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Thesis Is Presented for the Degree of Doctor of Philosophy of Curtin University of Technology

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School of Biomedical Sciences Analysis of Candidate Genes within the 3p14-p22 Region of the Human Genome for Association with Bone Mineral Density Phenotypes Benjamin H Mullin This thesis is presented for the Degree of Doctor of Philosophy of Curtin University of Technology February 2011 To the best of my knowledge and belief this thesis contains no material previously published by any other person except where due acknowledgment has been made. This thesis contains no material which has been accepted for the award of any other degree or diploma in any university. Preface The experimental work contained within this thesis was performed in the Department of Endocrinology & Diabetes at Sir Charles Gairdner Hospital under the supervision of Doctor Cyril Mamotte, Associate Professor Scott Wilson, and Professor Richard Prince. All experimental work in this thesis was performed by myself unless otherwise stated. Benjamin H. Mullin, B.Sc. Publications arising from this thesis Mullin, B. H., Prince, R. L., Dick, I. M., Hart, D. J., Spector, T. D., Dudbridge, F. & Wilson, S. G. 2008. Identification of a role for the ARHGEF3 gene in postmenopausal osteoporosis. American Journal of Human Genetics , 82 , 1262-9. Mullin, B. H., Prince, R. L., Mamotte, C., Spector, T. D., Hart, D. J., Dudbridge, F. & Wilson, S. G. 2009. Further genetic evidence suggesting a role for the RhoGTPase-RhoGEF pathway in osteoporosis. Bone , 45 , 387-91. Research grants received during completion of this thesis Wilson, S. G., Prince, R. L., Mamotte C., Mullin B. H. 2008. Influence of the ARHGEF3 gene on bone phenotypes. Arthritis Australia Project Grant ($14,500). i Presentations Mullin, B. H., Prince, R. L., Dick, I. M., Hart, D. J., Spector, T. D., Dudbridge, F., Mamotte, C. & Wilson, S. G. Polymorphisms within ARHGEF3 are associated with bone mineral density in women. • Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting . Rydges Lakeland Resort, Queenstown, NZ . 9th – 12 th September 2007 (Poster Presentation) . • Mark Liveris Health Sciences Research Student Seminar . Curtin University of Technology, Western Australia . 2nd November 2007 . Mullin, B. H., Prince, R. L., Mamotte, C., Dick, I. M., Hart, D. J., Spector, T. D., Dudbridge, F. & Wilson, S. G. Identification of a role for the ARHGEF3 gene in postmenopausal osteoporosis. • Western Australian Bone and Cartilage Program Journal Club . Dept. of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Western Australia . 23 rd May 2008 . • Australian Society for Medical Research - WA Scientific Symposium . Curtin University of Technology, Western Australia . 4th June 2008 . • Endocrinology Forum . Dept. of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Western Australia . 19 th June 2008 . • Sir Charles Gairdner Hospital Young Investigator Award . Western Australian Institute for Medical Research, Sir Charles Gairdner Hospital, Western Australia . 25 th June 2008 . Mullin, B. H., Prince, R. L., Mamotte, C., Dick, I. M., Hart, D. J., Spector, T. D., Dudbridge, F. & Wilson, S. G. Association between polymorphism in the RHOA gene and bone mineral density in Caucasian women. • Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting . Hilton on the Park, Melbourne, Victoria . 28 th – 30 th August 2008 (Poster Presentation) . • Mark Liveris Health Sciences Research Student Seminar . Curtin University of Technology, Western Australia . 6th November 2008 . ii Mullin, B. H., Mamotte, C., Prince, R. L., Dick, I. M., Hart, D. J., Spector, T. D., Dudbridge, F. & Wilson, S. G. Association between variation in two related genes, RHOA and ARHGEF3 , and bone mineral density in Caucasian women. • Western Australia Biomedical Research Institute Postgraduate Symposium . Curtin University of Technology, Western Australia . 30 th September 2008 . Mullin, B. H., Prince, R. L., Mamotte, C., Spector, T. D., Hart, D. J., Dudbridge, F. & Wilson, S. G. Further genetic evidence suggesting a role for the RhoGTPase- RhoGEF pathway in osteoporosis. • Australian Society for Medical Research - WA Scientific Symposium . Curtin University of Technology, Western Australia . 3rd June 2009 . • Endocrinology Forum . Dept. of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Western Australia . 2nd July 2009 . • Sir Charles Gairdner Hospital Young Investigator Award . Western Australian Institute for Medical Research, Sir Charles Gairdner Hospital, Western Australia . 19 th October 2009 . Mullin, B. H., Prince, R. L., Mamotte, C. & Wilson, S. G. Role of the ARHGEF3 and RHOA genes in postmenopausal osteoporosis. • Australian Society for Medical Research - WA Scientific Symposium . Curtin University of Technology, Western Australia . 9th June 2010 . • Endocrinology Forum . Dept. of Endocrinology & Diabetes, Sir Charles Gairdner Hospital, Western Australia . 15th July 2010 . Mullin, B. H., Mamotte, C., Prince, R. L. & Wilson, S. G. Identification of a role for the ARHGEF3 and RHOA genes in postmenopausal osteoporosis. • Mark Liveris Health Sciences Research Student Seminar . Curtin University of Technology, Western Australia . 8th November 2010 . iii Acknowledgements Firstly, I would like to express my gratitude to my three supervisors, Doctor Cyril Mamotte, Associate Professor Scott Wilson, and Professor Richard Prince. Thank you for your guidance and support; without it my project would not have been possible. Your expertise has proven to be invaluable and your help in everything from planning experiments to applying for funding has been immense and will not be forgotten. Secondly, I’d like to thank Professor Jiake Xu and Doctor Taksum Cheng from the Centre for Orthopaedic Research at UWA for performing the bone resorption assays, providing me with research materials and for instruction on how to differentiate and culture osteoclast-like cells. I must also thank Doctor Peter Dallas, Professor Rebecca Mason and Doctor Giuseppe Verdile for providing me with access to cell lines. Many thanks to Doctor Suzanne Brown for providing me with expert advice related to statistical analyses, particularly with regards to analysing the data from my microarray work. I would like to acknowledge the financial support provided to me by Curtin University of Technology in the form of a Curtin University Postgraduate Scholarship. I would also like to acknowledge Arthritis Australia for providing me with a Project Grant, which funded a significant amount of the laboratory consumables and reagents that were used during my project. I would like to say a big thank you to my family, particularly my Mother who passed away during my project. You always provided me with immense support and encouragement over the years and you always believed in me. Thanks Dad for nurturing my interest in science from a young age. Finally, I would like to say a very special thank you to my partner Shelby. You have kept me smiling when times were tough and motivated when I have been feeling flat. I am eternally grateful for your unwavering support. iv Summary Previous studies have identified the 3p14-p22 chromosomal region as a quantitative trait locus for bone mineral density (BMD). The overall aim of this thesis is to identify the gene or genes from this region that are responsible for the linkage observed. The studies described within are centred on the analysis of polymorphisms within candidate genes from 3p14-p22 for association with BMD phenotypes in Caucasian women. Some functional work has also been performed to provide information on the role of these genes in bone cells. It is hoped that by identifying the genes that regulate BMD, a better understanding of osteoporosis may be gained, more effective treatments can be developed and individuals at increased risk of developing the disease can be identified. Osteoporosis is a systemic bone disease characterised by low bone density and micro-architectural deterioration which results in fragile bones that are at increased risk of low-trauma fracture. Peak bone mass is attained in early adult life, but declines in postmenopausal women, in particular, due to a reduction in oestrogen production with effects on bone as well as intestinal and renal calcium handling. However, in addition to the effects of oestrogen, calcium and other environmental factors on bone structure and fracture, there is a strong genetic effect on peak bone mass, bone loss and fracture rates in postmenopausal women. Twin and family studies have suggested that 50 – 90 % of the variance in peak bone mass is heritable. Multiple genetic linkage studies have identified the 3p14-p22 region of the human genome as a quantitative trait locus for BMD. Therefore, it was hypothesised that one or more genes in this genomic region are significantly associated with BMD in Caucasian women. Based on a review of the literature, the ARHGEF3 gene was identified as a positional candidate in this chromosomal region. Common genetic variation within this gene was analysed by genotyping tagging single nucleotide polymorphisms (SNPs) in two populations of Caucasian women using the Illumina GoldenGate assay and matrix- assisted laser desorption/ionisation time-of-flight (MALDI-ToF) mass spectrometry techniques, the latter of which was performed in-house. Multiple associations were identified between polymorphism within the ARHGEF3 gene and BMD, with the strongest associations observed between the SNP rs7646054 and various BMD v phenotypes in both populations ( P < 0.001 – 0.038 ). This SNP was also found to be significantly
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  • The Toxic Peptides from Amanita Mushrooms

