Positional cloning has previously resulted in the identification of a gene which is disrupted by deletions in patients with the classic choroideremia (CHM) phenotype. More subtle mutations had been identified in 4 exons of the 3' portion but not elsewhere in the CHM gene. We have now isolated and characterized the complete open reading frame of the CHM gene and determined its exon-intron structure. The CHM gene encodes a protein of 653 amino acids, which is highly homologous to the mouse and rat CHM proteins, and, to a slightly lesser extent, to the human CHM-like (CHML) protein. The open reading frame (ORF) of the human CHM gene consists of 15 exons, spanning at least 150 kb of Xq21.2, and it is possible that there is an additional exon corresponding to the 5' non-coding region of the gene. Cloning of the 5' end of the CHM gene and the elucidation of its intron-exon structure enabled us to localize the X-chromosomal breakpoint in a CHM female with an X;7 translocation between exons 3 and 4.

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The recent isolation of the complete open reading frame of the choroideremia (CHM) gene and the characterization of the exon-intron boundaries has paved the way to mutation detection in patients with classical choroideremia. We have performed mutation screening in patients from 15 Danish and Swedish families by using Southern blot hybridization and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) technique. Causative mutations in the CHM gene were detected in at least 12 families, indicating that a substantial part of the mutations can be identified by this approach. In four of these families deletions of different sizes were found. Thus, in one patient, the deletion resulted in the absence of only one exon, while in another the deletion comprised the entire CHM gene. Mapping of the deletion endpoints in these four patients and in another 11 male patients with sizeable deletions enabled us to construct a very detailed map of intervals 2 and 3 of Xq21. In the remaining 11 Danish and Swedish families at least 8 causative mutations were found by PCR-SSCP analysis and direct sequencing. Interestingly, all CHM gene mutations detected thus far in choroideremia patients give rise to the introduction of a premature stop codon.

Choroideremia (CHM) is an X-linked recessive eye disease that results from mutations involving the Rab escort protein-1 (REP-1) gene. In 18 patients deletions of different sizes have been found. Two females suffering from CHM were reported to have translocations that disrupt the REP-1 gene. In 22 patients, small mutations have been identified* Interestingly, these are all nonsense, frameshift or splice-site mutations; with one possible exception, missense mutations have not been found. This comprises all the known mutations in the disease. Hum Mutat 9:110-117, 1997. © 1997 Wiley-Uss, Inc. key w o r d s : c h o r o i d e r e m i a (CHM), m u t a t i o n s , r a b e s c o r t p r o t e i n-1 (REP-1) gene by X-autosomal translocations (Kaplan et ab, 1989; Siu et al., 1990). In these cases, X-inacti-vation is nonrandom; the normal X is preferen­ tially inactivated, while both translocation frag­ ments remain active. The gene for CHM was localized to the X q l3-q22 region by linkage analysis (Lewis et a l., 1985; Nussbaum et alM 1985; Lesko et al., 1987; Sankila et al., 1989). This assignment was further refined by the characterization of cytogenetically visible deletions in patients with CHM, mental retarda-tion(M R), and deafness type 3 (DFN3) (Rosenberg et al.} 1987; Schwartz et al., 1988; Cremers et alM 1989a). Molecular analysis of deletions in patients with classic CHM (Cremers et al., 1987, 1989b, 1990a,b) formed the basis for the positional cloning of a part of the CHM gene from Xq21.2 (Cremers