89Phenotype in CRX mutations

Correspondence and reprintrequests to: Radouil Tzekov9900 N Central Expressway, #400Retina Foundation of the SouthwestDallas, TX 75231USATel: (214) 363 3911Fax: (214) 363 4538e-mail: rtzekov@retinafoundation.org

Acknowledgements: We acknowledgethe excellent technical assistance ofKirsten Locke in obtaining some ofthe ERG recordings and fundusphotographs. We thank William H.Swanson for performing some of thecolor vision testing and Edwin Stoneand Gerald Fishman for providingDNA for comparative testing. Thiswork was supported by NIH grantsEY05235 and EY07142 and by grantsfrom the George Gund Foundationand the Foundation FightingBlindness.

Research report

Ophthalmic Genetics 1381-6810/00/US$ 15.00

Ophthalmic Genetics – 2000, Vol. 21,No. 2, pp. 89-99© Swets & Zeitlinger 2000

Accepted 28 February 2000

Visual phenotype in patients with Arg41Glnand Ala196+1bp mutations in the CRX

gene

Radouil T. Tzekov1

Melanie M. Sohocki2

Stephen P. Daiger2

David G. Birch1

1Retina Foundation of the Southwest, Dallas, TX and 2HumanGenetics Center, School of Public Health, and Department of

Ophthalmology, University of Texas, Houston, TX, USA

Abstract Our aim was to describe the visual function characteris-tics of affected members from two unrelated families with differentdominant mutations in the CRX gene. Standard full-field ERGs andhigh-intensity a-wave series were obtained. In addition, in most sub-jects, dark-adapted (DA) thresholds, color vision function (arrangementtests), and static perimetry were assessed. A point mutation in codon 41of the CRX gene (Arg41Gln) was identified in family members fromthe RFS087 family who were tested on several occasions since 1983.Depending on age, affected members showed varying degrees of acuityloss, normal or slightly elevated DA thresholds, reduced cone a- and b-wave amplitudes, normal or minimally delayed cone b-wave implicittimes, and normal rod and cone phototransduction gain parameters. Aninsertion mutation (Ala196+1bp) was found in two members of anotherfamily (RFS014). Affected members showed reduced visual acuity,normal or slightly elevated DA thresholds, relatively preserved rodERG and substantially reduced or undetectable cone ERG, and normalrod phototransduction gain parameters. The Arg41Gln was associatedwith a late-onset, slowly progressing mild form of cone-rod dystrophywith cone loss but preserved rod and cone sensitivity until later in life.The Ala196+1bp mutation was associated with an early-onset, severeform of cone-rod dystrophy similar to that described in the originalCORD2 family (Evans et al., Arch Ophthalmol 1995;113:195-201).

Key words Full-field electroretinography; CRX; cone-rod dystro-phy; a-wave; phenotype-genotype correlation

Introduction The CRX (Cone-Rod homeoboX) gene was isolatedand cloned independently at about the same time by three groups.1-3 Itconsists of three exons and encodes an OTX-like paired homeodomainprotein of 299 amino acids with molecular mass of 32 kDa. It is ex-

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pressed in retina tissue and pineal gland, but not in any other of 10tissues or cells examined. More recently, it was shown that CRX mightplay a role in regulating pineal gene expression by interacting withpineal regulatory element.4 It has been shown that CRX binds to thesite of photoreceptor cell-specific genes such as rhodopsin, IRBP, andarrestin1,3 and is crucial for the formation of the outer segments and forphotoreceptor survival.2,3,5,6

Human CRX maps within the chromosome interval 19q13.3, whichcontains the locus responsible for the cone-rod dystrophy-2 phenotype(CORD2, #120970, OMIM database, 1). The original CORD2 familyshowed severe, early-onset chorioretinal atrophy with loss of visualacuity in the first decade, onset of night blindness after 20 years of age,and little visual function after the age of 50 years. To date, a specificCRX mutation has not been identified in the original family.7,8

Truncation mutations in the CRX gene have been associated with de-novo (presumed dominant) cases of Leber’s congenital amaurosis(LCA).9,10 In addition, a 12 base-pair deletion was found in a largedominant pedigree diagnosed with LCA.11 Dominant LCA can be con-sidered to lie at the extreme of a continuum of severity in CORD2.Toward the other extreme is a fairly mild, late-onset phenotype de-scribed for affected members of a family with the Arg41Trp mutation.6

Point mutations tend to be associated with milder forms of autoso-mal dominant cone-rod dystrophy,11 including the R90W mutation12

that causes mild disease in heterozygous state and LCA in homozygousstate.

