Molecular basis and characteristics of the polyclonal antibody response to exogenous coagulation ...

146 Pages·2008·4.31 MB·English

Molecular basis and
characteristics of the polyclonal antibody response
to exogenous coagulation factor VIII
in patients with hemophilia A

Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades

vorgelegt von

Christiane Mühle

aus Leipzig
Als Dissertation genehmigt von den Naturwissenschaftlichen
Fakultäten der Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 02.07.2008

Vorsitzender der Prüfungskommission: Prof. Dr. E. Bänsch

Erstberichterstatter: Prof. Dr. K. von der Mark

Zweitberichterstatter: Prof. Dr. T. Winkler
We are here to add what we can to life,
not to get what we can from it.

(William Osler)

To my parents

Satisfaction of one's curiosity
is one of the greatest sources of happiness in life.

(Linus Pauling)

We often think that when we have completed our study of one we know all about two,
because "two" is "one and one." We forget that we still have to make a study of "and."

(Arthur Eddington)
Abstract

Molecular basis and characteristics of the polyclonal antibody response
to exogenous coagulation factor VIII in patients with hemophilia A

Hemophilia A, the most common severe coagulation disorder in males, is caused by absence
or impaired activity of clotting factor VIII (FVIII) resulting from various mutations in the
large X-chromosomal FVIII gene (F8). Approximately half of the severely affected
hemophiliacs carry a genomic inversion originating from a hot spot intrachromosomal
recombination between one intragenic and either of two extragenic intron 22 homology
regions (int22h). Such inversions have been proposed to be associated with concomitant large
deletions in F8 which are responsible for an increased risk of an immune response against
therapeutic exogenous FVIII. Exact localization of the deletion breakpoints may facilitate
molecular diagnosis, allow identification of mutational hot spots and help to establish
understanding of mechanisms behind gross genomic rearrangements causing hemophilia and
other diseases.

To investigate the hypothesis that large deletions may originate from different mechanisms
and possibly involve additional mutation events, breakpoints were characterized in a group of
32 patients with severe hemophilia A and the diagnosis of a large deletion in F8. To this end,
genomic DNA was investigated by standard PCR, long-range PCR and primer walking
techniques. Based on the recently published human X chromosome sequence, a new
mechanism for int22h-related inversions was proposed in this study and a PCR was
established to discriminate the two inversion types. The identification of novel breakpoints
revealed that these patients in fact represent a heterogeneous group with a variety of mutation
types: mere deletions of a distinct DNA region but also complex deletions combined with
additional DNA microinsertions or with recurrent int22h-related inversions. The detection of
two surplus int22h copies in a hemophiliac with combined deletion and inversion events
implies for the first time the presence of a double int22h-related inversion. The identification
of such complex arrangements may help to explain unusual patterns observed in Southern
blots for detection of int22h-related inversions.
In a patient with persistent FVIII inhibitory antibodies, molecular analysis revealed a
combination of an int22h2-related inversion, a deletion and an insertion of a duplicated part of
the X-chromosomal MPP1 gene. The defective X-chromosome most likely originated through
a new complex recombination mechanism during single spermatogenesis facilitated by
homologous SINE and LINE elements.
In silico analysis of the influence of a patient’s single base pair substitution in F8 illustrated
diverse consequences at the RNA and protein levels besides alterations imposed by the
missense mutation effect.

Development of FVIII-inactivating antibodies (inhibitors) in approximately 30 % of patients
with severe hemophilia A represents a serious complication in hemophilia treatment, as it
renders FVIII supplementation therapy ineffective. To understand the FVIII inactivation
mechanisms and to pave the way towards modifications of recombinant clotting factors with
reduced immunogenicity, the exact localization of immunodominant epitopes is required. In a
group of 19 patients with hemophilia A, acquired hemophilia or von Willebrand disease,
immunoprecipitation of labeled FVIII domains by FVIII-specific antibodies was used to
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determine the spectrum of antigenicity. A random peptide phage display library was then
employed to identify epitopes of polyclonal FVIII antibodies isolated from patient’s plasma
by affinity chromatography. Biopanning on individual samples yielded specific phage clones
which were displaced from binding to antibodies by excessive FVIII. Similarity of their
peptide sequence with amino acid motifs of human FVIII, their accessibility in a three-
dimensional protein model and immunoprecipitation results suggested putative epitopes in the
A1, A2 and C1 domains for one, in the C2 domain for a second patient and overlapping
epitope sequences in the A2 domain for two further patients. Synthetic peptides corresponding
to the A2, C1 and C2 domain epitopes blocked antibody binding to FVIII and partially
neutralized the inhibitory activity of the respective plasma. These results provide the proof of
principle that random phage-displayed peptide libraries can be used for the mapping of
epitopes recognized by a polyclonal antibody preparation.

