oth eyes continued therefter. The ptient reported eing extremely sensitive to right light ut hd no sujective loss of night or peripherl vision. Difficulty with color perception hd lso een noted y the ptient. He hd undergone lser in situ kertomileusis (LASIK) procedure in oth eyes 4 yers previously. His medicl history ws significnt for hypertension, which ws well controlled with mediction. His fmily history reveled pedigree consistent with n X-linked pttern of inheritnce. On ophthlmic exmintion, est-corrected visul cuity on Snellen visul cuity chrt ws / OD nd / OS. He could red / OU for ner vision with n 7 mgnifier. On testing for color vision using Ishihr pseudoisochromtic pltes, he ws le to identify only single plte with either eye. Anterior segment exmintion using slitlmp iomicroscopy showed the presence of cornel scrs temporlly in oth eyes from the LASIK procedure flps. The introculr pressure ws 15 mm Hg OU. A fundus exmintion showed grossly norml optic discs nd retinl vessel ttenution in oth eyes. A ilterl ull s eye ppering mculr lesion ws present in ech eye with tpetl type sheen temporl to the mcul (Figure 2). isul field exmintion using Goldmnn kinetic perimetry showed only mild peripherl restriction with the -4-e, I-4-e, nd -4-e test trgets nd the presence of centrl scotom to the I-4-e nd -4-e trgets in the right eye nd to the -4-e trget in the left eye (Figure 3 nd Figure 4). An electroretinogrm (ERG) ws otined using unipolr Burin- Allen contct lens electrode, s descried previously. 18 Stimuli were presented in commercil recording unit (Nicolet Gnzfeld; Nicolet Biomedicl Inc, Mdison, Wisconsin), nd signls were cquired (Nicolet iking I system, Nicolet Biomedicl (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 38 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.
135 1 5 9 75 45 1-4-e 165 I-4-e 15 18 9 8 8 9 195-4-e 345 2 225 2 255 2 285 To chnge the side, swing index long this line Left 315 Right Oject 3 Density of filters No. 4 3 2 1 I I I Figure 3. Goldmnn kinetic perimetry of the right eye of ptient I-1 showing mild peripherl restriction of the visul field nd the presence of centrl scotom. 135 1 5 9 75 45 1 165-4-e I-4-e 15-4-e 18 9 8 8 9 195 345-4-e 2 225 2 255 2 285 To chnge the side, swing index long this line Left 315 Right Oject 3 Density of filters No. 4 3 2 1 I I I Figure 4. Goldmnn kinetic perimetry of the left eye of ptient I-1 showing centrl scotom nd mildly reduced peripherl oundries. (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 381 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.
A Norml ERG OD Blue flsh Amplitude, µ/division White flsh 8 1 1 8 1 1 Time, ms B Norml ERG OD -Hz white Amplitude, µ/division White flsh 8 1 8 1 Time, ms Figure 5. Electroretinogrphic (ERG) wveforms of the right eye of ptient I-1 showing reduction of the mximl drk-dpted response with n isolted rod response within the lower rnge of norml (A) nd mrkedly reduced cone responses (B). Inc). The ptient s drk-dpted -wve responses to short wvelength nd to mximl flsh stimuli were 296 µ (within the lower norml rnge of 273-684 µ) nd 359 µ (22% elow the lower rnge of norml, 461-98 µ), respectively. The lightdpted rief flsh -wve mplitude ws 34 µ (82% elow the lower rnge of norml, 133-3 µ), nd the mplitude for the light-dpted 32-Hz flicker ws 25 µ (81% elow the lower rnge of norml, 131-354 µ) (Figure 5). The clinicl phenotype nd ERG recordings were consistent with cone-rod dystrophy in this ptient. The presumed dizygotic twin rother of ptient I-1 (ptient I-2 in Figure 1) ws seen t the University of Illinois t the ge of 52 yers. He gve history of impired peripherl vision nd nyctlopi since the ge of yers. Retinitis pigmentos ws dignosed when the ptient ws 12 yers old. His visul cuity t ge 14 yers ws / OD nd / OS; the fundus hd tessellted myopic ppernce. At tht time, non- Gnzfeld ERG ws nondetectle under light-dpted nd drkdpted conditions. Progressive worsening of impired peripherl vision nd nyctlopi, more in the left eye, ws noted y the ptient, nd he reported hving very poor vision in the left eye for the pst yers. There ws less severe sujective impirment of centrl cuity in the right eye. His other symptoms included photosensitivity nd difficulty with color vision. His medicl history ws significnt for sthm, hypertension, nd gstric reflux disese. The ptient s most recent est-corrected visul cuity ws / 1 OD mesured y Snellen visul cuity chrt nd light perception in the left eye with temporl projection. He could red J1 on Jeger ner vision chrt using n 7 mgnifier with his right eye. He hd to of left exotropi. Anterior segment exmintion showed the presence of pseudophki in the right eye nd moderte posterior cpsulr, nucler, nd nterior corticl ctrct in the left eye. Fundus exmintion showed the presence of ilterl optic disc pllor nd retinl vessel ttenution. There ws n trophic ppernce of the retinl pigment epithelium within the posterior pole in oth eyes, with reltive spring of the fovel region in the right eye. Modertely extensive midperipherl one spicule pigment clumping ws lso seen in oth eyes (Figure 6). isul field testing (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 382 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.
