title: Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD).
abstract: KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene.
authors.affiliation: Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK; Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; St Vincent's Clinical School, UNSW Australia, Sydney, NSW, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McCabe
authors.affiliation: Centre for Neuroendocrinology, Royal Free Hospital and University College Medical School, University College London, London, UK; Department of Anaesthesiology, Nanjing Medical University First Affiliated Hospital, Jiangsu Province Hospital, Nanjing 210029, China.
authors.forename: Youli
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK.
authors.forename: Louise C
authors.initials: LC
authors.lastname: Gregory
authors.affiliation: Neural Development Unit, UCL Institute of Child Health, London, UK; Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, UK.
authors.forename: Carles
authors.initials: C
authors.lastname: Gaston-Massuet
authors.affiliation: Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK.
authors.forename: Kyriaki S
authors.initials: KS
authors.lastname: Alatzoglou
authors.affiliation: Division of Mathematical Biology, National Institute for Medical Research, London, UK.
authors.forename: José W
authors.initials: JW
authors.lastname: Saldanha
authors.affiliation: Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, UK.
authors.forename: Angelica
authors.initials: A
authors.lastname: Gualtieri
authors.affiliation: University of Cambridge, Addenbrookes Hospital, Cambridge, UK.
authors.forename: Ajay
authors.initials: A
authors.lastname: Thankamony
authors.affiliation: University of Cambridge, Addenbrookes Hospital, Cambridge, UK.
authors.forename: Ieuan
authors.initials: I
authors.lastname: Hughes
authors.affiliation: Princess Margaret Hospital for Children, Subiaco, Western Australia, Australia.
authors.forename: Sharron
authors.initials: S
authors.lastname: Townshend
authors.affiliation: Neural Development Unit, UCL Institute of Child Health, London, UK.
authors.forename: Juan-Pedro
authors.initials: JP
authors.lastname: Martinez-Barbera
authors.affiliation: Centre for Neuroendocrinology, Royal Free Hospital and University College Medical School, University College London, London, UK.
authors.forename: Pierre-Marc
authors.initials: PM
authors.lastname: Bouloux
authors.affiliation: Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK. Electronic address: m.dattani@ucl.ac.uk.
authors.forename: Mehul T
authors.initials: MT
authors.lastname: Dattani
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D016326:Extracellular Matrix Proteins / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism*; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D010375:Pedigree; D010902:Pituitary Gland / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D025962:Septo-Optic Dysplasia / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
chemical_list: C076266:ANOS1 protein, human; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D008156:Luciferases
keywords: Females; KAL1; Kallmann syndrome; Luciferase assay; Septo-optic dysplasia