Search

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in the skin and eyes. We have previously isolated the gene for OA1 and characterized its protein product as melanosomal membrane glycoprotein displaying structural and functional features of G protein-coupled receptors. We and others have identified mutations of various types within the OA1 gene in patients with this disorder, including deletions and splice site, frameshift, nonsense, and missense mutations. However, different prevalences of large intragenic deletions have been reported, ranging from 10% to 50% in independent studies. To determine whether these differences might be related to the geographic origin of the OA1 families tested, we performed a further extensive mutation analysis study leading to the identification of pathogenic mutations in 30 unrelated OA1 patients mainly from Europe and North America. These results, together with our earlier mutation reports on OA1, allow us to resolve the apparent discrepancies between previous studies and point to a substantial difference in the frequency of large intragenic deletions in European ( 50%) OA1 families. These observations and our overall refinement of point mutation distribution within the OA1 gene have important implications for the molecular diagnosis of OA1 and for the establishment of any mutation detection program for this disorder

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a major impairment of visual acuity, nystagmus, strabismus, photophobia and retinal hypopigmentation. From the analysis of patients carrying deletions and translocations involving the distal short arm of the X chromosome (Xp22.3) we have identified a region of approximately 110 kb in which the OA1 gene must lie. We have extensively searched for genes in this region using a variety of techniques which included exon amplification, cDNA selection and direct hybridization of cosmid inserts to cDNA libraries. Putative exons identified by exon amplification were used to screen a human retina cDNA library and several cDNA clones corresponding to an approximately 7.5 kb transcript were isolated and characterized. Transcripts of this newly identified gene were found to be abundant in retina and melanoma and could also be detected in brain, placenta, lung, kidney and pancreas. Interestingly, sequence analysis revealed that this new gene encodes a 1616 amino acid protein sharing significant similarities with the Apical Protein from Xenopus laevis (APX) which is implicated in amiloride-sensitive sodium channel activity. The gene, termed APXL (APX-Like), spans approximately 160 kb, contains 10 exons and covers over 70% of the 110 kb critical region for OA1. A truncated pseudogene sharing very high levels of homology with the rat eIF-5 gene, a eukaryotic translation initiation factor, was found to lie in the middle of intron 1. APXL was found deleted in two patients with contiguous gene syndromes including OA1 and in one patient with isolated OA1. Mapping, expression and patient analysis data led us to consider the APXL gene a strong candidate for the OA1 gene. DNA from 57 unrelated patients with OA1 was, therefore, scanned for mutations in the coding region, using both SSCP analysis and direct sequencing. No functionally significant mutation was identified, suggesting that APXL is not directly involved in OA1. Further studies are needed to clarify the physiologic role of this highly conserved gene.