Autosomal Dominant Cone-Rod Dystrophy with Mutations in the Guanylate Cyclase 2D Gene Encoding Retinal Guanylate Cyclase-1

OPHTHALMIC MOLECULAR GENETICS Autosomal Dominant Cone-Rod Dystrophy With Mutations in the Guanylate Cyclase 2D Gene Encoding Retinal Guanylate Cyclase-1

Susan M. Downes, MD; Annette M. Payne, PhD; Rosemary E. Kelsell, PhD; Frederick W. Fitzke, PhD; Graham E. Holder, PhD; David M. Hunt, PhD; Anthony T. Moore, FRCOphth; Alan C. Bird, MD

Objective: To describe the phenotype in 4 families with cal testing revealed a marked loss of cone function with dominantly inherited cone-rod dystrophy, 1 with an only minimal rod involvement, even in older subjects. R838C mutation and 1 with an R838H mutation in the Photopic and scotopic static perimetry demonstrated cen- guanylate cyclase 2D (GUCY2D) gene encoding retinal tral and peripheral cone-mediated threshold elevations guanylate cyclase-1. with midperipheral sparing.

Methods: Psychophysical and electrophysiological evalu- Conclusion: The phenotype associated with autosomal ation and confocal laser scanning ophthalmoscopic im- dominant cone-rod dystrophy with either an R838C or aging was performed on 10 affected members of 4 Brit- R838H mutation in GUCY2D is distinctive, with predomi- ish families. nantly cone system involvement. There is some variation in severity within the 3 families with the R838C mutation. Results: Although subjects had lifelong poor vision in bright light, a major reduction in visual acuity did not Clinical Relevance: Families with the R838C or R838H occur in most of them until after their late teens. Fun- mutation have a much milder phenotype than the fam- dus abnormalities were confined to the central macula, ily previously described that had 2 sequence changes, and increasing central atrophy was noted with age. In- E837D and R838S, in GUCY2D. creased background autofluorescence was observed surrounding the central atrophic area. Electrophysiologi- Arch Ophthalmol. 2001;119:1667-1673

ONE AND cone-rod dystro- tral atrophy occurs. In some cases, tem- phies are a subgroup of poral pallor of the optic nerve head is seen. the inherited retinal dys- Nystagmus may be present in early-onset trophies. Cone dystro- disease. In cone-rod dystrophy, function phies are characterized by that is dependent on rods, including vi- poorC central vision and an abnormal cone- sual fields and night vision, will be vari- isolated electroretinogram (ERG). In cone- ably affected according to the degree of in- rod dystrophy, the abnormality of rod volvement. Varying degrees of intraretinal function is less severe than that of cone pigment and vessel attenuation occur. function and may be detected later in the To date, studies have shown that cone course of the disease than cone dysfunc- dystrophy and cone-rod dystrophy are ge- tion.1-3 The diagnosis is established by elec- netically heterogeneous. Dominant, re- trophysiological evaluation; functional recessive, and X-linked inheritance pat- sults depend on the stage of the disease and terns have been reported.3 Seven loci have the age of the individual. The diagnosis of been identified in autosomal dominant cone-rod dystrophy may be reinforced by cone and cone-rod dystrophies.4 Loci as- the demonstration of peripheral as well as sociated with known genes include chro- central visual field loss. mosome 6p12 with mutations in periph- The most common symptoms of cone erin/RDS,5-7 chromosome 6p21 with From Moorfields Eye Hospital involvement include varying degrees of mutations in GUCA1A,8 chromosome 17p (Drs Downes, Holder, Moore, photophobia and loss of visual acuity, color with mutations in GUCY2D,9,10 and chro- and Bird) and the Institute of vision, and central visual fields. Retinal ap- mosome 19q with mutations in CRX.11 The Ophthalmology (Drs Downes, 12,13 Payne, Kelsell, Fitzke, Hunt, pearance may be normal in the early stages other loci include 2 in chromosome 6q Moore, and Bird), University of the disease, but with progression the and a presumed autosomal dominant lo- College London, retinal pigment epithelium (RPE) may take cus in chromosome 17q in association with London, England. on a bull’s-eye pattern of change; later, cen- neurofibromatosis.14 A sporadic case due

