British journal of Haematology. 1991, 78, 506-514

ADONIS

0007104891001730

Identification of two point mutations in the von Willebrand factor gene of three families with the 'Normandy' variant of von Willebrand disease GAUCHER,BERNARDM E R C I E R , S Y L V I E JORIEUX, DRISSO U F K I R A N D C L A U D I N E MAZURIER Laboratoire de Recherche sur Z'He'mostase, Centre Regional de Transfusion Sanguine (CRTS. Directeur: Dr 1. 1. Huart), Lille. France

CHRISTINE

Received 4 February 1991; acceptedfor publication 1 5 April 1 9 9 1

Summary. Plasma von Willebrand factor (vWf) is a multidomain multimerized glycoprotein which has a dual role in haemostasis: it promotes platelet adhesion to subendothehum and is the carrier of blood coagulation factor VIII (FVIII). We previously characterized a functional defect of vWf. limited to its ability to bind FVIII. in two families whose affected members have the same phenotype that mimics mild haemophilia A and was tentatively named von Willebrand's disease (vWD) 'Normandy'. A homozygous point mutation C-T converting Thr 28 to Met in mature vWf subunit was identified in one of these patients who was born of thirdcousin parents. In the present studies we report two unrelated new cases of vWD 'Normandy' and characterize, using the analysis of the vWf gene intron 40 region containing a variable number of tandem repeats, the recessive inheritance of the disease in two affected families without known consanguinity. Exons 18-24 of the vWf gene encoding for the first 31 1 amino acids of mature vWf subunit were amplified by the polymerase chain reaction method and

sequenced. Two new missense mutations, both corresponding to a C+T transition and predicting respectively an Arg 53 +Trp and an Arg 91 +Gin substitution, were characterized. The three patients from family 1 were homozygous for the first-mentioned mutation while the patient from family 3 was homozygous for the second. The patient from family 2 was found a compound heterozygote for the two mutations. None of the two point mutations reported, both destroying a MspI restriction site, could be detected in DNA from 50 normal controls screened by restriction endonuclease analysis. Our data show that different mutations may be found in patients with the 'Normandy' phenotype. The mutations characterized so far are all localized on the N-terminal region of mature vWf subunit, within or near the major FVIII binding domain, and some of them occur within the epitope of monoclonal antibodies inhibiting the vWf/FVIII interaction. These observations suggest a causal relationship between these mutations and the vWD 'Normandy' phenotype.

In addition to its major role in platelet adhesion to subendothelium, von Willebrand factor (vWf) is known to serve in plasma as a carrier for factor VIII (FVIII) (Owen & Wagner, 1977: Hoyer. 1981). This vWf/FVIII association has been shown to be of critical importance in stabilizing 'in vitro' FVIII coagulant activity (Weiss et al. 1977) and prolonging 'in vivo' FVIII half-life (Tuddenham et al. 1982). Along with platelet membrane glycoproteins. collagen and heparin binding domains (Mazurier, 1989). a major FVIII-binding site has been characterized on the 2050 amino acids (aa) of the mature vWf subunit. It is present on the SpIII (aa 1-1365)

but not on the SpI (aa 911-1365) or SpII (aa 1366-2050) fragments of vWf obtained by digestion with SV8 protease (Girma et al, 1986) and has been localized more precisely on the tryptic fragment Sp IILT4 corresponding to the first NH2terminal 272 aa of mature vWf (Foster et al, 1987). Von Willebrand disease (vWD). the most common inherited bleeding disorder, is highly heterogeneous. Both quantitative (vWDtypes I and 111)and qualitative (vWDtype 11) changes in vWf (Berkowitz et al, 1989) cause the disease and many phenotypic subtypes of the variant form of vWD (i.e. vWD type 11) have been defined, based mainly on the multimeric profiles of plasma and platelet vWf (Ruggeri & Zimmerman. 1987). As the human vWf gene spans 178 kb and contains 52 exons transcribed as a 9 kb messenger RNA (Ginsburg et al. 1985: Mancuso et al. 1989), only a few of the vWD

Correspondence: Dr Claudine Mazurier. Centre Regional de Transfusion Sanguine (CRTS).2 1 rue Camille Guerin, 5901 2 Lille Cedex. France.

