Molecular basis and characteristics of the polyclonal antibody response to exogenous coagulation factor VIII in patients with hemophilia A [Elektronische Ressource] / vorgelegt von Christiane Mühle

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• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

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
ivdetermine 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

vZusammenfassung

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 verschi