Patient #1 (c.G264T, Lys88Asn) Patient 1, a 2 month old female, presented with nystagmus mimicking opsoclonus, which prompted a work-up for neuroblastoma (negative) and a brain MRI (normal). At age 3 months, she would blink to light, but not fix and follow with either eye. At age 5 months, she underwent an electroretinogram under sedation, which showed absent scotopic and photopic responses. At age 8 months, her nystagmus had dampened slightly. Pupils were sluggishly reactive without paradoxical response. Anterior segment exam was normal. Dilated fundus exam showed bilateral pigment granularity, particular in the maculae. Her cycloplegic refraction was +6.50 OU. At 2 years of age, her binocular visual acuity is 20/300 using single HOTV optotypes. Fundus exam and refraction were stable. DNA sequencing of the CEP290, AIPL1, CRB1, GUCY2D, RPE65, RDH12 and RPGRIP1 exons and exon-intron boundaries revealed no likely pathological sequence changes. Sequencing of the CRX gene showed a c.G264T mutation, predicted to change lysine 88 to an asparagine in the third helix of the homeodomain (Figure 2). Neither parent possessed this sequence change. Patient #2 (c.413delT, Ile138fs48) Patient 2, a 10 month-old girl, presented with a history of nystagmus since 2-3 months of age. Her father had noted reduced visual behavior in dimly-lit surroundings;in normal lighting, she seemed to see up close, but not at a distance. The child was otherwise healthy and was on- or above-target for developmental milestones. Her parents were both of Ashkenazi Jewish descent and there was no family history of relevant eye disease. On exam, she fixed and followed well with either eye and had no ocular preference. Her binocular Teller acuity was 20/400. She had a high frequency, moderate amplitude nystagmus, but no anomalous head posture or strabismus. Anterior segment exam was normal. Dilated fundoscopic examination showed bilateral attenuation of her retinal arterioles, diffuse macular and extramacular pigment granularity, and early waxy pallor to the optic nerves bilaterally. An electroretinogram performed under anesthesia showed unrecordable photopic and scotopic responses. Cycloplegic refraction showed mild myopic astigmatism bilaterally. At 16 months of age, her exam was approximately stable, save that she saw 20/600 on binocular Teller acuity testing. At 2.5 years, her binocular visual acuity was 6/200 with crowded, single HOTV optotypes. Fundus examination showed some progression of pigment changes and significant macular atrophy with thinning of the temporal nerve fiber layer. The optic nerves exhibited mild pseudopapilledema. Fundus autofluorescence shows little retinal signal, consistent with little to no lipofuscin accumulation. Cirrus ocular coherence tomography (OCT) performed as a 5 line raster through the nerve and temporal retinaalthough limited due to the patients age and nystagmusconfirmed elevation of the optic nerve and diffuse retinal thinning, including thinning of the temporal nerve fibeer layer. However, an outer band of reflectivity seen in all images suggests the presence of some residual cells in the outer nuclear layer. DNA sequencing of the CEP290, AIPL1, CRB1, GUCY2D, RPE65, RDH12 and RPGRIP1 exons and exon-intron boundaries revealed no likely pathological sequence changes. Sequencing of the CRX gene showed a c.413delT mutation, predicted to frameshift the CRX protein sequence after Ile138, followed by 48 amino acids of unique sequence, shortening the OTX tail of the protein. Her mother did not possess this sequence change;her father was unavailable for genotyping. Functional Characterization of Mutations In silico modeling of patient #1's missense mutation shows that lysine88 forms a hydrogen bond with the major groove of DNA at the homeodomain recognition sequence. Changing this residue to an asparagine disrupts this interaction. In order to test how these two mutations affected CRX function in a model system, we transiently transfected a human retinal pigment epithelial-like cell like (h-ARPE-19 cells)with a expression constructs of human wild-type, c.G264T, or c.413delT CRX cDNA, along with a reporter construct consisting of the bovine rhodopsin promoter coupled to the luciferase gene. Expression of the wild-type CRX protein resulted in a 5-fold activation of the bovine rhodopsin promoter. When compared to the wild-type protein, the c.G264T and c.413delT constructs resulted in 10% (p= 1.5x10-11)and 30% (p=3.7x10-11) of normal CRX activity. Since the retinal leucine zipper transcription factor, NRL, cooperates with CRX to facilitate rhodopsin gene activation, we investigated the effect of co-expression of NRL in our experimental paradigm. As previously reported, expression of NRL by itself results in approximately a 5-fold activation of the bovine rhodopsin promoter and this effect is increased to 30-fold in the presence of CRX. Co-expression of NRL with the c.G264T mutant CRX resulted in a minimal increase in transactivation (2- fold) that is not likely to be biologically significant. However, co-expression of the c.413delT mutation (predicted to affect the WSP and OTX domains) with NRL resulted in 9-fold activation of the rhodopsin promoter, slightly more than we observed with NRL expression alone. In order to evaluate the effect that these mutations had on CRX and NRL expression in our cell culture system, we performed Western blot analysis on protein lysates from our transactivation experiments. While expression levels of wild-type and Lys88Asn CRX were comparable relative to the housekeeping gene GAPDH, the Ile138fs48 mutant had a higher steady-state level of protein expression. Co-expression of the wild-type NRL protein did not affect wild-type CRX protein expression, although levels of NRL protein were slightly lower in this co-expression paradigm than when NRL was expressed in the absence of CRX. In contrast, the steady-state levels of CRX and NRL were both drastically reduced in the presence of the Lys88Asn missense mutation. Co-expression of p.Ile138fs48 CRX with wild-type NRL resulted in a slight reduction of the steady-state levels of the truncated CRX protein and had no significant effect on the level of NRL expression (when compared to the expression of NRL in the absence of CRX). We next compared the sub-cellular localization of wild-type and mutant CRX proteins in human ARPE-19 cells. As previously reported in other cell types, wild-type CRX is predominantly a nuclear protein. The Lys88Asn protein remains mostly nuclear, but some faint cytoplasmic localization was detected. The Ilefs48 mutant also was predominantly nuclear, although the percentage of cytoplasmic localization was qualitatively greater than in the Lys88Asn mutation. These results are summarized in a paper published this year in "Human Mutation."