506

Two N e w Candidate Mutations in vWD Normandy

507

Table 1. Biological data for the five patients belonging to three families Von Willebrand factor

Factor VIlI (Itl/dl)

Family, member Family 1, patient 111.1 Family I , patient 111.3 Family 1 . patient 111.5 Family 2 . patient 11.2 Family 3 , patient 11.3’

Bleeding time (min) Coagulant activity Antigen vWf:Ag (one-stage) (IRMA) (IU/dl) (Duke’smethod) 1.5 -

2 3 3.5

Normal range ( 5 0 blood donors) Q 4

6.5 6.3 x.3 22 15

7.0 5.5 18.5 27.5

57-1 75

44-186

6.0

100 150 110 115 55 50-175

vWf:RCo HMW (%)t (IU/dl) 140 150 130 112 65 50-175

73 84 78 74 59

(70) (70) (70) (75) (63)

55-75

Ability to hind FVIII (

10-4)$

-7.6 (485) 1.9 ( 3 1 0 ) 1.9 (310) - 4 . 2 (485) - 1 . 1 (485)

120-560

* Studied only once.

t Percentages of high molecular weight (HMW) forms of vWf (multimers 2 5) are measured as described in Mazurier et al (1990a). The percentage obtained for a pool of citrated normal plasmas studied in the same electrophoretic run is in parentheses. $ The results are expressed as the slopes of the regression lines calculated with the grouped data obtained in a given experiment as reported in the Method section and detailed previously (Mazurier et a/. 1990b).The value obtained for a pool of citrated normal plasmas studied in the same experiment is in parentheses. variants have been characterized so far at the DNA or RNA level. For vWD type 111, total or partial deletions have been reported (Shelton-Inloes et al, 1987; Ngo et a / . 1988: Peake et al, 1990), while single point mutations have been found in vWD types IIA (Ginsburg et a / , 198 5; Lyons et a/, 1989) and IIB (Cooney et al. 1990: Murray et al. 1990). Recently we described two families (Mazurier et al, 1990a. b) with a new vWD variant form, tentatively named ‘Normandy’. characterized by a normal bleeding time and the absence of any plasma or platelet vWF quantitative change or multimerization abnormality, but associated with a FVIII deficiency resulting from the vWf inability to bind FVIII. This new vWf defect, strictly limited to its functional interaction with FVIII. may be responsible for the FVIII deficiency observed in some patients previously misdiagnosed as mild haemophiliacs or haemophilia A carriers with extreme lyonization and stresses the need to reinvestigate such cases (Mazurier et a/. 1990b). The first reported patient with ‘Normandy’ vWD was shown to have a single point mutation ACG+ ATG, changing Thr 2 8 of mature vWf into a Met, presumably responsible for its FVIII binding defect (Gaucher et al. 1991). This paper reports the characterization of two other point mutations, also altering the 272 aa NH2-terminal region of mature vWf subunit, which probably account for the FVIII binding defect of vWf observed in the three patients belonging to the second family previously described (Mazurier e t a / , 1990b) and in two other non-related patients with the ‘Normandy’ variant form of VWD. MATERIALS AND METHODS Patients. Family I. For a complete data report on this family of Portuguese origin living in Northern France refer to an earlier publication (Mazurier et a / . 1990b). The biological data for the three affected members are reported in Table I and their family tree is shown in Fig 1A. Family 2. The patient is a 24-year-old woman living in northern France whose mother is of Portuguese origin. She

has a personal history of bleeding: epistaxis, menorrhagia. haemorrhages after tooth extraction and after her first delivery. On the other hand, she did not bleed after tonsillectomy and after her second delivery. Her biological data are reported in Table I. Her two sons, her parents and her two sisters (see family tree in Fig 1B) have no haemorrhagic syndrome, normal levels of FVIII, vWf antigen (vWf:Ag) and vWf ristocetin cofactor activity (vWf:RCo) and normal bleeding time. Family 3. The patient is a 33-year-old woman living in central France. She has moderate bleeding symptoms including haematomas and long bleeding after injuries. Her biological data are reported in Table I. Her parents were second cousins. One of her two brothers died from a haemorrhage after a car accident. Her father, her second brother and her daughter (see family tree in Fig 1C) were said to have no haemorrhagic syndrome. Blood samples from her relatives were not available. Antibodies against vWF. Polyclonal antibodies against human vWf were raised in rabbits as previously described (Mazurier et al, 1979). Monoclonal antibody (MAb) 33E12 and MAb 333A9 directed against a n epitope localized on the SpII and Spl vWf fragments respectively are used for FVIII binding assays: neither influences the binding of FVIII to vWf in concentrations of up to 0.1 mg/ml. MAb 239, prepared in collaboration with Immunotech (Marseilles, France), recognizes all the multimeric forms of vWf (Mejan et al. 1988). Two other monoclonal antibodies prepared in our institute and reacting with SpIII-T4 tryptic fragment were also used: MAb 175-35A8 completely inhibits FVIII binding to vWf and recognizes both unreduced and reduced SpIILT4 while MAb 175-3183 reacts only with unreduced SpIILT4 fragment. Phenotypic analysis. Plasma from patients and relatives was studied for FVIII and vWf by the standard procedures already reported (Mazurier et a/, 1990a). FVIII binding to vWf was carried out as previously described (Mazurier et al. 1990b). In brief, immobilization of plasma vWf was performed by incubating serial dilutions of

