Journal: Investigative Ophthalmology & Visual Science

Article Title: Molecular Consequences of BEST1 Gene Mutations in Canine Multifocal Retinopathy Predict Functional Implications for Human Bestrophinopathies

doi: 10.1167/iovs.10-6385

Figure Lengend Snippet: Fundus phenotypes in cmr and BVMD. (A1, A2) Fundus photographs of an 18-month-old English Mastiff with cmr1 and a 15-month-old Coton de Tulear with cmr2 (A3). In both animals, multiple subretinal lesions (arrowheads) are located in the superior tapetal region of the fundus. Note that the color of the lesions ranges from gray to tan-brown, influenced by the color of the overlying tapetum lucidum. (A2, inset) Fundus picture of 12-month-old Great Pyrenees with a pseudohypopyon-like lesion (arrow). (B1) Fundus appearance of a 47-year-old patient with unifocal BVMD lesion in the pseudohypopyon stage. The patient carries a Y227N mutation in the BEST1 gene and has a visual acuity of 20/80. (B2) Multifocal BVMD (K194_A195insV) in a 15-year-old patient with visual acuity of 20/100; arrowheads indicate some of the multiple retinal lesions scattered throughout the fundus periphery. (B3) Fundus photograph of a 10-year-old patient with multifocal vitelliform macular dystrophy (Y29X/R141H). Irregular vitelliform degeneration is present in the fovea (arrow). Note the rounded, vitelliform, yolk-like degeneration superiorly (arrowhead) and the more subtle, somewhat diffusely demarcated, vitelliform alterations along the superior vascular arcade. Fundus photographs provided courtesy of William Beltran (A1, A2), Bruce Grahn (A2, inset), Camiel Boon (B1, B2),11 and Patrik Schatz (B3).25

Article Snippet: For qRT-PCR verification, commercially available canine (Cf02697409_gH, Cf02697407_g1) or human (Hs00188249_m1) BEST1 specific assays (TaqMan; Applied Biosystems) spanning 5′- or 3′- of the gene coding sequence were used.

Techniques: Mutagenesis

Journal: Investigative Ophthalmology & Visual Science

Article Title: Molecular Consequences of BEST1 Gene Mutations in Canine Multifocal Retinopathy Predict Functional Implications for Human Bestrophinopathies

doi: 10.1167/iovs.10-6385

Figure Lengend Snippet: In vivo evaluation of cmr1 premature termination mutation. (A) Total RNA from canine WT, cmr1+/− and cmr1−/− RPE/choroid was reverse transcribed and evaluated by RT-PCR using two overlapping primer sets corresponding to the 5′- (exon 2 − 7) or 3′- (exon 7 − 10) end of cBEST1. No variations in the abundance of mRNA transcripts or in amplicon sizes were noted; the control gene was GAPDH. (B) Two canine-specific BEST1 expression assays were used for quantifying the relative RNA expression levels and validated the RT-PCR findings (vertical bars) SEM values. (C, D) Immunohistochemistry and Western blot analyses demonstrated twofold reduction (∼50% of WT) of the bestrophin-1 protein in cmr1+/− and its absence in cmr1−/− animals. The control protein was ACTB. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer. Scale bar, 10 μm.

Article Snippet: For qRT-PCR verification, commercially available canine (Cf02697409_gH, Cf02697407_g1) or human (Hs00188249_m1) BEST1 specific assays (TaqMan; Applied Biosystems) spanning 5′- or 3′- of the gene coding sequence were used.

Techniques: In Vivo, Mutagenesis, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Expressing, RNA Expression, Immunohistochemistry, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Molecular Consequences of BEST1 Gene Mutations in Canine Multifocal Retinopathy Predict Functional Implications for Human Bestrophinopathies

doi: 10.1167/iovs.10-6385

Figure Lengend Snippet: Canine multifocal retinopathy in vitro models. (A, B) cDNA transcripts from cmr1 or cmr2 transiently transfected HEK-293T cells were analyzed by RT-PCR (A) or qRT-PCR assays (B) spanning the region with mutations of interest. No cBEST1 mRNA expression level changes were observed with either cmr mutant; error bars represent SEM. (C) Confocal images illustrating immunohistochemical detection of canine bestrophin-1 (red), WT, or cmr mutants on polarized MDCK cell monolayers. WT protein traffics to the plasma membrane of the cells, whereas the cmr2 mutant is mislocalized. Bestrophin-1 could not be detected in cells expressing cmr1 by either immunohistochemistry (C) or Western blot analysis (D). The in vitro model accurately recapitulates transcriptional and translational expression events observed in vivo. (E) Mislocalization of cmr2 mutant protein. Fluorescence confocal images showing colocalization (yellow-orange in merge) of cmr2 mutant Best1 protein (red) with the ER marker Calnexin (green). RPE, canine WT RPE/choroid tissue; mock, HEK cells mock transfected; WT, cBEST1 WT transfected cells; cmr1, cmr1 transfected cells; cmr2, cmr2 construct transfected cells. The control protein was ACTB. Nuclei were labeled with DAPI (blue). Scale bar, 10 μm, applies to all panels.

Article Snippet: For qRT-PCR verification, commercially available canine (Cf02697409_gH, Cf02697407_g1) or human (Hs00188249_m1) BEST1 specific assays (TaqMan; Applied Biosystems) spanning 5′- or 3′- of the gene coding sequence were used.

Techniques: In Vitro, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Mutagenesis, Immunohistochemical staining, Clinical Proteomics, Membrane, Immunohistochemistry, Western Blot, In Vivo, Fluorescence, Marker, Construct, Control, Labeling