Ingvild Aukrust1, Ragnhild W Jansson2,3, Cecilie Bredrup2, Hilde E Rusaas1, Siren Berland1, Agnete Jørgensen4, Marte G Haug5, Eyvind Rødahl2,3, Gunnar Houge1, Per M Knappskog1,6. 1. Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway. 2. Department of Ophthalmology, Haukeland University Hospital, Bergen, Norway. 3. Department of Clinical Medicine, University of Bergen, Bergen, Norway. 4. Division of Child and Adolescent Health, Medical Genetics Department, University Hospital of North Norway, Tromsø, Norway. 5. Department of Pathology and Medical Genetics, St. Olav's University Hospital, Trondheim, Norway. 6. Department of Clinical Science, University of Bergen, Bergen, Norway.
Abstract
PURPOSE: Despite being the third most common ABCA4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA4 variant c.5461-10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant. METHODS: Fibroblast samples from patients carrying the ABCA4 variant c.5461-10T>C were analysed by isolating total RNA, followed by real-time polymerase chain reaction (RT-PCR) using specific primers spanning the variant. For detection of ABCA4 protein, fibroblast samples were lysed and analysed by SDS-PAGE followed by immunoblotting using a monoclonal ABCA4 antibody. RESULTS: The ABCA4 variant c.5461-10T>C causes a splicing defect resulting in the reduction of full-length mRNA in fibroblasts from patients and the presence of alternatively spliced mRNAs where exon 39-40 is skipped. A reduced level of full-length ABCA4 protein is observed compared to controls not carrying the variant. CONCLUSIONS: This study describes the functional effect and the molecular mechanism of the pathogenic ABCA4 variant c.5461-10T>C. The variant is functionally important as it leads to splicing defects and a reduced level of ABCA4 protein.
PURPOSE: Despite being the third most common ABCA4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA4 variant c.5461-10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant. METHODS: Fibroblast samples from patients carrying the ABCA4 variant c.5461-10T>C were analysed by isolating total RNA, followed by real-time polymerase chain reaction (RT-PCR) using specific primers spanning the variant. For detection of ABCA4 protein, fibroblast samples were lysed and analysed by SDS-PAGE followed by immunoblotting using a monoclonal ABCA4 antibody. RESULTS: The ABCA4 variant c.5461-10T>C causes a splicing defect resulting in the reduction of full-length mRNA in fibroblasts from patients and the presence of alternatively spliced mRNAs where exon 39-40 is skipped. A reduced level of full-length ABCA4 protein is observed compared to controls not carrying the variant. CONCLUSIONS: This study describes the functional effect and the molecular mechanism of the pathogenic ABCA4 variant c.5461-10T>C. The variant is functionally important as it leads to splicing defects and a reduced level of ABCA4 protein.
Authors: Jane S Green; Darren D O'Rielly; Justin A Pater; Jim Houston; Hoda Rajabi; Dante Galutira; Tammy Benteau; Amy Sheaves; Nelly Abdelfatah; Donna Bautista; Jim Whelan; Terry-Lynn Young Journal: Eur J Hum Genet Date: 2020-05-28 Impact factor: 4.246
Authors: Kamron N Khan; Melissa Kasilian; Omar A R Mahroo; Preena Tanna; Angelos Kalitzeos; Anthony G Robson; Kazushige Tsunoda; Takeshi Iwata; Anthony T Moore; Kaoru Fujinami; Michel Michaelides Journal: Ophthalmology Date: 2018-01-06 Impact factor: 12.079
Authors: Di Huang; Jennifer A Thompson; Jason Charng; Enid Chelva; Samuel McLenachan; Shang-Chih Chen; Dan Zhang; Terri L McLaren; Tina M Lamey; Ian J Constable; John N De Roach; May Thandar Aung-Htut; Abbie Adams; Sue Fletcher; Steve D Wilton; Fred K Chen Journal: Mol Genet Genomic Med Date: 2020-04-23 Impact factor: 2.183
Authors: Riccardo Sangermano; Mubeen Khan; Stéphanie S Cornelis; Valerie Richelle; Silvia Albert; Alejandro Garanto; Duaa Elmelik; Raheel Qamar; Dorien Lugtenberg; L Ingeborgh van den Born; Rob W J Collin; Frans P M Cremers Journal: Genome Res Date: 2017-11-21 Impact factor: 9.043
Authors: Aneta Ścieżyńska; Marta Soszyńska; Michał Komorowski; Anna Podgórska; Natalia Krześniak; Aleksandra Nogowska; Martyna Smolińska; Kamil Szulborski; Jacek P Szaflik; Bartłomiej Noszczyk; Monika Ołdak; Jacek Malejczyk Journal: Int J Mol Sci Date: 2020-05-13 Impact factor: 5.923