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MOLECULAR ANALYSIS Static threshold perimetry in the dark- and light-adapted states was performed using a modified Humphrey Field Ana- The methods performed for linkage studies, mutation lyzer (Allergan Humphrey, Hertford, England). Photopic screening, heteroduplex electrophoresis, and direct ge- Humphrey visual fields were assessed using the standard 19 nomic sequencing have been described previously.9 protocol. For the scotopic perimetry, the pupil was di- lated with 2.5% phenylephrine hydrochloride and 1% cy- SUBJECTS clopentolate hydrochloride, and the eye was dark adapted for at least 45 minutes. An infrared source illuminated the Three families with an R838C mutation and 1 family with bowl, and an infrared monitor (Phillips, Eindhoven, The an R838H mutation were invited to participate in the study. Netherlands) was used to monitor fixation. Fields were re- Five blood samples were available from family A, 8 from corded using programs 30-2 (central), 30-2 and 60-2 (pe- family B, 4 from family C, and 3 from family D. Three af- ripheral), and the macular program. The target size corre- fected members from family A, 4 from family B, 2 from fam- sponded to Goldmann perimeter size V for peripheral testing ily C, and 1 from family D underwent phenotypic charac- and Goldmann size III for macular programs. Each pro- terization. This research was performed in accordance with gram was performed with both a red (predominant wave- the Declaration of Helsinki, developed by the World Medi- length, 650 nm) and blue (predominant wavelength, 450 cal Association, and was approved by the Moorfields Eye nm) filter in the stimulus beam to record cone and rod func- Hospital Ethics Committee (London, England). Informed tion, respectively. consent was obtained from all participants. Color contrast sensitivity was evaluated in 7 of the 11 subjects using published protocols,20 and Hardy-Rand- CLINICAL AND FUNCTIONAL INVESTIGATIONS Rittler color plate testing was performed in the others.

Phenotypic characterization included a full ophthalmic his- Imaging tory and detailed fundus examination. Fundus photogra- phy, confocal scanning laser ophthalmoscopy, and psy- Images of the central macular region were obtained using chophysical and electrophysiological evaluations were also a prototype confocal laser scanning ophthalmoscope performed. (cLSO SM 30-4024; Zeiss, Oberkochen, Germany). An argon laser (488 nm, 250 mW) was used for illumination. Electrophysiology Reflectance imaging was undertaken with a 40° field and the argon blue laser with the depth plane adjusted to Subjects underwent electrophysiological investigation us- maximize the visibility of the fundus features. A wide- ing techniques in accord with the recommendations of the band pass filter with a cutoff at 521 nm inserted in front International Society for Clinical Electrophysiology of Vi- of the detector was used to detect autofluorescence, sion.16-18 Electro-oculographic responses, full-field ERGs, which was recorded, measured, and analyzed using pub- and pattern ERGs were recorded for 10 subjects. lished techniques.21

to a balanced translocation between chromosomes 1 and common origin in a relatively distant ancestor. How- 6 has also been recorded.15 ever, it was not possible to prove this conclusively. The phenotype described in this article in 3 families with R838C and 1 family with R838H is compared CLINICAL FINDINGS with that previously described in a family with 2 sequence changes in GUCY2D (R838S and E837D) asso- Clinical characteristics are documented in the Table. Most ciated with intense photophobia, poor vision before 6 years subjects became symptomatic during the first 2 decades of age, loss of the peripheral visual field in middle life, of life, and the affected members of family B reported more and loss of detectable ERG responses.10 disabling symptoms in the early stages of their disease than other families. All subjects were aware of lifelong RESULTS poor vision in bright sunlight followed by reduced central vision and color vision. Eccentric fixation was com- DNA ANALYSIS mon. No subject complained of nyctalopia. Visual acuities in families A, C, and D were found An R838C mutation in GUCY2D was identified in 3 fami- to be better than those of family B at a comparable age. lies (families A, B, and C), and an R838H mutation was However, no subject older than 50 years had a visual acu- discovered in 1 family (family D) (Figure 1). The mu- ity better than 20/200. tation was found to segregate with disease in all 4 families. Within these 3 families and including the family FUNDUS APPEARANCE with the 2 sequence changes in GUCY2D (R838S and E837D),10 there is some evidence of linkage disequilib- Soon after the onset of visual symptoms, mild RPE granu- rium between the disease allele and one of the flanking lar abnormalities were apparent at the fovea (Figure 2A markers, indicating that this mutation may have had a and 2B). At this stage, autofluorescence imaging showed