In conclusion, this work provides insight into the heterogeneity of large deletion mutations in
F8 and into the underlying mechanisms. The discovery of concomitant large deletions
undetected by conventional PCR for int22h-related inversions highlights the need for routine
screening of patients with inversions for additional mutation events associated with an
increased risk of developing FVIII-inactivating antibodies. Characterization of such
antibodies by epitope mapping, e.g. with phage display technology, may help to identify
peptides capable of neutralizing patient’s FVIII-inhibitory antibodies and contributes to a
better understanding of pathogenic mechanisms.

Keywords:
Hemophilia A, F8 mutation analysis, large deletion breakpoints, intron 22 inversion,
FVIII-specific antibodies, inhibitors, phage display, immunoprecipitation

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Zusammenfassung

Molekulare Ursachen und Charakterisierung polyklonaler Antikörper
gegen den Blutgerinnungsfaktor VIII von Patienten mit Hämophilie A

Hämophilie A ist die häufigste schwere Blutgerinnungsstörung bei Männern. Sie wird durch
Fehlen oder eine verringerte Aktivität des Gerinnungsfaktors VIII (FVIII) aufgrund
verschiedener Mutationen im X-chromosomalen FVIII-Gen (F8) verursacht. Bei etwa der
Hälfte der Patienten beruht die schwere Hämophilie auf einer genomischen Inversion durch
intrachromosomale Rekombination zwischen einer innerhalb des Gens liegenden Intron 22-
Homologieregion (int22h) mit einer der beiden außerhalb des Gens liegenden Kopien. Schon
länger wird vermutet, dass solche Inversionen gemeinsam mit großen Deletionen auftreten
können, die für ein erhöhtes Risiko einer Immunantwort gegen therapeutischen exogenen
Faktor VIII verantwortlich sind. Eine genaue Lokalisierung von Deletionsbruchpunkten kann
die molekulare Diagnostik erleichtern, sowie zur Identifizierung von Mutations-Hotspots und
zum besseren Verständnis der Mechanismen großer genomischer Umstrukturierungen bei
Hämophilie und anderen Erkrankungen beitragen.

Zur Untersuchung der Hypothese, dass große Deletionen durch verschiedene Mechanismen
möglicherweise mit zusätzlichen Mutationsereignissen entstehen können, wurden die
Bruchpunkte in einer Gruppe von 32 Patienten mit schwerer Hämophilie A und der Diagnose
einer großen Deletion im F8 Gen charakterisiert. Dazu wurde genomische DNA mittels
Standard-PCR, Long-range-PCR und Primer Walking-Techniken untersucht. Basierend auf
der kürzlich veröffentlichten Sequenz des humanen X-Chromosoms wurde hier ein neuer
Mechanismus der int22h-abhängigen Inversion vorgeschlagen und eine PCR zur
Unterscheidung der zwei Inversionstypen etabliert. Die Identifizierung neuer Bruchpunkte
offenbarte, dass es sich bei diesen Patienten um eine heterogene Gruppe mit verschiedenen
Mutationstypen handelt: reine Deletionen bestimmter DNA-Bereiche, aber auch komplexe
Deletionen, die mit zusätzlichen DNA-Mikroinsertionen oder mit den wiederkehrenden
int22h-abhängigen Inversionen kombiniert sind. Der Nachweis zweier zusätzlicher int22h-
Kopien bei einem Hämophilen mit kombinierter Deletions-Inversions-Mutation weist
erstmals auf eine doppelte int22h-abhängige Inversion hin. Das Erkennen solcher
komplizierter Anordnungen kann bei der Erklärung ungewöhnlicher Bandenmuster im
Southern Blot zur Untersuchung auf Inversionen helfen.
Bei einem Patienten mit persistierenden FVIII-inaktivierenden Antikörpern wurde eine
Kombination aus einer int22h2-abhängigen Inversion, einer Deletion und einer Insertion eines
verdoppelten Bereichs des X-chromosomalen MPP1-Gens nachgewiesen. Das defekte X-
Chromosom entstand wahrscheinlich während einer einzigen Spermatogenese durch einen
neuen komplexen Rekombinationsmechanismus, der durch homologe SINE- und LINE-
Elemente ermöglicht wurde.
Die In-silico-Analyse des Einflusses eines Einzelbasenaustausches im Gen F8 eines Patienten
veranschaulicht neben den Auswirkungen einer Missense-Mutation die verschiedenartigen
Konsequenzen auf RNA- und Proteinebene.

