Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

Mark J McCabe; Youli Hu; Louise C Gregory; Carles Gaston-Massuet; Kyriaki S Alatzoglou; José W Saldanha; Angelica Gualtieri; Ajay Thankamony; Ieuan Hughes; Sharron Townshend; Juan-Pedro Martinez-Barbera; Pierre-Marc Bouloux; Mehul T Dattani

doi:10.1016/j.mce.2015.09.010

Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

Mol Cell Endocrinol. 2015 Dec 5:417:63-72. doi: 10.1016/j.mce.2015.09.010. Epub 2015 Sep 14.

Affiliations

¹ 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.
² 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.
³ Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, UCL Institute of Child Health, London, UK.
⁴ 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.
⁵ Division of Mathematical Biology, National Institute for Medical Research, London, UK.
⁶ 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.
⁷ University of Cambridge, Addenbrookes Hospital, Cambridge, UK.
⁸ Princess Margaret Hospital for Children, Subiaco, Western Australia, Australia.
⁹ Neural Development Unit, UCL Institute of Child Health, London, UK.
¹⁰ Centre for Neuroendocrinology, Royal Free Hospital and University College Medical School, University College London, London, UK.
¹¹ 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.

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.

Keywords: Females; KAL1; Kallmann syndrome; Luciferase assay; Septo-optic dysplasia.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
COS Cells
Chlorocebus aethiops
Extracellular Matrix Proteins / genetics*
Extracellular Matrix Proteins / metabolism*
Female
Humans
In Vitro Techniques
Luciferases / metabolism*
Models, Molecular
Nerve Tissue Proteins / genetics*
Nerve Tissue Proteins / metabolism*
Pedigree
Pituitary Gland / metabolism
Polymorphism, Single Nucleotide*
Septo-Optic Dysplasia / genetics*
Septo-Optic Dysplasia / metabolism
Septo-Optic Dysplasia / pathology

Substances

ANOS1 protein, human
Extracellular Matrix Proteins
Nerve Tissue Proteins
Luciferases

Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

Authors

Affiliations

Abstract

Publication types

MeSH terms

Substances

Grants and funding