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II:1 II:2 II:3 II:4 II:5 II:6 II:7 II:8 II:1 II:2 II:3 II:4

III:1 III:2 III:3 III:4 III:5 III:6 III:7

III:1 III:2 III:3 III:4 III:5

IV:1 IV:2

C D I:1 I:2 I:1 I:2

? ? II:1 II:2 II:3 II:4 II:5 II:6

II:1 II:2 II:3 II:4

III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10

III:1 III:2 III:3 III:4 III:5

IV:1 IV:2 IV:3 IV:4 IV:5 IV:6 IV:7 IV:8

V:1 V:2 V:3 V:4 V:5 IV:1 IV:2 IV:3 IV:4 IV:5

Figure 1. Pedigrees of families A, B, C, and D. Roman numerals indicate generations; solid symbols, affected family members; open symbols, unaffected family members; squares, males; circles, females; and slash, deceased.

Clinical Characteristics and Electrophysiological Results in 4 Families With Dominantly Inherited Cone-Rod Dystrophy*

Age of Bright Photopic Cone Color Symptom Rod-Specific White Flash ERG b Flicker Contrast Pedigree Age, y Onset, y P Visual Acuity EOG ERG, µV ERG, µV Wave, µV Response, µV Sensitivity A:II/3 60 Ͻ3 +++ Counting NP NP NP NP NP NP fingers OU A:III/3 32 17 + 20/60 OU 170 170 a, 305; b, 360 30 35 Absent A:III/4 29 3 + 20/120 OU 225 160 a, 250; b, 350 a, 15; b, 15 20 NP ↑IMP A:III/5 25 20 + 20/30 OU 205 140 a, 220; b, 305 b, 45 45 ↑↑↑ Threshold B:II/2 49 2 ++ Counting N 165 a, 290; b, 350 a, 10; b, 5 13 ↑IMP Absent fingers OS ↑↑IMP 20/400 OD B:II/4 46 6 ++ 20/400 OU 180 90 a, 130; b, 210 Absent Absent ↑↑↑ Threshold DϾPϾT B:III/1 28 6 +++ 20/220 OU 185 140 a, 195; b, 280 b, 10 15 ↑↑↑IMP ↑↑↑ Threshold DϾPϾT B:III/3 16 14 ++ 20/40 OU 260 120 a, 200; b, 325 b more 14 ↑IMP ↑↑↑ abnormal Thresholds than a DϾPϾT C:IV/2† 68 40s + Counting 185 N N Absent Absent NP fingers OU C:IV/6 78 20s + 20/220 OS Flat 65 a, 115; b, 135 Absent Absent NP perception of light OD D:IV/3 35 20s + 20/120 OU N 120 a, 200; b, 380 70 ↑IMP 55 ↑IMP Grossly elevated

*No patient had nyctalopia. P indicates protan; EOG, electro-oculogram; ERG, electroretinogram; NP, not performed; a, a wave; b, b wave; IMP, implicit times; N, normal; D, deutan; and T, tritan. Plus signs indicate the presence of photophobia. One arrow indicates mild increase; 2 arrows, moderate increase; and 3 arrows, severe increase. †This value was obtained without using the standard of the International Society for Clinical Electrophysiology of Vision.

