Histopathologic and Immunohistochemical Study of an Autopsy Eye with X-Linked Cone Degeneration

CLINICOPATHOLOGIC REPORT Histopathologic and Immunohistochemical Study of an Autopsy Eye With X-linked Cone Degeneration

King W. To, MD; Michael Adamian; Frederick A. Jakobiec, MD; Eliot L. Berson, MD

e performed a histopathologic and immunohistochemical study of eyes obtained at autopsy of an 84-year-old man from a family with X-linked cone degeneration in which affected members have a 6.5-kilobase deletion in the red cone pigment gene. At his most recent ocular examination, at age 71 years, this patient had Whad a visual acuity of 20/200 OU, fundus changes suggestive of macular degeneration, borderline- normal full-field rod electroretinograms, and profoundly reduced full-field cone electroretinograms. Histopathologic examination demonstrated marked loss of cone and rod photoreceptors and the retinal pigment epithelium in the central macula. The peripheral cone population was reduced, while the peripheral rod population was relatively preserved. Immunohistochemical examination with an antibody to both red and green cone opsin and an antibody to blue cone opsin disclosed a prominent loss of the red and green cone population and preservation of the blue cone population. These findings show that a defect in the red cone pigment gene can result in extensive degeneration of the red and green cone population across the retina. Arch Ophthalmol. 1998;116:100-103

Hereditary conditions that result in gener- tion. No treatments are known for pa- alized cone dysfunction may be divided into tients with hereditary cone dysfunctions. 2 groups. The first group is considered non- We studied eyes obtained at autopsy progressive and consists of cases recog- of an 84-year-old affected man from a fam- nized near birth because of decreased vi- ily with X-linked cone degeneration. This sual acuity, sensitivity to light, nystagmus, patient and other affected family members and abnormal color vision. Patients in this had a known 6.5-kilobase deletion in the red group include those with autosomal reces- cone pigment gene.4 To our knowledge, this sive complete achromatopsia and X- report is the first histopathologic and im- linked blue cone monochromacy. While the munohistochemical study of X-linked cone retina usually appears normal or nearly nor- degeneration with a known gene defect. mal in early life, the electroretinogram (ERG) demonstrates markedly dimin- REPORT OF A CASE ished cone responses with normal rod responses. The second group is considered The donor with X-linked cone degenera- progressive and consists of cases typically tion was an 84-year-old man whose clini- identified in young adulthood because of cal findings have been previously de- gradual deterioration of visual acuity and scribed (see Reichel et al,4 patient II-4). At decline in color vision. Patients in this group our most recent ocular examination, at age include those with autosomal dominant, au- 71 years, he had a best-corrected Snellen tosomal recessive, and X-linked cone de- visual acuity of 20/200 OU with a 10° cen- generations.1-3 The macula may have an ab- tral scotoma with full peripheral visual fields normal appearance ranging from mild on Goldmann perimetry; a nonspecific axis granular changes to geographic atrophy. As of confusion on the Farnsworth D15 panel; in the nonprogressive forms, the diagno- and normal full-field rod ERGs and non- sis is confirmed by ERG testing, which detectable (ie, Ͻ10 µV) full-field cone shows diminished full-field cone ampli- ERGs. His eyes were enucleated 71⁄2 hours tudes with normal or near-normal rod func- after he died of cardiac arrest. The donor’s brother also had a visual acuity of 20/200, From the Berman-Gund Laboratory for the Study of Retinal Degenerations (Drs To impaired color vision, normal rod ERGs, and Berson and Mr Adamian) and the Ophthalmic Pathology Service (Dr Jakobiec), and diminished cone ERGs. The donor’s 2 Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston. daughters had normal visual acuity and re-

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IPL OPL H B

Figure 1. Fundus of the right eye of an 84-year-old man with X-linked cone degeneration, showing an atrophic macular lesion and the optic nerve head with a temporal conus.