A B Figure 6. Fundus photogrphs of ptient I-2 with retinitis pigmentos phenotype showing the presence of midperipherl one spicule pigmenttion, retinl vsculr ttenution, nd retinl pigment epithelil cell trophy in the midperipherl retin, with reltive spring of the mcul. 135 1 5 9 75 45 1 165 15-4-e I-4-e 18 9 8 8 9-4-e 195 345 2 225 2 255 2 285 To chnge the side, swing index long this line Left 315 Right Oject 3 Density of filters No. 4 3 2 1 I I I Figure 7. Goldmnn kinetic perimetry of the right eye in ptient I-2 showing severe peripherl field loss. using Goldmnn perimeter showed severe peripherl field loss in ech eye to even -4-e test trget (Figure 7). An ERG recording showed nondetectle cone or rod responses. This ptient s phenotype ws consistent with n X-linked form of RP. GENETIC ANALYSIS Blood smples were otined from the twins for the purpose of genetic nlysis. Lymphocyte DNA ws used for mplifiction of exon ORF15 of RPGR using 1 forwrd nd 4 reverse primers s descried y Demirci et l. 11 High-fidelity DNA Tq polymerse (AccuPrime; Invitrogen, Sn Diego, Cliforni) ws used to mplify frgment of pproximtely 1.9 kilose (k). Polymerse chin rection (PCR) regents were 5 µl of the enzyme PCR uffer, nm of ech forwrd nd reverse primer, nd 1 µl of DNA t to ng/µl. The rection volume ws mde to µl with PCR wter. Rection tues were plced in PCR mchine (model 9; Applied Biosystems, Norwlk, Connecticut) nd were run on the following progrm: 94 C for 2 minutes, followed y cycles t 92 C for seconds, nd then 56 C nneling nd 68 C for 2 minutes. This ws followed y 25 cycles t 92 C for (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 383 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.
seconds, nneling t C for seconds, nd extension t 68 C for 2.5 minutes. Further extension ws done t 68 C for minutes nd then held t 4 C. The PCR-mplified products were run on 1% grose gels with 1-k ldder to check the product size nd qulity efore sending the PCR products for sequencing. Sequencing ws performed y the Biomedicl Reserch Core Fcility t the University of Michign. RPGR- ORF15 PCR products were sequenced using the reltive primers s in the referenced rticle. 11 Chromtogrms were red using demonstrtion version of the sequencer softwre to help in identifying the sequence vrints compred with norml sequences. ThemuttionidentifiedinechsujectwsORF15 1339delA. This muttion is predicted to result in frmeshift nd premture trunction of the protein. It ws previously identified y Bder et l 15 nd y Snderg et l 19 in ptients with X-linked RP. COMMENT Previous studies 1-6,11-16,19 identified tht ORF15 muttions cn cuse cone-rod dystrophy or n RP phenotype. Our findings show tht even mong individuls of the sme fmily the sme genetic muttion my present distinct phenotypes. In study y Demirci et l, 11 2-nucleotide deletion involving the sme loction, ORF15 1339_13delAG, ws shown to e ssocited with X-linked cone-rod dystrophy. A more severe form of X-linked RP ws elieved to e ssocited with muttions t the 5 region of RPGR-ORF15. 2 However, 1 of our ptients demonstrtes tht muttion t the 3 end of exon ORF15 my lso express severe form of X-linked RP, s mesured y visul field nd ERG testing. A similr oservtion ws reported y Snderg et l. 19 It would e vlule to identify the fctors responsile for the different phenotypes tht cn occur with the sme genotype. Our findings in the 2 presumed dizygotic twins crrying the sme RPGR-ORF15 muttion suggest tht modifier genes re likely to significntly contriute to n individul s phenotype. It is possile tht environmentl fctors my lso modulte disese phenotype to some degree. Nevertheless, dditionl genetic nlysis would seem prudent in fmilies tht show intrfmilil vrition in the phenotype of their retinl disese yet crry similr custive muttions. Sumitted for Puliction: June 8, 7; finl revision received July 26, 7; ccepted July, 7. Correspondence: Gerld A. Fishmn, MD, Deprtment of Ophthlmology nd isul Sciences, University of Illinois, Mil Code 648, Room 3.85, 1855 W Tylor St, Chicgo, IL 612-7234. Finncil Disclosure: None reported. Funding/Support: This study ws supported y funds from the Foundtion Fighting Blindness, Grnt Helthcre Foundtion (Dr Fishmn), nd Reserch to Prevent Blindness (Dr Fishmn). Additionl Contriutions: Shryn Ferrr, BA, provided dministrtive ssistnce. REFERENCES 1. Breuer DK, Yshr BM, Filippov E, et l. A comprehensive muttion nlysis of RP2 nd RPGR in North Americn cohort of fmilies with X-linked retinitis pigmentos. Am J Hum Genet. 2;(6):1545-1554. 2. Shron D, Snderg MA, Re W, Stillerger M, Dryj TP, Berson EL. 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Genomics. 2;8(2):166-171. 18. Pechey NS, Fishmn GA, Derlcki DJ, Alexnder KR. Rod nd cone dysfunction in crriers of X-linked retinitis pigmentos. Ophthlmology. 1988;95(5): 677-685. 19. Snderg MA, Rosner B, Weigel-DiFrnco C, Dryj TP, Berson EL. Disese course of ptients with X-linked retinitis pigmentos due to RPGR gene muttions. Invest Ophthlmol is Sci. 7;48(3):1298-14. (REPRINTED) ARCH OPHTHALMOL / OL 126 (NO. 3), MAR 8 384 Downloded From: on /17/18 8 Americn Medicl Assocition. All rights reserved.