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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Autosomal dominant macrothrombocytopenia in Italy is most frequently a type
of heterozygous Bernard-Soulier syndrome
Anna Savoia, Carlo L. Balduini, Maria Savino, Patrizia Noris, Maria Del Vecchio, Silverio Perrotta,
Simona Belletti, Vincenzo Poggi, and Achille Iolascon
A form of autosomal dominant macrothrombocytopenia is characterized by
mild or no clinical symptoms, normal platelet function, and normal megakaryocyte
count. Because this condition has so far
received little attention, patients are subject to misdiagnosis and inappropriate
therapy. To identify the molecular basis of
this disease, 12 Italian families were studied by linkage analysis and mutation
screening. Flow cytometry evaluations of
platelet membrane glycoproteins (GPs)
were also performed. Linkage analysis in
2 large families localized the gene to
chromosome 17p, in an interval containing an excellent candidate, the GPIb␣
gene. GPIb␣, together with other proteins, constitutes the plasma von Willebrand factor (vWF) receptor, which is
altered in Bernard-Soulier syndrome
(BSS). In 6 of 12 families, a heterozygous
Ala156Val missense substitution was
identified. Platelet membrane GP studies
were performed in 10 patients. Eight were
distinguished by a reduction of GPs comparable to that found in a BSS heterozygous condition, whereas the other 2, without the Ala156Val mutation, had a normal
content of platelet GPs. In conclusion, the
current study provides evidence that most
(10 of 12) patients with an original diagnosis of autosomal dominant macrothrom-
bocytopenia shared clinical and molecular features with the heterozygous BSS
phenotype. The remaining 2 affected subjects represented patients with “true” autosomal dominant macrothrombocytopenia; the GPIb/IX/V complex was normally
distributed on the surface of their platelets. Thus, the diagnosis of heterozygous
BSS must always be suspected in patients with inherited thrombocytopenia
and platelet macrocytosis. (Blood. 2001;
97:1330-1335)
© 2001 by The American Society of Hematology
Introduction
Inherited thrombocytopenias were, in the past, considered
exceedingly rare. Today the widespread diffusion of electronic
cell counters has rendered the identification of these conditions
more common, and many asymptomatic patients are discovered
during routine blood analysis, often in adulthood. The hereditary
nature of the illness may be missed, and patients are subject
to misdiagnosis of autoimmune thrombocytopenic purpura
and inappropriate therapy, such as steroid treatment and
splenectomy.1-3
Different diagnostic parameters have been used to classify these
disorders, including degree of bleeding, inheritance trait, platelet
function and kinetics, and concomitant clinical abnormalities.4 The
simplest classification relies on mean platelet volume (MPV),
which separates macrothrombocytopenia from thrombocytopenia
with normal MPV. Despite the lack of epidemiologic data, clinical
practice suggests that the former are more frequent than the latter.
Patients with increased platelet size have been reported since
the nineteenth century.5 In the first half of twentieth century,
authors reported the genetic basis of some platelet macrocytoses
associated with thrombocytopenia and described 2 distinct entities,
Bernard-Soulier syndrome (BSS)6 and May-Hegglin anomaly
(MHA).7,8 Over the following 5 decades, the molecular basis of
BSS9 and MHA was clarified.10
Moreover, other forms of inherited macrothrombocytopenia
have been described—gray platelet syndrome, platelet-type von
Willebrand disease, Montreal platelet syndrome, macrothrombocytopenia with mitral valve insufficiency, familial macrothrombocytopenia with glycoprotein (GP) IV abnormality, Epstein, Fechtner,
and Sebastian syndromes—all with their typical distinguishing
features.11 However, these well-defined forms are rare, and most
hereditary macrothrombocytopenias remain poorly characterized
despite several case series. In 1975, von Behrens12 examined
platelet count and MPV in 145 apparently healthy subjects from
Italy and the Balkan peninsula, many of whom were affected by an
undefined form of macrothrombocytopenia. Because this abnormality was not detected in control subjects from northern Europe, the
condition was named Mediterranean macrothrombocytopenia. Subsequently, Najean et al2 retrospectively analyzed a series of patients
with thrombocytopenia who underwent platelet kinetic studies and
isolated 54 patients with chronic macrothrombocytopenia of unknown origin. This condition had an autosomal dominant transmission and was characterized by mild or no clinical symptoms,
normal bone marrow megakaryocytosis and platelet survival, and
normal in vitro platelet function. They named this condition genetic
thrombocytopenia with autosomal dominant transmission. More
recently, Fabris et al13 described 47 patients from 13 families with
From the Medical Genetics Service, IRCCS Hospital CSS, Foggia; the
Department of Internal Medicine, IRCCS San Matteo-University of Pavia; the
Department of Pediatrics, II University of Najoli; the Department of Pediatric
Hematology, Azienda Santobono, Pausilipon, Najoli; and the Department of
Biomedicine of Evolutive Age, University of Bari, Italy.
