Identification of mutations in the factor VIII gene in children with severe haemophilia A

Doctoral Dissertation uoadl:1305694 108 Read counter

Τομέας Υγείας - Μητέρας - Παιδιού
Library of the School of Health Sciences
Deposit date:
Καψιμάλη Ζωή
Dissertation committee:
Καναβάκης Εμμανουήλ, Καλπίνη-Μαύρου Αριάδνη, Παπαγεωργίου Ευστάθιος
Original Title:
Ανίχνευση μεταλλάξεων στο γονίδιο του παράγοντα VIII σε παιδιά με βαριά αιμορροφιλία Α
Translated title:
Identification of mutations in the factor VIII gene in children with severe haemophilia A
Haemophilia A is the most common X-linked bleeding disorder which is caused by
a spectrum of mutations in the factor VIII gene (FVIII). Point mutations,
deletions and insertions have been detected in all 26 exons of the FVIII gene
of Haemophilia A patients and cause phenotypes of variable severity. Most
mutations are individual ones but some variants are recurrent because they
occur at specific hot spots. Two types of recurrent inversions in the FVIII
gene are known to cause severe haemophilia A: intron 22 and intron 1 inversions
with an incidence of 40-50% and 2-5%, respectively. The type of mutation plays
an important role in the FVIII inhibitor formation. The aim of the present
study was to determine the spectrum of mutations in the FVIII gene in patients
with severe haemophilia A. Genomic DNA samples of 96 unrelated severe
haemophilia A children were analyzed by Southern blotting and Long-Distance PCR
for the detection of intron 22 inversion. Two complementary multiplex PCRs were
used for the detection of intron 1 inversion in patients without intron 22
inversion. Direct sequencing of the coding region of the FVIII gene was used to
identify the mutations in 14 unrelated patients who were negative for both
inversions. The sequences were compared with the (HAMSTeRs;
database. The intron 22 inversion occurred in 50% (48/96) of patients with
severe haemophilia A in the present study. All patients tested for intron 1
inversion were negative for this mutation. Direct sequencing of the coding
regions and the exon /intron bounderies of FVIII gene revealed 13 different
mutations, and five had not been previously described in the international
database which supports the diversity of FVIII gene mutation. These novel
mutations included one large deletion, two small deletions, one nonsense and
one splice-site mutation. Among the 13 mutations, four were nonsense, one was
large deletion four were small deletions, two were missense and two were
splice-site mutations. Five patients out of the 13 developed FVIII inhibitors,
one patient with large deletion, two patients with small deletions and two
patients with nonsense mutations. Also, in the patients with intron 22
inversion the occurrence of inhibitor-positive cases was 34% and the clinical
phenotype in 92% of patients was severe. Knowledge of the disease-causing
mutation may have clinical significance for appropriate patient management, as
it may help predict patients at risk of developing inhibitors to FVIII
replacement therapy. It may also accurately determine the carrier status of at
risk family members and prenatal diagnosis.
Severe haemophilia A, FVIII gene, Mutations, Inhibitors
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