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Christine Gaucher et a1

I

II

Ill Familv 2

G 'H

I

4 Family 3

Q+

0

Fig 1. Trees of families 1 . 2 and 3. The members with haemorrhagic diathesis are shown with solid symbols: those whose plasma vWf gave FVIII binding values intermediate between the values for normal individuals and affected patients are represented with halffilled symbols: t:deceased. Blood samples from the family 3 members were not available but note that the patient's parents were second cousins (- -). The capital letters are allelic codes for the vWf polymorphism identified after Alu I digestion of the PCR-amplified VNTR region of vWf intron 40 as detailed in Materials and Methods. Stars indicate the intron 40 alleles associated with a mutation of the vWf gene.

plasma on a microtitration plate coated with MAb 33812. Following washing with 0.4 M CaCL purified FVIII was added and incubated for 2 h at 37°C. The amount of FVIII bound onto immobilized vWf was then quantified after washing by adding the reagents of a chromogenic assay of FVIII :C (Diagnostica Stago, France). Lastly, after washing, the amounts of vWf bound to the wells were controlled by incubation overnight at 4°C with lz5I-MAb 333A9. The results were expressed in 10-4IU FVIII: C bound as a function of the radioactivity bound (cpm) reflecting vWf immobilized.

In each experiment the regression lines for normal and tested plasmas were established and their slopes were compared. Analysis oj plasmin vwjfragments. vWf was immunoisolated from normal and patients' plasmas with anti-vWf MAb 239 which was coupled to benzoquinone-activated Sephacryl-S1000 beads (Brandt et a/, 1975) at a ratio of 0.8 mg of antibody/ml of beads. 100 pI of beads were incubated with plasma samples containing 0.75 U vWf:Ag. for 2 h at 4OC with gentle stirring. After incubation, the beads were washed using the same buffers as described by Zimmerman et a1 (1986). The beads were then suspended in 100 j t l 0.02 M Tris. 0.15 M NaCI, pH 7.4, and heated for 1 5 min at 60°C. After cooling. plasmin (0.046 U ) was added to each gel sample which was then incubated overnight at 37OC. After boiling for 5 min with 2% sodium dodecyl sulphate (SDS),the beads were removed by centrifugation at 12 000 g. The resulting supernatants were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 3-1 6% gradient gels under unreduced conditions. The gels were blotted electrophoretically onto nitrocellulose (Towbin et al, 1979) and the transferred proteins were specifically revealed using either '251-radiolabelled polyclonal antibodies or MAbs against vWf SpIILT4 followed by 'z51-radiolabelledgoat antimouse IgG (Amersham. England) and autoradiography. Analysis of v W j intron 40 VNTR polymorphism. Direct 'labelled' amplification (Mercieret al. submitted) of the region of vWf gene intron 40 containing a variable number of tandem repeats (VNTR) was performed under the following conditions: for a 100 PI reaction mixture 0.3-0.5 pg of genomic DNA, 50 pmol ofeach specific primer, 185 kBq of j5S dATP and 1 5 PM of each deoxynucleotide triphosphate (dNTP) were mixed with the Taq polymerase l o x buffer provided by the manufacturer (Amersham, England). After the initial denaturation step, at 94OC for 5 min, 2.5 U of Taq polymerase were added before starting the 30 PCR cycles (94OC for 1 min, 6OoC for 1 min, 72OC for 1 min). The PCRlabelled products were digested with Alu I restriction endonuclease (Appligene. Illkirch. France) according to the manufacturer's instructions, electrophoresed on a 6% sequencing gel and autoradiographed overnight. Because of the presence of two constant Alu I restriction sites in the amplied product, each vWf intron 40 allele corresponded to the combination of three restriction fragments. In order to facilitate the allele identification and segregation analysis, each allele was identified by an arbitrary letter code depending on the length of each restriction fragment. PCR amplification of genomic DNA. All PCR amplifications (Saiki et a / , 1988)were performed on genomic DNA extracted from peripheral blood leucocytes according to the method described by Miller et a1 (1988). Amplifications were performed using Taq polymerase in a reaction mixture containing 0.5 pg of genomic DNA, 100 pmol of each primer, 200 p~ of each dNTP and the Taq polymerase 10 x buffer provided by the manufacturer (Amersham, England). Before addition of the Taq polymerase (2-2.5 U / l O O pl reaction), the reaction mixture was heated at 94°C for 5 min to allow proper genomic DNA denaturation. Thirty amplification cycles were subsequently carried out under standard conditions: 94OC for 1 min, 55OC for 1 min, and 72°C for 2 min.