duced cone ERGs. The daughters’ cone responses, although diminished, were still greater than those of their father or paternal uncle. One daughter’s son, at age 15 years, had nearly normal visual acuity N (20/30 OU), a protan deficiency on G the Farnsworth D15 panel, a normal rod ERG, and a reduced cone ERG.4 IPL The right eye was immediately fixed by immersion in 2.5% glutaral- INL dehyde and 1% formaldehyde in 0.1- mol/L phosphate buffer at pH 7.4. H The left eye was fixed in 4% formal- ONL dehyde in 0.1-mol/L phosphate buffer IS at pH 7.4 and stored wet at 4°C for OS immunohistochemical studies. RPE B The right eye was processed for light and electron microscopy and em- Figure 2. Light micrograph of the eye obtained at autopsy from the donor affected with X-linked cone beddedinepoxyresin(Taab812,Mari- degeneration (top), showing an area within the macular lesion where Henle fiber layer abuts Bruch membrane, contrasted with an age-matched control (bottom). Nerve fiber layer is thinned and vacuolated vac, Ltd, Halifax, Nova Scotia) blocks in the patient compared with the control. B indicates Bruch membrane; RPE, retinal pigment epithelium; forsectioning.Sectionsforlightmicros- OS, outer segment; IS, inner segment; ONL, outer nuclear layer; H, Henle fiber layer; copywerecutfromthemaculaandpe- INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; G, ganglion cell layer; ripheral retina at a thickness of 1 µm and N, nerve fiber layer. Bar indicates 60 µm. and stained with a 1:1 solution of 2% methylene blue and 2% azure II. Sil- antibodies diluted 1:10 in PBS and PATHOLOGIC FINDINGS ver or gold sections from the same specific to either red and green cone regions were stained with uranyl opsin in combination or to blue cone Gross examination of the right eye acetate–lead citrate and examined opsin. The sections were then rinsed from the patient with X-linked cone with an electron microscope. in PBS and labeled with a secondary degeneration disclosed a large atro- With respect to the left eye, 4- goat anti–rabbit fluorescent anti- phicmacularlesionandanunremark- mm trephine punches were taken be- body named CY3 (Jackson Immuno- able fundus periphery (Figure 1). tween 10° and 20° eccentric to the fo- research Laboratories, West Grove, Histologically, there was loss of cone vea in the near temporal peripheral Pa), diluted 1:200 in PBS. Labeled and rod photoreceptors, retinal pig- retina along the horizontal meridian. sections were then viewed on an epi- ment epithelium, and choriocapillaris Retinal punches were then frozen in fluorescent photomicroscope (Carl in the central macula (Figure 2, top) a pentane slush in liquid nitrogen Zeiss, Oberkochen, West Germany). compared with an age-matched con- and embedded in OCT compound For morphological comparison, trol (Figure 2, bottom). In this area (Miles, Inc, Elkhart, Ind) for cryosec- we also evaluated an eye obtained at the retinal pigment epithelium and tioning.Cryostatsections(6µm)were autopsy, with a death-to-fixation in- outer nuclear layer were absent, and cut and mounted on slides immersed terval of 2 hours, from an 87-year-old the Henle fiber layer was atrophic and for 10 minutes in a phosphate- male donor with no history of eye dis- abutted the Bruch membrane. The in- buffered saline (PBS) solution con- ease. Another male donor, 84 years ner nuclear layer and ganglion cell taining 0.3% Triton X-100, blocked of age, with no history of ocular dis- layer showed a reduction in cell num- with normal goat serum for 20 min- ease and with a death-to-fixation ber compared with an age-matched utes, and then incubated for 1 hour interval of 24 hours, was used for control. Just anterior to these abnor- at room temperature5 with polyclonal immunohistochemical comparison. malities, abnormal rod and cone pho-

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©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 normal full-field rod ERGs, and profoundly reduced full-field cone ERGs at 71 years of age. If the macular changes were age related and con- fined to the macula, a normal or borderline-normal full-field cone ERG would be expected,6 since the major- ity of the cones are outside the macula. Because of the abnormal full- Patient Control field cone ERG responses and the history of declining visual acuity and color vision, a progressive general- ized degenerative process involving the cones was diagnosed clinically. A Our histological findings confirm that this patient with a large deletion in the red cone pigment gene had extensive cone photoreceptor degeneration not only in the macula but also across the retina. On the basis of the prolonged vi- sualevokedpotentiallatenciesinsome C male patients with X-linked cone degeneration, it has been hypothesized thattranssynapticdegenerationofgan- C glion cells can occur in cone dystro- R phies.1 Our histological observation oftheatrophicchangesintheganglion cell layer confirm that transsynaptic degenerationdoesoccurinassociation with cone degeneration. The mechanism by which a deletion in the red conepigmentgeneaffectsganglioncell structure is not known. B The immunohistochemical re- Figure 3. Light micrograph (A) and electron micrograph (B) of the temporal edge of the macular lesion of sults show a significant diminution the eye obtained at autopsy with X-linked cone degeneration. In the light micrograph, a transition from of the red and green cone population absent photoreceptors (right) to intact photoreceptors (left) can be seen. The electron micrograph shows when compared with an age-matched degenerating rods and cones in this region. R indicates rod photoreceptor; C, cone photoreceptor. Bar indicates 50 µm (A) and 6 µm (B). control eye. Some remaining red and green cones without outer segments toreceptors could be seen by light mi- Immunohistochemicalexamina- in the eye with X-linked cone degen- croscopy (Figure 3, A) and electron tion with an antibody to both red and eration did stain weakly with the an- microscopy (Figure 3, B). The mid- green cone opsin and an antibody to tibody to red and green opsin in the peripheral retina (Figure 4, A) also blue cone opsin showed, outside the inner segments. This contrasts with showed a reduction in the number atrophic macular lesion, a specific loss thestrongstainingpatternoftheouter of cones compared with the control ofmanyredandgreenconeswithspar- segments of the red and green cones (Figure 4, B). The retinal pigment epi- ing of blue cones. In the patient eye, in the control eye. We hypothesize thelium was attenuated over drusen the antibody to the red and green cone that the remaining red and green (Figure 4, A). In the peripheral retina opsin weakly stained remaining inner cones have accumulated opsin in re- there was an absence of cones and segments (Figure 6, A) in contrast to maining cone inner segments as part relatively preserved rods; the re- the control eye, where this antibody of the pathogenesis of cone degenera- maining rods showed vacuolization strongly stained outer segments (Fig- tion. Alternatively, there may exist a of the inner segments and short- ure6,C).Incontrast,antibodyspecific defect in the transport of cone opsin ened outer segments. In the periph- for blue cone opsin similarly stained from the inner segment to the outer eral retina, the retinal pigment epi- the outer segments of the patient and segment in X-linked cone degenera- thelium showed a scarcity of melanin control eyes (Figure 6, B and D). tion, resulting in loss of outer seg- granules and numerous melanoly- ments. By an unclear mechanism, the sosomes; the cells lacked both api- COMMENT deletion in the red cone pigment gene cal processes and basal infoldings. adversely affects the green cone popu- Drusen were prominent, and the Our patient with X-linked cone de- lation and rods in the macula; simi- elastica of Bruch membrane was generation had fundus changes of larly, in retinitis pigmentosa a mutant granular and thickened (Figure 5). macular degeneration, borderline- rhodopsin gene in rods is associated

