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Genetic testing in a multisite clinical trial network for inherited eye conditions is described in this retrospective review of data collected through eyeGENE®, the National Ophthalmic Disease Genotyping and Phenotyping Network. Participants in eyeGENE were enrolled through a network of clinical providers throughout the United States and Canada. Blood samples and clinical data were collected to establish a phenotype:genotype database, biorepository, and patient registry. Data and samples are available for research use, and participants are provided results of clinical genetic testing. eyeGENE utilized a unique, distributed clinical trial design to enroll 6,403 participants from 5,385 families diagnosed with over 30 different inherited eye conditions. The most common diagnoses given for participants were retinitis pigmentosa (RP), Stargardt disease, and choroideremia. Pathogenic variants were most frequently reported in ABCA4(37%), USH2A(7%), RPGR(6%), CHM(5%), and PRPH2(3%). Among the 5,552 participants with genetic testing, at least one pathogenic or likely pathogenic variant was observed in 3,448 participants (62.1%), and variants of uncertain significance in 1,712 participants (30.8%). Ten genes represent 68% of all pathogenic and likely pathogenic variants in eyeGENE. Cross‐referencing current gene therapy clinical trials, over a thousand participants may be eligible, based on pathogenic variants in genes targeted by those therapies. This article is the first summary of genetic testing from thousands of participants tested through eyeGENE, including reports from 5,552 individuals. eyeGENE provides a launching point for inherited eye research, connects researchers with potential future study participants, and provides a valuable resource to the vision community.

Molecular genetics is revolutionizing the diagnosis and treatment of inherited eye diseases. The National Eye Institute of the National Institutes of Health (NIH), in an effort to facilitate future basic and clinical research in inherited eye disease, created The National Ophthalmic Disease Genotyping and Phenotyping Network (eyeGENE). This review describes the process and utility of the eyeGENE program as it relates to ophthalmic clinical practice.

Scanning electron microscopy of the lenses from transgenic mice (TG72) containing the HIV-1 protease linked to the lens αA-crystallin promoter showed structural changes around postnatal day 16. Frank opacification of the lens was observed at day 24. To relate the biochemical and biophysical changes that occur during the process of cataract development, high-resolution two-dimensional gel electrophoresis (2D), quantitative image analysis and ion measurements were carried out on lenses from postnatal day 10 and on days 15–24. The phase separation temperature (Tc), a measure of molecular interactions between proteins, was also determined for normal and transgenic lenses. A comparison of the transgenic and normal lenses on day 10 revealed no significant differences in any of the measured parameters. However, starting around day 16 or the first stage of observed structural changes, the TG72crystallin profiles of the αA- αB-, βA3-, βA4-, βB3 and one γ-crystallin began to deviate from the normal. By postnatal day 20, a second stage was initiated with an influx of calcium and sodium ions that was accompanied by modifications of βB1- and βB2-crystallin. In the third and final stage of the cataract process, a large increase in the proteolysis of crystallins was accompanied by the appearance of the frank cataract on day 24. The Tc initially increased in all of the mouse lenses until just prior to eyelid opening. After that time, the Tc decreased in all lenses. Although the Tc continued to decrease in the normal lenses with age, for the homozygous transgenic mice it exhibited a dramatic increase that began on day 20. Thus, in the TG72transgenic mouse, cataract formation occurs in a three-stage process. Tc and other biophysical parameters previously measured appeared to be insensitive to the modifications that occur during stage 1. However, during the second stage of cataract formation, there was a correspondence between abnormal Tc and the abnormal interactions between cellular constituents apparently resulting from lens hydration, the loss of ion homeostasis and continued proteolysis. The last stage of cataract formation results in a total loss of lens transparency and leakage of lens proteins.

Protein-nucleic acid interactions which occur during Escherichia coli 50 S ribosomal subunit assembly between 23 S rRNA, 5 S rRNA and a complete set of 34 L-proteins were monitored by high resolution scanning transmission electron microscopy (STEM). This approach made it possible to visualize and quantitatively analyze conformational changes induced in the rRNAs during E. coli 50 S ribosomal subunit assembly. The reconstituted RNA-protein complexes, the control 23 S rRNA and native 50 S subunits were characterized by their mass and morphology. Association of 23 S rRNA with the first assembly protein, L24, led to the formation of a distinct nucleus of mass ("cluster") on the filamentous and loosely coiled molecule of the 23 S rRNA. This structural feature was preserved and enhanced in 23 S rRNA after its association with the rest of the early assembly proteins, namely L3, L20, L13, L4 and L22. Since the above proteins, with the exception of L3, bind to the 5' end of the 23 S rRNA, the cluster seems to be formed predominantly by interactions of L24, L13, L20, L22 and L4 with this segment of the 23 S rRNA molecule. Association with the rest of the primary binding proteins (L2, L23, L9, L1), which interact with the 3' end of the 23 S rRNA, appears to result in the formation of a second mass center. Binding of additional proteins led to the formation of compact particles with an apparent similarity to the 50 S subunit. However, particles with defined structural features characteristic of the native 50 S subunit requires the interactions of both 23 S rRNA and 5 S rRNA with all of the L-proteins. STEM image analysis demonstrated that 50 S subunit reconstitution proceeds by the immediate folding of the 23 S rRNA into a single mass center followed by the formation of a second mass center. These mass centers merge into one central body, which is gradually enhanced and decorated with structural elements characteristic of the 50 S subunit in the latter stages of assembly. Copyright 1994, 1999 Academic Press