Two N e w Candidate Mutations in vWD Normandg After amplification. the PCK products were electrophoresed on a 2% agarose gel and purified, when needed, on DEAE cellulose paper during the electrophoresis (Mack et al. 1990) to remove unincorporated primers and dNTP. Each set of primers was designed according to the sequence previously published (Mancuso et al. 1989) to allow amplification of the intron sequences adjacent to the exon coding sequences. PCR product direct sequencing. Single-stranded DNA suitable for sequencing was obtained by performing a second PCR amplification of 2 5-30 cycles on double-stranded PCK product as a template with only one of the two primers used in the first PCK reaction. Before sequencing, the singlestranded products were purified on Centricon 100 (Amicon Corp.. Lexington. [J.S.A.) in order to remove unincorporated primers and dNTP. Sequencing was performed with a Sequenase kit (IISB. Cleveland) using PCR primers and $’SdATP (Amersham. England) as a label. The samples were analysed on a by, sequencing gel. Restriction errdonuclease analysis. PCK-amplified exon 19 and 20 fragments were directly digested after amplification with Msp I restriction endonuclease (Boehringer. Mannheim. Germany) according to the manufacturer’s instructions and separated on a 12% polyacrylamide gel. After electrophoresis, the gels were stained with ethidium bromide for direct visualization under UV light. RESULTS Phenotypic arrdysis The main biological data for the patients are reported in Table I. All of them are characterized by FVIII deficiency with normal bleeding times and normal levels of vWf:Ag and vWf: KCo. Furthermore, the multimeric pattern of vWf and its ability to bind to either normal platelets in the presence of ristocetin or to collagen were normal for all five patients (data not shown). On the other hand, the vWf of all affected patients failed to bind FVIII (right column, Table I). All the patients’ relatives studied had a negative bleeding history, normal bleeding time and normal vWf levels. Except for the father (11.1) of the three patients belonging to family 1 (for detailed results see Mazurier et a / , 1 990b), all of them also had normal FVIll levels. However, FVIII binding assays performed on plasma samples showed in some of them (represented by half-filled symbols in Fig 1) a reduced ability of their vWf to bind normal purified FVIII. The slopes of the regression lines obtained with their grouped data in the FVIII binding assays were about half those obtained with normal plasmas and similar to the slope values obtained with the mixture (1 vol: 1 vol) of normal plasmas and affected patients’ plasmas (Fig 3 from Mazurier et a / , 1990b).

Analysis of vwfplasmin fragments The fragments resulting from plasmin digestion of immunopurified plasma vWf were analysed for the three patients of family 1 and for the patient of family 2 , for whom a sufficient amount of plasma was available. As shown in Fig 2. the vWf of these patients displayed a plasmin digest electrophoretic pattern similar to normal vWf. The smaller peptides revealed by either anti-vWf polyclonal antibodies or anti-SpIII-T4