Die Bildung von FVIII-inaktivierenden Antikörpern (Inhibitoren) bei ca. 30 % der Patienten
mit schwerer Hämophilie A stellt eine ernsthafte Komplikation in der Hämophiliebehandlung
dar, weil sie die Wirkung der FVIII-Supplementation drastisch vermindert. Um die
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Inaktivierungsmechanismen besser zu verstehen und den Weg für die Entwicklung
veränderter rekombinanter Gerinnungsfaktoren mit geringerer Immunogenität zu bereiten, ist
die genaue Bestimmung immundominanter Epitope notwendig. Immunpräzipitation
markierter FVIII-Domänen durch FVIII-spezifische Antikörper von 19 Patienten mit
Hämophilie A, erworbener Hämophilie oder von Willebrand Syndrom erlaubte die
Bestimmung des Antigenitätsspektrums. Eine Bakteriophagenbank mit Zufallspeptiden wurde
benutzt, um die Epitope polyklonaler FVIII-Antikörper zu identifizieren, welche aus
Patientenplasma mittels Affinitätschromatographie isoliert wurden. Phagenselektion auf den
einzelnen Patientenproben führte zur Anreicherung spezifischer Phagen, die durch
überschüssigen FVIII von der Bindung an den Antikörper verdrängt wurden. Der Vergleich
der phagenspezifischen Peptidsequenz mit der Aminosäuresequenz von FVIII, deren
Zugänglichkeit im dreidimensionalen FVIII-Modell sowie Immunpräzipitationsdaten wiesen
bei einem Patienten auf mögliche Epitope in der A1-, A2- und C1-Domäne, bei einem
zweiten auf ein Epitop in der C2-Domäne und bei zwei weiteren Hämophilen auf
überlappende Epitopsequenzen in der A2-Domäne hin. Synthetische Peptide, die den
Epitopen in der A2-, C1- und C2-Domäne entsprechen, blockierten die Bindung der
Antikörper an FVIII und konnten die inhibitorische Aktivität des zugehörigen Plasmas zum
Teil neutralisieren. Diese Ergebnisse zeigen, dass Zufallspeptid-Phagenbanken prinzipiell zur
Identifikation von Epitopen für polyklonale Antikörperproben geeignet sind.

Die vorliegende Arbeit gewährt einen Einblick in die Heterogenität großer Deletionen im Gen
F8 sowie in die zugrunde liegenden Mechanismen. Die Entdeckung, dass Inversionen mit
großen Deletionen einhergehen können, welche mittels konventioneller PCR für die int22h-
abhängigen Inversionen nicht detektierbar sind, unterstreicht die Notwendigkeit der
Routineuntersuchung von Patienten mit Inversionen auf zusätzliche Mutationen mit einem
erhöhten Risiko für die Entwicklung FVIII-inaktivierender Antikörper. Die Charakterisierung
solcher Antikörper, z.B. mittels der Phage-Display-Technologie, kann bei der Identifizierung
von Peptiden helfen, die FVIII-inhibierende Antikörper von Patienten neutralisieren, und trägt
sicherlich zu einem besseren Verständnis der pathogenen Mechanismen bei.