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C D

E F

G H

I J

Figure 2. A, The left macula of subject A:III/3, aged 32 years, shows minimal granular retinal pigment epithelial abnormalities. B, Reflectance imaging of the right macula of subject B:III/3, aged 16 years, shows a depressed foveal reflex. C, Autofluorescence imaging of the right macula of subject D:IV/3, aged 35 years, shows an area of decreased autofluorescence surrounding a small central area of retained autofluorescence at the fovea. D, Autofluorescence imaging of the left macula of the same subject with similar findings. E, The right macula of subject B:II/4, aged 46 years with a visual acuity of 20/400 OU, shows central atrophy. F, Autofluorescence imaging of the same eye shows a ring of autofluorescence surrounding this atrophy. G, The left macula of subject B:II/2, aged 49 years with a visual acuity of counting fingers OU, shows central atrophy with pigmentation. H, Autofluorescence imaging of the same fundus shows an annulus of increased autofluorescence surrounding the atrophic center shown on the left of Figure 2G. I, Macula of subject C:IV/2, aged 68 years with a visual acuity of counting fingers OU, shows central atrophic changes. J, Autofluorescence imaging in the same subject with increased autofluorescence surrounding the atrophic area.

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400 µV 300 µV Scotopic Rod 200 µV 100 µV 0 V

O s 100 ms O s 100 ms O s 100 ms O s 100 ms O s 100 ms

500 µV 400 µV µ Maximum Rod/Cone 300 V 200 µV 100 µV 0 V

O s 100 ms O s 100 ms O s 100 ms O s 100 ms O s 100 ms

150 µV

100 µV 30-Hz Flicker 50 µV

0 V

O s 50 ms O s 50 ms O s 50 ms O s 50 ms O s 50 ms

150 µV

100 µV Photopic 50 µV

0 V

O s 50 ms O s 50 ms Os 50 ms Os 50 ms O s 50 ms

Figure 3. The electrophysiology is shown of 2 subjects from family B: B:II/2, aged 49 years, and B:III/3, aged 16 years; 1 subject is from family A, and 1 is from family D. The youngest subject from family B has a rod electroretinogram (ERG) with a subnormal amplitude, as does subject D:IV/3, aged 35 years. The bright white flash ERG is subnormal in amplitude in all but subject B:II/2. Photopic and flicker ERGs are grossly delayed and reduced in amplitude in all subjects.

little or no abnormality (Figure 2C and 2D). All other PSYCHOPHYSICS subjects had well-defined atrophy centered on the fovea without central sparing. No gross abnormalities of the Sixty-degree photopic visual fields showed primarily cen- peripheral retina were detected; in particular, there was tral losses within 20° of fixation of greater than 30 dB. no intraretinal pigmentation. Striking abnormalities of At 50° to 60°, a band of decreased sensitivity was also autofluorescence were visible including an annulus of in- observed during photopic testing in all patients, the loss creased autofluorescence around the perimeter of the at- of sensitivity being less severe in family A (Figure 4A rophy (Figure 2E-J). and B). Dark-adapted perimetry demonstrated losses of both ELECTROPHYSIOLOGY rod and cone function primarily localized to the central macular region and extending to about 20° from fixa- The electrophysiological results are presented in the Table tion. The extent and magnitude of central rod and cone- and Figure 3. Pattern ERGs were extinguished in all pa- mediated losses were similar (Figure 4C). The youngest tients; only 1 subject (A:II/3) did not have a pattern ERG. individual (B:III/3) showed early losses of photopic sen- The electro-oculographic light rise was normal in each sitivity in the central macula of between 8 and 17 dB at subject except for subject C:IV/6, who was the oldest one the age of 16 years. His dark-adapted perimetry to both examined. The responses in each family were broadly simi- the red and blue stimuli showed sensitivities within 5 dB lar, although family B had the more severe disease. Cone of normal values throughout the visual field. function was consistently more affected than rod func- Color vision was assessed in all subjects, and pro- tion. All subjects had abnormal full-field cone re- tan, deutan, and tritan thresholds were found to be grossly sponses characterized by markedly reduced amplitudes elevated with no axis predominantly affected. of photopic ERG a and b waves, and some had flicker responses with prolonged implicit time. Rod function was COMMENT outside the normal range in only 3 subjects, including a 16-year-old boy. In the remaining subjects, the rod- The phenotype of cone-rod dystrophy reported in this specific ERG was at the lower end of the normal range. article would be classified as type 1a according to Szlyk

