|Molecular Vision 2004;
Received 23 October 2003 | Accepted 22 January 2004 | Published 26 January 2004
Mutation screening of the phosducin gene PDC in patients with retinitis pigmentosa and allied diseases
Koji M. Nishiguchi, Eliot L. Berson,
Thaddeus P. Dryja
Ocular Molecular Genetics Institute and the Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA
Correspondence to: Thaddeus P. Dryja, Ocular Molecular Genetics Institute, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA, 02114; Phone: (617) 573-3319; FAX: (617) 573-3168; email: firstname.lastname@example.org
Purpose: To search for a phenotype associated with mutations in the phosducin gene PDC.
Methods: We screened 853 patients with retinitis pigmentosa or an allied disease diseases, including groups of 61 to 212 patients, each with dominant retinitis pigmentosa (RP), recessive RP, Leber congenital amaurosis, or cone-rod degeneration, for mutations in the PDC gene using direct genomic sequencing of the three coding exons and their flanking intron splice sites.
Results: We found one polymorphism in the 5' untranslated region (minor allele frequency of 0.149) and three rare single-base sequence variants (one missense change, one isocoding change, and one in the 3' untranslated region). The rare variants were found in one heterozygous patient each and none was interpreted as pathogenic.
Conclusions: Phosducin mutations are not a major cause of dominant or recessive RP, Leber congenital amaurosis, or cone-rod degeneration. The human phenotype associated with phosducin defects remains unknown.
Phosducin (PDC, OMIM 171490; Locus ID 51320) is a protein abundantly expressed in the retina and is found in both the inner segments and the outer segments of rod and cone photoreceptors [1-3]. While its function is not completely known, it interacts with the βγ complex of transducin [4,5]. In the dark, phosducin is phosphorylated by protein kinase A or calmodulin-dependent protein kinase II, and the phosphorylated form of phosducin more avidly binds to βγ transducin [5-9]. Recently, the binding of phosducin to βγ transducin was shown to inhibit phosducin's degradation by an ubiquitin-proteasome pathway . This function of phosducin may be a potential mechanism for regulating the level of βγ transducin complexes; this, in turn, may modulate the phototransduction cascade.
Mutations in genes encoding many other members of the phototransduction cascade are known causes of retinitis pigmentosa or allied diseases (RetNet). The gene encoding phosducin is located within 1q25-32.1 . There is evidence of a gene causing age-related macular degeneration in this region ; however, no mutations were found in the sequence of PDC in affected members of families showing linkage to this region . Thus far, no phenotype has yet been associated with mutations in the PDC gene. The only published analysis of the PDC gene in patients with retinitis pigmentosa or an allied disease found no disease-causing mutations among 83 patients . We report here our extended analysis of this gene in a much larger set of patients.
This study involved human subjects and conformed to the Declaration of Helsinki. Leukocyte DNA was extracted from peripheral blood of 853 unrelated patients with various hereditary retinal diseases. Almost all patients resided in the United States and Canada, and they were of mixed ethnic background comparable to what is found in these countries. Among these patients, 212 patients were diagnosed with autosomal recessive retinitis pigmentosa (RP), 193 with autosomal dominant RP, 93 with autosomal recessive cone-rod degeneration, 61 with Leber congenital amaurosis (LCA), 40 with cone dysfunction syndrome, 37 with achromatopsia, 27 with isolate RP, 26 with macular malfunction, and the remaining 164 with atypical forms of retinal disease, including sector RP  and clumped pigmentary retinal degeneration  among others, each category of which was composed of small numbers of patients. Patients with autosomal recessive RP included in this study were from families with two or more affected siblings and no previous generations with RP or were the affected offspring of a consanguineous mating of parents without a history of RP; isolate RP patients had no affected relatives and had parents who had no known blood relationship. Patients with autosomal dominant RP came from families with two or more consecutive affected generations and, in most cases, three consecutive affected generations or evidence of father-to-son transmission. Patients with cone-rod degeneration had substantially greater loss of cone function compared to loss of rod function . Patients with LCA came from families exhibiting a recessive inheritance pattern. The diagnosis of cone dysfunction syndrome was given to patients with features of cone malfunction but with no definite indication of a progressive cone degeneration. Patients with macular malfunction had abnormal foveal ERGs and normal full-field ERGs . None of the relatives of patients with cone dysfunction syndrome or macular malfunction was affected. Patients with identified causative mutations in other genes were excluded from this study except for two patients with recessive RP, 27 with dominant RP, two with LCA, one with cone dysfunction syndrome, 13 with achromatopsia, and three with atypical forms of retinal disease whose causative mutations in genes other than phosducin were discovered during the course of this study.
All three coding exons of the PDC gene (GenBank accession number NM_002597) [19,20] and the immediate intronic sequences including splice sites were amplified by the polymerase chain reaction (PCR) and analyzed by direct DNA sequencing using a dye-terminator cycle sequencing kit (version 3.1) and an ABI automated sequencer. The sequences of the primers used for this study are in Table 1.
One missense sequence variant (c.200 G>A; Arg67Gln, CGA>CAA) was observed in a heterozygous patient with macular malfunction; both parents and the only sibling, all unaffected by history, were deceased and thus segregation analysis of this mutation was not possible. An iso-coding sequence variant (c.177 G>A; Arg59Arg, AGG>AGA) was found in a heterozygous isolate patient with RP. Two other patients, one with isolate RP and the other with clumped pigmentary retinal degeneration, were heterozygotes for an intron change in the 3' UTR region of exon 4 (c.*7 A>T) respectively. None of these three changes create or destroy splice donor or acceptor sites based on splice-site prediction software (NN SPLICE, version 0.9) available at Berkeley Drosophila Genome Project . No second change in the phosducin gene was found in any patient.
A polymorphism in the 5' UTR was also identified (c.-9 G>A). The minor allele frequency (the "A" allele) was 14.9% based on an analysis of 820 individuals (1640 chromosomes) in whom this polymorphism could be reliably determined.
In this study, mutation screening of 853 patients with RP or an allied retinal disease failed to reveal mutations that convincingly account for any photoreceptor disease. Only three rare sequence variations and a common polymorphism were identified. Among the three rare variants, only one (Arg67Gln) is expected to alter the wild-type amino acid sequence. We were unable to determine whether or not this change was pathogenic. It was found heterozygously in an isolate patient with macular malfunction, and no family members were available for a segregation analysis. While Arg67 is a highly conserved residue among the mammalian phosducins ranging from mouse to man, so are 88-91% of the residues in this protein, so it is not possible to estimate, based on sequence conservation, how important Arg67 is for phosducin's function. The residue corresponding to position 67 in phosducin is not Arg in the human phosducin-like protein PhLP .
It is possible that pathogenic mutations might exist outside of the coding exons and the flanking intron splice sites, or that large deletions or gene rearrangements involving the coding exons might exist that were not detected among the patients evaluated in this study. Nevertheless, based on the numbers of patients evaluated, one can estimate that, with 90% confidence, pathogenic PDC mutations in the regions of the gene screened occur in fewer than 1.1% of cases of autosomal recessive RP, 1.2% of autosomal dominant RP, 2.4% of cone-rod degeneration, 3.7% of LCA, and 5.6% of cone dysfunction syndrome. Thus, our results suggest that PDC is probably not a major cause of these clinical entities in the population from which the patients are derived.
This work was supported by grants from the Foundation Fighting Blindness (TPD, ELB) and the National Eye Institute (EY08683, EY00169).
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