1 2 3 4 5

1 2 3 4 5

509

1 2 3 4 5 kDa

-100

-68

-43

-29

PAb

17535~8

175 3 1 ~ 3

Fig 2. Electrophoretic analysis of the plasmin digests of different plasma samples immunoadsorbed on anti-vWf MAb 2 39-coupled gel. 1: pool of 50 normal plasmas. 2: plasma from the first reported case of vWD ‘Normandy’ (Mazurier ct al. 1990a). 3: individual normal plasma. 4: plasma from patient 111.1 from family 1 (similar patterns were obtained with the plasma from his sisters, 111.3 and 111.5).5: plasma from patient 11.2 from family 2. Electrophoresis was performed in 3-16% polyacrylamide gradient gel and under nonreducing conditions.Separatedproteins were then electroblotted and revealed with either radiolabelled anti-vWf polyclonal antibodies (PAb)or MAbs 175-35A8 and 175-3183 directed against the SpIIIT4 fragment of vWf. Note that the mobility shift (apparent molecular weight 31-29 kDa) observed in the fastest migrating plasmin fragment of the patient previously reported (Mazurier et al. 1990a: Gaucher et al. 199 1) is not characterized in the family 1 and 2 patients’ plasma samples. MAbs migrated as a single band or a doublet with an apparent molecular weight of approximately 3 1 kDa. On the other hand, like the trypsin fragment (Gaucher et a / , 1 99 1 ), the plasmin fragment of the first reported case of vWD ‘Normandy’ exhibited a mobility shift and an apparent molecular weight of around 29 kDa.

vWf gene segregation analysis In the three families the Alu I restriction pattern of the VNTK region of the vWf gene intron 40 was determined for all the family members whose blood samples were available. From both pedigree data and restriction pattern analysis we were able to attribute to each individual a combination of two alleles and study their segregation (Fig 1). Family 1 . The three affected members 111.1. 111.3 and 111.5 were all characterized by the association of the alleles Band E. inherited respectively from their mother (11.2). BC, and their

5 10

Christine Gaucher et a1

Fig 3 . Part of the nucleotide sequence gels of PCR-amplified vWf exon 19 in (a)normal control individual: (b) family 1 patient, 111.1: (c)family 1 patient’smother, 11.2: (d)family 2 patient, 11.2:and (e)family 2 patient’smother, 1.2.The asterisk indicatesthe point mutation: the numbers refer to the position of correspondingamino acids in the mature protein subunit.

Fig 4. Part ofthe nucleotide sequence gels of PCR-amplified vWfexon 20 in (a)normal control individual,(b)family 3 patient, (c)family 2 patient’s father, 1.1, (d) family 2 patient, 11.2. The asterisk indicatesthe point mutation: the numbers refer to the position of correspondingamino acids in the mature protein subunit.

father (ILl), EF. On the other hand, the two non-bleeding children were found to have a different allelic combination with genotypes CE (111.2) or CF (111.4).From both this vWf gene segregation data and the previous phenotypic analysis we can conclude that the disease in this family is associated with the alleles B and E and transmitted according to a recessive mode. Family 2. The patient was characterized by the alleles G and I. inherited respectively from her father (1.1) GH, and her mother (1.2),IJ, while her two unaffected sisters shared the same genotype HJ. The data suggest that the defect is associated in this family with both alleles G and I and that the disease was transmitted according to a recessive mode, given the absence of FVIII deficiency in the two patients’ sons. Family 3. The only member available in this family was found to display a three band Alu I restriction pattern (276. 252 and 236 bp) characterizing only one single allele. This profile is consistent with either an homozygous status for this patient or an heterozygous status with one allele null. one of the two vWf genes being partially or totally deleted. Unfortunately the lack of material from the patient’s relatives prevents us for the moment from verifying our hypothesis. DNA sequencing Direct sequencing of each of the PCR-amplified exons 18-24. covering the N-terminal region of mature vWf up to a a 3 1 1, was performed on both sense and antisense strands for all the patients of each family as well as their parents when available.

