Human Molecular Genetics Advance Access originally published online on July 23, 2009
Human Molecular Genetics 2009 18(20):3997-4006; doi:10.1093/hmg/ddp339
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Rare missense variants of neuronal nicotinic acetylcholine receptor altering receptor function are associated with sporadic amyotrophic lateral sclerosis
1 Istituto di Neurologia and 2 Istituto di Genetica Medica, Università Cattolica del Sacro Cuore, Roma, Italy, 3 I.CO.M.M. Association for ALS research, Rome, Italy, 4 Dipartimento di Fisiologia e Farmacologia-Centro di Eccellenza BEMM and 5 Dipartimento di Biologia Cellulare e dello Sviluppo—CRIN, Sapienza Università di Roma, Italy, 6 Neuromed I.R.C.C.S, Isernia, Italy, 7 MRC Centre for Neurodegeneration Research, King's College London Institute of Psychiatry, London, UK and 8 Fondazione Don C. Gnocchi I.R.C.C.S, Roma, Italy
* To whom correspondence should be addressed. Tel: +39 0630154303; Fax: +39 0635501909; Email: msabatelli{at}rm.unicatt.it
Received May 23, 2009; Accepted July 20, 2009
Sporadic amyotrophic lateral sclerosis (SALS) is a motor neuron degenerative disease of unknown etiology. Current thinking on SALS is that multiple genetic and environmental factors contribute to disease liability. Since neuronal acetylcholine receptors (nAChRs) are part of the glutamatergic pathway, we searched for sequence variants in CHRNA3, CHRNA4 and CHRNB4 genes, encoding neuronal nicotinic AChR subunits, in 245 SALS patients and in 450 controls. We characterized missense variants by in vitro mutagenesis, cell transfection and electrophysiology. Sequencing the regions encoding the intracellular loop of AChRs subunits disclosed 15 missense variants (6.1%) in 14 patients compared with only six variants (1.3%) in controls (P = 0.001; OR 4.48, 95% CI 1.7–11.8). The frequency of variants in exons encoding extracellular and transmembrane domains and in intronic regions did not differ. NAChRs formed by mutant 
3 and 4 and wild-type (WT) β4 subunits exhibited altered affinity for nicotine (Nic), reduced use-dependent rundown of Nic-activated currents (INic) and reduced desensitization leading to sustained intracellular Ca2+ concentration, in comparison with WT-nAChR. The cellular loop has a crucial importance for receptor trafficking and regulating ion channel properties. Missense variants in this domain are significantly over-represented in SALS patients and alter functional properties of nAChR in vitro, resulting in increased Ca2+ entry into the cells. We suggest that these gain-of-function variants might contribute to disease liability in a subset of SALS because Ca2+ signals mediate nAChR's neuromodulatory effects, including regulation of glutamate release and control of cell survival.
M.S., M.Z. and F.E. are lead investigators and contributed equally to this work.