Several lines of transgenic mice were generated with either active or inactive forms of the human immunodeficiency virus type 1 (HIV-1) protease gene under the control of the mouse lens αA-crystallin promoter. Mice bearing the inactive protease coding sequence displayed no gross abnormalities in the lens, while mice with the active protease developed time-dependent bilateral cataracts. One line, TG61, developed cataracts in uterowhile the second line, TG72, developed cataracts postnatally. TG61mice, homozygous for the transgene, developed severe microphthalmia and were significantly smaller than the control mice at postnatal day 30. two-dimensional-polyacrylamide gel electrophoresis analysis of the protein profiles of TG72and TG61lenses revealed extensive modifications in the lens crystallins. Proteolysis in the homozygous TG72mouse lenses began at postnatal day 20 with the disappearance or partial loss of βB1-, βB3-, and βA3-crystallins and the appearance of crystallin fragments. Protein leakage and the gradual breakdown of cytoskeletal elements also occurred. In contrast, the opacification of the homozygous TG61lenses appeared to have been influenced by differentiation and developmental processes. It apppears that HIV-1 protease expression activates other proteases, and these enzymes, in concert with the HIV-1 protease, are responsible for the protein modifications that eventually result in the opacification of the lens.

Transgenic mice, homozygous for HIV-1 protease expression in the eye lens, display degradation of some lens crystallins and cytoskeletal proteins prior to cataract formation on postnatal days 23-25. Alterations to the internal lens hydration state also occur; therefore, the status of the aquaporin protein MIP26 was examined over postnatal days 16-25 to determine if it was altered during cataractogenesis. The MIP was identical in transgenic and control lenses until day 21. By postnatal day 25 (frank cataract), in the lenses obtained from transgenic animals, the 26-kDa band was absent and there was a concurrent increase in the proportion of MIP23. Immunoblotting demonstrated cleavage at the C terminus. Lenses were also maintained in an organ culture system to demonstrate that the cataractogenic process is inherent to the isolated lens and to determine the contribution of cysteine protease action. Organ culture experiments revealed a similar progression to nuclear cataract formation as seen in vivo.Two-dimensional gel analysis of the soluble lens crystallin fraction of organ cultured lenses revealed the same cleavage pattern as occurs in vivo. Organ culture of transgenic lenses with E64, a cysteine protease inhibitor, dramatically delayed cataractogenesis and prevented proteolytic cleavage of both MIP26 and crystallins. HIV-1 protease, while the trigger of cataract formation, does not appear to be the protease responsible for cleavage of MIP or lens crystallins. These results suggest that activation of endogenous cysteine protease activity is involved in the cleavage of these proteins and occurs downstream of HIV-1 protease action.

Two constructs of transgenic mice, TG61and TG72, containing the HIV-1 protease linked to the lens αA-crystallin promoter develop cataract. The TG61construct develop cataract in utero, while the TG72construct exhibit frank opacities on the 24th day (homozygotes) and 26th day (hemizygotes) post natum. Polarized light scattering measurements were performed on cortical and nuclear sections of TG72lenses from day 19 to day 26 as a function of scattering angle. The parallel components of the scattered light intensity increase gradually during opacification, the perpendicular components show very low values from day 19 to day 22 after which they increase exponentially. Analysis of the scattered light intensities yielded parameters describing the size of the protein aggregates, the size of the domains exhibiting optical anisotropy/birefringence, the difference in refractive index between (a) the protein aggregates and their surroundings and (b) the birefringent units and their surroundings. The last parameter accounts for the major portion of the increase in lens turbidity.

A method to quantitate protein leakage was developed to assess the integrity of mammalian lenses in organ culture. Protein leakage into the culture medium was found to be a strong indicator of lens integrity. Rat lens incubation medium that contained more than 14 μg protein per lens after 1 hr was indicative of lens damage. Damaged lenses almost always exhibited lens opacities within 24 hr. However, on occasion, a lens predicted by this method to be damaged would maintain clarity at the 24 hr timepoint. In these instances, damage was confirmed by the inability of the lens to accumulate [3H]choline and 86RbCl from the medium at levels equivalent to undamaged lenses. When the protein concentration of the incubation medium was greater than 14 μg per lens, there was a concomitant impairment of lens transport function. This indicates that quantitation of protein leakage is a better predictive measure of lens viability than is lens clarity.

Two constructs of transgenic mice, TG61and TG72, containing HIV-1 protease linked to lens αA-crystallin promoter develop cataract. The TG61construct exhibits cataractogenesisin utero,while in the TG72construct frank opacities appear 24 days (homozygotes) and 26 days (hemizygotes) after birth. Differential scanning calorimetry and thermogravimetric analysis studies indicate that the hydration of lenses is strongly correlated with cataractogenesis. In all clear lenses (normal and precataractous) the total water content was the same, 68%, and increased upon opacification. The bound water, measured as percentage nonfreezable water of the total water, decreased upon cataract formation, indicating a syneretic process. On the other hand, the bound water expressed as grams of nonfreezable water per gram dry weight increases upon opacification. This implies that proteolysis and subsequent enhanced hydration is the primary supramolecular event in cataractogenesis and that syneresis in the lens of transgenic mice is of secondary importance.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2gse ( m2GMT), while the other one makes m5C967 (m5C RAT). The m2G RAT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C RflT functions reciprocally, using free rRNA but not 30S subunits (Negre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid itfethylation (Alan Liss, New York), pp. 1–17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C RAT, while simultaneously inducing methylation by the m2G RAT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G RAT but not by the m5C RAT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitute of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G RAT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960–975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-RA., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309–319). Our results show that the two ribosomal RNA RflTs studied In this work are exquisitely sensitive to this small but nevertheless functionally Important structural change.