Journal of Neuro-Ophthalmology, September 2001, Volume 21, Issue 3

The Photoreceptor Cell-Specific Nuclear Receptor is an Autoantigen of Paraneoplastic Retinopathy

OBJECTIVES: To report a novel antibody associated with paraneoplastic retinopathy and to characterize the retinal autoantigen. METHODS: Immunohistochemistry of rat and human tissues was used to identify antiretinal antibodies. Serologic screening of a bovine retinal cDNA expression library was performed to clone the target antigen. RESULTS: A 72-year-old woman presented with a 6-month history of progressive visual loss, bilateral central scotomas, light flashes, and night blindness. Visual acuity was 20/40 OD and 20/30 OS. There was generalized loss of retinal pigment and narrow arterioles; discs were normal in appearance. The electroretinogram showed no response. Chest computed tomograph scan demonstrated a right lung mass; biopsy revealed poorly differentiated carcinoma. The patients' serum contained antibodies that immunolabeled nuclei of cells of the outer--and to a lesser extent, the inner--nuclear layer of the adult rat retina. No reactivity was identified with nonretinal adult human or rat tissues. Reactivity was seen in the developing rat embryo. Serologic screening of a bovine retinal library resulted in the isolation of three overlapping clones, encoding a protein highly homologous to the human photoreceptor cell-specific nuclear receptor gene product. CONCLUSIONS: The target antigen of an antibody associated with paraneoplastic retinopathy is the photoreceptor cellspecific nuclear receptor, a member of a conserved family of nuclear receptors involved in photoreceptor cell development or maintenance.

Journal of Neuro- Ophthalmology 21( 3): 168- 172, 2001. © 2001 Lippincott Williams & Wilkins, Inc., Philadelphia Original Contribution The Photoreceptor Cell- Specific Nuclear Receptor is an Autoantigen of Paraneoplastic Retinopathy Joseph G. Eichen, MS, Josep Dalmau, MD, PhD, Alexis Demopoulos, MD, Deborly Wade, BS, Jerome B. Posner, MD, and Myrna R. Rosenfeld, MD, PhD Objectives: To report a novel antibody associated with paraneoplastic retinopathy and to characterize the reti­nal autoantigen. Methods: Immunohistochemistry of rat and human tissues was used to identify antiretinal antibodies. Serologic screening of a bovine retinal cDNA expression library was performed to clone the target antigen. Results: A 72- year- old woman presented with a 6- month his­tory of progressive visual loss, bilateral central scotomas, light flashes, and night blindness. Visual acuity was 20/ 40 OD and 20/ 30 OS. There was generalized loss of retinal pigment and narrow arterioles; discs were normal in appearance. The elec-troretinogram showed no response. Chest computed tomograph scan demonstrated a right lung mass; biopsy revealed poorly differentiated carcinoma. The patients' serum contained anti­bodies that immunolabeled nuclei of cells of the outer- and to a lesser extent, the inner- nuclear layer of the adult rat retina. No reactivity was identified with nonretinal adult human or rat tissues. Reactivity was seen in the developing rat embryo. Se­rologic screening of a bovine retinal library resulted in the isolation of three overlapping clones, encoding a protein highly homologous to the human photoreceptor cell- specific nuclear receptor gene product. Conclusions: The target antigen of an antibody associated with paraneoplastic retinopathy is the photoreceptor cell-specific nuclear receptor, a member of a conserved family of Manuscript received April 3, 2001; accepted June 25, 2001. Supported in part by the Charles A. Dana Neuroscience Research Program ( JD); Medical Research Endowment Awards, University of Arkansas for Medical Sciences ( MRR, JD), and NS02664 ( JBP, JD). From the Department of Neurology and Laboratory of Neuro- Oncology ( JGE, JD, MRR), and the Department of Immunology and Microbiology ( JD, DW, MRR), University of Arkansas for Medical Sciences and the Arkansas Cancer Research Center, Little Rock, Ar­kansas; and the Department of Neurology ( AD, JBP), Memorial Sloan- Kettering Cancer Center, New York, New York. Address correspondence and reprint requests to Myrna R. Rosenfeld, MD, PhD, Department of Neurology, University of Arkansas for Medi­cal Sciences, 4301 W. Markham, Slot 500, Little Rock, AR 72205: e- mail: rosenfeldmyrnar@ uams. edu nuclear receptors involved in photoreceptor cell development or maintenance. Key Words: Cancer- Retinopathy- Paraneoplastic- Photoreceptor cell- specific nuclear receptor- Antibodies. Paraneoplastic visual syndromes comprise a heteroge­neous group of disorders involving retina and, less fre­quently, uvea and optic nerves ( 1- 5). Some of these syn­dromes are associated with serum antibodies that specifically react with the subset of retinal cells under­going degeneration, suggesting an immune- mediated pathogenesis ( 6,7). Studies with these antibodies