Abstract
Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A , domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and ON WILLEBRAND factor (vWF) is a high-molecular V weight disulfide-linked glycoprotein that circulates in plasma as a series of multimers ranging from 400,000 to greater than 20 million daltons.'.2 vWF plays a dual role in hemostasis: it mediates platelet adhesion to the subendothelium at sites of vascular and it serves as a camer protein for coagulation factor VIII, permitting normal survival of this factor in the cir~ulation.~ Quantitative and qualitative defects of vWF cause the most common hereditary bleeding disorder in humans, known as von Willebrand disease (vWD 0006-49 7 I/93/8203-0040$3.00/0 chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)+Gln in one patient, Gly(846)hArg in one patient, and Va1(902)+Glu in three ostensibly unrelated patients. By AS0 hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Va1(902)+Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.

By Aida Inbal, Talma Englender, Nurit Kornbrot, Anna M. Randi, Giancarlo Castaman, Pier M. Mannucci, and J. Evan Sadler Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A , domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and ON WILLEBRAND factor (vWF) is a high-molecular V weight disulfide-linked glycoprotein that circulates in plasma as a series of multimers ranging from 400,000 to greater than 20 million daltons.'.2 vWF plays a dual role in hemostasis: it mediates platelet adhesion to the subendothelium at sites of vascular and it serves as a camer protein for coagulation factor VIII, permitting normal survival of this factor in the cir~ulation.~ Quantitative and qualitative defects of vWF cause the most common hereditary bleeding disorder in humans, known as von Willebrand disease (vWD). The most common qualitative disorder of vWF, type IIA vWD, is characterized by autosomal-dominant inheritance, a decrease in large and intermediate vWF multimers, and diminished vWF binding to platelet glycoprotein Ib ( G P I~) .~ Thirteen missense mutations causing type IIA vWD have been rep~rted,~-" and all but one are within the A, domain 0 1993 by The American Society of Hematology.
0006-49 7 I/93/8203-0040$3.00/0
chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)+Gln in one patient, Gly(846)hArg in one patient, and Va1(902)+Glu in three ostensibly unrelated patients. By AS0 hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Va1(902)+Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified. of vWF. Expression of selected mutant vWF sequences by transfection in mammalian cells showed that type IIA vWD is caused by at least two distinct mechanisms in specific subgroups of these mutations." In one group, represented by the Arg(834)+Trp mutation, a full range of vWF multimers is produced, but they are abnormally sensitive to proteolytic degradation in the circulation, resulting in a type IIA multimeric pattern and decreased vWF function. In the second group, represented by the Va1(844)-.Asp mutation, there is abnormal intracellular processing and consequent secretion of vWF with a type IIA multimeric pattern and decreased function."
0006-49 7 I/93/8203-0040$3.00/0
The sensitivity and specificity of the current standard diagnostic tests for vWD may be as low as 60%.13 Additional variability is contributed by a number of other factors, including AB0 blood group and estrogen l e~e l . '~-'~ The complexity of vWD classification and the limitations of the available diagnostic tests present major problems for the practicing clinical hematologist.
0006-49 7 I/93/8203-0040$3.00/0
These problems could be overcome by the direct detection of vWF mutations. Since the mutations that are known to cause type IIA vWD are localized to vWF exon 28, differential hybridization with a panel of oligonucleotide probes containing type IIA mutations would permit accurate diagnosis and classification at the DNA level for this common vWD subtype.
0006-49 7 I/93/8203-0040$3.00/0
Nonradioactive hybridization with sequence-specific oligonucleotide probes has become a widely used technique for the detection of genetic mutations and polymorphism~."~'~ An efficient approach suggested by Saiki et alZ0 is to cross-link the oligonucleotides onto a nylon membrane with UV light after addition of a long deoxyribothymidine homopolymer tail to the 3' end of the oligonucleotide. The target segment DNA is amplified by polymerase chain reaction (PCR) with biotin-labeled primers and then hybridized to the immobilized oligonucleotides. Hybridization signals are detected after binding of streptavidin-horseradish peroxidase to the biotinylated primers. Thus, in a single hybridization reaction, a DNA sequence can be analyzed for many mutations simultaneously.
