New VMD2 gene mutations identified in patients affected by Best Vitelliform Macular Dystrophy

Dominique Marchant ¹, Kuai Yu ², Karine Bigot ¹, Olivier Roche ³, Aurore Germain ¹, Dominique Bonneau ⁴, Valérie Drouin-Garraud ⁵, Daniel Schorderet ⁶, Francis Munier ⁷, Dominique Schmidt ³, Philippe Le Neindre ³, Cécile Marsac ¹, Maurice Menasche ¹, Jean Louis Dufier ³, Rodolphe Fischmeister ⁸, Criss Hartzell ² and Marc Abitbol ^1*

¹ Centre de recherche thérapeutique en ophtalmologie, Université René Descartes, France
² Department of Cell Biology, The Center for Neurodegenerative Disease, Emory University School of Med, United States
³ Département d’ophtalmologie, Centre Hospitalier Universitaire Necker-Enfants Malades, France
⁴ Département de génétique, Centre Hospitalier Universitaire d’Angers, 49033 Angers, France
⁵ Département de génétique, Centre Hospitalier Universitaire de Rouen, 76031 Rouen, France
⁶ IRO - Institut de Recherche en Ophtalmologie, Université de Lausanne et EPFL - Lausanne, Switzerland
⁷ Service d’ophtalmologie, Hôpital Jules Gonin - Lausanne, Switzerland
⁸ INSERM U769, Châtenay-Malabry, F-92296, France

^* To whom correspondence should be addressed. E-mail: abitbol{at}necker.fr.

Accepted 17 August 2006

Abstract

Purpose: The mutations responsible for Best Vitelliform Macular Dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin-1 (hBest1) which is a Ca2+-sensitive chloride channel. This study was performed to identify disease-specific mutations in 27 patients with BVMD. Because this disease is characterized by an alteration of the Cl- channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variant is indeed causing the disease.

Methods: Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK-293 cells and the associated Cl- current was examined using whole-cell patch clamp.

Results: We identified six new VMD2 mutations, located exclusively in exons four, six and eight. One of these mutations (Q293H) is particularly severe. Patch clamp analysis of HEK cells expressing the Q293H mutant shows that this mutant channel is non-functional. Furthermore, the Q293H mutant inhibits the function of wild-type bestrophin-1 channels in a dominant negative manner.

Conclusions This study provides further support to the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in family C which carries the Q293H mutation, it is also clear that a disease-linked mutation in VMD2 is not sufficient to produce BVMD. Our finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.

Keywords: Best macular dystrophy, Patch clamp, VMD2, chloride channel

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