PMC3314005_01
Pedigree of the family with bleeding disorder.. Squares and circles indicate males and females, respectively, and arrow indicates the proband. Black color denotes affected individuals. A slash through the symbol indicates decreased individuals.
pone.0033263.g001.jpg
PMC3314005_01_pone.0033263.g001.jpg
pone-0033263-g001
The proband was a 9-year-old boy, whose family pedigree is shown in Fig. 1.
Analysis of plasma and platelet VWF multimers.. Plasma (A) and platelet (B) VWF multimers were assessed by 1.6% SDS-agarose gel electrophoresis and Western blotting. Normal plasma samples were diluted 1:20 and patient plasma samples were diluted 1:5. Samples from family members and a normal control (N) were indicated. Platelet samples were not available from IV-4.
pone.0033263.g002.jpg
PMC3314005_01_pone.0033263.g002.jpg
pone-0033263-g002
III-3, IV-2, IV-3 and IV-4 lacked high or intermediate MW multimers, whereas VWF from the proband's father (IV-5) had a similar multimer pattern to that of a normal control (Fig. 2A). In contrast, platelet VWF from these individuals all had HMW multimers that were indistinguishable from those of normal controls (Fig. 2B).
Identification of a VWF gene mutation.. Sequence analysis of the VWF gene in the proband detected a 6-bp nucleotides deletion in exon 28. The mutation caused D1529V1530 deletion (DeltaD1529V1530) in VWF A2 domain. The ADAMTS13 cleavage site is indicated by an arrow.
pone.0033263.g003.jpg
PMC3314005_01_pone.0033263.g003.jpg
pone-0033263-g003
Sequencing analysis of the resulting plasmids showed that the patient had one normal VWF allele and a mutant allele with a 6-bp deletion in exon 28, which encoded amino acids D1529 and V1530 in VWF A2 domain (Fig. 3).
Multimer analysis of recombinant VWF expressed in HEK293 cells.. Multimeric analysis of recombinant VWF was performed with the conditioned medium from HEK293 cells transfected with plasmids for WT and the mutant, individually or in combination. Samples from the vector-transfected cells were used as a negative control.
pone.0033263.g004.jpg
PMC3314005_01_pone.0033263.g004.jpg
pone-0033263-g004
As shown in Fig. 4, recombinant WT VWF from the conditioned medium had HMW multimers. In the conditioned media from the cells transfected with both the mutant and WT plasmids or one-half amount of WT plasmid alone, VWF protein expression levels were lower but HMW multimers were clearly present (Fig. 4).
Detection of WT and mutant VWF in transfected HEK293 cells by immunostaining.. HEK293 cells were transfected with a control vector or plasmids expressing WT or mutant VWF. The cells were stained for VWF (red), PDI (green) or DAPI (nucleus, blue). In the right column panels, merged pictures of green, red and blue channels are shown. The images were obtained with a confocal microscope. Scale bars: 10 microm.
pone.0033263.g005.jpg
PMC3314005_01_pone.0033263.g005.jpg
pone-0033263-g005
As shown in Fig. 5 (middle row panels), recombinant WT VWF was detected in transfected HEK293 cells. In similarly transfected HEK293 cells, the mutant VWF was detected mostly around the nucleus and the expression pattern overlapped with that of PDI (Fig. 5, lower panels).