0006-49 7 I/93/8203-0040$3.00/0
We used such a method to screen for eight type IIA mutations in representative patients from 12 unrelated families. Five unrelated patients were negative for the panel of tested type IIA mutations, and among them three additional mutations were identified by further DNA sequencing. One mutation occurred independently in three unrelated families. Patients. The patients studied were from 12 unrelated families from Italy affected with type IIA vWD. Each of the affected family members showed a prolonged bleeding time, decreased vWF antigen and ristocetin cofactor activity, and a plasma vWF multimer pattern consistent with type IIA vWD.
MATERIALS AND METHODS

Materials
PCR. The PCR was performed with a Perkin Elmer-Cetus DNA Thermal Cycle?' as previously described." Briefly, 100 ng of peripheral blood leukocyte DNA was amplified with 2.5 U of Taq polymerase and approximately 70 pmol of each nucleotide primer 
Materials
Antisense denoted by "a." Names indicate in single-letter code the amino acid residues they represent.
Materials
t Numbers indicate the position of the first nucleotide of the corresponding primer within the vWF sequence, numbering from the first nucleotide of the initiation codon. in a total volume of 100 pL. Reactions were preheated at 99°C for 5 minutes before addition of Taq polymerase and then at 96°C for an additional 5 minutes after addition of Taq polymerase. DNA was amplified for 28 cycles of I minute at 96°C (denaturing), 1 minute at 53 to 55'C (annealing), and 2 minutes at 72°C (extension), with a final extension time of 5 minutes at 72°C.
Materials
The primers used for amplification of part of exon 28 are listed in Table I . For nonradioactive allelle-specific oligonucleotide (ASO) hybridization, the primers were 5'-biotinylated before amplification? ' Tailing of oligonucleotides. Oligonucleotides used are listed in Table 1 . Tailing of oligonucleotides with dTTP was accomplished using TdT. Briefly, to 30 pmol oligonucleotides were added 500 pmol/L dTTP, 2 fiL 10X OPA, and 40 U TdT in a total 20-pL vol reaction. According to the manufacturer's protocol (TdT protocol: simplified tailing of DNA in OPA), those conditions provide a nominal tail length of approximately 400-dT residues. After a I-hour incubation at 37"C, 1/10 vol of 0.5 mol/L EDTA was added to stop the reaction. The tailed oligonucleotides were transferred onto nylon membrane (Genescreen, New England Nuclear) using a slot manifold (Schleicher & Schuell, Keene, NH; 5 pmol/well) and immobilized by UV light in a UV cross-linker (Stratalinker; Instruction Manual, Stratagene, La Jolla, CA). Approximately 120 to 150 mJ/cm' was applied. The filters were air-dried and stored at room temperature.
Materials
Nonradioactive A S 0 hybridization of biotinylated DNA. The membrane was prehybridized for 15 minutes at 50°C in 8 mL of hybridization buffer (5X SSPE/5X Denhardt's/O.S% SDS [5X SSPE = 0.9 mol/L NaCI, 0.05 mol/L NaH'PO, pH 6.8, 0.005 mol/L EDTA; 5X Denhardt's = 0.1% polyvinyl-pyrrolidon, 0.1% Ficoll, 0.1% bovine serum albumin; SDS = sodium dodecyl sulfate]); Biotinylated amplified DNA (50 pL of a 100-pL PCR reaction, -150 ng) was denatured in a boiling water bath for 10 minutes and then added to the membrane. Hybridization continued for 2 hours at 50°C. The membrane was washed twice for 20 minutes in 2X SSPE/O. I% SDS/3 mol/L tetramethyl ammonium chloride at 50°C. To permit visualization of all immobilized oligonucleotides, control hybridizations were performed under low-stringency conditions: 20-minute wash in 2X SSPE/3 mol/L tetramethyl ammonium chloride at 37°C. Positive signals were detected using avidinalkaline phosphatase-conjugate and chemiluminescent substrate (Lumigen PPD) from Stratagene (Flash Detection System).
Materials
Sequencing. Peripheral blood leukocyte DNA was amplified with primers 373 and 375a as described above, except that only one of the primers was biotinylated. The amplified DNA was electrophoresed on 0.8% Sea-Plaque agarose gels (FMC Bio Products, Rockland, ME) and visualized by staining with ethidium bromide. The desired fragment was excised and purified on Geneclean glass beads (Bio Lab, La Jolla, CA). Biotinylated strands were separated from nonbiotinylated by binding to streptavidin-magnetic beads complex (Dynabeads, M-280; Dynal AS, Oslo, and sequenced directly with Sequenase. Sequences were identified on products from several independent PCRs, and were confirmed by sequencing both strands. Additional mutations were excluded by DNA sequencing of representative cloned PCR products. The sequence obtained corresponded to cDNA nucleotides 4504 t o 5033, which encode amino acid residues 739 to 921. This segment includes the entire domain A2.
