Vitelliform Macular Dystrophy Research Paper

Clinical investigation

This study was approved by the Institutional Review Board of the Yonsei University College of Medicine (IRB No. 4-2017-0097) and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from the participant. A patient with AVMD was investigated in this study. The patient underwent a detailed ophthalmological examination, including best-corrected visual acuity, intraocular pressure measurement, slit-lamp examination, indirect ophthalmoscopy, fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography, and full-field electroretinography (ERG), and EOG. Full-field ERG and EOG were performed according to the guidelines of the International Society for Clinical Electrophysiology of Vision (www.iscev.org). The normal range of the Arden ratio of the EOG (ratio of the light peak to the dark trough) is more than 1.8 for our laboratory.


Genetic analysis

Genetic analysis was performed as reported previously22. Genomic DNA was isolated from the subject’s blood using QIAamp RNA Blood Mini Kit (Cat. No. 51106, QIAGEN, Hilden, Germany). Each exon of the BEST1 gene was amplified from genomic DNA by polymerase chain reaction (PCR) using the intronic oligonucleotide primers and PCR conditions described previously34. Each exon of the PRPH2, IMPG1, and IMPG2 genes was also amplified from genomic DNA by PCR using the intronic oligonucleotide primers (Supplementary Table S3). Each PCR was performed by using Maxime PCR PreMix (Cat. No. 25167, iNtRON Biotech., Seoul, Korea). PCR products were analyzed by direct sequencing using an Applied Biosystems (ABI) 3730 DNA sequencer (ABI, Foster City, CA, USA).

We examined the most recent versions of dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), exome variant server (EVS; http://evs.gs.washington.edu/EVS/), and exome aggregation consortium (ExAC; http://exac.broadinstitute.org/). We examined the NBK control database (397 healthy individuals) and our in-house whole exome sequencing data (59 subjects), which were described in our previous study21.


Plasmids and cell culture

Human embryonic kidney 293 T cells (HEK293T) and HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM)-HG (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The mammalian expression plasmids for hBEST1 WT, p.Ala195Val, and p.Trp93Cys were previously described22. The hBEST1 p.Ile38Ser mutant plasmids were generated by using PCR-based site-directed mutagenesis. Plasmids were transiently transfected into cells using Lipofectamine Plus (Invitrogen). For electrophysiological experiments, hBEST1 plasmids without tags were transfected at a 9:1 ratio with a plasmid expressing the green fluorescence protein (pEGFP-N1). An average transfection rate over 90% was confirmed by transfection with a plasmid expressing green fluorescence protein (Supplementary Fig. S3). For surface biotinylation, immunocytochemistry, and immunoblotting, hBEST1 plasmids containing a Myc-tag were used.


Electrophysiology in cultured cells

Anion channel activities were measured in HEK293T cells using the whole-cell patch clamp techniques reported previously22, 35. Briefly, cells were transferred into a bath mounted on a stage with an inverted microscope (IX-70; Olympus, Tokyo, Japan). Conventional whole-cell clamp was achieved by rupturing the patch membrane after forming a gigaseal. The bath solution was perfused at 5 mL/min. The voltage and current recordings were performed at room temperature (22–25 °C). Patch pipettes with a free-tip resistance of approximately 2–5 MΩ were connected to the head stage of a patch-clamp amplifier (Axopatch-700B; Molecular Devices, Sunnyvale, CA, USA). pCLAMP software v. 10.2 and Digidata-1440A (Molecular Devices) were used to acquire data and apply command pulses. AgCl reference electrodes were connected to the bath via a 1.5% agar bridge containing 3 M KCl solution. Voltage and current traces were stored and analyzed using Clampfit v. 10.2 and Origin v. 8.0 (OriginLab Corp., Northampton, MA, USA). Currents were sampled at 5 kHz. All data were low pass-filtered at 1 kHz.

The bath solution for the whole-cell patch clamp contained 146 mM N-methyl-d-glucamine-Cl (NMDG-Cl), 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4). The pipette solution contained 148 mM NMDG-Cl, 1 mM MgCl2, 3 mM MgATP, 10 mM HEPES, and 5 mM ethylene glycol tetra-acetic acid (EGTA) (pH 7.2).The free Ca2+ concentrations of the buffer solutions were fixed to 1 μM by adjusting the Ca2+ chelator EGTA (5 mM) and CaCl2 concentrations using WEBMAX-C software (http://www.stanford.edu/~cpatton/maxc.html). The osmolarity of the bath solution was set to be 10 mOsm higher than that of the pipette solution by adding sorbitol to suppress the volume-activated anion channels. To determine the current-voltage (I-V) relationship, the clamp mode was shifted to voltage clamp mode, and the I-V curve was obtained by applying step pulses from −100 to 100 mV (voltage interval: 20 mV; duration: 2 s; holding potential: 0 mV).