In patients 111.1. 111.3 and 111.5 from family 1 the DNA sequence analysis showed a single base mutation CGG-+TGG in exon 19 a t codon 816, predicting a n Arg to Trp substitution at aa 53 of the mature vWf. The patients were found to be homozygous for this substitution (for example see 111.1 in Fig 3b) while their parents were both heterozygous for the mutation (for example see 11.2 in Fig 3c). The same base mutation in exon 19 was also found in one allele of the patient (11.2)from family 2 (Fig 3d). Furthermore, a second single base mutation CGG-CAG in exon 20. at codon 854. changing Arg 91 of mature vWF into a Gln (Fig 4d), was identified in this patient. This patient appears to be a double heterozygote. having inherited both mutations from her parents: her father (1.1) is heterozygous for the mutation in exon 2 0 (Fig 4c) and her mother (1.2)is heterozygous for the mutation in exon 19 (Fig 3e). Finally, the exon 20 CGG-CAG mutation was also detected in the patient from family 3 who is apparently homozygous for this G-*A transition (Fig 4b). Restriction endonuclease analysis The two mutations CGG-TGG and CGG-CAG both destroying a Msp I restriction site CCGG, the PCR-amplified exons 19 and 2 0 of all the patients, their relatives and controls were digested with Msp I in order to confirm and complete the sequencing data and also search for possible identical mutations in normal individuals. On the basis of the published DNA sequence (Mancuso et al. 1989) both PCK

511

Two New Candidate Mutations in vWD Normandy i n t r o n 19

exon 19

i n t r o n 18

I

I

r

Msp I 8

Msp I polymorphic

I

I

A bp

4 2 bp

125 bp

5 9 bp

3

1 2 3 4 5 6 7 E

3

I

C

B

1 2 3 4 5 6 78

€ 1 2 3 4 5

bp -184 -1 6 7

164-

167-1 2 5

125-

-59

59-42 42

-

Fig 5. Msp I restriction fragment analysis of PCR-amplified vWf exon 19. Top = restriction pattern, the asterisk indicates the site destroyed by the mutation. Bottom =digested fragments run on a 12% polyacrylamide gel; mw=pBR 322 Hae 111 molecular weight marker. A: family 1 patients It1.5. 111.3 and 111.1 (lanes 1-3). patients' unaffected brother. 111.2 (lane 4). patients' mother, 11.2 (lane 5). patients' unaffected sister, 111.4 (lane 6) and patients' father, 11.1 (lane 7). B: family 2 patient, 11.2 (lane 1 ) . patient's unaffected sisters, 11.3 and 11.4 (lanes 2 and 3). patient's mother, 1.2 (lane 4). patient's father. 1.1 (lane 5). C: lanes 1-8: normal control individuals (the controlled DNA in lane 2 is homozygous for the absence of the Msp I polymorphic site in intron 19. as shown by the absence of the 12 5 bp restriction fragment).

3 E1

2 3 4 567

3 E

bP -260 -163

e x o n 20

i n t r o n 19

i n t r o n 20

I

-97198

I

Msp I

Msp I

I I

I

I

1

163 bp

97 bp

98 bp

Fig 6. Msp I restriction fragments analysis of PCR-amplified vWfexon 20. Left = restriction pattern. the asterisk indicates the site destroyed by the mutation. Right=digested fragments run on a 12% polyacrylamide gel; mw=pBR 322 Hae 111 molecular weight marker. Family 2 patient 11.2 (lane 1 : the bands, not clear on thc photograph, are the same as on lane 5 = 260. 163. 98 and 97 bp). patient's unaffected sisters 11.3 and 11.4 (lanes 2 and 3). patient's mother 1.2 (lane 4). patient's father 1.1 (lane 5). family 3 patient (lane 6). normal control individual (lane 7).