Materials
The Va1(902)+Glu, Gly(846)+Arg, and Arg(834)+Gln substitutions were evaluated by AS0 hybridization. Oligonucleotides (1 7 mer) were synthesized with either normal sequences (49947,48256, and 47906, Table 1 ) or mutant sequences (4994A, 4825A, and 4970A, Table 1 ).
Materials
Samples of genomic DNA from patients and controls were am-A S 0 hybridization using [ y-'*P]ATP.
Materials
For vWD. When these studies were performed, nine candidate type IIA vWD mutations had been and eight of them were selected for study. Preliminary comparisons indicated that specific hybridization signals were easily obtained with 17-mer oligonucleotides, but not with 15-mer, and 17-mer oligonucleotides were used for subsequent experiments. Oligonucleotides ( I7 mers) containing each of these mutations or the corresponding normal sequences (Table l ) were elongated with (dT)n tails using terminal transferase and cross-linked to nylon membranes by UV irradiation. All eight tested mutations are within the A, domain of vWF, which is encoded by a portion of exon 28. This segment was amplified from genomic DNA by PCR with biotinylated primers 373 and 375a, yielding a 627-bp product that encodes amino acid residues 732 to 92 I of the mature vWF subunit. These primers permit selective amplification of vWF exon 28 without interference from the highly conserved sequences of the vWF p~e u d o g e n e .~~*~~ The PCR products were hybridized with membranes containing immobilized target oligonucleotides. Membranes were washed in the presence of tetramethyl ammonium chloride, which permits the discrimination of mismatches independent of base composition,26 and thus allows the simultaneous analysis of multiple hybridization reactions with a single highstringency wash condition. An avidin-alkaline phosphatase conjugate was then bound to the biotinylated primers ofthe hybridized PCR probe, and hybridization signals were visualized with a chemiluminescent phosphatase substrate.
RESULTS

Screening
Control hybridizations under low-stringency conditions confirmed that all target oligonucleotides were successfully immobilized on the membrane and were available for annealing with the PCR probes (data not shown).
Screening
Genomic DNA samples from 12 families with type IIA vWD were screened with this assay, and four previously reported mutations5,'*' I were identified in seven unrelated patients (Fig I) . All patients gave hybridization signals with every normal oligonucleotide, but only one mutant oligonucleotide, suggesting that they were heterozygous with one apparently normal and one mutant allele. The Val(844)-Asp and Arg(834)+Trp mutations occurred independently in three and two unrelated families, respectively. The For personal use only. on October 23, 2017 . by guest www.bloodjournal.org From known mutations. Therefore, the PCR products from affected family members were sequenced, and additional candidate mutations were identified in all five families: Va1(902)+Glu in three families and Gly(846)+Arg and Arg(834)+Gln in one family each.
Screening
In three unrelated families (representative members BA, PG, and CR), a T+A transversion was identified at position 4994 resulting in Val+Glu substitution at amino acid residue 902 ofthe mature vWF subunit (Fig 2) . This substitution was confirmed by AS0 hybridization (Fig 3) . All of the affected patients from the three families were heterozygous for this candidate mutation, consistent with the dominant inheritance of type IIA vWD. This substitution was not observed either in the unaffected family members or in 50 normal controls.
Screening
In one type IIA vWD family, the Va1(902)+Glu substitution was not observed in the unaffected father (CC), mother (CCV), or sister (CV) (Fig 3) . The assessment of patemity in this family was performed by HLA-DR genetic typing" and with the polymorphic marker VNTR pYNZ22 (DI 7S5).'* The results showed that patient CR inherited two different HLA-DR alleles (one paternal and one maternal) and the same VNTR allele from both parents, indicating that she is almost certainly the daughter of parents CC and CCV. Thus, the Va1(902)+Glu change appears to be a de novo mutation in this family. Candidate mutation Arg(S34)+Gln. A representative affected member of the fourth type IIA vWD family was heterozygous for the transition of G(4790)+A. This changes the encoded amino acid sequence from Arg to Gln at residue 834 (Fig 4) . This candidate mutation was detected only in the affected patient and not in 50 normal controls by AS0 hybridization (Fig 5) .
