The golden era of ocular disease gene discovery: race to the finish.

Within the last decade, technological advances have led to amazing genetic insights into Mendelian and multifactorial ocular diseases. We provide a perspective of the progress in gene discovery and discuss the implications. We believe that the time has come to redefine the goals and begin utilizing the genetic knowledge for clinical management and treatment design. The unbelievable opportunities now exist for those nimble enough to seize them.

Mutations in the gene encoding lecithin retinol acyltransferase are associated with early-onset severe retinal dystrophy.

The chromophore of the visual pigments, 11-cis retinal, is derived from vitamin A (all-trans retinol) through a series of reactions that take place in retinal pigment epithelium (RPE); (ref. 1). The first of these reactions is catalyzed by lecithin retinol acyltransferase (LRAT); (ref. 2). We screened 267 retinal dystrophy patients for mutations in LRAT and identified disease-associated mutations (S175R and 396delAA) in three individuals with severe, early-onset disease. We showed that the S175R mutant has no acyltransferase activity in transfected COS-7 cells. Our findings highlight the importance of genetic defects in vitamin A metabolism as causes of retinal dystrophies and extend prospects for retinoid replacement therapy in this group of diseases.

Nrl is required for rod photoreceptor development.

The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.

Retinopathy induced in mice by targeted disruption of the rhodopsin gene.

Retinitis pigmentosa (RP) represents the most common mendelian degenerative retinopathy of man, involving death of rod photoreceptors, cone cell degeneration, retinal vessel attenuation and pigmentary deposits. The patient experiences night blindness, usually followed by progressive loss of visual field. Genetic linkage between an autosomal dominant RP locus and rhodopsin, the photoreactive pigment of the rod cells, led to the identification of mutations within the rhodopsin gene in both dominant and recessive forms of RP. To better understand the functional and structural role of rhodopsin in the normal retina and in the pathogenesis of retinal disease, we generated mice carrying a targeted disruption of the rhodopsin gene. Rho-/- mice do not elaborate rod outer segments, losing their photoreceptors over 3 months. There is no rod ERG response in 8-week-old animals. Rho+/- animals retain the majority of their photoreceptors although the inner and outer segments of these cells display some structural disorganization, the outer segments becoming shorter in older mice. These animals should provide a useful genetic background on which to express other mutant opsin transgenes, as well as a model to assess the therapeutic potential of re-introducing functional rhodopsin genes into degenerating retinal tissues.

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

Bioetics Issues of Artificial Placenta and Artificial Womb Technology.

Abstract: The worldwide infertility crisis and the increase in mortality and morbidity among infants, due to preterm births and associated complications, have stimulated research into artificial placenta (AP) and artificial womb (AW) technology as novel solutions. These technologies mimic the natural environment provided in the mother's womb, using chambers that ensure the supply of nutrients to the fetus and disposal of waste substances through an appropriate mechanism. This review aims to highlight the background of AP and AW technologies, revisit their historical development and proposed applications, and discuss challenges and bioethical and moral issues. Further research is required to investigate any negative effects of these new technologies, and ethical concerns pertaining to the structure and operation of this newly developed technology must be addressed and resolved prior to its introduction to the public sphere.

In Memory of Professor Derek Pheby.

Abstract: Professor Derek Pheby's passing in November 2022 marked a profound loss for the scientific community. Professor Derek Pheby, a stalwart figure in the fields of autoimmune diseases and bioethics, was known for his dedication to scientific research and patients' support, particularly for those affected by paraneoplastic autoimmune syndromes. Professor Pheby made significant contributions to research, especially about Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). His leadership of the ME Biobank and scientific coordination of EUROMENE demonstrated his commitment to pushing boundaries and fostering international collaborations. Professor Pheby's scientific work addressed various aspects of ME/CFS, from physician education to patient needs, the development of a post-mortem tissue bank, and effective treatments. Beyond his medical career, Professor Pheby was a crucial member of the Independent Ethics Committee of MAGI, he was a poet, humanitarian, and advocate for child protection. His generosity and boundless spirit left an enduring legacy, fostering innovative research in the pursuit of combating autoimmune diseases.

Human Cloning: Biology, Ethics, and Social Implications.

Abstract: This scholarly article delves into the multifaceted domains of human cloning, encompassing its biological underpinnings, ethical dimensions, and broader societal implications. The exposition commences with a succinct historical and contextual overview of human cloning, segueing into an in-depth exploration of its biological intri-cacies. Central to this biological scrutiny is a comprehensive analysis of somatic cell nuclear transfer (SCNT) and its assorted iterations. The accomplishments and discoveries in cloning technology, such as successful animal cloning operations and advances in the efficiency and viability of cloned embryos, are reviewed. Future improvements, such as reprogramming procedures and gene editing technology, are also discussed. The discourse extends to ethical quandaries intrinsic to human cloning, entailing an extensive contemplation of values such as human dignity, autonomy, and safety. Furthermore, the ramifications of human cloning on a societal plane are subjected to scrutiny, with a dedicated emphasis on ramifications encompassing personal identity, kinship connections, and the fundamental notion of maternity. Culminating the analysis is a reiteration of the imperative to develop and govern human cloning technology judiciously and conscientiously. Finally, it discusses several ethical and practical issues, such as safety concerns, the possibility of exploitation, and the erosion of human dignity, and emphasizes the significance of carefully considering these issues.

Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis.

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse.

Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse.

X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11-15 weeks after the application.

Mutations in the cone-rod homeobox gene are associated with the cone-rod dystrophy photoreceptor degeneration.

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.

Constitutive "light" adaptation in rods from G90D rhodopsin: a mechanism for human congenital nightblindness without rod cell loss.

A dominant form of human congenital nightblindness is caused by a gly90-->asp (G90D) mutation in rhodopsin. G90D has been shown to activate the phototransduction cascade in the absence of light in vitro. Such constitutive activity of G90D rhodopsin in vivo would desensitize rod photoreceptors and lead to nightblindness. In contrast, other rhodopsin mutations typically give rise to nightblindness by causing rod cell death. Thus, the proposed desensitization without rod degeneration would be a novel mechanism for this disorder. To explore this possibility, we induced mice to express G90D opsin in their rods and then examined rod function and morphology, after first crossing the transgenic animals with rhodopsin knock-out mice to obtain appropriate levels of opsin expression. The G90D mouse opsin bound the chromophore and formed a bleachable visual pigment with lambda(max) of 492 nm that supported rod photoresponses. (G+/-, R+/-) retinas, heterozygous for both G90D and wild-type (WT) rhodopsin, possessed normal numbers of photoreceptors and had a normal rhodopsin complement but exhibited considerable loss of rod sensitivity as measured electroretinographically. The rod photoresponses were desensitized, and the response time to peak was faster than in (R+/-) animals. An equivalent desensitization resulted by exposing WT retinas to a background light producing 82 photoisomerizations rod(-1) sec(-1), suggesting that G90D rods in darkness act as if they are partially "light-adapted." Adding a second G90D allele gave (G+/+, R+/-) animals that exhibited a further increase of equivalent background light level but had no rod cell loss by 24 weeks of age. (G+/+, R-/-) retinas that express only the mutant rhodopsin develop normal rod outer segments and show minimal rod cell loss even at 1 year of age. We conclude that G90D is constitutively active in mouse rods in vivo but that it does not cause significant rod degeneration. Instead, G90D desensitizes rods by a process equivalent to light adaptation.

Scotopic threshold response (STR) of the human electroretinogram.

We recorded the human ERG using full-field stimuli at light intensities near absolute threshold to examine characteristics of the scotopic threshold response (STR). The human STR was seen below PII threshold and was the only component of the ERG evident near absolute rod psychophysical threshold. The human STR was detectable in the corneal ERG at stimulus intensities 0.6-1.0 log units above psychophysical threshold and had about a 2 log unit range to apparent saturation. Maximum STR amplitude was 12-20 microV. The STR latency ranged from 100-185 msec, depending on stimulus duration and intensity. The STR returned to baseline by 300 msec after onset, for very brief flashes, but it was prolonged with longer flashes. The spectral characteristics of the human STR matched rods and not cones. The STR followed Bloch's law and exhibited temporal integration for at least 80 msec. At 2.5-3 log units above visual threshold, corneal positive PII (b-wave and d.c. component) progressively obscured the negative STR. We propose that the human STR reflects post receptor processing in the retina. This is based on the similarity of the human STR to the STR of the cat and monkey, both of which originate at postreceptoral sites. Thus the human STR may find clinical use to evaluate the rod pathway in the proximal retina.

Contribution from proximal retina to intraretinal pattern ERG: the M-wave.

Intraretinal electroretinographic (ERG) responses were recorded from cat to spatial square wave gratings that were reversed in contrast (pattern ERG, PERG). Maximum 8 Hz PERG amplitudes occurred in proximal retina. Intraretinal ERGs to circular spots (photopic) also were maximal in proximal retina and resembled the M-waves of cold-blooded retinas. The temporal transforms of M-wave responses to 8 Hz flicker (to simulate contrast reversal conditions) imitated the 8 Hz PERG, suggesting that the M-wave may contribute significantly to the PERG.

A proximal retinal component in the primate photopic ERG a-wave.