from the Italian Foundation of Cancer Research.
Submitted August 28, 2000; accepted November 7, 2000.
Supported by grants from the Italian Ministry of Health (A.S., C.B., A.I.),
MURST, and 60% University of Bari (A.I.). M.S. is the recipient of a fellowship
1330
Reprints: Carlo Balduini, Medicina Interna, IRCCS San Matteo, Piazzale
Golgi, 27100 Pavia, Italy; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
HETEROZYGOUS BERNARD-SOULIER SYNDROME
similar features who were reported as having chronic isolated
hereditary macrothrombocytopenia.
The molecular mechanisms that cause hereditary giant platelet
disorders are not known in detail except those for BSS. In this case,
giant platelets are caused by defects in the platelet glycoprotein
(GP) Ib/IX/V complex, a platelet receptor for von Willebrand
factor (vWF) and the major membrane GP system that interacts
with the platelet cytoskeleton.9 Patients with BSS were found to be
homozygotes or compound heterozygotes for mutations in the
GPIb␣, GPIb␤, or GPIX genes, 3 of the 4 distinct gene products of
the GPIb/IX/V complex. Only one family characterized by
mild clinical symptoms was described as having autosomal dominant BSS.14
To identify the molecular defect of autosomal dominant hereditary macrothrombocytopenia, we studied 12 consecutive patients
and their relatives. By evaluating platelet surface GPs and using
molecular biology techniques, we concluded that macrothrombocytopenia was compatible with a heterozygous status of BSS in 10
patients. Of particular interest, a missense mutation of the GPIb␣
gene was responsible for the illness in 6 families.
Patients, materials, and methods
Clinical findings and laboratory evaluation of patients
Twelve consecutive patients (TP-1 to TP-12) who had hereditary macrothrombocytopenia that was transmitted through an autosomal dominant
mechanism but who received no definitive diagnoses were enrolled in this
study from January 1999 to March 2000 (Table 1). The patients came from
12 unrelated families and required medical attention because of a mild
bleeding diathesis, incidental discovery of thrombocytopenia, and platelet
macrocytosis. Investigated subjects gave informed consent to the studies,
which were carried out in accordance with the Principles of the Declaration
of Helsinki.
1331
Platelet aggregation
For the study of in vitro platelet function, blood was collected in 3.8%
(wt/vol) sodium citrate (blood–anticoagulant ratio 9:1). To minimize the
loss of denser platelets, platelet-rich plasma (PRP) was obtained by
sedimentation rate of blood at 1g for 20 to 30 minutes; platelet-poor
plasma (PPP) was obtained by centrifugation of the remaining blood at
4500g for 20 minutes. The platelet count of PRP was adjusted to
150 ⫻ 109/L with PPP. Platelet aggregation was evaluated by the
densitometric method of Born15 after stimulation with collagen (4 and
20 ␮g) (Mascia Brunelli, Milan, Italy), adenosine diphosphate (ADP, 5
and 20 ␮M), and ristocetin (1.5 and 3.0 mg/mL), both from Sigma
Chemical, St Louis, MO, and the maximal extent of aggregation was
recorded. Values obtained in 20 healthy subjects were used to identify
the normal range.