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Figure 5. Electron micrograph of the retinal pigment epithelium in the peripheral retina of the eye obtained at autopsy with X-linked cone A degeneration. Retinal pigment epithelial cells are filled with lipofuscin and melanolysosomes, and they lack apical processes and basal infoldings. Extensive drusen can be seen. Bruch membrane is abnormally thickened. D indicates drusen; B, Bruch membrane. Bar indicates 6 µm.

Presented in part at the annual meeting of the Association for Re- search in Vision and Ophthalmology, Fort Lauderdale, Fla, April 23, 1996. We thank Thaddeus P. Dryja, MD, for his thoughtful suggestions B and comments, and Dorothy J. Roof, PhD, for her assistance in the immu- Figure 4. Light micrographs of the midperipheral retina of the eye obtained at autopsy with X-linked cone nohistochemical studies. Polyclonal degeneration (A) and the eye of an age-matched normal donor (B). Some cones are designated with arrows. antibodies were provided by Connie The numberof cones is reduced in the affected patient compared with the normal subject. Barindicates 40 µm. Lerea, PhD. Reprints: King W. To, MD, Ber- Patient Control man-Gund Laboratory for the Study of Retinal Degenerations, Harvard OS Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles St, Red/ Boston, MA 02114. Green IS IS REFERENCES

A C 1. Pinckers A, Deutman AF. X-linked cone dystrophy: an overlooked diagnosis? Int Ophthalmol. 1987;10:241-243. OS OS 2. Jacobson DM, Thompson S, Bartley JA. X-linked progressive cone dystrophy: clinical characteris- Blue tics of affected males and female carriers. Oph- IS thalmology. 1989;96:885-895. 3. Keunen JEE, van Everdingen JAM, Went LN, et al. IS Color matching and foveal densitometry in patients and carriers of an X-linked progressive cone B D dystrophy. Arch Ophthalmol. 1990;108:1713- Figure 6. Localization, by indirect immunofluorescence, of red and green cone opsin (A) and blue cone 1719. opsin (B) in a patient with X-linked cone degeneration. The antibody to red and green cone opsin does 4. Reichel E, Bruce AM, Sandberg MA, et al. An elec- not recognize any cone outer segments within the field but weakly reacts with some cone inner segments troretinographic and molecular genetic study of (IS) slightly above background labeling (A). The blue cone opsin antibody recognizes cone outer X-linked cone degeneration. Am J Ophthalmol. segments (OS with arrowhead) (B). For comparison, localization of red and green cone opsin in an 1989;108:540-547. age-matched control (C) shows that most of the cone outer segments in the field are labeled. Localization 5. Curcio CA, Allen KA, Sloan KR, et al. Distribution of blue cone opsin in the age-matched control is seen in D. Bar indicates 10 µm. and morphology of human cone photoreceptors stained with anti-blue opsin. J Comp Neurol. 1991; with death of cones. The reason for Accepted for publication July 25, 1997. 312:610-624. 6. Berson EL. Electrical phenomena in the retina. In: the pathologic changes in the retinal This work was supported by a Moses RA, Hart WM Jr, eds. Adler’s Physiology pigment epithelium also remains to grant from The Foundation Fighting of the Eye. 9th ed. St Louis, Mo: CV Mosby Co; be clarified. Blindness, Baltimore, Md. 1992:641-707.

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