Post-LASIK exacerbation of granular corneal dystrophy type 2 in members of a chinese family.

PurposeThe post-LASIK exacerbation of corneal dystrophy, otherwise asymptomatic, is almost exclusively associated with the TGFBI gene mutations at codon 124 in exon 4 and codon 555 in exon 12. It is our intention to demonstrate that the pre-operative genetic screening for TGFBI mutations should be mandatory for refractive surgery candidates.Patients and MethodsIn this study, we reviewed the proband's post-LASIK slit-lamp and in vivo confocal microscopy images and genetic testing results, and performed genetic testing on eleven additional members of the family to investigate the penetrance of corneal dystrophy in asymptomatic members who carry the mutation.ResultsThe proband demonstrated a post-LASIK exacerbation of Granular Corneal Dystrophy type 2 (GCD2), identified as a TGFBI R124H mutation. Three of the 11 family members tested positive for the same R124H mutation as the proband.ConclusionThe lesson learned from this case is that the genetic screening of TGFBI mutations must be incorporated into the pre-operative screening procedures to prevent exacerbation and recurrence, which eventually could lead to the need for a corneal transplant.

Analysis of an oligonucleotide N3'-->P5' phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis.

N3'-->P5' phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, "second generation" chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18-mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3'-->P5' phosphoramidate (amidate) linkages on the 5'-end and five amidate linkages ont he 3'-end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3'-->P5' phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase - high performance liquid chromatography (RP-HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.

An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification.

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.

Capillary gel electrophoresis and antisense therapeutics. Analysis of DNA analogs.

Capillary gel electrophoresis (CGE) has become an effective tool for the analysis of antisense oligonucleotides. As these compounds begin to show promise in the pharmaceutical field, CGE is often used to determine the quality of chemically synthesized DNA analogs, which are presently being studied as potential antisense therapeutics. The demand for gel capillaries to process high resolving power and provide statistically meaningful data has indirectly provided a better understanding of what is required to denature single-stranded oligonucleotides. For CGE to be useful for the analysis of oligonucleotides in general, an internal standard is often employed; however, apart from being a strictly quantitative tool, CGE has the capability to be useful in a wide range of applications within the field of antisense therapeutics. CGE can be used in conjunction with HPLC to determine an effective method for the purification of crude oligonucleotide solutions. It has also proven useful in determining whether or not a DNA analog can promote the ribonuclease H-mediated hydrolysis of RNA. An understanding of the interactions between antisense oligonucleotides and nucleases in general is critical for determining how antisense oligonucleotides function within a biological system.

Oligonucleotide N3'-->P5' phosphoramidates.

Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3'-->P5' phosphoramidates containing 3'-NHP(O)(O-)O-5' internucleoside linkages and the study of their hybridization properties. Thermal dissociation experiments show that these compounds form very stable duplexes with single-stranded DNA, RNA, and with themselves following Watson-Crick base pairing. The duplex thermal stability was enhanced by 2.2-2.6 degrees C per modified linkage compared with phosphodiesters. The structure of complexes formed by phosphoramidates closely resembles that of RNA oligomers and corresponds to an A form, as judged by CD spectroscopy. N3'-->P5' phosphoramidates also form stable triplexes with double-stranded DNA under near-physiological conditions when natural phosphodiesters fail to do so. Physicochemical characteristics of the amidates are similar to those of RNA oligomers, even though they are composed of 2'-deoxyfuranose-based nucleosides.