    The Toxic Peptides from Amanita Mushrooms

    INVITED REVIEW In?. J. Peptideprotein Res. 22,1983,251-216 The toxic peptides from Amanita mushrooms THEODOR WIELAND Max-Planck-Institute for Medical Research, Heidelberg, West Germany Received 13 December 1982, accepted for publication 15 January 1983 The results of 50 years of effort in the chemistry of Amanita toxins are reviewed. The phallotoxins, fast acting components, but not responsible for fatal intoxi- cations after ingestion, are bicyclic heptapeptides. They combine with F-actin, stabilizing this protein against several destabilizing influences. The virotoxins likewise fast acting are monocyclic heptapeptides. The amatoxins which are the real toxins lead to death within several days by inhibiting the enzymatic synthesis of m-RNA. They are bicyclic octapeptides. The‘ structures of all of these com- pounds are described, as well as conformations, chemical reactions and modifi- cations, syntheses and correlations between structures and biological activities. Key words: amatoxins; cyclic peptides; peptide thioethers; phallotoxins; tryptathionine; virotoxins Heinrich Wieland (photograph), born in 1877, was one of the most productive and versatile chemists during the first part of this century. In the thirties, at his institute the Chemical Laboratory of the Bavarian Academy of Science at Munich, Germany, students and post doctorals worked on such different themes as steroids, pigments of butterflies, tocd and snake venoms, radical reacfions, strychnos alkaloids, alkaloids from _calabash curare and mechanism of biological oxidation. Natural substances that had any striking properties were of interest. 50 years of Amanita research In the early thirties, H. Wieland suggested to Hans A. Raab that he attempt the isolation of the poisonous principal of the most danger- ous mushroom Amanita phalloides, “der griine Knollenblatterpilz”, whose erroneous ingestion yearly accounted for numerous victims.