Objective: This study aimed to establish a practical diagnostic test for choroideremia (CHM) and to show its application in a family with the clinical diagnosis of choroideremia.
Design: Case series.
Participants: Sixteen males from 13 families with clinically documented CHM and unaffected normal males were enrolled in this study.
Methods: Protein extracted from either leukocytes or Epstein-Barr virus-transformed lymphocytes was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of the protein was performed with two monoclonal antibodies, one against the CHM gene product, Rab escort protein-1 (REP-1), and the other against the alpha-subunit of farnesyl transferase. DNA was extracted from peripheral leukocytes and subjected to polymerase chain reaction-single stranded conformation polymorphism analysis using primers for the exons of the CHM gene. Where altered mobility of the DNA fragments was detected, direct sequencing of that exon was compared with the published normal sequence.
Results: The authors detected REP-1 in protein samples extracted from lymphoblastoid cell lines from female carriers but not from CHM males. The authors also showed the absence of REP-1 in the peripheral leukocytes of males affected with CHM. In one male who lacked REP-1, direct sequencing of exon 7 showed a cytosine-to-thymine transition mutation (Arg293X) in the CHM gene.
Conclusions: The clinical diagnosis of CHM can be confirmed simply by immunoblot analysis with anti-REP-1 antibody, showing the absence of REP-1 protein in peripheral blood samples. Because all known mutations in the CHM gene create stop codons that truncate the protein product and result in absence of REP-1, the authors predict that most patients with CHM can be diagnosed by this procedure.