We recently identified disease-causing CRX mutations in four ped-igrees.11 Phenotypic information for two of the families was publishedprior to identifying the mutation.13,14 The purpose of the present paperis to present a detailed phenotypic description of the other two families.

Subjects and methods

patients The proband from family RFS087 was first seen in 1983.Based primarily on fundus appearance and field loss, the patient wasdiagnosed with atypical retinitis pigmentosa by a retinal specialist. Thepedigree of the family (Fig. 1) shows clear evidence of an autosomaldominant mode of inheritance. Six members of the family were avail-able for clinical testing. Genetic analysis revealed an Arg41Gln muta-tion in the CRX gene in four family members.

The proband from family RFS014 was first seen in 1992. Based pri-marily on uncorrectable visual acuity since childhood, she was diag-nosed with progressive cone degeneration. Four members of this familywere available for genetic and electrophysiological examination (Fig.1).Genetic analysis revealed a 1-bp insertion in codon 196 (Ala196+1bp)in two family members. Since there was no consanguinity in the familyand the grandmother of the proband died at an early age, the mostlikely mode of inheritance is autosomal dominant.

clinical examination Ophthalmologic examination included best-corrected visual acuity, direct and indirect ophthalmoscopy, and fundusphotography. Fluorescein angiography was performed on selected cases.

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91Phenotype in CRX mutations

Dark-adapted thresholds in the parafovea with an 11-degree test wereobtained using the Goldman-Weekers adaptometer. In infants too youngfor detailed psychophysical testing, dark-adapted thresholds were ob-tained with full-field pupillometry.15 Automated perimetry was per-formed with a Humphrey Field Analyzer (model 640) and kinetic pe-rimetry with a Goldman perimeter.

Color vision was tested with pigment (Ishihara plates) and arrange-ment tests (D-15 and Adams desaturated D-15). In a few cases, anom-aloscopy was performed with specially designed equipment.16

electroretinography Standard full-field ERGs were elicited bymethods previously described.17 Responses were obtained from the anes-thetized cornea with a bipolar gold contact lens electrode (Doran In-struments, Littleton, MA, USA). The analysis of rod and cone a-wavesfollowed a recently described protocol.18 Briefly, a set of four whiteflashes (3.2 to 4.4 log scot td.s) were first presented in the dark. Thesame stimuli presented against a rod-saturating background (30cd/m2)elicited cone a-waves. These were subtracted from the dark-adaptedresponses to produce rod-only a-waves. Rod-only responses and cone-only responses were then fit with computational models.19,20 Rod pho-toreceptor recovery kinetics were assessed with the paired-flash para-digm.21,22 Briefly, a saturating probe flash was used to follow recoveryfrom a test flash of 2.3 log scot td.s. An exponential recovery functionwas used to determine the period of complete photoreceptor saturation.

Results

rfs087 family

History Visual symptoms in all four affected family members wererelated to age. The proband (II:2) was asymptomatic until his late fif-

Fig.1. Pedigrees of the RFS087 andRFS014 families. The black arrowindicates the proband. A solid linecrossing the symbol indicatesdeceased person.

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ties. He complained initially of bilateral temporal deficits in his visualfield and minor problems related to night blindness. The son (III:2) wasseen initially at age 44, when he did not have any visual complaints.On the second visit, when he was 49, he reported occasional ‘distor-tions and blurry spots’ in his visual field. The granddaughter (IV:2)reported minor night vision problems, side vision distortion, and occa-sional blind spots in the periphery when she was initially tested at age19. The great-grandson (V:I) was asymptomatic at age 4.

Genetic testing The initial results from genetic testing of this familywere reported previously.11 More recently, two DNA samples fromaffected members of the RFS087 family were compared to a samplefrom the Arg41Gln individual reported by Swain et. al.6 using threemarkers tightly linked to the CRX locus: D19S902, D19S606, andD19S412.23 For each marker, an allele was shared by affected membersof the two families. These data suggest that the mutations in the twofamilies are likely to have arisen in a common ancestor.

Fundus appearance The proband (II:2) at age 58 showed mild vesselattenuation, normal optic disk appearance, and three round regions ofsubtle depigmentation in the upper part of the macular region in botheyes (Fig. 2A,B). The fundus appearance of the posterior pole changedduring the follow-up examinations and at his latest visit, at age 72,there was a region of perimacular atrophy in both eyes. The son (III:2)at age 44 showed minor pigment clumping in the periphery (Fig. 2C).The granddaughter (IV:2) showed a pale optic disk head and irregulardisk margin related to peripapillary atrophy. Macular appearance wasnormal (Fig. 2D). In both the son (III:2) and the granddaughter (IV:2),there was no appreciable change in fundus appearance over six yearsof follow-up. The great-grandson (V:I) showed no fundus abnormali-ties at age 4.

