Blood, 1 July 2001, Vol. 98, No. 1, pp. 248-250
CORRESPONDENCE
To the editor:
Homozygous gene conversion in von Willebrand factor gene as a
cause of type 3 von Willebrand disease and predisposition to
inhibitor development
Von Willebrand disease (VWD) is the commonest congenital
bleeding disorder, with a prevalence, estimated from population
studies, of about 1%,1,2 although the prevalence of
patients requiring treatment is much lower.3 This
autosomally inherited disorder is caused by either qualitative (type 2)
or quantitative (type 1 and 3) deficiency of von Willebrand factor
(VWF). The human VWF gene is located on chromosome
12 and consists of 52 exons.4 A pseudogene on chromosome
22 has also been identified, which is 21-29 kb in length and
corresponds to exons 23-34 of the VWF gene. It has 97%
homology to the authentic VWF gene, but the presence of
multiple stop codons on the pseudogene indicates that this is not a
functional gene in humans.5
In recent years, many molecular defects of the VWF gene have
been identified in patients with VWD.6,7
Molecular defects in types 1 and 3 VWD are not confined to
specific regions of the gene, as in type 2 VWD.7-9 The
reported molecular defects that have caused autosomal recessive severe
type 3 VWD are large gene deletions, frameshift mutations, nonsense
mutations, splice-site mutations, defects at the level of mRNA
expression, and some candidate missense mutations.6
Heterozygotes could present as mild type 1 VWD individuals and may be
asymptomatic.10-12
We have investigated a boy who was diagnosed as having severe type 3 VWD at the age of 13 months. His parents are Asian Indians and are
first cousins. No previous family history of a bleeding disorder
existed. The patient was initially treated for his bleeding episodes
with intermediate purity factor VIII concentrate (BPL 8Y). At 6 years
of age, he developed a high-titer anti-VWF inhibitor that was
identified after a poor response to treatment. This inhibitor caused
complete inhibition of von Willebrand factor activity and has persisted
at high titer having been unaffected by an immune tolerance treatment regime.
Laboratory investigations of the patient have shown that FVIII:C,
VWF:Ag, and VWF:Ricof were all below 0.01 U/mL (normal range, 0.5-1.5 U/mL), and VWF:Ag multimers were absent. The VIII:C, VWF:Ag, and
VWF:RiCof values for the patient's mother were 2.04 U/mL, 0.96 U/mL,
and 0.60 U/mL, respectively, and for the patient's father were 1.36 U/mL, 0.84 U/mL, and 0.64 U/mL respectively. Both parents have normal
plasma VWF:Ag multimers. DNA and RNA were extracted from the propositus
and parents to carry out genotypic studies. Haplotypes of parents and
propositus were determined by analysis of 2 polymorphic regions of the
tetranucleotide (ATCT) simple repeat in intron 40 (VNTR I and II) of
the VWF gene, along with 2 restriction fragment length
polymorphisms (RFLP) of the VWF gene. These gene-tracking
studies showed that the boy had inherited the same affected allele from
each parent. The whole VWF gene was screened for mutations
using chemical cleavage mismatch detection (CCMD)
analysis.13 Any indication of sequence changes was
investigated by sequencing.
CCMD of exon 28 revealed multiple mismatched bands. The sequencing of
this exon revealed a gene conversion to have taken place, for which the
boy is homozygous and the parents are heterozygous (see
Figure 1). Five base changes
corresponding to the pseudogene sequence were identified: 4181C>T,
4201C>T, 4277A>G, 4329T>C, and 4355T>A. The maximal length of the
conversion is approximately 299 bp, and the minimal length is
173 bp. The first base change (4181C>T) results in a stop codon, which
would result in premature termination of the protein. The nucleotides
are numbered from the major transcription cap site (+1), which is
located 250 nucleotides upstream of the first nucleotide in the ATG
initiation codon. Amino acid residues are numbered from the ATG
initiation codon (residue 1) to the carboxy-terminal lysine (residue
2813) of pre-pro-VWF. All changes identified in the VWF gene
of the parents and the propositus were confirmed by DNA sequencing and
then by restriction enzyme analysis. Two possible factors may
contribute to the occurrence of a gene conversion. First, the
divergence between the pseudogene and the VWF gene is
3.14%, and the divergence between them for sequences corresponding to
exons 23-34 is 2.47%.5 Homology between the 2 genes is
greatest in these exonic regions and may contribute to recombinations.
Second, it is hypothesized that a chi sequence or sequence
similar to a chi sequence can regulate homologous
recombination.14 There are 2 consensus chi sequences (5'-GCTGGTGG-3') around the region of this gene conversion (one in exon
28 and the other in intron 27), which may be responsible for the
relatively frequent gene conversion events in exon 28.15 Six other cases of homologous gene conversion have been reported previously to be the cause of VWD type 1 or type 2B in 5 unrelated families.15,16,17 A Q1311 mutation has also
recently been detected in the homozygous state in 4 Spanish patients
from 2 apparently unrelated families of gypsy origin.18
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| Figure 1.
Schematic presentation of the 3'- end of the intron 27 and 5'- end of the exon 28 of the
VWF gene, showing the base pair differences detected
in propositus investigated in this study and other reported cases of
gene conversion between VWF and pseudogene.
Selected nucleotides and their numbers for the wild-type VWF
gene are shown together with the corresponding bases of the
pseudogene at these positions. Nucleotides in bold in the third line
(from the top) shows those that match with bases in the pseudogene in
our own and the other reported patients.15-17 Below each
changed nucleotide, the resulting amino acid substitution is shown, and
finally at the bottom the predicted maximal and minimal length of the
putative converted sequences and the reported VWD phenotype are shown.
The location and the nucleotides of the chi-like sequence is
indicated.19
|
|
The rest of the VWF gene was screened by investigating six
overlapping mRNA segments. Another point mutation was found in exon 49, a 8363G>A substitution, which would predict G2705R. This was confirmed
in the DNA to be homozygous in the propositus and heterozygous in the
parents, suggesting that it be on the same allele as the gene
conversion. As the gene conversion is upstream of this mutation and
generates a stop codon, this point mutation would not be translated.
In conclusion, we have investigated a boy with severe type 3 VWD who is
homozygous for a gene conversion in exon 28 that results in premature
termination of the protein. The asymptomatic parents, who are first
cousins, are heterozygous for the same mutation. It can be speculated
that this mutation produces a truncated dysfunctional protein lacking
many of the essential functional sites of VWF. In a patient homozygous
for this mutation, the protein cannot be assembled and, therefore, will
not be secreted and will be absent from the patient's plasma.
Gurcharan K. Surdhar, Mohammad S. Enayat, Sarah Lawson, Michael
D. Williams, and Frank G. H. Hill
Correspondence: Gurcharan K. Surdhar, Molecular Haemostasis
Laboratory, Department of Haematology, Birmingham Children's Hospital
NHS Trust, Steelhouse Lane, Birmingham, B4 6NH, United Kingdom
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