Retinal Structure in Pre-Clinical Age-Related Macular Degeneration.

PURPOSE: To determine, if there are identifiable retinal structural changes associated with genetic risk for age-related macular degeneration (AMD). MATERIALS AND METHODS: Seventy-three subjects (range 51.5 to 68.9 years) participated in this prospective study. Subjects were recruited based on the presence of a family history of AMD in one or both parents. All participants underwent a complete ophthalmic exam and imagery for staging of disease severity and genetic testing to assess genetic risk for AMD development. Optical coherence tomography (OCT) imaging was performed on all participants. Semi-automated retinal layer segmentation was performed to assess retinal structural changes. RESULTS: Of 73 subjects, 47 subjects had normal appearing retina with no evidence of drusen or other changes consistent with AMD, 16 subjects were classified as early AMD, and 13 were designated as intermediate AMD. Retinal volume measures of total retina, outer retina, outer nuclear layer and the retinal pigment epithelium, were not related to AMD classification, genetic risk scores, or age. The thickness of the outer retina showed statistically significant thickening in the foveal region in only the intermediate AMD group and a statistically significant thickening of the RPE in early and intermediate AMD groups in the central retina. CONCLUSION: No consistent changes were observed in retinal structure at multiple locations that are associated with pre-clinical AMD, based on AMD genetic risk or with aging within the age range of our cohort.

Analysis of autofluorescent retinal images and measurement of atrophic lesion growth in Stargardt disease.

Current retinal imaging techniques using scanning laser ophthalmoscopy (SLO) provide a powerful mechanism for characterizing the topographical distribution of lipofuscin fluorophores and atrophic lesions (ALs) in retinal disease. In this paper we describe a novel Edge-Flow-Driven Variational Image Segmentation analysis to measure and evaluate progressive change in the area of ALs as well as regions of hyperfluorescence (HF). The algorithm is embedded in a series of almost completely automated image processing steps that allow rapid comparison of serial images. The sensitivity of the methodology to detect change was evaluated by measuring progression of AF lesion size in a cohort of Stargardt Macular Dystrophy (STGD) patients. Fifty-two STGD subjects (mean age = 41.0 +/- 16.6 years, range 9-78 yrs) at varying stages of disease participated in this prospective study. Twenty-four of the 52 subjects presented with atrophic lesions in one or both eyes on first evaluation. For this subgroup of subjects, the mean (+/-1 sd) follow-up time was 2.92 (+0.26) years (range 0.57-3.26 years) and the mean (+/-1 sd) rate of change was found to be approximately 0.94 (+/-0.87) mm(2)/year (range 0.2-2.13 mm(2)/yr). With this methodology, progressive enlargement of AL area was detectable in as little as one year, while regions of HF generally decreased, although there was considerable variability in the appearnce of HF, presumably reflecting the combined effects of the creation or expansion of lipofuscin deposits and resorption and loss associated with retinal cell death. Our findings suggest that this methodology is sufficiently sensitive to detect change and provides a clinically relevant tool to monitor progression not only with regards to natural history, but also to evaluate the efficacy of potential therapeutic interventions in STGD. Finally, we evaluated the association between AL area and measures of rod- and cone-mediated retinal function, as assessed with electroretinography (ERG). In general, the larger the AL, the poorer the ERG response, with a greater impact of lesion size on cone- rather than rod-mediated retinal function, a finding that was expected on the basis of the location and size of the AL and the distribution of rod- and cone-photoreceptors.

Retinal function in X-linked ocular albinism (OA1).

PURPOSE: To characterize retinal function in human recessive X-linked ocular albinism (OA1) across the normal lifespan. METHODS: Retinal function was evaluated in 14 OA1 patients (ages 11 to 71 years) and five obligate carriers (ages 41 to 50 years) and compared to normal controls using full-field and multi-focal electroretinograms (ERG and mERG, respectively) and electro-oculography (EOG). RESULTS: No consistent differences in ERG response parameters were observed when OA1 patients were compared as a group to normal controls. A trend in the direction of better correlations of response parameters with age was, however, observed in OA1. EOG Arden ratios were normal or hypernormal for all patients, but were uncorrelated with age. Central retinal function measured with the mERG suggested a flat response topography with depressed macular function compared to normal controls. CONCLUSIONS: Panretinal function in OA1 is within normal limits at all ages, consistent with previous reports in generalized albinism. The stronger correlations with age in OA1 may suggest a different rate of age-related change in OA1 compared to normal populations, but the precise nature of this change must await an appropriate prospective study. The topography of mERG amplitudes in OA1 is relatively flat across the central retina with a reduction in amplitude in the macular region consistent with anatomical studies demonstrating an underdeveloped macular region in albinism.