PURPOSE: The monkey photopic ERG was studied during administration of glutamate analogs to determine whether the photopic a-wave derives exclusively from photoreceptors. METHODS: Monkey photopic ERGs were elicited using 200-msec flashes or 30-microseconds xenon photostrobe flashes on a steady light-adapting background of 40 cd/m2 (3.3 log scotopic troland). Intravitreal injections of APB, PDA, or both were given to block transmission to depolarizing and hyperpolarizing second-order retinal neurons, respectively. RESULTS: After injecting PDA to block light responses of horizontal cells and hyperpolarizing bipolar cells, part of the photopic a-wave was eliminated. The PDA-sensitive component, presumed to be due to activity postsynaptic to cones, was responsible for the photopic a-wave threshold and dominated the response over the initial 1 to 1.5 log units of intensity. For brighter stimuli, this component made a constant contribution to the photopic a-wave. A non-PDA-sensitive contribution to the a-wave, presumed to originate directly from cones, was first evident 1 to 1.5 log units above photopic a-wave threshold. It progressively dominated the a-wave at higher intensities, particularly at early time points after the flash. Injecting PDA almost eliminated the photopic a-wave elicited with bright xenon photostrobe flashes that are commonly used for human clinical ERG diagnostic testing, indicating that this a-wave may contain significant postreceptoral activity. CONCLUSION: The primate photopic ERG a-wave derives, in part, from retinal activity postsynaptic to cone photoreceptors, particularly for stimuli near the photopic ERG threshold that are typically used for human clinical studies.

Primate photopic sine-wave flicker ERG: vector modeling analysis of component origins using glutamate analogs.

PURPOSE: To study how the photoreceptoral and postreceptoral ON- and OFF-components contribute to the photopic sine-wave flicker ERG in the monkey by isolating the components with glutamate analogs. METHODS: Monkey photopic flicker ERGs were elicited with sine wave stimuli (mean luminance, 2.66 log cd/m(2); 80% modulation depth, on a 40 cd/m(2) white background) and were recorded for stimulus frequencies of 4 Hz to 64 Hz, before and after intravitreal injection of DL-2-amino-4-phosphonobutyric acid (APB) and cis-2, 3-piperidinedicarboxylic acid (PDA) that block ON- and OFF-bipolar activity, respectively. The amplitude and phase of the fundamental component were analyzed. RESULTS: The flicker response amplitudes increased after APB, for frequencies of 6 Hz to 32 Hz. The further addition of PDA to isolate the photoreceptor component resulted in a relatively small residual response that decreased monotonically from 4 Hz to 32 Hz. The postsynaptic APB (ON-) and PDA (OFF-) sensitive components were isolated by subtraction and were characterized by amplitude and phase vectors. The ON- and OFF-components were larger than the initial control responses for stimuli of 8 Hz to 40 Hz. These two components had a frequency-dependent phase difference of 160 degrees to 230 degrees; normally, they interfere with each other and reduce their net contribution. The phase difference between ON- and OFF-components was nearly 180 degrees for a 10-Hz stimulus, and the phase cancellation caused a prominent dip in amplitude at this frequency. CONCLUSIONS: These results indicate that postreceptoral ON- and OFF-components contribute substantially to the sine-wave flicker ERG, especially at higher stimulus frequencies. Because of phase cancellation, they mask each other in the net response in a frequency dependent fashion. The photoreceptor contribution is greater than the net postsynaptic component only for frequencies of approximately less than or equal to 10 Hz. These results can be summarized by a vector model that may be useful for interpreting changes resulting from retinal disease.

Aspartate separation of the scotopic threshold response (STR) from the photoreceptor a-wave of the cat and monkey ERG.

We recorded ERG responses at the cornea of cat and monkey and identified the initial negative wave elicited by very dim stimuli as the scotopic threshold response (STR) comparable to that previously recognized by intraretinal recordings of cat. The STR, but not the photoreceptor a-wave, was eliminated by intravitreal aspartate in both cat and monkey, which demonstrated that the STR origin was post-photoreceptoral. Intraretinal recordings before and after aspartate confirmed that the a-wave of cat with bright light was fast-PIII from photoreceptors, and further showed that there was minimal or no extracellular activity recordable near the photoreceptors with very dim stimuli after aspartate. This study showed practical ways to separate the STR from the photoreceptor a-wave in corneal records, by the range of stimulus intensity (STR with dim stimuli; photoreceptor a-wave with bright stimuli) and by response latency (STR, long latency; photoreceptor a-wave, short latency). These recordings provide the first evidence that the monkey has an STR, and that it is post-photoreceptoral like the STR of cat. Further, this provides support to consider that the corneal negative STR wave of the human ERG with dim light may also be post-photoreceptoral.

Autosomal dominant macular atrophy at 6q14 excludes CORD7 and MCDR1/PBCRA loci.