Genome-wide search and linkage analysis of candidate loci
High-molecular–mass DNA was extracted from peripheral blood leukocytes in an automatic DNA extractor, according to the manufacturer’s
recommendations (ABI 341 GenePure; Applied Biosystems, Foster City,
CA). For genome-wide search, genomic DNA samples from patients
from 2 large families, TP-1 and TP-2, were used for linkage analysis
(Figure 1). Genotyping was performed using an ABI Prism 377 DNA
sequencer (ABI 377A) and the Linkage Mapping Set Version 2 (both
Applied Biosystems). This set comprises 400 fluorescently labeled
markers that define a 10-cM resolution human map. Prescreening was
performed using only the 8 affected DNA samples from family TP-1
(Figure 1). Microsatellite mapping excluded most of the genome and
identified a few loci at which patients had the same allele. The markers
of candidate regions were then tested in all pedigree members, including
those of family TP-2. Classic 2-point LOD score analysis was conducted
in these pedigrees under the assumption of autosomal dominant
inheritance and complete genetic penetrance. We used the MLink
program included in the Linkage package (Applied Biosystems) to
perform linkage analysis.16
Table 1. Main features of the patients studied
Family
(patients in the family)
Platelet count
⫻ 109/L mean
(range)
MPV fL mean
(range)
Clinics* (symptomatic
patients)
GPIb/IX/V
complex†
Bolzano
mutation
P, E, M (4)
Defective
Yes
P, E (6)
Defective
Yes
E (2)
Defective
Yes
M (1)
nd
Yes
P (1)
Normal
No
No
Defective
No
E (2)
Normal
No
E, M (3)
Defective
Yes
P, E (2)
nd
Yes
No
Defective
No
No
Defective
No
E (1)
Defective
No
TP-1 (8)
69
14.0
Figure 1
(50-82)
(13.7-14.5)
TP-2 (9)
88
12.9
Figure 1
(21-127)
(12.2-13.6)
TP-3 (2)
TP-4 (2)
TP-5 (3)
TP-6 (7)
TP-7 (3)
TP-8 (3)
TP-9 (2)
TP-10 (3)
TP-11 (2)
TP-12 (2)
94
11.7
(77-111)
(11.5-11.9)
74
12.7
(68-80)
(12.3-13.2)
49
16.1
(22-82)
(14.0-17.2)
140
11.6
(100-178)
(11.2-12.3)
90
11.4
(68-135)
(10.4-11.9)
58
13.9
(42-87)
(12.1-14.3)
45
12.9
(37-53)
(12.7-13.1)
119
11.4
(109-133)
(10.7-11.9)
143
14.1
(128-158)
(13.8-14.5)
120
11.5
(116-125)
(11.2-11.8)
*P, petechias; E, epistaxis; M, mucosal bleeding; No, no clinical findings.
†Defective means a reduction detected by flow cytometry; nd, nondetermined.
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1332
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
SAVOIA et al
Figure 1. Family pedigrees with haplotype reconstruction for informative markers on 17p. The at-risk haplotype is boxed.
Molecular analysis of the GPIb␣ gene
The GPIb␣ gene (GenBank accession numbers NM000173 and
AC004771) was completely sequenced in one patient from families
TP-1 and TP-2. The coding region, the untranslated exon 1, the donor
and acceptor splicing sites,17 and the promoter18 were amplified using
the following oligonucleotide sets: 1F (5⬘-TGGAGAGGTTTTTAAAAGATG-3⬘) and 2R (5⬘-ACCTTGCTTCCATACGTAGAC-3⬘); 3F
(5⬘-CCCCTGGTTATGCAACTGTG-3⬘) and 3R (5⬘-TGGATGCAAGGAGGAGGGCAT-3⬘); 4F (5⬘-GATTACTACCCAGAAGAGGACA-3⬘)
and 5R (5⬘-CACAGGCTCTTCTCTCAAGG-3⬘). To analyze the region containing the VNTR polymorphism better, two new primers
were synthesized, 7F (5⬘-ACACTTCACATGGAATCCAT-3⬘) and 7R
(5⬘-GGATTCTAAGAGTGATACGGGT-3⬘). To separate the alleles, the
polymerase chain reaction (PCR) product was electrophoresed on 3%
agarose gel before sequencing.
