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Transcript of Genetic Studies of Familial Myeloproliferative Disorders â€؛ 930 â€؛ 1 â€؛...

  • Genetic Studies of Familial

    Myeloproliferative Disorders

    INAUGURALDISSERTATION

    ZUR

    ERLANGUNG DER WÜRDE EINES DOKTORS DER

    PHILOSOPHIE

    VORGELEGT DER

    PHILOSOPHISCH-NATURWISSENSCHAFTLICHEN

    FAKULTÄT DER UNIVERSITÄT BASEL

    VON

    KUN LIU

    AUS BENXI, CHINA

    BASEL, 2007

  • Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät

    auf Antrag von

    Professor Radek Skoda Professor Markus Affolter Professor Andreas Papassotiropoulos Professor Mike Hall

    Basel, den 13th November 2007

    Professor Dr. Hans-Peter Hauri Dekan

  • To my parents

  • Acknowledgements

    I am very grateful to my supervisor Prof Radek Skoda for giving me the opportunity to

    do my PhD study in his laboratory and for his patient instructions and constant

    encouragement throughout this course of research. I thank Dr. Robert Kralovics for his

    supervision and invaluable suggestions on my projects. I thank Prof Markus Affolter,

    Prof Andreas Papassotiropoulos and Prof Mike Hall for joining my thesis committee.

    My thanks go to all the lab members during my PhD years: Teo Soon Siong for

    introducing me to the lab and his friendship in all these years; Ralph Tiedt for correcting

    my thesis and many help at work; Hui Hao-Shen, Franz Schaub, Pan Dejing, Li Sai for

    their kind help and company.

    I specially thank my friends Wang Xuejuan, Liu Kenan, Zhou Haiyan and Zhang Xin for

    their kind support in my time of need, and I thank Philip Fung for the happy time.

    My deepest appreciations belong to my parents, my sister and brother-in-law for their

    loving supports through all these years.

    Finally I thank the Swiss National Foundation for supporting my study.

  • 3

    TABLE OF CONTENTS

    SUMMARY….………......……………………………………………………….………5

    GENERAL INTRODUCTION……………………………………………….………….8

    Hematopoiesis……………………………………………………………………….….8

    Megakaryopoiesis and thromobopoiesis……………………………………………..8

    Thrombopoietin and thrombopoietin receptor c-MPL…………………….……….8

    Other growth factors in megakaryopoiesis………………………………………12

    Transcription factors regulating megakaryopoiesis……………………………..12

    Platelet biogenesis……………………………………………..………………..13

    Erythropoiesis………………………………………………………………….………15

    Erythropoietin and erythropoietin receptor………………………………………16

    Hypoxia induction of EPO………………………………………………………..17

    Familial Myeloproliferative disorders………………………………………………...19

    Genetic approaches to identify human disease genes…………………………….20

    Linkage studies………………………………………………………………….22

    Association studies……………………………………………………………..23

    RESULTS I Mutations of thrombopoietin and MPL gene in hereditary

    thrombocythemia…………………………………………………………………….25

    A de novo splice donor mutation in the thrombopoietin gene causes hereditary

    thrombocythemia in a Polish family………………………………………………….26

    Abstract………………………………………………………………………….27

    Introduction……………………………………………………………………...28

    Design and methods……………………………………………………………..29

    Results…………………………………………………………………………...34

    Discussion……………………………………………………………………….44

    Mutation analysis in families with hereditary thrombocythemia and identification

    of a founder effect for a MPL mutation………………………………………..…..46

    Abstract…………………...……………………………………………………..47

    Introduction…………………………………………………………...…………48

  • 4

    Design and methods……………………………………………………………..49

    Results and discussion…………………………………………………………..50

    RESULTS II Genetic studies of a hereditary thrombocythemia family with

    normal thrombopoietin and MPL gene…………………………………………...55

    Abstract………………...………………………………………………………..56

    Introduction……………………………………………………………………...57

    Design and methods……………………………………………………………..58

    Results…………………………………………………………………………...62

    Discussion and perspective….…………………………………………………..70

    RESULTS III Genetic analysis of a family with congenital secondary

    polycythemia………………….………………………………………………………72

    Abstract………...………………………………………………………………..73

    Introduction…………………...…………………………………………………74

    Design and methods……………………………………………………………..76

    Results…………………………………………………………………………...79

    Discussion……………………………………………………………………….86

    PERSPECTIVES………………………………………………………………………88

    Mutations causing hereditary thrombocythemia……………………………..……..88

    TPO and MPL gene mutations……………………………………………………88

    Origins of the mutations…………………………………………………………89

    Novel genetic components……………………………………………………………90

    Genetic regions identified by microsatellites and SNPs………………………....90

    Functional studies for the candidate gene gelsolin……………………..……....91

    Sequencing candidate genes…………………………………………………....92

    REFERENCES………………………………………………………….……..……....93

    CURRICULUM VITAE………………………………………….……………………102

  • 5

    SUMMARY

    Genetic studies using families have successfully identified many disease genes

    causing Mendelian diseases. Familial myeloproliferative disorders (MPD) offer

    interesting opportunities to identify disease genes involved in the thrombopoiesis

    and erythropoiesis, and some undiscovered genetic components might also

    contribute to the etiology of the sporadic MPD.

    Hereditary thrombocythemia (HT) is an autosomal dominant disorder with clinical

    features resembling sporadic essential thrombocythemia. HT families share

    similar clinical symptoms caused by heterogeneous genetic alterations. Inherited

    germ-line mutations in the thrombopoietin (TPO) gene and its receptor MPL have

    been found causing thrombocytosis in a number of HT families. Five reported

    mutations in the thrombopoietin gene are all located in the 5 prime untranslated

    region (5’UTR) and cause overproduction of Tpo protein by the same

    mechanism: increased translation efficiency for the mutant mRNAs. One

    mutation identified in the MPL gene is located at the transmembrane domain and

    results in a hyperactive receptor, thereby leading to thrombocytosis. All these

    germ-line mutations have not been found in sporadic patients and are only

    responsible for the etiology of some HT families, indicating that the occurrence of

    these germ-line mutations is a rare event. The disease-causing genes for many

    HT families remain unknown. Identifying genetic lesions in these families will

    increase our knowledge of the physiology of thrombopoiesis and some of these

    unknown genetic components may contribute to the pathogenesis in sporadic

    MPD patients.

    In the first part of the project for genetic studies of HT families, the TPO and MPL

    genes were analyzed by genomic DNA exon sequencing and linkage analysis. A

    splice donor mutation in the TPO gene was identified in a Polish family. This

    mutation was previously identified in a Dutch family and the reoccurrence of this

    rare mutation has not been reported to