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Christine Gaucher et al

products were expected to contain a second Msp I restriction site providing an internal control for complete enzyme digestion (Figs 5 and 6). However, the second Msp I restriction site of the exon 19-PCR fragment, localized in the intron 19 portion of the PCR product, was found to be polymorphic in the DNAs tested (Mercier et a / , 1990). Exon 19-PCRJrugrnent. An undigested pattern (two restriction fragments of 184 and 42 bp) was found in patients 111.1. 111.3 and 111.5 from family 1 keeping with their homozygous state for the mutation (Fig 5A). Meanwhile the patients’ mother (11.2), father (11.1) and unaffected brother (111.2), exhibited both undigested ( 1 8 4 + 4 2 bp) and digested (125+ 5 9 + 4 2 bpor 167+59 bp)restrictionpatternsconsistent with a heterozygous state for the mutation. The patient’s unaffected sister (111.4) showed both digested alleles ( 1 2 5 + 5 9 + 4 2 bpand 1 6 7 + 5 9 bp)(Fig 5A)consistentwith the absence of the mutation. The presence of the 167 bp restriction fragment in both 11.1 and 111.4indicated that their common normal vWf gene does not carry the polymorphic Msp I site. After Msp I digestion, the patient 11.2 from family 2 and her mother (1.2) displayed a heterozygous digested/ undigested pattern consistent with our sequencing data (Fig 5B). On the other hand, her sisters (11.3 and 11.4),her father (1.2) (Fig 5B) and all the 5 0 control DNAs tested (Fig 5C) presented homozygous digested patterns foi, this restriction site: three fragments of 125. 59 and 4 2 bp or two fragments of 167 and 59 bp being observed depending on the presence or absence of the polymorphic MspI site in intron 19. Exon 20-PCRJragrnent. MspI digestion of amplified exon 2 0 in the family 2 (Fig 6) also confirmed our sequencing data: the patient (11.2) and her father (1.1) were found to be heterozygous with both undigested (260 and 98 bp restriction fragments) and digested (163 97 98 bp) patterns. Meanwhile the patient’s mother (1.2). mutated in exon 19, exhibited a homozygous digested pattern for exon 2 0 (163 + 9 7 + 98 bp) also found in the patient’s unaffected sisters, 111.3 and 111.4. On the other hand, the patient from family 3 showed a n undigested restriction pattern (260 98 bp) in keeping with her possible homozygous status (Fig 6). Finally, all the 5 0 PCR-amplified fragments from control DNAs presented a digested pattern with three restriction fragments of 163. 97 and 98 bp.

+ +

+

DISCUSSION The present study describes two new unrelated cases of vWD ‘Normandy’ together with the further investigation of a previously reported family. It enabled us to define the general mode of inheritance as well as the phenotypic and genotypic expressions of this new variant form of vWD that mimics haemophilia A. Two complete family studies were performed, combining the biological and biochemical analysis of plasma vWf, the vWf gene segregation study using intron 40 polymorphism, and the identification of the potential mutations both by direct sequencing and by MspI digestion of PCR amplified exons. We thus confirmed the recessive mode of transmission of vWD ‘Normandy’ suggested in earlier reports (Mazurier et al. 1990a, b; Gaucher et nl, 1991)as only homozygotes or compound heterozygotes exhibited a FVIII

deficiency. Indeed. all the characterized heterozygotes but one (family 1. member 11.1). showed normal FVIII levels associated with intermediate values regarding the ability of their plasma vWf to bind FVIII. From the phenotypic data it appears that some individual variability may occur in the expression of vWD ‘Normandy’, as revealed by the comparison of the FVIII levels measured in the different individuals tested. These variations do not appear to be linked to the vWf defect involved, as the vwfcapacity to bind FVIII did not differ significantly from one family to another. Some heterozygotes for the same mutation, such as family 1 patients’ father (11.1 ) and brother (111.2), present different phenotypic expressions (i.e. FVIII level, see Mazurier et a / . 1990b), suggesting that other parameters, influencing the FVIII synthesis and/or catabolism, may be responsible for this individual variation. The analysis of the N-terminal plasmin vWf fragment of the five patients investigated so far also demonstrates the heterogeneity of their defect: unlike the first reported example of ‘Normandy vWf (Gaucher et a!, 1991). no abnormal electrophoretic mobility was detected when analysing the affected members of families 1 and 2. This finding indicated that different mutations must cause the disease and this was confirmed by our sequencing data. Indeed, the sequencing of exons 18-24 in these patients revealed two missense mutations, Arg 5 3 to Trp and Arg 91 to Gln. different from the Thr 28 to Met substitution found in the first reported case (Gaucher et a/, 1991). These two mutations, changing a CGG codon into a TGG or CAG codon. both result from the C to T transition a t a potential CpG dinucleotide mutational hotspot (Barker et al, 1988). either on the sense or antisense DNA strands. As both mutations destroy a CCGG MspI restriction site, the accuracy of the sequencing data was conveniently verified by restriction digest analysis of the DNA of all members of the two families investigated. The three patients from family 1 were found to be homozygous for the Arg 5 3 to Trp substitution while their parents and unaffected brother were heterozygous for the mutation. Their remaining unaffected sister inherited a normal vWf gene from both parents. as confirmed by the intron 40 polymorphism segregation data. It was quite surprising to find the same mutation in both parents, as no consanguinity was known in this family and affected alleles of the vWf gene were different when characterized using the intron 40 polymorphism. However, we cannot rule out the existence of a remote common Portuguese ancester bearing this mutation, both parents originating from the same village in Portugal. Indeed, the observed sequence variations within the intron 40 VNTR might have arisen after the Arg 53 to Trp mutation occurred. The patient of family 2 was found to harbour the two mutations. inheriting the Arg 5 3 to Trp substitution from her mother and the Arg 9 1 to GLn change from her father. Here again, the presence of the mutation found in family 1 in the family 2 propositus’s mother, also originating from Northern Portugal, may indicate common ancestors. The last patient investigated was homozygous for both the Arg 91 to Gln substitution and the intron 4 0 polymorphism. In light of the consanguinity of this patient’s parents, she is likely a homozygote for the defective vWf gene. The failure to find similar mutations at Arg 53 and 91 in the 5 0 normal