PCR was performed in 50 ␮L containing 50 ng DNA, 15 pmol each
primer, 2.5 mM MgCl2, 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.01%
Tween-20, 0.01% gelatin, 0.01% NP40, and 2 U Taq polymerase. Initial
denaturation was 5 minutes at 94°C followed by amplification for 30 cycles
with denaturation at 94°C for 30 seconds, annealing at 60°C for 45 seconds,
and extension at 72°C for 45 seconds. After electrophoresis, PCR products
were isolated and purified by GFX DNA and a Gel Band Purification Kit
(Amersham, Pharmacia, Biotech, Buckinghamshire, England). Sequence
analysis was performed using a fluorescence-labeled dideoxy-nucleotide
termination method (dye terminator) in the automated DNA sequencer ABI
377A (Applied Biosystems). Both PCR oligonucleotides and specific
internal primers were used in the sequencing reactions.
To confirm and detect the Ala156Val substitution, genomic DNA was
amplified using primers 2F (5⬘-CACCCCATCTGTGAGGTCTCC-3⬘) and
3R (see above). Because the substitution creates a restriction enzyme site
for HpaI,19 PCR products were purified, digested with the specific enzyme,
and electrophoresed on 2% agarose gel.
Flow cytometry
The expression of platelet membrane GPs was investigated by flow
cytometry with the following monoclonal antibodies (mAbs): SZ21 (Immu-
notech SA, Marseille, France), which recognizes GPIIIa (CD61); AP2
(Immunotech), which recognizes GPIIb/IIIa (CD41/61); MB45 (CLB,
Amsterdam, The Netherlands) and SZ2 (Immunotech) against GPIb␣
(CD42b); FMC25 (kindly provided by Zola H, Adelaide, Australia) and
SZ1 (Immunotech), which recognizes GPIX (CD42a); SW16 (CLB) against
GPV (CD42d); FA6-152 and Gi9 (both from Immunotech) against GPIV
(CD36) and GPIa (CD49b), respectively. MO2 (Coulter, Miami, FL) was
used as the negative control. Fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (GAM-FITC) was also purchased from Coulter.
PRP and PPP were obtained from blood anticoagulated with EDTA
(final concentration, 10 mM) as described above for the platelet aggregation
studies. PRP was adjusted to 50 ⫻ 109 platelets/L with PPP and fixed with
0.2% paraformaldehyde for 5 minutes at room temperature. Samples of 100
␮L were incubated for 30 minutes at room temperature with 10 ␮L of the
working solution of each mAb. Platelets were then washed with phosphatebuffered saline (PBS) containing 3.0 mM EDTA and 5% (wt/vol) bovine
serum albumin (Sigma), resuspended in 100 ␮L PBS and incubated for 30
minutes at room temperature in the dark with an equal volume of
GAM-FITC diluted 1/50 in the same buffer. After an additional wash, cells
were resuspended in 300 ␮L PBS containing 0.3 mM EDTA and 0.1%
bovine serum albumin and were analyzed with an Epics XL flow cytometer
(Coulter). Platelets were identified on the basis of their size (linear forward
scatter) and granularity (log side scatter), and an electronic gate was drawn
around the platelet cloud to exclude residual erythrocytes and white cells.
Ten thousand platelet events were collected, and the value of mean
fluorescence, expressed in arbitrary units, was recorded. A sample from a
healthy donor was run with the patients’ samples (the same healthy donor
was used for all patients).
Results
Clinical and laboratory data
Twelve patients from unrelated Italian families affected by hereditary macrothrombocytopenia were enrolled in this study (Table 1).
Clinical and laboratory investigations of their available relatives
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
identified 34 additional subjects with quantitative or qualitative
platelet defects. All had larger than normal platelets, and a platelet
count lower than 150 ⫻ 109/L was observed in 29 of 34 subjects.
