Clinical, laboratory, and genetic studies of families with Charcot- Marie-Tooth type 2C disease Guida Landouré A thesis submitted to the University College London for the degree of Doctor of Philosophy August 2011 Department of Medicine University College London 1 „I, Guida Landouré, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.‟ 2 Dedication and acknowledgements I dedicate this work to: GOD the Almighty, the clement and merciful who gave me the chance and courage to accomplish this work, and to his Prophet Muhammad, Peace upon Him. My parents: thanks for your unshakable love and support throughout my life. All my relatives and friends: thanks for being there whenever I needed you. My wife and son for their love and support. My friend Addis Taye and our patient Kelly Clever (in memoriam). First, my thanks to my supervisor Professor Robert Kleta. You have given me the chance to accomplish a dream that would be unlikely in other situations. It has sometimes been hard, but you have been patient and understanding to make this happen. Your sense of good and high standard work is remarkable. I am thankful to my supervisor Professor John Hardy. Your guidance and encouragement throughout this work were most helpful. To my scientific Godfather Dr. Kenneth Fischbeck: I met you when I did not know that a gene was made of exons and introns, but you have trusted me from the beginning and gave me an opportunity that not many would give to unskilled people like me. Thank you for your continued support. I will always be grateful to you. To my mentor Dr. Charlotte Sumner: I still remember the day you handed me this project, and I was wondering why to a layperson as me. Thank you for all the knowledge learned. Thank you for all your teaching, and above all for your friendship. A special thanks to Professor Moussa Traoré for his continued support. Many thanks to my co-workers at UCL and NIH, in particular Horia Stanescu and Barrington Burnett. Your presence and support helped me pass difficult moments. I thank all the other people who worked in this project from the beginning to the end: Dr. Michael Caterina, Shelly Choo, Dr. Rachelle Gaudet, Dr. Henry Houlden, Hitoshi Inada, Lingling Kong, Dr. Christy Ludlow, Tara Martinez, Clare Munns, Rima Paudel, Christopher Phelps, Yijun Shi, and Dr. Anselm Zdebik. 3 Abstract Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. We studied five unrelated families with CMT2C, of which two showed significant linkage to chromosome 12q24.11. Linkage analysis excluded this locus in one of the remaining families, suggesting genetic heterogeneity within this CMT subtype. SNP genotyping of samples from affected individuals in the three linked families did not reveal any deletion or copy number variation. All genes in this region were sequenced and two heterozygous missense mutations were identified in the transient receptor potential, subfamily V, member 4 (TRPV4), at positions c.805C>T and c.806G>A in two families, predicting the amino acid substitutions R269C and R269H, respectively. Two other mutations (R186Q, R232C) and one change of uncertain significance (D584N) were subsequently identified. The R269C and R269H variants were not present in more than 200 controls. TRPV4 is a well known member of the TRP superfamily of cation channels. We confirmed that it is expressed in motor and sensory neurons. The R186, R232, and R269 residues are located in the intracellular N-terminal portion of the TRPV4 protein in the ankyrin repeat domain (ARD); functional domains known to mediate protein-protein interactions. When expressed in HEK293 cells and Xenopus oocytes, R269C and R269H TRPV4 trafficked normally to the cell surface, but caused increased cell toxicity and increased constitutive and activated channel currents compared to wild-type TRPV4. Other mutations in TRPV4 have been previously associated with inherited forms of skeletal dysplasia. When mapped onto the crystal structure of the TRPV4 ARD, the mutations found in bone diseases lie on the opposite face of the domain compared to those causing neuropathy. Our findings indicate that TRPV4 mutations cause a degenerative disorder of peripheral nerves. Thus, there is striking phenotypic variability with different mutations in this important channel protein. 4 Table of contents 1 Introduction ............................................................................................................... 10 1.1 The nervous system............................................................................................ 10 1.1.1 Neuron......................................................................................................... 10 1.1.2 The central nervous system ......................................................................... 12 1.1.3 The peripheral nervous system ................................................................... 17 1.1.4 Histopathological responses of peripheral nerves to injury ....................... 21 1.1.5 Diseases of the peripheral nervous system ................................................. 22 1.1.6 Approach to evaluation of peripheral neuropathies ................................... 26 1.1.7 Treatment of peripheral neuropathies ........................................................ 29 1.2 Charcot-Marie-Tooth disease (CMT) ................................................................ 29 1.2.1 Historical notes ........................................................................................... 29 1.2.2 General findings.......................................................................................... 30 1.2.3 Classification .............................................................................................. 30 1.2.4 Therapeutic approaches.............................................................................. 47 1.3 Transient Receptor Potential (TRP) Channels ................................................... 48 1.3.1 The canonical transient receptor potential channels (TRPC) .................... 52 1.3.2 Transient receptor potential vanilloid channels (TRPV) ............................ 54 1.3.3 Transient receptor potential melastatin channels (TRPM) ........................ 58 1.3.4 Transient receptor potential ankyrin channel (TRPA) ............................... 61 1.3.5 Transient receptor potential polycystin channels (TRPP) .......................... 62 1.3.6 Transient receptor potential mucolipin channels (TRPML) ....................... 64 1.4 Objectives .......................................................................................................... 65 2 Materials and Methods .............................................................................................. 66 2.1 Patients ............................................................................................................... 66 2.2 Laboratory studies .............................................................................................. 72 2.2.1 Muscle and nerve biopsy ............................................................................. 72 2.2.2 Nerve conduction studies (NCS) and electromyography (EMG) ................ 72 2.2.3 Fiberoptic nasolaryngoscopy...................................................................... 74 2.2.4 Radiological evaluation .............................................................................. 75 2.2.5 DNA extraction ........................................................................................... 75 2.2.6 Skin biopsy .................................................................................................. 75 2.2.7 Immortal cell lines establishment ............................................................... 75 5 2.3 Genetic analysis ................................................................................................. 76 2.3.1 Linkage analysis/Fine mapping .................................................................. 76 2.3.2 Single Nucleotide Polymorphism (SNP) array analysis ............................. 77 2.3.3 Selection of candidate genes ....................................................................... 77 2.3.4 Mutation detection ...................................................................................... 78 2.3.5 Exome sequencing ....................................................................................... 79 2.4 Functional studies of mutants TRPV4 ............................................................... 80 2.4.1 TRPV4 expression in the nervous system .................................................... 80 2.4.2 Expression of WT and mutant TRPV4 in Xenopus oocytes and mammalian cells ...................................................................................................................... 81 2.4.3 TRPV4 blockers .......................................................................................... 82 2.4.4 Cell death quantification............................................................................. 82 2.4.5 Immunohistochemistry ................................................................................ 83 2.4.6 Cell surface biotinylation ............................................................................ 83 2.4.7 Endoplasmic Reticulum (ER) stress experiment ......................................... 84 2.4.8 Intracellular