Mutated alleles of the rod and cone Na-Ca+K-exchanger genes in patients with retinal diseases.
OBJECTIVE
To study the possible involvement of the rod (SLC24A1) and cone (SLC24A2) Na-Ca+K exchanger (NCKX) genes in retinal diseases.


METHODS
DNA was collected from unrelated patients with retinal disease, mainly from North America. A human genomic library was screened with the cone NCKX cDNA, and hybridizing clones were sequenced to determine the genomic organization of the SLC24A2 gene. The single-strand conformation polymorphism (SSCP) technique and direct sequencing were used to screen the patients' DNA for mutations in SLC24A1 and SLC24A2. The effect of selected missense changes on protein function was tested by measuring potassium-dependent Na-Ca exchange of the mutant proteins expressed in insect cells.


RESULTS
Twenty-seven novel sequence changes were found in the rod NCKX gene, 21 of which are unlikely to be pathogenic, because they did not cosegregate with the disease or did not affect conserved regions of the protein. Of the remaining six, two were frameshift mutations found in one patient each. If translated, these alleles would encode nonfunctional proteins. Three of the six possibly pathogenic mutations were missense changes located in conserved regions, and their protein functions were assayed. Only one (Ile992Thr) had a significantly low level of exchanger function, but it was found in two unrelated patients who were heterozygotes with different retinal diseases, and this mutation could not be unequivocally associated with either disease. The last of the six changes is likely to create a new splice acceptor site. The genomic organization of the cone NCKX gene was determined, and it contained 11 exons with a few splice variants. Fifteen novel sequence changes were identified in the cone exchanger gene in patients with a cone dysfunction or degeneration. Only three of these sequence changes, all missense changes found in heterozygous patients, were considered possibly pathogenic. Functional analysis showed only a slight reduction in the activity of the corresponding mutant proteins.


CONCLUSIONS
Although variant alleles of the rod and cone NCKX genes were found, none could be definitively associated with a specific retinal disease. The human phenotype associated with mutant exchanger alleles remains unknown.
Sharon
Dror
D
Ocular Molecular Genetics Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.
Yamamoto
Hiroyuki
H
McGee
Terri L
TL
Rabe
Vivian
V
Szerencsei
Robert T
RT
Winkfein
Robert J
RJ
Prinsen
Clemens F M
CF
Barnes
Claire S
CS
Andreasson
Sten
S
Fishman
Gerald A
GA
Schnetkamp
Paul P M
PP
Berson
Eliot L
EL
Dryja
Thaddeus P
TP
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000032:analysis; D004289:Dolphins; D050612:Fatty Acid Transport Proteins; D005260:Female; D015870:Gene Expression; D015698:Genomic Library; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D018807:Polymorphism, Single-Stranded Conformational; D051381:Rats; D012164:Retinal Diseases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D050612:Fatty Acid Transport Proteins; C493572:Slc27a4 protein, mouse; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D002118:Calcium