The disease was transmitted within the families as an autosomal
dominant trait (Figure 1).
The male–female ratio of the 46 affected subjects was 1:1, and
their ages ranged from 1 to 70 years. They had platelet counts from
22 to 178 ⫻ 109/L. The mean value of MPV was 12.6 fL, with a
range from 10.4 to 17.2 fL. In vitro platelet aggregation after ADP,
collagen, and ristocetin was normal in all investigated patients
(data not shown). Twenty-two patients had mild to moderate
bleeding tendency consisting of frequent episodes of epistaxis,
gingival bleeding, menorrhagia, easy bruising, or prolonged bleeding after dental surgery. Six patients from 5 families (TP-1, TP-2,
TP-4, TP-5, and TP-7) underwent bone marrow examination that
showed normal numbers of megakaryocytes. In some patients,
however, the analysis did reveal mild and aspecific dysmegakaryocytopoietic phenomena, such as increased fraction of precursor
cells and reduced megakaryocytes that appeared to be producing
platelets.
Genome-wide search
To localize the gene(s) responsible for hereditary macrothrombocytopenia, we considered 2 large pedigrees, TP-1 and TP-2, for the
genome-wide search (Figure 1). Under the assumption of complete
genetic penetrance, the gene was mapped to chromosome 17q.
Marker D17S938 gave a maximum 2-point LOD score of 7.51 at
recombination fraction of 0.00.
Haplotype analysis was performed in both families (Figure 1).
In TP-1, a telomeric recombination was evident between D17S831
and D17S938 in subjects I-1, II-4, III-1, and III-6. In TP-2, 2
recombination events in 2 healthy subjects, IV-6 and IV-7, established the centromeric limit of the disease region between D17S938
and D17S1852. Therefore, the candidate region was defined in an
interval of approximately 18 cM between microsatellites D17S831
and D17S1852. Among possible disease candidates, GPIb␣, one of
the gene responsible for BSS, was of particular interest.
Mutation analysis
The GPIb␣ gene was completely sequenced in 2 probands from
each of the TP-1 and TP-2 families. The screening identified a
heterozygous C ⬎ T transition at position 515 in both patients (data
not shown). This mutation, previously reported as the Bolzano
variant, causes an Ala156Val amino acid substitution and creates a
restriction enzyme site for HpaI.19 Restriction analysis in all family
members showed that only the affected subjects were heterozygous
for the missense mutation, indicating a full concordance within
families between the phenotypic expression and the protein variant.
Restriction analysis in the remaining 10 families allowed us to
identify the same variant in the heterozygous state in 4 of them. In
conclusion, 6 of 12 affected families had the Bolzano variant. The
change was absent in 50 unaffected, unrelated control subjects.
Flow cytometry
To investigate for possible defects of the platelet surface, the major
membrane GPs were investigated in 10 of 12 patients and some of
their relatives (the 2 unavailable patients were carriers of the
Bolzano variant). The most abundant surface protein, GPIIb-IIIa,
which acts as a receptor mainly for fibrinogen during platelet
activation,20 was normal or increased in all subjects (Figure 2).
Similar results were obtained for GPIa and GPIV, which are
HETEROZYGOUS BERNARD-SOULIER SYNDROME
1333
Figure 2. Flow cytometry analysis of platelet GPs. Tracings of flow cytometry with
mAbs against GPIIb/IIIa, GPIb␣ (SZ2 and MB45, the conformational-dependent and
-independent mAb, respectively), GPIX, and GPV of 3 patients, each representative
of a distinct group. At the genotype level patient A carries the Bolzano mutation,
whereas patients B and C do not have the mutation. In each patient (white curve), the
expression of GPIIb/IIIa on the platelet surface was increased compared to that of the
healthy control (gray curve), as shown by shifting of the curves to the right. In
contrast, single components of the GPIb/IX/V complex were variously reduced in
patients A and B. These patients have a GP profile consistent with a heterozygous
BSS phenotype. Of particular interest is the different fluorescence obtained with SZ2
and MB45 mAbs in the Bolzano patient, suggesting the presence of a conformationally changed GPIb␣ protein. Patient C shows a clear increase of all GP because of
the larger volume of platelets and thus represents the “true” thrombocytopeniaaffected patient.
