11-cis-retinal and Cystic-Fibrosis

Detection of small alterations or abnormalities in genomic DNA (eg, point mutations or small deletions) has become increasingly important in the diagnosis of genetic disease and polymorphism. When a mutation or polymorphism creates a new restriction endonuclease site, it can easily be identified by polymerase chain reaction (PCR) amplification of the DNA region of interest, followed by digestion with the restriction endonuclease. However, useful restriction sites are the exception, and a variety of specialised techniques have been developed to identify subtle DNA abnormalities. We have shown that where a DNA mutation does not create a useful novel restriction site, such a site can be introduced by PCR and specially chosen primers. The approach is simple and inexpensive and should be broadly applicable in the diagnosis of genetic polymorphism and mutation. The technique is illustrated here by the three base-pair deletion responsible for most cases of cystic fibrosis and by detection of the point mutation in the rhodopsin gene that has been associated with some cases of autosomal dominant retinitis pigmentosa.

Topics: Alleles; Base Sequence; Blood Proteins; Calgranulin A; Chromosome Deletion; Cystic Fibrosis; DNA, Single-Stranded; Evaluation Studies as Topic; Humans; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping; Retinitis Pigmentosa; Rhodopsin

Patients with cystic-fibrosis (CF) often have low plasma concentrations of vitamin A. We have measured dark-adapted scotopic thresholds of 56 patients with CF, ages 4 to 34 years, either with a two-alternative forced-choice procedure or as the final threshold after a full dark-adaptation curve. Fasting plasma vitamin A alcohol (retinol) was measured in 34 of the 56 patients. The average thresholds were higher and retinol values lower in patients than in controls. In two patients with very low (less than 7 micrograms/dl) initial retinol levels and elevated thresholds, decreased rhodopsin densities were observed; rhodopsin density and thresholds returned to normal after treatment with oral vitamin A. Rhodopsin density and log sensitivity were linearly related. Only marked decreased in plasma retinol were associated with elevations of dark-adapted threshold and decreases in rhodopsin density, suggesting that the tissues of patients with CF sequester vitamin A to maintain retinal function.

Topics: Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Dark Adaptation; Humans; Psychophysiology; Rhodopsin; Vision, Ocular; Vitamin A