Psychophysical measurements Best-corrected central visual acuity inthe proband (II:2) was normal at the time of his initial visit (OD=20/15; OS=20/20, age=58). On three consecutive visits, 7, 13, and 15years later, visual acuity deteriorated substantially (to <20/1000). Dark-adapted thresholds in the parafovea showed progressive elevation –from borderline abnormal at the first visit to 1.4 log units elevation atthe last visit (Table 1). Static perimetry in the proband revealed a smallparacentral scotoma and a temporal field defect in each eye. This visualfield defect did not demonstrate significant change over time.

The son (III:2) was tested for the first time when he was 45. At hisinitial visit, he had small (about 10 deg) paracentral scotoma in theupper temporal portion of each visual field. This visual field defect didnot change over nine years of follow-up. Color vision testing withIshihara plates revealed a moderate protan defect (Table 1).

The granddaughter (IV:2) was tested initially when she was 19 yearsold. At that time, her visual acuity was normal (OD=20/20, OS=20/20)and dark-adapted thresholds were within the normal range. Standardpigmented and arrangement tests were unremarkable. However, anom-aloscopy revealed a red-green defect, consistent with reduced foveal

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Fig. 2. Fundus photographs frommembers of both families. (A)Proband in RFS087 (II:2, 58 yrs),right eye, nasal and inferior region ofthe near periphery. (B) Proband inRFS087 (II:2, 58 yrs), left eye,posterior pole. (C) Son in RFS087(III:2; 41yrs), left eye, posterior pole.(D) Granddaughter in RFS087 (IV:2,25 yrs), left eye, posterior pole. (E)Proband in RFS014 (III:1, 30 yrs), lefteye, posterior pole.

A B

CD

E

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cone optical density. These findings were unchanged over six years offollow-up.

The great-grandson of the proband (V:1) has normal visual acuity atage 4. Dark-adapted rod thresholds obtained with full-field pupillome-try were within normal limits.

Electroretinography Standard full-field ERGs from one non-affectedmember (IV:1) and three affected members at three different ages areshown in Figure 3A. The top row shows rod responses and revealstiming and amplitude within normal limits at age 19 (IV:2) and clearamplitude loss with age (III:2 and II:2) with normal timing until late indisease. The second row shows mixed rod and cone responses to ascotopically matched red stimulus. The amplitude of the dark-adaptedcone component is severely attenuated by age 58 (II:2). The third rowshows the maximal response to an ISCEV standard white stimulus. Inall affected members, an abnormally low b/a-wave ratio was present.The 30 Hz flicker response (4th row) shows normal cone b-wave im-plicit time at least until age 50. Both the flicker response and the singleflash cone b-wave (5th row) show progressive decrease in amplitudewith age. The ratio between the amplitude of the rod b-wave and coneb-wave shows slightly greater loss of cone than rod ERG.

Progression within a patient was roughly the same as progressionwith age in the family. For example, the proband (II:2) had ERG char-acteristics in his 50s similar to those of his son (III:2) at a comparableage.

High-intensity a-wave series were obtained from three affected fam-

Age Follow-up Visual acuity Fundus Visual field Color DA thresholds(initial (years) Initial–last visit appearance vision (range)

RFS087 Family

II:2 58 15 OD=20/15–20/700 Perimacular Central scotomas Moderate red- Borderline–OS=20/20–20/1000 atrophy green defect 1.4 log ↑

III:2 51 6 OD=20/20–20/20 Pigment clumping Reduction of Slight red- WNLOS=20/20–20/20 in the periphery sensitivity, green defect

paracentralscotomas

IV:2 19 6 OD=20/20–20/20 Peripapillar WNL Slight red- WNLOS=20/20–20/20 atrophy green defect1

V:1 2 2 OD=N/A–20/20OS=N/A–20/20 WNL WNL WNL

RFS014 FamilyII:1 54 3 OD=20/200-20/200 Depigmented WNL

OS=20/250-20/250 posterior poleIII:1 24 6 OD=20/50–20/160

OS=20/200–20/200 Pigment Paracentral Tritan WNLclumping – scotomas defectperiphery

1With anomaloscopy.WNL, within normal limits.

table 1. Basic clinical findings infamily members carrying CRX muta-tions (RFS087 and RFS014 families).