Screening
Candidate mutation GI.v@44)+Arg. A representative affected member from the fifth type IIA vWD family was heterozygous for the transition of G(4825)+A. This changes the encoded amino acid sequence from Gly to Arg at residue 846 (Fig 6) . This candidate mutation was detected only in the affected patient and not in 50 normal controls by AS0 hybridization (Fig 7) .
DISCUSSION
Screening of genomic DNA from 12 patients with type IIA vWD by a nonradioactive AS0 hybridization method identified five type IIA patients who did not contain a previously reported mutation. By further sequencing, three additional candidate missense mutations were detected within the Az domain encoded by exon 28. The mutations were confirmed by AS0 hybridization in the affected patients and excluded in unaffected relatives and 50 normal controls. One potential missense mutation resulting in Va1(902)+Glu substitution occurred independently in three unrelated patients. The Gly(846)+Arg and Arg-(834)+Gln substitutions each occurred in a single patient.
DISCUSSION
Since these studies were performed, four additional vWD type IIA mutations were reported: one ofthem was identical to the Arg(834)+GIn mutation identified in one ofour families, suggesting that it occurred independently. This mutation could be explained by deamination of methylcytidine in a CG dinucleotide, causing a C+T transition on the noncoding strand. Such methylated CG dinucleotides are often hot spots for mutation in the human genome.29 C+T transition in the same CG dinucleotide, but on the coding strand, results in the substitution Arg(834)+Trp, which is the most common vWD type IIA The Gly(846)+Arg candidate mutation can also be explained by a C+T transition in a CG dinucleotide on the noncoding strand.
DISCUSSION
In one type IIA vWD family (Figs 2 and 3, Table I ), the Va1(902)+Glu substitution was not detected in the unaffected parents. Since correct assignment of parentage was supported by studies with DNA polymorphic markers, the Va1(902)+Glu mutation in the affected daughter (CR) may represent a new mutation. Germinal mosaicism for the mutation in one of the parents could also account for the de novo appearance of the mutation, as has been reported for one type IIB vWD family.m Among the three families in the present study, the Va1(902)+Glu candidate mutation appears to have occurred independently at least twice.
DISCUSSION
The results of this study together with previous reportsSI2 indicate that the vast majority of type IIA vWD mutations cluster within A, domain of vWF. These mutations appear to cause vWD by at least two mechanisms. Some directly impair the biosynthesis of vWF multimers." Others are . . . expected to impair vWF multimer biosynthesis or stability, and thereby cause a phenotype-like vWD type IIA, this a p parent clustering of type IIA mutations requires an explanation. It is possible, for example, that sequence variations in domain A, may not be causative mutations, but may simply be linked to mutations elsewhere. This is not a problem for mutations that affect multimer biosynthesis, because the corresponding mutant recombinant proteins have an a p propriate abnormal phenotype. It is a potential problem for candidate mutations that cause no apparent abnormal phenotype upon expression as recombinant mutant proteins.
DISCUSSION
However, substantial indirect genetic evidence indicates that mutations in the A, domain of vWF do cause vWD type IIA without directly impairing vWF multimer biosynthesis. The evidence includes the following: (1) candidate mutations have not been found in a single unaffected relative or normal control; (2) identical candidate mutations have been found repeatedly in unrelated patients who otherwise have distinguishable vWF genes, indicating multiple independent origins; (3) candidate mutations and vWD type IIA have appeared together de novo in a single generation; and (4) amino acid residues affected by candidate mutations are highly conserved. For example, sequence for pig vWF recently was reported that corresponds to amino acid residues 473 to 891 of human vWF.~' Fourteen of 15 type IIA mutations (including the mutations in the present study) occur in this region, and all 14 of them affect amino acid residues that are identical in human and pig vWF. Similarly, a partial bovine vWF cDNA sequence was reported that corresponds to amino acid residues 262 to 777 of human vWF" within this segment are five amino acid residues altered by type IIA vWD mutations, and all are conserved in the bovine sequence.
DISCUSSION
vWD is an extremely heterogeneous disorder, with more than 20 distinct clinical subtypes d e~c r i b e d . '~.~~~' The complexity of vWD classification and the limitations of the current diagnostic tests suggest that a convenient screening assay for known DNA mutations could be useful. As described herein, a nonradioactive "reverse" AS0 hybridization method permits the rapid screening of patient samples 

{'paper_id': '46176325', 'title': 'Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method.', 's2_url': 'https://api.semanticscholar.org/CorpusID:46176325', 'source': 'S2ORC'}