Schlagwörter:
Hämophilie A, F8 Mutationsanalyse, Bruchpunkte großer Deletionen, Intron-22-Inversion,
FVIII-spezifische Antikörper, Inhibitoren, Phage Display, Immunpräzipitation

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Table of contents

Abstract ....................................................................................................................................iv
Zusammenfassung...................................................................................................................vi
Abbreviations...........................................................................................................................xi

1. Introduction...............................................................................................................1
1.1 Blood coagulation.......................................................................................................1
1.2 Bleeding disorders – hemophilia A.............................................................................4
1.3 Genomic organization of F8 and molecular basis of hemophilia A...........................6
1.4 Structure and interaction sites of the coagulation factor VIII.....................................9
1.5 FVIII-inhibitory antibodies in hemophiliacs.............................................................13
1.6 Objectives and experimental strategy.......................................................................17

2. Results......................................................................................................................19

2.1 Mutation mechanisms in patients with hemophilia A..........................................19

2.1.1 Elucidation of a complex rearrangement with deletion, insertion and
inversion events in F8............................................................................................19
2.1.1.1 Analysis of F8 cDNA revealing absence of expression...................................19
2.1.1.2 Gross localization of deletion breakpoints.......................................................20
2.1.1.3 Identification of an additional insertion...........................................................21
2.1.1.4 Determination of a breakpoint within the int22h1 region................................25
2.1.1.5 Cytogenetic analysis suggesting gross chromosomal integrity........................27
2.1.1.6 Identification of a concomitant int22h-related inversion.................................29
2.1.1.7 Determination of the paternal origin of the defective X chromosome.............31

2.1.2 Slipped strand mispairing leading to large deletions in F8 and concomitant
deletions/insertions.................................................................................................34
2.1.3 Previously undetected large deletions in F8 combined with recurrent int22h-
related inversions....................................................................................................36
2.1.4 Evaluation of the effect of substitution mutations in silico....................................38
2.1.5 Overview of analyzed mutations and mechanisms................................................43

2.2 Characterization of FVIII antibody epitopes in hemophilia A patients............44

2.2.1 Characterization of patients’ material....................................................................44
2.2.2 Determination of the antigenicity profile of FVIII domains..................................45
2.2.2.1 Production of recombinant FVIII fragments....................................................45
2.2.2.2 Immunoprecipitation of labeled FVIII domains with patients’ antibodies......46
2.2.3 Epitope mapping by phage display technology......................................................50
2.2.3.1 Phage display strategy............................................................................................50
2.2.3.2 Establishment and optimization of the phage display method.........................52
2.2.3.3 Isolation of FVIII-specific antibodies from plasma of hemophiliacs..............53
2.2.3.4 Selection of phage-displayed peptides binding to patients’ FVIII-specific
antibodies.........................................................................................................54
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2.2.3.5 Identification of overlapping peptide sequences for different patients’
samples.............................................................................................................57
2.2.4 Localization of putative epitopes in a three-dimensional FVIII model.................58
2.2.5 Functional tests for putative epitopes.....................................................................60
2.2.5.1 Synthetic peptides displacing patient’s antibodies from FVIII in
competitive ELISA...........................................................................................60
2.2.5.2 Synthetic peptides partially neutralizing FVIII-inhibitory antibodies in
Bethesda Assay................................................................................................61
2.2.6 Summary of antigenic domains and identified epitopes........................................63

3. Discussion.................................................................................................................64
3.1 Heterogeneity of large deletion mutations in F8: combination with insertions
and inversions............................................................................................................64
3.2 New inversion polymorphism underlying the recurrent int22h-related
inversion....................................................................................................................67
3.3 New mechanism of a pathogenic X-chromosomal rearrangement...........................70
3.4 Influence of genetic and exogenous factors on the immune response of
hemophiliacs to FVIII...............................................................................................72
3.5 Broad spectrum of FVIII domain antigenicity and diverse role of specific
identified epitopes.....................................................................................................75
3.6 Evaluation of methods for the identification of epitopes..........................................81
3.7 Novel therapeutic including epitope-based approaches to FVIII inactivating
antibodies in hemophilia...........................................................................................83
3.8 Conclusion and Outlook............................................................................................89