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ily members. Values of maximum amplitude (log RmP3) and amplifi-cation (log S) were derived from ensemble fits to rod-only and cone-only responses to four intensities. The rod-only (Fig. 3B) and cone-only (Fig. 3C) responses to the highest intensity (along with the modelprediction) in patient II:2 were smaller than the responses in a repre-sentative normal. For all affected family members (Fig. 3D), rod max-imum amplitude (log RmP3) was reduced by less than 0.5 log unitsfrom mean normal and the rod amplification parameter (log S) waswithin the normal range for all except the oldest family member (II:2at age 72). The high-intensity cone a-wave data revealed a clear trendof decrease in cone RmP3 with age despite normal values for the Sparameter.

Fig. 3. (A) Standard full-field ERGs from one non-affected (IV:1) and three affected members of the RFS087 family. Horizontalrows show computer-averaged responses. Scotopic blue: a rod response to blue test; scotopic red: mixed rod and cone response tored test; maximal response: mixed rod and cone response to ISCEV standard white test; 30 Hz flicker: cone responses to a rapidlyrepeated stimulus (30 Hz white flicker); photopic white: cone response to standard white on background (30 cd/m2). Two tracesare presented for the proband (II:2) for each stimulus condition: the upper one is from his initial visit (age 52) and the lower one isfrom a more recent visit (age 72). (B) Rod-only a-waves (solid curves) for highest intensity flash in RFS 087 patient II:2 and rep-resentative normal subject. Dotted curves show the fit of the model. (C) Cone-isolated photoresponses and computational modelsfrom the same patient and normal subject. (D) Difference from mean normal values for rod and cone RmP3 and S. Dotted linesrepresent the upper and lower limit of 95% confidence interval for the mean. For cone responses, S was calculated only if theRmP3 amplitude was greater than 10 µV. For further details, see text.

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The period of complete suppression of the photoresponse followinga saturating flash of 2.3 log scot td.s was 229 msec for the proband(II:2) and 231 ms for his granddaughter (IV:2). Neither refractory timederived from the paired-flash paradigm was substantially different froman average normal refractory time of 306 ms.

rfs014 family

History The proband (III:1) was initially diagnosed in 1992. She re-ported low central vision since childhood, increased sensitivity to brightlight, and poor color vision. The father (II:1) was seen for the first timein 1995 at age 54. He reported low central vision since childhood,extreme bright light sensitivity since the second decade, poor colordiscrimination, and normal night vision.

Fundus appearance Fundus examination of the proband showed al-most normal appearance (Fig. 2E), except for a few locations withpigment clumping in the periphery of the retina and slightly irregularmacular appearance. The fundus examination of the father (II:1) showeda depigmented appearance of the posterior pole and moderate vesselattenuation (not shown).

Psychophysical measurements On their initial visit, both affectedmembers had reduced visual acuity. The daughter (III:1) had 20/50 inthe right eye and 20/200 in the left eye (Table 1). There was a consid-erable deterioration in the central visual acuity in the right eye of thedaughter (III:1) over the six years of follow-up (to 20/160). The visualacuity of the father (II:1) was 20/200 in each eye; this had not changedthree years later. Dark-adapted thresholds in the parafovea were normalin both affected members on both visits. Automated perimetric fieldswere obtained from the daughter. There were small (5 deg) paracentralscotomas in the inferior and nasal portion of each visual field. Colorvision testing revealed a moderate tritan defect in the proband. Colorvision testing of the father was not performed.

Electroretinography Standard full-field ERGs for one non-affected andtwo affected members of the family are presented in Figure 4A. For bothaffected members, the rod response (top row) showed decreased b-waveamplitude but normal implicit time. The mixed rod and cone response to ascotopically matched red response (second row) was very similar in shapeto the rod response, consistent with profound loss of the dark-adapted coneresponse. The maximal response (third row) in both affected membersshowed an abnormally low b/a-wave ratio. The 30 Hz flicker response wasseverely attenuated and delayed in the daughter (III:1) and within thesubmicrovolt range in the father (II:1). The single flash cone response hadgreatly reduced a- and b-wave amplitudes and delayed b-wave implicit timein the daughter and was nondetectable (less than 1 µV) in the father (fifthrow).

Fits of the computational model to the high intensity a-wave series inboth affected individuals demonstrated substantial reduction in cone Rmp3response, more pronounced than the reduction of rod Rmp3 response (Fig.