PURPOSE: Localization of the gene responsible for autosomal dominant atrophic macular degeneration (adMD) in a large pedigree UM:H785. METHODS: Standard ophthalmologic examinations were performed. Microsatellite markers were used to map the disease gene by linkage and haplotype analyses. RESULTS: The macular degeneration in this family is characterized by progressive retinal pigment epithelial atrophy in the macula without apparent peripheral involvement by ophthalmoscopy or functional studies. Acuity loss progressed with age and generally was worse in the older affected individuals. The rod and cone function remained normal or nearly normal in all tested affected members up to 61 years of age. The phenotype in our family has characteristics similar to Stargardt-like macular degeneration with some differences. Haplotype analysis localized the disease gene in our adMD family to an 8-cM region at 6q14, which is within the 18-cM interval of STGD3 but excludes cone-rod dystrophy 7 (CORD7; centromeric) and North Carolina macular degeneration and progressive bifocal chorioretinal atrophy (MCDR1/PBCRA; telomeric). The mapping interval overlaps with that of recessive retinitis pigmentosa (RP25). CONCLUSIONS: These results implicate at least three genetically distinct loci for forms of macular degeneration that lie within a 30-cM interval on chromosome 6p11-6q16: CORD7, adMD, and MCDR1/PBCRA. Because the critical interval for the adMD family studied overlaps with STGD3 and RP25, these loci could be allelic.

The effect of calcium channel blocker diltiazem on photoreceptor degeneration in the rhodopsin Pro213His rat.

PURPOSE: To determine whether the calcium channel blocker D-cis-diltiazem promotes photoreceptor survival in rats with the Pro23His rhodopsin mutation. METHODS: Heterozygous Pro23His rhodopsin line 1 rats (n = 11) were treated daily, according to a protocol applied successfully in rd mice, with D-cis-diltiazem hydrochloride increased incrementally from 21 to 54 mg/kg in a divided dose (8 AM and 8 PM) administered by intraperitoneal (i.p.) injection for 21 days, beginning on days of age 10 through 12. Saline-treated line 1 rats (n = 6) received i.p. injections of an equal volume of 0.9% saline. Analysis on day 35 of age included dark-adapted corneal electroretinogram (ERG) b- and a-waves recorded from threshold to 0.63 log candela-seconds [cd-s]/m2, saturated a-waves elicited with a 2.1 log cd-s/m2 flash, and morphometry of the outer nuclear layer (ONL) and rod outer segments (ROS). RESULTS: ONL width and cell counts of diltiazem-treated and saline-treated animals at 35 days were reduced to 64%-68% of 15-day-old untreated P23H line 1 retinas. No photoreceptor rescue was found by measuring ONL width (P = 0.84), cell count (P = 0.42), or ROS length (P = 0.85). Functional assays by ERG b-wave threshold (P = 0.57), b-wave maximum amplitude (P = 0.46), and saturated a-wave amplitude (P = 0.59) also showed no rescue. CONCLUSIONS: D-cis-Diltiazem did not rescue photoreceptors of Pro23His rhodopsin mutation line 1 rats treated according to the protocol used in rd mouse.

Human melanoma-associated retinopathy (MAR) antibodies alter the retinal ON-response of the monkey ERG in vivo.

PURPOSE: Melanoma-associated retinopathy (MAR) is a paraneoplastic condition that causes visual symptoms of night-blindness and photopsias. The electroretinogram (ERG) of MAR patients is characteristically abnormal in a way that implicates retinal depolarizing bipolar cell (DBC) dysfunction. Whether an injection of IgG from MAR patients into the vitreous of monkeys would alter the ERG acutely as a demonstration of a functional basis for patients' visual symptoms was explored. METHODS: MAR IgG was isolated from three visually symptomatic melanoma patients. Control IgG was from melanoma patients with no vision problems. The ERG was monitored after intravitreal injections into monkey eyes. One eye was injected with 2-amino-4-phosphonobutyric acid (APB), which is known to block DBC ON-pathway responses. Retinal immunocytochemistry was performed using fluorescein isothiocyanate-labeled goat anti-human IgG. RESULTS: Within 1 to 3 hours after MAR IgG injection, the ERG photopic b-wave was diminished, with far less effect on the a- and d-waves. These changes are characteristic of DBC dysfunction and were similar to the effects of APB. The scotopic ERG b-wave, which reflects activity of rod-driven DBCs, showed a loss of amplitude and threshold sensitivity after MAR IgG. Retinal immunocytochemistry with anti-IgG antibody showed IgG penetration throughout the retinal layers, but staining was not specific for a single type of retinal neuron. CONCLUSIONS: Intravitreal injection of human MAR IgG altered the monkey ERG acutely in ways that implicate functional disruption of retinal DBC signaling. These results support the hypothesis that MAR IgG circulating antibodies are responsible for the reported visual symptoms. Bipolar cells in the ON-pathway appear to be affected more than OFF-pathway bipolar cells of the cone pathway in this acute preparation.