P\ ■Studies of the Molecular Basis of the Lysosomal Storage Disorder Fucosidosis by Helen Marie Cragg Thesis submitted for the degree of Doctor of Philosophy University of London Division of Biochemistry and Genetics Institute of Child Health ProQuest Number: U541539 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U541539 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Fucosidosis is a rare, autosomal recessive, lysosomal storage disorder resulting from a deficiency of the enzyme a-fucosidase. This defect leads to the accumulation in the lysosomes of tissues and excretion in urine of fucose-containing oligosaccharides, glycoasparagines, and glycolipids. The gene encoding lysosomal a-fucosidase has been mapped to the short arm of chromosome 1 at position lp34.1-36.1 and consists of eight exons spanning 23kb ofDNA. In this thesis the molecular basis of the enzyme defect has been investigated in thirteen fucosidosis patients. The residual a-fucosidase activity in extracts of fibroblasts, leukocytes and plasma has been characterised and the urinary oligosaccharides investigated by thin layer chromatography. Patient DNA was haplotyped using two previously described restriction fragment length polymorphisms observed with the restriction enzymes, PvuQ. and BgH. Single strand conformation polymorphism analysis (SSCP) was used to screen the 13 fucosidosis patients for mutations. 8 of the 9 classic fucosidosis patients revealed conformational alterations (89%). These alterations were subsequently sequenced resulting in eight new mutations being identified. One mutation, P5R, was located in the signal peptide of a-fucosidase. A lObp deletion, G96fs (TAG), resulting in a frame shift and the production of a stop codon was also identified in exon 1. Analysis of exon 3 revealed three frirther mutations, W138X, Y21IX and a Ibp deletion in the donor splice site, S216fs(TGA). A g^a point mutation was found in the highly conserved splice site sequences of intron 5. Exon 6 contained two mutations, a missense point mutation N329Y and a Ibp insertion, Y330fs(TAA), which results in the production of a stop codon. The latter mutation was found in the same patient as the P5R mutation, both were homozygous. A new polymorphism has also been identified in exon 5, Q281R, which has been shown to be responsible for the Ful/Fu2 polymorphism of a-fucosidase isoenzymes observed by starch gel electrophoresis. Transient expression of the normal a-fucosidase cDNA in COS cells was established which allowed the further investigation of the mutations identified. W138X, S216fs(TAG), and both alleles of the polymorphism, Q281R, were created by site directed mutagenesis of normal a- fucosidase cDNA and characterisation of the expressed protein was carried out following transient expression. To Andrew 111 Preface Parts of the work described in this thesis were presented in the following pubhcations; 1) Cragg H, O'Brien J, Kretz K, Willems P, Williamson M, Winchester B (1991) The molecular genetics of fucosidosis. Abst. 15. 8th Workshop of the European Study Group of Lysosomal Storage Disorders (ESGLD), Annecy, France. 2) Cragg H, Grant J, Kretz K, O'Brien J, Willems P, Williamson M, Young E, Winchester B (1992) A splice site mutation in fucosidosis. Abst P212. 30th Annual Symposium of the Society for the study of inborn errors of metabolism (SSIEM) Leuven, Belgium. 3)Wilhamson M, Cragg H, Grant J, Kretz K, O'Brien JS, Willems PJ, Young E, Winchester B (1993) A 5'splice site mutation in fucosidosis. J Med Genet 30:218-223. 4) Cragg H, Winchester B, Seo HC, O'Brien J, Swallow D (1994) Molecular basis of the common electrophoretic polymorphism (Ful/Fu2) in human a-L-fucosidase. J Med Genet 31:8:659-660 5)Vellodi A, Cragg HM, Winchester B, Young E, Young J, Downie CJC, Hoare D, Stocks R, Baneijee GK (in press) Allogeneic Bone Marrow Transplantation for fucosidosis. 