4. Materials and Methods...........................................................................................90
4.1 General materials.......................................................................................................90
4.2 Biological samples....................................................................................................90
4.3 Equipment.................................................................................................................90
4.4 Databases and Programs............................................................................................91

4.5 General procedures....................................................................................................91
4.5.1 Agarose gel electrophoresis...................................................................................91
4.5.2 Transformation of bacteria.....................................................................................91
4.5.3 Isolation and purification of bacterial plasmid DNA.............................................92
4.5.4 Cloning of PCR products.......................................................................................93
4.5.5 Sequencing.............................................................................................................93
4.5.6 SDS-PAGE.............................................................................................................93
4.5.7 Western blot...........................................................................................................94

4.6 Mutation analysis......................................................................................................94
4.6.1 Isolation of total RNA and genomic DNA from blood..........................................94
4.6.2 RNA analysis..........................................................................................................95
4.6.2.1 cDNA synthesis................................................................................................95
4.6.2.2 RT-PCR............................................................................................................96
4.6.3 DNA analysis: standard methods...........................................................................96
4.6.3.1 Southern blot, CSGE and DHPLC...................................................................96
ix
4.6.3.2 PCR amplification of exons in the F8 gene.....................................................97
4.6.4 DNA-analysis: methods for inversion/deletion/insertion mutations......................97
4.6.4.1 PCR for gross localization of the deletion in the gene F8...............................97
4.6.4.2 PCR amplification to determine the extent of the MPP1 insertion..................98
4.6.4.3 Long range PCR across the deletion and inside int22h1..................................98
4.6.4.4 Quantitative real-time PCR..............................................................................99
4.6.4.5 Analysis of sequence variations in the intron 22 homology regions..............100
4.6.4.6 Long-range PCR for detection of int22h-related inversions..........................101
4.6.5 DNA-analysis: identification of unknown flanking sequences............................101
4.6.5.1 Whole genome amplification with phi29 DNA polymerase..........................101
4.6.5.2 Inverse PCR....................................................................................................101
4.6.5.3 Primer walking with the Seegene-Kit............................................................102
4.6.5.4 TAIL-PCR......................................................................................................103
4.6.5.5 Unpredictably primed PCR............................................................................104
4.6.6 DNA-analysis: polymorphic markers...................................................................104
4.6.7 Cytogenetic methods............................................................................................105

4.7 Production of recombinant FVIII fragments...........................................................105
4.7.1 Vector construction..............................................................................................105
4.7.2 Cell culture...........................................................................................................106
4.7.3 Generation of stable cell lines producing recombinant proteins..........................106
4.7.4 Production and purification of His-tagged FVIII domains..................................106

4.8 Processing and analysis of plasma samples............................................................107
4.8.1 Bethesda Assay....................................................................................................107
4.8.2 Radio-immunoprecipitation.................................................................................107
4.8.3 Antibody purification...........................................................................................108
4.8.4 ELISA for total and FVIII-specific immunoglobulins.........................................109

4.9 Phage Display Methods...........................................................................................109
4.9.1 Selection of random peptides displayed by phage...............................................109
4.9.2 Phage titering........................................................................................................111
4.9.3 Amplification and purification of phage..............................................................111
4.9.4 Plaque amplification and rapid purification of sequencing templates.................111
4.9.5 Evaluation of sequences.......................................................................................111
4.9.6 Phage binding assays............................................................................................112
4.9.7 Competitive ELISA with peptides.......................................................................112

5. References..............................................................................................................113

Appendix...............................................................................................................................127

Additional tables....................................................................................................................127
List of figures.........................................................................................................................129
Acknowledgments..................................................................................................................130
Curriculum vitae....................................................................................................................131
List of publications.................................................................................................................132
x