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4B,C). The S parameter for the rod response was normal in both patients.Due to the small amplitude of the cone a-wave response, reliable S valuescould not be obtained.

Photoresponse recovery time was determined with the paired-flash para-digm in the daughter (data not shown). The refractory time was 539 ms,comparable to the average normal refractory time of 306 ms.

Discussion Several lines of evidence are consistent with the diagnosisof cone-rod dystrophy in our two families. The main electroretinographicfeatures of both families suggest that they can be considered as type 1acone-rod dystrophy.24 There was cone ERG reduction greater than rod ERGreduction, and most showed pericentral scotomas, reduced acuity, and colorvision defects among the earliest symptoms. Normal log S for the rod a-wave, normal rod b-wave implicit time, and minimal elevations in the dark-adapted visual threshold despite rod ERG amplitude reductions are all con-sistent with localized regions of rod photoreceptor loss. Patients retainedreadily measurable ERGs at all ages. These characteristics support the di-agnosis of CRD and help differentiate the phenotype from the classical phe-notypic features of retinitis pigmentosa, especially in the first pedigree(RFS087) where there are few, if any, fundus abnormalities until late inlife. When previously reported, this family was classified as having retinitispigmentosa, late onset with cone-rod involvement, autosomal dominant.11

Fig. 4. (A) Standard full-field ERGsand high-intensity a-wave recordingsfrom one non-affected (IV:2) and twoaffected members of the RFS014 fam-ily. See Figure 3 for details. (B) Rod-isolated photoresponse and models.(C) Cone-isolated photoresponse andmodels. (D) Difference from meannormal for rod and cone RmP3 and S.NR, nonrecordable. See Figure 3 fordetails.

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The missense mutation (Arg41Gln) found in RFS087 was described previ-ously in the literature.6 No phenotypic data, however, were presented. Theauthors described the phenotypic expression of another mutation at the samelocus (Arg41Trp). Similar to patients in RFS087, dark-adapted thresholdswere within normal limits or slightly elevated (except for later in life), con-sistent with normal photon catch and normal or near normal rod numbers inthe regions tested. There are several differences, however, in the pheno-typic expression between the Arg41Trp family they reported and RFS087.First, the fundus abnormalities were much more pronounced in their fam-ily. Secondly, they describe an ‘electronegative’ response from their sc-otopic white flashes, with a b-wave that is absent. This degree of electrone-gativity was not present in RFS087 ERG recordings, although the b/a-waveamplitude ratio of the maximal response was reduced. Thirdly, in our casesthere was no impairment of the S-cone sensitivity (revealed by D15 test) incontrast to tritan errors in three of the four affected members described bySwain et al.6 Fourthly, RFS087 affected members showed gradual progres-sion with age and consistent phenotype in contrast to considerable variationin disease expression in the Arg41Trp family.

Molecular biological studies of the Arg41Trp mutation have shown thatthere is a reduced binding activity for the rhodopsin promoter.6 Althoughno direct evidence exists that Arg41Gln can cause the same effect, it washypothesized that the mutation causes changes in the net charge of the mol-ecule and, as a result, the mutant CRX protein probably reduces promoteractivity. Since the rod log S parameter is within normal limits in youngpatients, the reduced levels of rhodopsin appear not to affect local concen-tration or initial stages of phototransduction. Rather, the reduced produc-tion of rhodopsin may result in shorter than normal outer segments, consis-tent with lower than normal values of RmP3.25

The phenotype of the mutation in the RFS014 family (Ala196+1bp) ismuch more severe than that with the missense mutation. In the RFS087family, some cone function was present until late in life, whereas with theinsertion mutation, cone function was severely impaired by the age of 24.This could be attributed to the fact that Ala196+1bp causes a frame shift,leading to 39 altered amino acids, followed by a premature stop codon.11

The resulting protein lacks the conserved OTX tail, which, apparently, af-fects the binding ability of the protein to a much greater extent than in thecase of the missense mutation. The severity of the Ala196+1bp mutation iscloser to that described originally as the dominant CORD2 phenotype.7

The phenotypic expression of these two mutations is related to the sever-ity of the genotype disturbance. The moderate visual functional deficit afteradolescence and slow degeneration in the case of the missense mutationsuggests that there is a slight disturbance in the protein function and thiseffect accumulates slowly over time. In contrast, the deletion mutation isassociated with phenotypic features of moderate to severe cone-rod dystro-phy even at age 20.

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