6) Cragg H, Winchester B (submitted for publication) Mutation analysis in patients with fucosidosis. IV The experimental work presented in this thesis was all carried out by the candidate with the exception of some of the enzyme assays which were carried out prior to the commencement of the project or as routine by either Magali Williamson or Elisabeth Young, to whom I am very grateful. The Bone marrow transplantation and any related clinical procedures were performed by Dr. Vellodi and his colleagues. My contribution to the BMT study was genetical and some biochemical analysis of the patient before and after the transplantation. Any work included in this thesis that is not my own has been clearly stated and the appropriate colleagues acknowledged. Acknowledgements I would like to thank my supervisor Bryan Winchester for the help and support he has provided through out this project. Many thanks go to all the members of Club 310 (AKA 5022), especially Barbara, David, Jess B and Jess T for all their help, friendship and endless hours of amusement. I would especially like to thank Barbara for both the proof reading of this thesis and her unlimited source of encouragement and fim, an essential addition to any PhD. I would like to thank the numerous clinicians who have provided the patients for this study and Dr Vellodi who has encouraged our involvement in the study of the first bone marrow transplant in fucosidosis. I would also like to thank Richard Jones for volunteering his help and advios which resulted in a more interesting and productive final year. I would like to acknowledge the work and help that has been provided by the clinical biochemistry laboratory, especially Elizabeth Young and Tony Whitfield. I would also like to acknowledge the supply of patients and cDNA probes that were essential to this project by Patrick Willems, John O'Brien and Heo-Chan Seo. Thanks must also go to my family, especially my mother, for all their support and encouragement which they have given me from the beginning to the very end of this project. Thanks also to my father whose own personal battle has provided an endless source of both inspiration and determination which has helped me during this project and will always remain with me. Finally the biggest thanks must go to Andrew for all his help, enthusiasm and support during the last 4 years. Also I would like to thank him for his role as assistant producer of this thesis. V Contents Pagel Title page Abstract i Dedication iii Preface iv Acknowledgements V Contents vi List of figures XV List of tables XX List of abbreviations xxi CHAPTER 1-Introduction 1 1.1 Lysosomes, lysosomal enzymes and lysosomal storage disorders 2 1.1.1 Lysosomes 2 1.1.2 Biosynthesis and processing of lysosomal enzymes 3 1.1.3 Lysosomal catabolism of glycoproteins and glycosphingolipids 7 1.1.3.1 Glycoproteins 7 1.1.3 .2 Glycosphingolipids 7 1.1.4 Lysosomal storage disorders 11 1.2 Fucosidosis 12 1.2.1 Definition 12 1.2.2 History 12 1.2.3 Incidence 13 1.2.4 Clinical features 14 1.2.5 Pathology 16 1.2.6 Diagnosis 16 1.2.7 Treatment 19 1.3 a-Fucosidase 21 1.3.1 Definition 21 1.3.2 Occurrence 21 1.3.3 Substrate specifity 22 VI 1.3.4 Properties of a- fucosidase 22 1.3.4.1 pH optimum 22 1.3.4.2 Michaelis-Menten constant 22 1.3.4.3 The active site of a-fucosidase 22 1.3.5 Biosynthesis and processing | of a-fucosidase 23 1.3.6 Polymorphisms of a- fucosidase 25 1.3 .6.1 Common electrophoretic polymorphism of a-fucosidase 25 1.3 .6.2 Low plasma activity polymorphism 26 1.4 Molecular Genetics 27 1.4.1 Chromosomal localisation 27 1.4.1.1 FUCAl 27 1.4.1.2FUCA1L 28 1.4.1.3FUCA2 28 1.4.2 a-fucosidase cDNA 29 1.4.3 Gene structure 29 1.4.3 .1 Restriction endonuclease mapping of genomic FUCAl 29 1.4.3 .2 Restriction fragment length polymorphisms (RFLP) 3 0 1.4.4 Known mutations in FUCAl 31 1.4.4.1 Obliteration of EcoRl restriction site 31 1.4.4.2 Deletion of the 3' region of FUCAl 32 1.5 Aims of the project 32 CHAPTER 2-Materials and methods 33 2.1 Materials 34 2.2 Solutions and media 35 2.2.1 General solutions 3 5 2.2.2 Sequencing solutions 35 2.2.3 Cycle sequencing solutions 36 2.2.4 Bacterial media 36 2.2.5 Tissue culture solutions 37 2.2.6 Southern blotting and hybridisation solutions 37 VII 2.2.7 Qiagen plasmid preparation solutions 38 2.2.8 Small scale plasmid preparation solutions 38 2.2.9 Solutions for site-directed mutagenesis 38 2.3 Methods 39 2.3.1 Tissue culture 39 2.3.1.1 Tissue culture of fibroblast and COS cell lines 39 2.3.1.2 Sub culturing and harvesting of fibroblasts and COS cells 39 2.3 .1.3 Storage of cells for subsequent reculture 3 9 2.3 .1.4 Storage of fibroblasts for enzyme assay or DNA extraction 39 2 .3 .1.5 Transformation of lymphocytes with Epstein Barr Virus 40 2 .3 .1.6 Culture of lymphoblast cell lines 40 2.3.2 Enzyme studies 41 2.3.2.1 Preparation cells for assay of enzymes 41 2.3.2.2 Assay of a-fucosidase activity 41 2.3.2.3 Assay of p -hexosaminidase activity 41 2.3.2.4 Protein determination 42 2.3.3 Thin layer chromatography of urinary oligosaccharides 42 2.3.4 Extraction and precipitation of DNA 44 2.3.4.1 Extraction of human