have led to the identification of two retinal- specific target an­tigens, recoverin and tubby- like protein 1 ( 8,9). We re­port a patient with paraneoplastic retinopathy whose serum contained antiretinal antibodies against the photoreceptor- specific nuclear receptor gene product ( PNR) ( 10). Inherited mutations of the PNR gene ( also known as NR2E3) have been found in patients with retinal degen­eration associated with enhanced S- cone syndrome ( ESCS), a disorder of retinal cell fate ( 11). In the mouse, deletions of the PNR homolog result in late- onset retinal degeneration ( 12). These data suggest that the retinal degeneration in our patient was likely caused by the im­mune- mediated knockout of PNR function. PATIENT AND METHODS Case report For several months, a 72- year- old woman complained of transient black spots before her eyes, sensation of light flashes, and progressive decrease of vision that was worse at night. Her past medical history was unremarkable. Evaluations by several ophthalmologists revealed no pathology, and the diagnosis of hysterical blindness was considered. After 6 months of symptom 168 NOVEL PARANEOPLASTIC RETINOPATHY ANTIGEN 169 development, a repeat evaluation demonstrated a cor­rected visual acuity of 20/ 40 OD and 20/ 30 OS. Extraocular motility was full. Pupils were 2.5 mm in bright light, normally reactive to light with no relative afferent defect. Goldmann visual fields were marked­ly constricted. Ophthalmoscopy revealed narrow retinal arterioles and a generalized loss of pigment throughout the retina in both eyes; the optic discs had a normal appearance. A subsequent electroretinogram ( ERG) showed absent pho-topic and scotopic responses OU. The rest of the neuro­logic and systemic examination results were normal, as was a magnetic resonance image ( MRI) of the brain without and with contrast. Blood cell count and general chemistry were normal except for elevated lactic dehy­drogenase ( LDH) ( 279 U/ L, normal 60- 200 U/ L). Chest CT showed a large right upper lobe paramediastinal mass with adjacent small nodules suspicious for bronchogenic tumor spread. A fine- needle aspirate of the lung mass showed poorly differentiated carcinoma. The patient re­fused treatment and was lost to follow- up. Sera and tissues The serum of the patient was obtained 6 months after the onset of visual symptoms; at this time, the ERG showed no responses. Control sera included four patients with paraneoplastic retinopathy associated with antire-coverin antibodies ( 3,8), three patients with melanoma-associated retinopathy ( MAR) ( 5), ten patients without cancer with idiopathic ( etiology unknown) retinopathy, eight patients with paraneoplastic syndromes of the cen­tral nervous system not involving the visual system ( four with encephalomyelitis associated with anti- Hu antibod­ies ( 13), two patients with paraneoplastic cerebellar de­generation and anti- Yo antibodies ( 14), two patients with limbic encephalitis and anti- Ma2 antibodies ( 15), and 27 healthy people. Sera were stored at - 70° C. Normal hu­man and Wistar rat tissues were processed and stored as reported ( 16). Frozen bovine retina was obtained from Pel- Freez, Inc. ( Rogers, AR). Immunohistochemistry Frozen 7-( xm- thick tissue sections were fixed in cold 30% methanol- 70% acetone and incubated with pa­tient's serum ( diluted 1: 4,000) using an avidin- biotin peroxidase immunoassay, as reported ( 17,18). To avoid reactivity with endogenous IgG, all immunohistochemi-cal studies with human tissues used IgG purified from patient sera and labeled with biotin ( 19). To determine whether the serum reactivity seen in immunohistochem-ical studies resulted from the expression of the cloned protein by retinal cells, sections of adult rat retina were incubated with the patient's serum preabsorbed with the recombinant protein, as reported ( 15). Cloning, isolation, and sequence analysis The patient's serum was used to screen a X ZAP bo­vine retinal library ( Stratagene, La Jolla, CA) at a density of 5 x 104 pfu/ 150- mm plate of Escherichia coli XL1- Blue ( Stratagene) La Jolla, CA. After a 6- hour incuba­tion at 37° C, plates were overlaid with filters soaked in 10 mmol/ L of isopropyl ( 3- D- thiogalactopyranoside ( IPTG) and incubated for 12 hours at 37° C. Filters were removed; blocked with 5% Blotto; incubated with the serum from the patient ( 1: 4,000) for 2 hours at room temperature; washed in 50 mmol/ L Tris ( pH 7.4), 100 mm/ L NaCl, and 0.2% triton ( TBST buffer); incubated at room temperature with [ I] 125 protein A ( 0.1 ( iCi/ mL) for 1 hour; washed with TBST; dried; and exposed to XAR5 film overnight at - 70° C. Clones giving positive results were purified by several rounds of antibody screening until a yield of 100% positive plaques was obtained and then subcloned into pBluescript using the in vivo exci­sion phage rescue protocol ( Stratagene). Double- stranded cDNA was purified