Temporal response properties of the primary and secondary rod-signaling pathways in normal and Gnat2 mutant mice.

Multiple signaling pathways have been proposed for rod vision in the mammalian retina. The primary and secondary rod pathways have been characterized in humans with the scotopic 15-Hz flicker electroretinogram (ERG). The purpose of this study was to determine whether the response properties of these pathways in the mouse are similar to those of humans. C57BL/6J and Gnat2(cpfl3) mutant mice lacking functional cones were used in these experiments. Standard ERG recording techniques were employed. Response functions were obtained for a range of flash intensities (-4.7logcd-s/m(2) to -0.2logcd-s/m(2)) and temporal modulation frequencies (1-30Hz). The mouse intensity-response functions to 15-Hz flickering stimuli possessed the same features as that of humans - a local amplitude minimum and a rapid phase change in the intensity region where the primary and secondary pathways are mutually inhibitory. However, the secondary pathway in the mouse did not achieve the same level of sensitivity as previously shown for humans, suggesting inter-species differences in post-receptoral signal processing. In Gnat2(cpfl3) mutant mice, the secondary pathway was completely abolished. Measurements of temporal acuity indicated that the primary and secondary rod pathways could mediate temporal frequencies as high as 30 and 50Hz, respectively. The response functions for mice are similar to those of humans, although the evidence suggests that the primary rod pathway dominates all rod-mediated signal processing in the mouse. Nevertheless, these results demonstrate the feasibility of measuring non-invasively the performance characteristics of the primary and secondary rod retinal pathways in the mouse and provide a mechanism for testing hypotheses about the action of disease where post-receptoral cells are differentially affected.

Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene.

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Cortical visual function in the rd12 mouse model of Leber Congenital Amarousis (LCA) after gene replacement therapy to restore retinal function.

One eye of rd12 mice received a sub-retinal injection of a vector carrying normal human RPE65 cDNA at post-natal day 18, and at 6- and 13-months of age. Electroretinograms (ERGs) and visual-evoked potentials (VEPs) were recorded to luminance, and to spatially and temporally modulated stimuli to assess the consequences of delayed treatment on visual pathway function. Early treatment resulted in better overall retinal rescue and better rescue of cone-mediated function. VEPs to low temporal frequency luminance modulation were well preserved at all but the oldest treatment age and corresponded to predictions based on the amount of retinal rescue. In contrast, VEPs to high frequency spatially and temporally modulated stimuli were impaired even at the earliest age. These results provide further support that early treatment in human LCA will have the most hope for optimal visual performance.

The visual evoked potential in the mouse--origins and response characteristics.

The visual evoked potential (VEP) in the mouse is characterized and compared to responses obtained with the electroretinogram (ERG). The results indicate that: 1, the VEP originates in the visual cortex; 2, the rod and cone pathways contribute separately to the VEP; 3, temporal tuning functions for rod and cone ERGs are low pass and band pass, respectively; VEP tuning functions are both band pass; and 4, VEP acuity is 0.62+/-0.156 cycles/degree. The differences in the spatial and temporal tuning functions obtained from the retina and visual cortex provides a tool to investigate signal processing through the visual system.

Mouse models of ocular diseases.

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to discover genetically determined eye variations and disorders. In this paper, we list and describe mouse models for ocular research available from Mouse Eye Mutant Resource at The Jackson Laboratory. While screening mouse strains and stocks at The Jackson Laboratory (TJL) for genetic mouse models of human ocular disorders, we have identified numerous spontaneous or naturally occurring mutants. We characterized these mutants using serial indirect ophthalmoscopy, fundus photography, electroretinography (ERG) and histology, and performed genetic analysis including linkage studies and gene identification. Utilizing ophthalmoscopy, electroretinography, and histology, to date we have discovered 109 new disorders affecting all aspects of the eye including the lid, cornea, iris, lens, and retina, resulting in corneal disorders, glaucoma, cataracts, and retinal degenerations. The number of known serious or disabling eye diseases in humans is large and affects millions of people each year. Yet research on these diseases frequently is limited by the obvious restrictions on studying pathophysiologic processes in the human eye. Likewise, many human ocular diseases are genetic in origin, but appropriate families often are not readily available for genetic studies. Mouse models of inherited ocular disease provide powerful tools for rapid genetic analysis, characterization, and gene identification. Because of the great similarity among mammalian genomes, these findings in mice have direct relevance to the homologous human conditions.

