n 2.13 MOLECULAR ANALYSES OF ALPHA l.ANTITRYPSIN VARIATION AND DEFICIENCY A thesis submitted for the Degree of DOCTOR OF MEDICINE tn The Depafiment of Medicine The University of Adelaide, South Australia by i Mark D. Holmes, MBBS, FRACP July, 1992. Delicøtel to Alrienne, E[eøtLor ønl Aftco[ø, CONTENTS Page ABSTRACT vilt DECLARATION ix ACKNOWLEDGEMENTS X LIST OF ILLUSTRATIONS xi LIST OF TABLES xvi AIMS AND GENERAL INTRODUCTION xvii 1. LITERATURE REVIEW 1.1. Early History 1 1.2. The Normal crlAT Gene 4 1.2.1 al AT Gene Locus 4 1 .2.2. crl AT Gene Structure 5 1 .2.3 crl AT Gene Expression I 1 .3 The Normal crl AT Protein 10 1.3.1 crlAT Protein Structure 10 1.3.2 crl AT Biosynthesis 12 1 .3.3 al AT Function 13 1.4 c¡1AT Variation 16 1.4.1 Classification of crlAT Variants 17 1.4.2 Detection of alAT Variation 18 Page 1.4.2.1 GlAT Serum Levels 18 1.4.2.2 Electrophoretic Variation of a1 AT 19 1.4.2.3 Analysis of cr,1AT Gene Mutations 20 1.4.3 Normal alAT Variants 22 1.4.4 ct1 AT Deficiency Variants 25 1.4.5 Dysfunctional cr,1 AT Variants 29 1.5 Molecular and Cellular Basis of crl AT Deficiency 30 1.6 olAT Variation and Disease 33 1.6.1 Lung Disease 33 1.6.2 Liver Disease 36 1.6.3 Other Disease Associations 37 1.7 Treatment of ol AT Deficiency 38 2. MATERIALS AND METHODS 2.1 lntroduction 41 2.2 Reagents, Radiolabels, and DNA Restriction Enzymes 42 2.3 Study Population 42 2.3.1 Source 42 2.3.2 Cli n ical C haracteristics 43 2.3.3 Bronchoalveolar Lavage 43 2.3.4 Monocytapheresis 44 2.3.5 Liver Bioosv 45 2.3.6 Drug Therapy 45 Page 2.4 Analysis of olAT Phenotype 45 2.4.1 General 45 2.4.2 cr,1AT Levels 45 2.4.3 al AT Function 45 2.4.4 lsoelectric Focusing of Serum 46 2.5 ol AT Gene Sequence Analysis 47 2.5 1 General 47 2.5 2 Extraction of High Molecular Weight DNA 48 2.5 3 lsolation of the Human cx,lAT Gene by Cosmid Cloning 49 2.5 4 Single Stranded DNA Generation by the Polymerase Chain Reaction 53 2.5 5 DNA Sequence Analysis of ol AT Variants 56 2.6 ldentification of crlAT Gene Point Mutations 58 2.6.1 General 58 2.6.2 Allele Specific Oligonucleotide Analysis 59 2.6.3 Allele Specific Amplification with the Polymerase Chain Reaction 59 2.7 Analysis of ln Vivo cll AT Biosynthesis 60 2.7.1 olAT mRNA Analysis 60 2.7.2 crlAT Protein Biosynthesis 61 2.8 Analysis of slAT Biosynthesis Using ln Vitro Svstems 63 2.8.1 General 63 2.8.2 Production of Recombinant Plasmids Containing Variant crl AT Genes 63 Page 2.8.3 crl AT mRNA Cell FreeTranslation ln Vitro ...... 66 2.8.4 Transient Expression of Human olAT cDNAs in COS I Monkey Kidney Cells 66 2.8.5 Permanent Production of Human crlAT in Murine NIH-3T3 Fibroblasts by lnsertion of a Human alAT cDNA by Retroviral Gene Transfer 68 3 ALPHA 1-ANTITRYPSIN NULLsran¡1s 1¿¡¡e: THE INTRACELLULAR MECHANISM CAUSING THE ABSENCE OF DETECTABLE ALPHA l.ANITRYPSIN 3.1 lntroduction 70 3.2 Methods 70 3.3 Results 75 3.3.1 Patient Characteristics ....... 75 3.3.2 cr1 AT Phenotype Analysis 75 3.3.3 Analysis of crl AT Genotype 75 3.3.4 Evaluation of a Nullgranire rals Homozygote for cl1 AT mRNA Transcripts ............... 77 3.4 Discussion 77 4 THE MOLECULAR BASIS OF THE LUNG AND LIVER DISEASE ASSOCIATED WITH THE ALPHA l.ANTITRYPSIN DEFICIENCY ALLELE Mmalton 4.1 lntroduction 82 iv Page 4.2 Methods 83 4.3 Results 84 4.3.1 Patient Characteristics 84 4.3.2 ldentification of the ø1AT M¡¡s¡1en Protein . 87 4.3.3 Functional Activity of the crl AT Mmatton...... 87 4.3.4 ldentification of the M¡¿¡sn Mutation 87 4.3.5 lnheritance of the Mmahon Gene...... 92 4.3.6 Evaluation of alAT mRNA Transcripts from Cells Expressing the Mmatron Gene 92 4.3.7 Synthesis and Secretion of olAT by Blood Monocytes Expressing the Mmatron Gene...... 97 4.4. Discussion 99 5 ANALYSIS OF THE MOLECULAR BASIS OF THE ALPHA l.ANTITRYPSIN DEFICIENCY VARIANT Wbethesda 5.1 lntroduction 104 5-2 Methods 105 5.3 Results 106 5.3.1 Patie nt Characteri stics 106 5.3.2 Phenotypic Analysis of u1 AT W6s1¡ssda .............. 106 5.3.3 Sequence of the crl AT Wbethesda Gene 106 5.3.4 Confirmation of lnheritance of crl AT Wbethesda 109 5.3.5 Biosynthesis of olAT Directed by the c¡1 AT Wbethesda Allele........ 