title: Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.
abstract: Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants.
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Rimoldi
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Straniero
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Rosanna
authors.initials: R
authors.lastname: Asselta
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Mauri
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Manfredini
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Silvana
authors.initials: S
authors.lastname: Penco
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Giovanni P
authors.initials: GP
authors.lastname: Gesu
authors.affiliation: Pediatric Ophthalmology Department, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Del Longo
authors.affiliation: Pediatric Ophthalmology Department, Niguarda Ca' Granda Hospital, Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Piozzi
authors.affiliation: Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Soldà
authors.affiliation: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy. Electronic address: paola.primignani@ospedaleniguarda.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Primignani
mesh_terms: D016115:Albinism, Oculocutaneous / Q000209:etiology / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010375:Pedigree; D022821:RNA Splice Sites; D012326:RNA Splicing; D035781:Siblings
chemical_list: D026901:Membrane Transport Proteins; C489600:OCA2 protein, human; D022821:RNA Splice Sites
keywords: ECD; ENT; ERG; Ears, Nose and Throat; Electroretinogram; Extra-Cellular Domain; IVS; In vitro analysis; Intervening Sequences; MLPA; Messenger RNA; Multiplex Ligation-dependent Probe Amplification; OA; OCA; OCA2; OCT; Ocular Albinism; Oculocutaneous Albinism; Oculocutaneous albinism; Optical Coherence Tomography; PCR; Polymerase Chain Reaction; RT-PCR; Reverse Transcription PCR; Splicing mutations; TMD; Trans-Membrane Domain; VEP; Visual Evoked Potential; mRNA