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 A Photopic Full-Threshold B Photopic Full-Threshold C Photopic Full-Threshold Sensitivities, Central 60° Sensitivities, Central 60° Sensitivities, Central 60° A:III/5 A:III/3 B:II/2 Scotopic Red, 30° D:IV/3 Aged 49 y

30° 30°

60° 60° 60° 60° 60° 60° Scotopic Blue, 30° 60° 60°

30° 30°

Figure 4. A, Photopic combined 30° and 60° field of the right eye in 2 subjects: A:III/5, aged 25 years with a visual acuity of 20/30 OU, and A:III/3, aged 32 years with a visual acuity of 20/60 OU, in whom minimal losses of central sensitivity exist within 20° of fixation. At 50° to 60°, a band of decreased sensitivity was also observed during photopic testing. B, Photopic combined 30° and 60° field in subject B:II/2, in whom there is a central loss in sensitivity of greater than 30 dB, eccentric fixation, and loss of the midperipheral photopic field at 30° out. The central 30° of the scotopic fields are shown for comparison, with blue and red stimuli demonstrating comparatively less central rod loss. C, Photopic combined 30° and 60° perimetry for subject D:IV/3, aged 35 years, in whom central and midperipheral losses in sensitivity are noted.

et al,22 having relatively mild involvement of the periph- failure to recycle products of phagosomal degradation that eral field and rod-driven ERG responses. The cone-rod are essential for outer-segment renewal.29 dystrophy phenotype we reported was less severe than Retinal guanylate cyclase-1 (retGC-1) is a key com- that reported in a family with sequence changes at adja- ponent in the phototransduction cascade and synthesis cent codons (R838S and E837D) of GUCY2D,10 which of cyclic guanosine monophosphate (cGMP) from 5Ј- was categorized as type 2b using the classification of Szlyk GMP. Cyclic GMP levels are responsible for increasing and colleagues. Although difficulty seeing in bright light the proportion of open cGMP-gated channels in the dark- was present at an early age, the photophobia was much adapted state. Therefore, mutations in GUCY2D- less disabling; in addition, our patients denied nyctalo- encoding retGC-1 could give rise to retinal disease. Leber pia. Peripheral visual field loss was mild, and intrareti- congenital amaurosis is also associated with mutations nal pigmentation was not seen in the midperipheral or in this gene, but in recessive disease.30 peripheral retinas in our families. Finally, the electro- The phenotype of a dystrophy predominantly of the physiological abnormalities were less marked than those cone variety with some involvement of rods is consis- associated with sequence changes at codons R838S and tent with the tissue localization studies of retGC-1.31 Im- E837D.10 munoimaging with confocal and electron microscopy in Of the genes described to date in association with human and monkey retinas has demonstrated that cone-rod dystrophies, mutations in RDS also exhibit early retGC-1 is primarily located in the photoreceptor layer, predominant cone involvement and have been classi- and outer segments of cones are more densely labeled fied as a type 2a cone-rod dystrophy; however, the phe- with antibodies than those of rods. This finding has been notype associated with mutations in CRX exhibits ear- confirmed by others.32,33 lier and more severe rod involvement and is classified as Possible factors causing the variability observed in type 2b.6,7,23 subjects with mutations in the same gene include the char- Autofluorescence imaging demonstrates the distri- acter or position of the mutation, the effect of the sec- bution of disease at the level of the RPE better than any ond allele exerting either a protective or deleterious in- other technique and sheds light on the cellular changes. fluence, and the susceptibility of the genotype to In vivo imaging and histopathologic investigations have environmental or stochastic phenomena as yet unrecog- provided evidence that the autofluorescent material is li- nized. In the original family with autosomal dominant pofuscin located in the RPE.24-26 Abnormally high levels cone-rod dystrophy, adjacent R838S and E837D muta- of autofluorescence may occur if the metabolic demand tions were present,10 and Perrault et al34 reported a fam- is increased or the RPE function is compromised. The ily with early-onset and severe retinal degeneration in earliest abnormality of increased autofluorescence at the which the disease gene encoded a protein with 3 amino fovea implies that this is the site of initial dysfunction in acid substitutions at positions 837, 838, and 839. The com- our families, in contrast to bull’s-eye dystrophies in which mon feature in these 2 latter cases and in all of the single there is central sparing in the early stages of the disease. mutants is the presence of a substitution at position 838; The lack of autofluorescence at the site of atrophy has this substitution would appear to be the disease- been well recorded and is indicative of the loss of pho- determining change, with severity depending on the pre- toreceptor cells, or at least their outer segments.27 In- cise amino acid replacement and the presence of addi- creased autofluorescence at the edge of atrophy is likely tional substitutions in adjacent codons. Codon 838 seems to indicate an area destined to become atrophic. The dys- particularly prone to mutational change; the R838H mu- function causing the autofluorescence may directly ac- tation described in this article as well as mutations in this count for cell death. Alternatively, the high levels of li- codon in a French and Swiss family all support this ob- pofuscin may contribute to cell loss due to the release of servation.34,35 free radicals, as has been claimed by Rozanowska et al.28 Sites 837 to 839 are located within the putative Finally, high levels of autofluorescence may indicate a dimerization domain of the retGC-1 protein. An inves-

