Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

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•Electroretinogram: Nonrecordable.

•Dilated fundal examination: Typical changes of RP, including waxy pallor of the optic nerve heads, attenuated retinal vasculature, marked depigmentation, and choroidal atrophy in the macular area and peripheral fundus.

Bone spicule pigmentary deposits were distributed through all four segments of the retinas. No cataracts were present, but marked cellularity of the vitreous was observed.

The macular showed cystoid changes with marked retinal epithelial thinning.

The RP17 locus was initially mapped to the 17q22 chromosomal region by Bardien et al. in 1995 (2). This region was refined and a number of candidate genes were excluded by Bardien et al. in 1997 (3). The locus was fine mapped into a 1-cM region by Bardien-Kruger in 1999 (4), whereafter the intensive hunt for a candidate gene in the region was started. Also in 1999, den Hollander et al. (5) published mapping results based on a Dutch family which mapped into the RP17 region. Because the original South African family was of Dutch extraction, it was assumed that the Dutch family and the South African families were related, albeit only by the RP17 mutation.

The hunt for the RP17 gene after 1999 was assisted by intensive bioinformatic effort to characterize candidates in the region and to construct sequence-based maps across the candidate region. Initially sequence-based maps were limited by the paucity of completed sequence in the region, although this situation quickly changed and it was possible to create a physical map to compare to the linkage map that had formed the backbone of the gene hunting effort up until that time.

In order to focus the effort of gene screening by DNA sequencing, which was at that time a time-limiting step, we needed to further refine the candidate region and this was performed by creating new STS markers based on the published sequence in the region. A number of steps allowed us to reduce the region from 3 Mb to a much more manageable 410 kb. Within this region, a number of genes were screened by sequencing in a couple of affected South African RP17 individuals from each of the families. Finally, a previously undescribed sequence change was detected in exon 1 of the CA4 gene. The change is at base 40 of the complementary DNA sequence has been detected in all affected individuals; this change has not been detected in 36 unaffected relatives and 100 unrelated individuals from the same population. The C to T transition mutation leads to a change from an arginine to a tryptophan in the signal sequence at position –5 relative to the signal peptidase cleavage site (R14W). This signal sequence variant is predicted not to alter the sequence of the mature CA4. The R14W mutation creates an MscI restriction endonuclease site which was used to screen for the mutation in the extended family.

The gene, CA4, is a member of the carbonic anhydrase family of genes the products of which catalyse the reversible reaction between carbon dioxide and carbonic acid, according to the following reaction:

H2O + CO2 ↔ HCO−3 + H+.

The CAIV enzyme is membrane bound and functions in the transport of CO2 across membranes and into, or out of, solution in various tissues of the body. CA4 is expressed in the luminal surfaces of vessels in a number of tissues including; the proximal renal tubules (6), the lungs (7), and the choriocapillaris of the eye (8). Hageman et al. (8) demonstrated expression of CA4 in only two tissues in the eye, namely the vessels of the choriocapillaris, and in the lens.

Functional studies on the R14W CA4, and comparisons with wild-type CA4 in Cos7 cells have shown that the R14W mutant leads to upregulation of markers of the unfolded protein response (UPR), namely, upregulation of the Endoplasmic Reticulum (ER) chaperone BiP, upregulation and activation of the ER kinase, PERK, and induction of CHOP (GADD153) (1). Secondarily, the cells expressing the mutant CA4 have been shown to induce apoptosis as measured by TUNEL staining and annexin V binding.

This evidence leads us to speculate that the R14W mutation is leading to cell death by the UPR and apoptosis. This will, in turn, lead to defective CO2 and HCO3 transport and eventual retinal destruction.

Subsequent work by Bonapace et al. (9) demonstrated in cell models that a number of nonspecific chemical chaperones influence the processing of the mutant (R14W) CAIV enzyme, and reduce the level of apoptotic loss associated with the mutation. Interestingly, other proteins that bind to the defective molecule also assist with rescuing the biological defect/process. In this respect, Bonapace et al. (9) investigated various carbonic anhydrase inhibitors, which bind to carbonic anhydrase. These drugs/reagents are a proven treatment for glaucoma. Bonapace et al. (9) showed that immunohistochemical staining for BiP, PERK, and CHOP were positive in 82.5, 69.0, and 85.7% of the R14W mutant-expressing cells, respectively, (these markers were detected in fewer than 5% of cells expressing wildtype CA4). Expression of each of these markers in R14W expressing cells was reduced to around 20% by 10 M acetazolamide. Similarly, the markers of apoptosis, TUNEL staining, and annexin V binding were reduced from 65 and 85% of R14W expressing cells respectively to 25 and 20%, respectively, in the presence of acetazolamide.

This work demonstrates that, at least in vitro, the phenotype of the R14W CA4 mutant may be rescued to a large degree. Before these results an be applied in a possible treatment trial in R14W mutant RP sufferers, three issues remain to be addressed:

1.Route of administration.

2.Dosage.

3.Duration of treatment.

Route of Administration

Clearly, the best approach will be to try to evaluate the treatment that enables the lowest dose of active ingredient to be used. This limits the choice of carbonic anhydrase inhibitors because acetazolamide is systemically administered, and dorzolamide and brinzolamide are topically delivered, thereby requiring much lower doses.

Dosage

The concentration of active ingredient evaluated by Bonapace et al. (9) was 10M and the only evidence for tissue concentration post treatment is available for dorzolamide in which Sugrue examined the tissue layers in rabbit eyes after 14 ds of twice daily administration and showed that the concentration of dorzolamide in the various layers were: retina, – approx 3.6 M; choroid, approx 4.0 M; and Sclera, approx 3.6 M (10). These concentrations are less that half the desired 10 M and clinical efficacy, on this evidence alone, would be unsure. When this reservation was communicated to W. Sly (the group leader who carried out the work reported in the Bonapace et al. article [9]), they performed a second analysis using Dorzolamide at different concentrations and measuring the percentage of TUNEL positive cells.

This work showed that at concentrations from 0.1 M and up, the percentage of TUNEL positive cells was 5% or less. This is strongly suggestive, assuming that rabbit and human eyes behave similarly, that treatment with dorzolamide drops will deliver an effective dose of active ingredient to the cell in the choriocapillaris.

Duration of Treatment

An equally important aspect of the design of a potential trial is that the metrics used in the assessment of vision will be able to yield detectable changes that may then be attributed to the treatment. In order to evaluate this we are in the process of starting a 2-yr evaluation of our patient cohort to determine the baseline values for measures of vision, and to determine the rate of change in these parameters over time.

CONCLUSION

It is obvious that the most trustworthy drug-response experiments will be derived from work on an animal model of the disease, and this is in progress. We are working to set the stage so that when the animal work has been concluded we will be in a position to move to a treatment trial without further delay. Thereby, we are fulfilling our promise to our subjects, and the implicit promise of molecular genetics as a whole, to use the genetic discoveries to bring some meaningful change to the lives of sufferers.

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