109 V Page 5.4 Discussion 114 6 MOLECULAR HETEROGENEITY AMONGST THE P.FAMILY OF ALPHA l.ANTITRYPSIN VARIANTS 6.1 lntroduction 120 6.2 Methods 121 6.3 Results 126 6.3.1 Patient Characteri stics 126 6.3.2 cr1 AT Phenotype Analysis 126 6.3.3 Genetic Basis of al AT Pbwel and ol AT Psainr atbans Alleles 129 6.3.4 Confirmation and lnheritance of the Ptowe¡ and Ps¿¡.1 atbans crl AT cDNAs 132 6.3.5 Biosynthesis of crlAT Directed by Prowe¡l and Ps¿¡n1 atbans crl AT cDNAs ........ 135 6.3.6 olAT mRNA Transcripts in the PtowellZ lndividual 139 6.3.7 Effect of Tamoxifen Therapy on crlAT Serum Levels in a P¡e,¡,s¡¡Z lndividual ............._ 139 6.4 Discussion 139 7 CHARACTERIZATION OF THE NORMAL ALPHA 1. ANTITRYPSIN VARIANT Vmunich : A VARIANT ASSOCIATED WITH A UNIQUE PROTEIN ISOELECTRIC FOCUSING PATTERN 7.1 lntroduction 146 VI Page 7.2 Methods 148 7.3 Results 148 7.3.1 ldentification of the al AT V¡u¡¡s¡ Variant . 148 7.3.2 Elucidation of the Molecular Basis of the Vmunich Variant..... 150 7.3.3 Confirmation of the lnheritance of the Vmunich Allele 150 7.4 Discussion 156 8. CONCLUDING REMARKS 159 9. BIBLIOGRAPHY 161 10. APPENDICES A. Publications Arising From This Thesis 212 B. Curriculum Vitae 214 vil ABSTRACT Analyses of six rare alpha 1-antitrypsin (a1AT) alleles were undertaken to determine their molecular basis, assess the ol AT biosynthesis directed by these alleles and, examine their clinical correlation. The absence of detectable cr,1AT in the serum, resulting in premature emphysema, due to the Nullsranire 1¿¡¡s allele was demonstrated to be due to absent alAT mRNA. cr,1AT Mmatton (Phesz TTC "" delete TTC), a severely deficient variant, is associated with emphysema and liver disease. Examination of al AT biosynthesis in blood monocytes from an Mr¿¡1on homozygote revealed intracellular aggregation of a1AT, a process which not only gives rise to serum alAT deficiency but is also likely to be critical in the genesis of the hepatic damage due to this cx,1AT variant, The W6s¡rres¿a alAT allele (Alagse GCf ", Thr ACT), a moderately deficient variant with the potential of risk of emphysema if inherited in a heterozygous fashion with a severely deficient allele, directs the synthesis of a protein which is degraded intracellulady as demonstrated by in vitro experiments in COS I monkey kidney cells transfected with a Wbethesda ul AT cDNA. cr,1AT Pbwe¡ (Aspzse GAT "" Val GTT), gives riseto serum crlAT levels of 24/" predicted and is associated with emphysema. Analysis of alAT biosynthesis directed the crlAT P¡e,¡s¡¡ cDNA inserted into NIH-3T3 cells by retroviral transfer revealed intracellular degradation of cr1AT. ln contrast, another olAT "P qAC ,,,, is a Variant", Psaint atbans (Asps+t Asn Af,C; Asp2s0 GAT "" Asp GAC) normal level allele with normal cx,lAT biosynthesis. Elucidation of the ø1AT biosynthetic defect associated with the P¡syys¡ allele directed successful treatment with tamoxifen to raise deficient crl AT levels. The unique isoelectric focusing pattern for the Vmunich crlAT variant, a normal level alAT can be explained by its molecular basis (Asp2 GAT "" Ala GCT). The position of the amino acid substitution in the crlAT molecule changes the isoelectric point of only some of the microheterogenous forms of V¡y¡¡"¡ q1AT. These studies demonstrate the utility of molecular analyses to discover the genetic basis of slAT variation, determine the resultant defects of crlAT biosynthesis, help to explain the clinical correlates associated with variant alleles and aid in a rational approach to therapy. vill. DECLARATION I declare that this thesis is a record of original work and that it contains no material which has been accepted for the award of any other Degree or Diploma in any University. To the best of my knowledge and belief, this thesis contains no material previously published or written by any other person, except where due reference is given in the text of the thesis. I consent to this thesis being made available for photocopying or loan. Mark D. Holmes July, 1992. tx" ACKNOWLEDG EM ENTS The work presented in this thesis was carried out between 1987 and 1989 during a three-year tenure as a Fogarty lnternational Visiting Associate in the Pulmonary Branch, National Heart, Lung and Blood lnstitute, National lnstitutes of Health in Bethesda, Maryland, United States of America. I am indebted to my External Supervisor, Dr. Ronald G. Crystal, not only for providing me with an opportunity to pursue these studies, but also for his inspiration, enthusiasm and encouragement during my time in his laboratory.