Two New Candidate Mutations in vWD Normandy individuals screened by Msp I restriction analysis suggests that these substitutions do not represent non-pathogenic polymorphisms. Furthermore, the lack of a n y other change within the N-terminal 31 1 a a vWf region investigated also suggests that these non-conservative substitutions are likely the cause of the FVIII binding defect observed in these patients. Both mutations a r e localized in the 272 aa SpllLT4 fragment of vWf containing the major FVllI binding domain (Foster et a/. 1987). and in the 106 a a N-terminal polypeptide (Pietu et d.1 9 8 9 ) harbouring the epitope of MAb 418 which inhibits the FVIII/vWf interaction (Takahashi f f al. 1 9 8 7 ) . Furthermore, the Gln for Arg 9 1 substitution occurs within theepitope(aa 78-96)ofMAb W5-6Aalsoaninhibitorofthe FVIII binding to vWf (Bahou et a/. 1989). Nevertheless it is not yet possible to state whether these mutations are located within or near the N-terminal FVIII binding site of the mature vWf subunit. Indeed these alterations may be either functional, changing an aa directly involved in the FVIII interaction, or conformational. indirectly modifying the protein folding required for FVIII binding. Characterization of mutations in additional patients with the vWD ‘Normandy’ phenotype and the study of the corresponding recombinant mutant vWf will be required to elucidate this particular aspect of the vWf structure/function relationship. ACKNOWLEDGMENTS We thank Professor M. Goudemand for his interest and encouragement. We thank Drs P. Boulanger and A. ParquetGernez from CKTS, Lille, for their collaboration in the study of family 1. We thank Professor J. L. Demory from St Philibert Hospital, Lille. for referring the patient from family 2 to us and Dr J, Reynaud from CHU Hospital, Saint-Etienne, for sending the blood sample from the patient of family 3. We are indebted to Dr E. Sadler (Howard Hughes Institute. St Louis, Mo., [J.S.A.) for providing partial vWf gene sequence before publication (Mancuso et al, 1989). W e are grateful to Dr H. Broly (Laboratory of Cellular Engineering, CRTS. Lille) for his collaboration in preparing MAbs. We thank Mrs V. Duretz and Mr D.Hoguet for their excellent technical assistance and Mrs V. Dieval for typing the manuscript. REFERENCES Bahou. W F.. Ginsburg. I).. Sikkink. R.. Litwiller. R. & Fass. D.N. (1989) A monoclonal antibody to von Willebrand factor inhibits factor Vlll binding. lourrlal of Clinical hastigution. 84, 56-6 1. Barker. D.. Schafer. M. & White, R. (1988) Restriction sites containing CpC show a higher frequency of polymorphism in human DNA. Cell. 36, 131-138. Berkowitz. S.D..Ruggeri. Z.M. & 7immerman. T.S. (1989) von Willebrand disease. Coagulation and Bleeding Disorders. The role o/ factor VIII and von Willebrandjactor (ed. by T. S. Zimmerman and Z. M. Ruggeri), p. 2 1 5. Marcel Dekker. New York. Brandt. 1.. Anderson, LO. & Porath. J. ( 1 975) Covalent attachment of proteins to polysaccharide carriers by means of benzoquinone. Bioc‘hirnica et Biophysica Acta. 396, 196-202. Cooney. K.A.. Nichols, W.C.. Bruck. M.E.. Bowie. E.J.W.,Shapiro. A.. Gralnick. H.R. (G Ginsburg. D. (1990)The molecular defect in type IIB von Willebrand disease (vWD):identification of two potential missense mutations within the putative GPlb binding domain. Blood, 76, 418A (abstract 1661).

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