Surface biotinylation, immunoblotting, and detergent solubility assay

Surface biotinylation and immunoblotting were performed as described previously22. Transfected HEK293T cells were washed three times with ice-cold phosphate-buffered saline (PBS). The cells were then treated with sulfo-NHS-SS-biotin-containing buffer (Pierce, Rockford, IL, USA) for 30 min to biotinylate the plasma membrane proteins. After biotinylation, the cells were washed with quenching buffer to remove the excess biotin and washed twice again with PBS. The cells were harvested and incubated overnight with avidin solution (UltraLink Immobilized NeutrAvidin Beads 10%, Pierce) at 4 °C. Avidin-bound complexes were washed three times and the biotinylated proteins were eluted in a 2X sample buffer. The protein samples were suspended in a sodium dodecyl sulfate (SDS) buffer and separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blotted with the appropriate primary and secondary antibodies. The anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody was used as the primary antibody, and an anti-mouse IgG (HRP) (Thermo Scientific, Rockford, IL, USA) antibody was used as the secondary antibody. Protein bands were detected by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK).

Detergent solubility assay was performed as described previously36. Transfected HEK293T cells were washed twice with ice-cold PBS and lysed with 0.5% Triton X-100 (Tx) containing lysis buffer for 2 min on ice. After centrifugation, the Tx-soluble fraction was collected and denatured in SDS buffer. The pellet containing Tx-insoluble proteins was sonicated and denatured in SDS buffer containing 9 M urea. The samples were analyzed by western blotting as described previously.


Immunocytochemistry

Immunocytochemistry was performed as described previously22. Cells grown on coverslips and tissue sections were fixed in 10% formalin for 10 min and permeabilized with 0.1% triton X-100 for 10 min at room temperature. Nonspecific binding sites were blocked by incubation for 1 h at room temperature with 0.1 mL of phosphate buffered saline (PBS) containing 5% horse serum, 1% bovine serum albumin, and 0.1% gelatin (blocking medium). After blocking, cells were stained by incubation with appropriate primary antibodies and then treated with fluorophore-tagged secondary antibodies. Fluorescent images were obtained with a Zeiss LSM 780 confocal microscope (Carl Zeiss, Berlin, Germany). Anti-myc (Santa Cruz Biotechnology), and anti-Na+-K+ ATPase (Abcam, Cambridge, MA, USA) were purchased from commercial sources.


Structure modeling

Structure modeling was performed as previously described37, 38. The template structure that was sequentially most similar to the hBEST1 was selected by using Blast sequence search against PDB. The calcium-activated chloride channel bestrophin-1 from chicken (pdb ID: 4RDQ) was selected. SWISS-MODEL was used to generate tertiary structure of the domain (SWISS-MODEL, http://swissmodel.expasy.org/). Molecular graphics and analyses were performed with the UCSF Chimera package (Chimera, http://www.cgl.ucsf.edu/chimera).


Statistical analysis

The results are presented as the means ± SEM. Statistical analysis was performed with ANOVA followed by Newman-Keuls multiple comparison test as appropriate. P < 0.05 was considered statistically significant.

JOURNAL ARTICLES

Blodi CF and Stone EM. Best’s vitelliform dystrophy. Opthalmic Paediatr Genet 1990;11:49-59.

Eksandh L, Bakall B, Bauer B, et al. Best’s vitelliform macular dystrophy caused by a new mutation (Val89Ala) in the VMD2 gene. Opthalmic Genet 2001; 22:107-15.

Fishman GA, Baca W, Alexander KR, et al. Visual acuity in patients with best vitelliform macular dystrophy. Opthalmology 1993;100:1165-70.

Loewenstein A, Godel V, Godel L, et al. Variable phenotypic expressivity of Best’s vitelliform dystrophy. Opthalmic Paediatr Genet 1993;14:131-6.

Marano F, Deutman AF, Leys A, et al. Hereditary retinal dystrophies and choroidal neovascularization. Graefes Arch Clin Exp Opthalmol 2000;238:760-4.

Palmowski AM Allagayer R, Heinemann-Vernaleken B, et al. Detection of retinal dysfunction in vitelliform macular dystrophy using the multifocal ERG (MF-ERG). Doc Opthalmol 2003;106:145-52.

Petrukhin K, Koisti MJ, Bakall B, et al. Identification of the gene responsible for Best Macular dystrophy. Nat Genet 1998;19:241-7.

FROM THE INTERNET

MacDonald IM, Lee T, and Mah DY, (Updated 10/27/03). Best Vitelliform Macular Dystrophy. In: GeneReviews at GeneTests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle. 1997-2005. Available at http://www.genetests.org. Accessed 5/05.

Altaweel M. Best Disease. EMedicine. Last Updated: 10/4/04.

McKusick VA, ed. Online Mendelian Inheritance in Man (OMIM). Baltimore, MD: The Johns Hopkins University; Entry No. 153700; Last Update: 3/9/04.

Comments

Leave a Reply

Your email address will not be published. Required fields are marked *