using the Qiagen ( Santa Clarita, CA) plasmid midi- prep system and se­quenced on both strands using an ABI377 automated DNA sequencer and the dye terminator fluorescence method. T3, T7, and internal oligonucleotide primers were used. Western blot analysis Recombinant fusion proteins, E. coli proteins, and pro­teins from rat, bovine, and human tissues were obtained as previously described ( 18), resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The nitrocellulose strips were incubated with the patient's sera ( diluted 1: 4,000), and the reactivity was demonstrated by an enhanced chemiluminescence method ( Amersham, Arlington, IL) ( 16). RESULTS The serum of the patient contained antibodies ( titer 1: 500,000) that reacted with proteins expressed in the outer and inner nuclear layers of rat retina ( Fig. 1). These autoantigens were not identified in nonretinal adult hu­man or rat tissues ( data not shown). Reactivity with hu­man retinal tissue was not examined. In the developing rat, reactivity was identified in the migratory cells of the ventricular zone of the brain, the limb bud apical ecto­dermal ridge, and the lamina propia of the gut ( El 7) ( data not shown). Similar antibody reactivities were not demonstrated in the sera from patients with paraneoplastic retinopathy and recoverin antibodies, MAR, or in any of the other control and normal sera. In immunoblots of retinal pro­tein extracts, no reactivity was found at 23 kd ( compat­ible with recoverin reactivity) or at 37 to 40 kd ( com­patible with Hu reactivity) ( 8,17). The absence of recoverin antibodies was confirmed with immunoblots of recombinant recoverin protein ( data not shown). To determine the identity of the target retinal protein of these autoantibodies, we screened a bovine retinal cDNA expression library and isolated three clones, called Del, Del, and De3 ( Fig. 2). Sequence analysis demonstrated that Del contained a complete open reading frame coding for a 46.5- kd protein. Del and De3 contained the same sequences but lacked varying amounts of the 5' terminal region. De3 also contained additional 3' sequence. The three cDNA sequences were J Neuro- Ophthalmol, Vol. 21, No. 3, 2001 170 J. G. EICHEN ETAL. - == r = -^ ' : " = FIG. 1. Immunohistochemical analysis of the patients' serum with rat retina. Section of rat retina incubated with the patient's serum antibodies demonstrates immunolabeling of the outer nuclear ( ONL) and inner nuclear layers ( INL). No reactivity is seen with the photoreceptor ( PR), outer plexiform ( OPL), inner plexiform ( IPL), and the ganglion cell layers ( GCL) ( hematoxylin counter-stain, x400). combined into a consensus cDNA sequence that was used in a homology search of GenBank databases ( Be-thesda, MD). This search revealed almost complete iden­tity to the recently described human retinal- specific gene, PNR ( AF121129) ( 10), with the exception of the E * *- I FIG. 3. Del ( photoreceptor cell- specific nuclear receptor) con­tains the retinal epitopes targeted by antibodies associated with paraneoplastic retinopathy. Section of rat retina incubated with serum preabsorbed with A: bovine serum albumin and B: Del protein. Preabsorption with Del abrogated the serum reactivity with retina, indicating that Del and retina express the same epitopes ( hematoxylin counterstain, x200). HxnaiPtJ= FIG. 2. Homology alignment of the De clones with nucleotides 1- 2035 of the photoreceptor cell- specific nuclear receptor ( PNR). Clone De2 lacks the zinc- finger domain that is present in clone Del ( complete sequence) and De3 ( partial sequence). presence of a bovine- specific short interspersed element ( SINE) ( 20) near the 3' end of the De clones. Western blots of recombinant proteins derived from the Del, Del, and De3 clones revealed that the patients' serum recognized all three fusion proteins. Based on the homology alignment of the three clones with PNR ( Fig. 2), these data suggest that the immunodominant epitope does not reside in the zinc- finger domain, which is lack­ing in De2, but could reside in the ligand- binding do­main. None of the control or normal sera demonstrated reactivity with any of the three De recombinant proteins. Preabsorption of serum antibodies with Del resulted in abrogation of serum reactivity with retina, indicating that Del and retina express similar epitopes ( Fig. 3). DISCUSSION In this article, we demonstrate that the retinal- specific protein PNR is the target antigen of the autoimmune response in a patient with paraneoplastic retinopathy. The paraneoplastic retinopathies are a group of syn­dromes characterized by cone or rod dysfunction associ­ated with photosensitivity, progressive loss of vision and color perception, central or ring scotomas, night blind­ness, and attenuation of photopic and scotopic responses in the ERG ( 1,2,21,22). Patients with paraneoplastic ret­inopathy often develop recoverin antibodies and suffer from a small