Disrupted compaction of CNS myelin in an OSP/Claudin-11 and PLP/DM20 double knockout mouse.

OSP/claudin-11 and PLP are both tetraspan proteins concentrated in CNS myelin. It has been proposed that they have a structural role in myelin formation and maintenance due to their localization and concentration in membrane sheaths. This hypothesis is not supported by the fact that both OSP/claudin-11- and PLP-null mice have relatively normal-appearing myelin and mild neurological deficits. Since both OSP/claudin-11 and PLP are abundant in myelin and have similar structures, the mild phenotypes of the knockout mice are likely due to compensatory mechanisms. Here we show that when both OSP/claudin-11 and PLP genes are knocked out, mice had severe neurological deficits, markedly abnormal myelin compaction, and smaller axon diameters. Interestingly, when either of these genes was knocked out, the expression of the other protein was increased. These data demonstrate that OSP/claudin-11 and PLP have essential structural functions in maintaining normal compact myelin and there is redundancy in their functions.

Retinal degeneration mutants in the mouse.

The Jackson Laboratory, having the world's largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure.

BACKGROUND: Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. RESULTS: Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. CONCLUSIONS: We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

The reelin pathway modulates the structure and function of retinal synaptic circuitry.

The formation of synaptic connections requires the coordination of specific guidance molecules and spontaneous neuronal activity. The visual system has provided a useful model for understanding the role of these cues in shaping the precise connections from the neural retina to the brain. Here, we demonstrate that two essential genes in the Reelin signaling pathway function during the patterning of synaptic connectivity in the retina. Physiological studies of mice deficient in either reelin or disabled-1 reveal an attenuation of rod-driven retinal responses. This defect is associated with a decrease in rod bipolar cell density and an abnormal distribution of processes in the inner plexiform layer. These results imply that, in addition to its essential role during neuronal migration, the Reelin pathway contributes to the formation of neuronal circuits in the central nervous system.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is approximately 60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Maternal germ-line transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice.

We report a method for introducing mtDNA mutations into the mouse female germ line by means of embryonic stem (ES) cell cybrids. Mitochondria were recovered from the brain of a NZB mouse by fusion of synaptosomes to a mtDNA-deficient (rho degrees ) cell line. These cybrids were enucleated and the cytoplasts were electrofused to rhodamine-6G (R-6G)-treated female ES cells. The resulting ES cell cybrids permitted transmission of the NZB mtDNAs through the mouse maternal lineage for three generations. Similarly, mtDNAs from a partially respiratory-deficient chloramphenicol-resistant (CAP(R)) cell line also were introduced into female chimeric mice and were transmitted to the progeny. CAP(R) chimeric mice developed a variety of ocular abnormalities, including congenital cataracts, decreased retinal function, and hamaratomas of the optic nerve. The germ-line transmission of the CAP(R) mutation resulted in animals with growth retardation, myopathy, dilated cardiomyopathy, and perinatal or in utero lethality. Skeletal and heart muscle mitochondria of the CAP(R) mice were enlarged and atypical with inclusions. This mouse ES cell-cybrid approach now provides the means to generate a wide variety of mouse models of mitochondrial disease.

Retinal degeneration 6 (rd6): a new mouse model for human retinitis punctata albescens.

PURPOSE: To characterize the genetics and phenotype of a new mouse mutant with retinal degeneration, rd6, that is associated with extensive, scattered, small white retinal dots seen ophthalmoscopically. METHODS: The phenotype was characterized using ophthalmoscopy, fundus photography, electroretinography, light microscopy, immunocytochemistry, and electron microscopy. Genetic characterization and linkage analysis studies were performed using standard methods. RESULTS: The inheritance pattern of rd6 is autosomal recessive. Linkage analysis mapped rd6 to mouse Chromosome 9 approximately 24 cM from the centromere, suggesting that the human homolog may be on chromosome 11q23. Ophthalmoscopic examination of mice homozygous for rd6 revealed discrete subretinal spots oriented in a regular pattern across the retina. The retinal spots appeared by 8 to 10 weeks of age and persisted through advanced stages of retinal degeneration. Histologic examination revealed large cells in the subretinal space, typically juxtaposed to the retinal pigment epithelium. The white dots seen on fundus examination corresponded both in distribution and size to these large cells. By 3 months of age, the cells were filled with membranous profiles, lipofuscin-like material, and pigment. These cells reacted strongly with an antibody directed against a mouse macrophage-associated antigen. Photoreceptor cells progressively degenerated with age, and an abnormal electroretinogram was initially detected between 1 and 2 months of age. CONCLUSIONS: The fundi of mice homozygous for rd6 exhibit phenotypic similarities to the human flecked retinal disorder retinitis punctata albescens. Thus, rd6/rd6 mice may be a model for understanding the etiology of this or similar disorders. The relationship between the aberrant subretinal cells and the concomitant photoreceptor degeneration remains to be established.