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 tigation of the Ca2+ sensitivity and the catalytic activity brary of Medicine. Available at: http://www3.ncbi.nlm.nih.gov/omim. Accessed of the R838C, R838H, and R838S mutants and wild- August 30, 1999. 14. Klystra JA, Aylsworth AS. Cone-rod retinal dystrophy in a patient with neurofi- type retGC-1 has shown that significantly higher con- bromatosis type 1. Can J Ophthalmol. 1993;28:79-80. 2+ centrations of Ca are required to deactivate the en- 15. Tranebjaerg L, Sjo O, Warburg M. Retinal cone dysfunction and mental retarda- zyme. The overall effect is the constitutive activation of tion associated with a de novo balanced translocation 1;6 (q44;q27). Ophthal- mutant retGC-1 by GCAP-1 at physiological Ca2+ con- mic Paediatr Genet. 1986;7:167-173. 36,37 16. Marmor MF, Zrenner E. Standard for clinical electroretinography (1994 update). centrations. This gain in function may result in el- Doc Ophthalmol. 1995;89:199-210. 2+ evated levels of cyclic GMP and Ca in the photorecep- 17. Marmor MF, Holder GE, Porciatti V, Trick G, Zrenner E. Guidelines for pattern tors. Precisely which effect is more detrimental to electroretinography: recommendations by the International Society for Clinical photoreceptor survival remains to be established. Electrophysiology of Vision. Doc Ophthalmol. 1995;91:291-298. 18. Marmor MF, Zrenner E. Standard for clinical electro-oculography. Arch Ophthal- mol. 1993;111:601-604. Accepted for publication May 10, 2001. 19. Jacobsen SG, Boight WJ, Parel JM. Automated light and dark adapted perim- This study was supported by grant 053405 from the etry for evaluating retinitis pigmentosa. Ophthalmology. 1986;93:1604-1611. Wellcome Trust, London, England; the British Retinitis Pig- 20. Arden GB, Gunduz K, Perry S. Colour vision testing with a computer graphics mentosa Society, Surrey, England; the Medical Research system. Clin Vis Sci. 1988;2:303-320. Council, London; and the Foundation Fighting Blindness, 21. von Ru¨ckmann A, Fitzke FW, Bird AC. Distribution of fundus autofluorescence with a scanning laser ophthalmoscope. Br J Ophthalmol. 1995;79:407-412. Baltimore, Md. 22. Szlyk JP, Fishman GA, Alexander KR, Peachey NS, Derlacki DJ. Clinical sub- We thank all the families participating in this study. types of cone-rod dystrophy. Arch Ophthalmol. 1993;111:781-788. Corresponding author and reprints: Susan M. Downes, 23. Jacobson SG, Cideciyan AV, Huang Y, et al. Retinal degenerations with trunca- MD, Oxford Eye Hospital, Radcliffe Infirmary, Oxford OX2 tion mutations in the cone-rod homeobox (CRX ) gene. Invest Ophthalmol Vis 6HE, England (e-mail: susan.downes@ophthalmology Sci. 1998;39:2417-2426. 24. Eldred G, Katz ML. 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