cell lung cancer ( SCLC), although other tumors have been reported ( 7,21,23- 25). Recoverin an­tibodies have also been detected in the serum of patients without cancer but with a clinically similar retinopathy ( 26,27). In these cases, the immunologic staining pattern is diffuse and suggests an interaction with multiple non­specific retinal antigens ( 26). There are reports of pa­tients with paraneoplastic retinopathy and antibodies to human enolase ( 28) or neurofilaments ( 29), although re­cent studies have suggested that these are not specific immune responses ( 30). Patients with melanoma can de­velop a paraneoplastic retinopathy, MAR, associated with serum antibodies directed against an unknown an­tigen localized to bipolar cells ( 31,32). Our patient's symptoms were similar to those reported in paraneoplastic retinopathy associated with antirecov-erin antibodies, but the serum contained antibodies that targeted PNR. The PNR gene product is a member of the nuclear receptor superfamily ( 33,34). Nuclear receptors are ligand- dependent transcription factors that mediate a wide variety of physiologic and regulatory processes. Many nuclear receptors, including PNR, are orphan re­ceptors with unknown ligands. The PNR gene is closely / Neuro- Ophthalmol, Vol. 21, No. 3, 2001 NOVEL PARANEOPLASTIC RETINOPATHY ANTIGEN 171 related to the TLX gene, the human homolog of the Drosophila terminal/ gap gene tailless, which plays an essential role in fly eye development ( 35). A remarkable feature of PNR is its highly restricted expression in the retina. Kobayashi et al. ( 10) reported expression limited to the outer nuclear layer, whereas Chen et al. ( 36) found expression in the inner nuclear layer and retinal pigment epithelium. Our study suggests that PNR is expressed in the inner and outer nuclear layers of adult rat retina. Determination of the exact site of the immune response in our patient, however, would require studies with hu­man retinal tissue. Highly restricted expression patterns are relatively unique among transcription factors and have only been demonstrated for ERX and CRX ( 37,38). CRX is essential for the maintenance of mammalian pho­toreceptors and, in humans, represents a gene responsible for the autosomal dominant cone and rod dystrophy, CORDII ( 37,39). Interestingly, a potential CRX binding site has been noted upstream of the PNR promoter, sug­gesting regulation of PNR expression by CRX. Studies suggest that PNR is likely involved in sup­pressing genes whose expression is refractory to photo­receptor function ( 10,12). The pattern of expression in the developing rat embryo found in this study also suggests a role in early development of neural and nonneural structures. Finding PNR mutations in a cohort of patients with ESCS demonstrates that PNR is important in retinal function ( 11). Patients with ESCS develop enhanced sen­sitivity to blue light because of increased numbers of S cones and eventually progress to retinal degeneration ( 40,41). A deletion of the mouse PNR homolog is the cause of retinal degeneration in the rd7 mouse, a model of hereditary retinal degeneration ( 12). These data suggest a mechanism whereby anti- PNR antibodies interfere with PNR function, leading to retinal degeneration. This mechanism could occur by blockade of ligand binding, because our data suggest that the an­tibody recognition site may reside within the ligand-binding domain. This would be similar to several para­neoplastic syndromes of the peripheral nervous system, such as the Lambert- Eaton myasthenic syndrome asso­ciated with anti- voltage- gated calcium channel antibod­ies ( 42) and myasthenia gravis associated with anti-acetylcholine receptor antibodies ( 43). Alternatively, the occurrence of high- titer anti- PNR antibodies in the serum of our patient could represent an immunologic reaction against antigens released by reti­nal cells damaged by unknown mechanisms. In this case, one would expect the patient to also have serum anti­bodies against other highly immunogenic retinal proteins ( such as recoverin or Hu), which were not found in our patient ( 8,44). More likely, the presence of PNR anti­bodies resulted from an immunologic reaction triggered by the tumor. The exact mechanisms of how a tumor breaks immune tolerance for retinal or neuronal proteins is unknown, but for other models of paraneoplastic syn­dromes ( i. e., anti- recoverin associated retinopathy, anti- Hu or anti- Yo central nervous system syndromes), the associated tumors express the retinal or neuronal target antigens ( 44,45). In our patient, no tumor was available for antigen studies. Our findings indicate that PNR is a novel autoantigen of paraneoplastic retinopathy. Future studies should be aimed at elucidating the pathogenicity of the antibodies and the possible role of T- cell responses. Acknowledgments: The authors thank Dr. C. 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