A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse.

The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells.

Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene.

The tub gene is a member of a small, well conserved neuronal gene family of unknown function. Mutations within this gene lead to early-onset blindness and deafness, as well as late-onset obesity and insulin resistance. To test the hypothesis that mutations within other members of this gene family would lead to similar phenotypes as observed in tubby mice, and hence have similar functional properties, we have generated null mutants of the tubby-like protein ( Tulp ) 1 gene by homologous recombination. Similarly to tubby mice, Tulp1 (-/-)mice exhibit an early-onset retinal degeneration with a progressive, rapid loss of photoreceptors, further supporting the notion that previously identified mutations within the human TULP1 gene are indeed causative of retinitis pigmentosa. However, in contrast to tubby mice, Tulp1 (-/-)mice exhibited normal hearing ability and, surprisingly, normal body weight despite the fact that both TUB and TULP1 are expressed in the same neurons within the hypothalamus in areas known to be involved in feeding behavior and energy homeo stasis. However, TUB and TULP1 show a distinctly different staining pattern in the nucleus of these neurons, perhaps explaining the difference in body weight between the Tulp1 (-/-)and tubby mutant mice.

Rod multifocal electroretinograms in mice.

PURPOSE: To test the feasibility of recording rod multifocal electroretinograms (ERGs) from the mouse eye. METHODS: Multifocal ERGs were recorded from normal mice (C57BL/6J) using an array of equal-sized hexagons. Local stimuli were blue (W47A), and the number of blank frames between successive flashes at the same location was fixed at 14 (minimum 200 msec between flashes). Flash and surround intensity, and the number of hexagons, were varied to optimize the stimulus conditions for the mouse, and alterations in adaptation level were used to assess cone intrusion. Local response isolation was evaluated by comparing multifocal responses to full-field ERGs and by mapping local defects in laser-treated mice. RESULTS: Rod multifocal ERGs, although small, were clearly recordable and well formed under many conditions. Decreasing flash intensity or the size of stimulus elements, and/or increasing the surround intensity or adaptation level, decreased local response amplitudes. At the dimmest flash intensity (-0.70 log scotopic trolands [scot td]/s) and the smallest stimulus element (2.9 degrees x 3.5 degrees), local responses were nondetectable. Comparisons with full-field ERGs supported the hypothesis that the local responses were not contaminated by contributions from dark-adapted retinal areas surrounding the multifocal display. With sufficiently bright (0.30 log scot td-s) and relatively large (5.6 degrees x 6.9 degrees) stimulus elements, multifocal responses clearly revealed local retinal defects created with laser treatment. CONCLUSIONS: Rod multifocal ERGs can be recorded from the mouse eye to provide topographical maps of retinal function that have sufficient spatial resolution to be of practical use. The technique will be useful in characterizing the natural history of regional loss in mouse models of human retinal disease and in evaluating some forms of interventional therapy.

Genetic and physical maps of the mouse rd3 locus; exclusion of the ortholog of USH2A.

The rd3 retinal degeneration gene was previously mapped 10 +/- 2.5 cM distal to Akp1 on mouse Chromosome (Chr) 1 (Chang et al., 1993), a region that may be homologous to the locus of the human USH2A gene, which carries mutations responsible for Usher IIa retinal degeneration/hearing loss syndrome. An intercross from an Rb(11, 13)4Bnr(rd3/rd3) x C57BL/6J mating was set up, 428 F2 meioses were analyzed, and the rd3 gene was placed between the markers D1MIT292/D1MIT209 and D1MIT510, a distance of 1.40 +/- 0.57 cM. These flanking markers and the mouse ortholog of USH2A (Mush2a) were mapped in the T31 mouse radiation hybrid (RH) panel, with the result that D1MIT292/D1MIT209 and D1MIT510 were 7.9 cR3000 apart ( approximately 800 kb), and Mush2a was > 30 cR3000 proximal to the pair, excluding it from the rd3 locus. A contig spanning the rd3 locus and consisting of 2 YACs and one BAC was generated, and Mush2a was absent from it, confirming its exclusion from the locus. Comparison of adjacent marker pairs in the Whitehead genetic map and our genetic map showed some discrepancies in order of markers and genetic distances. Comparison of our genetic map and the RH map showed some highly skewed relationships between genetic and physical distances.