receptors for collagen21 and thrombospondin,22 respectively (data
not shown). Conversely, the patterns of binding to the single
components of the GPIb/IX/V complex differed between the
affected subjects. Representative flow cytometry tracings are
shown in Figure 2. Fluorescence was clearly reduced in 8
(including all those with the Bolzano variant) of 10 patients and in
their respective affected relatives (Figure 2A-B). GPIb/IX/V complex was normal or increased in the other 2 patients (Figure 2C).
Among platelets with reduced fluorescence, alternative mAbs
against GPIb␣ reacted differentially. In particular, binding of SZ2
was lower than that of MB45 in the 4 patients with Bolzano-type
BSS (Figure 2A), whereas it was similarly reduced in the others
(Figure 2B). In fact, SZ2, a conformational-dependent mAb, is
specific for an N-terminal epitope not recognized when GPIb␣
undergoes a conformational change due to variants, including the
Bolzano protein.23,24 In contrast, the binding of MB45 is less
sensitive to these changes and better defines the surface content of
GPIb␣, even if conformationally altered.25 On the basis of these
results, we confirm that Bolzano BSS platelets had both qualitative
and quantitative defects in GPIb␣.19,23 Therefore, the Bolzano
protein is expressed, albeit with a reduced number of copies, on the
platelet surface, but it is unable to bind vWF because of a
conformational change.
On the basis of these studies, we grouped patients into 3
categories. The first group consisted of subjects with the Bolzano
mutation and typical Bolzano platelet GP profile (8 patients from 4
families). The second group consisted of patients without the
Bolzano variant but with a defective GPIb/IX/V complex characterized by a BSS profile of fluorescence (12 subjects from 4 families).
The third group consisted of patients without the Bolzano mutation
and a non-BSS profile defined by normal or increased binding of
anti-GPIb/IX/V mAbs (3 subjects from 2 families).
The mean of the fluorescence measurements obtained from
patients of the 3 distinct groups was normalized to that from control
subjects (Figure 3A). The histogram showed a clear difference in
vWF receptor content in heterozygous subjects with BSS and in
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1334
SAVOIA et al
Figure 3. Mean of flow cytometry results of platelet membrane GPs from
patients of each group (see legend to Figure 2). (A) Mean fluorescence intensity
(arbitrary units) obtained with mAbs against GPIIb/IIIa, GPIb␣, GPIX, and GPV was
normalized to the mean from control platelets. (B) Mean values for single components of the GPIb/IX/V complex were normalized to those of GPIIb/IIIa and expressed
as percentage of control platelets. Numbers on the right of histograms refer to the
range of values.
thrombocytopenic patients without the BSS phenotype. The GPIb/
IX/V defect appeared to be more significant when we normalized
the data to the GPIIb/IIIa (Figure 3B).
Discussion
To understand the molecular basis of chronic macrothrombocytopenia transmitted as an autosomal dominant trait, we studied 12
consecutive Italian patients and their relatives. Family investigation identified another 34 subjects with either macrothrombocytopenia or platelet macrocytosis. Clinical manifestations were absent or
mild; when they were present, common clinical findings were
excessive ecchymoses, frequent epistaxis, gingival bleeding, prolonged menstrual periods, or prolonged bleeding after tooth
extraction. No patients or their relatives reported serious surgical or
obstetric hemorrhagic complication.
Genome-wide search in 2 large families localized a gene
responsible for macrothrombocytopenia in a region of chromosome
17p containing the gene for GPIb␣. This platelet GP forms a
membrane complex with GPIb␤, GPIX, and GPV. Its amino
terminus globular domain binds vWF, whereas its cytoplasmic tail
contains a binding site for actin-binding protein.26 The former
interaction initiates the arrest of platelets at sites of vascular injury,
whereas the latter anchors membrane-associated cytoskeleton and
is possibly involved in normal megakaryocytic maturation, demarcation membrane system production, and platelet size.11 A quantitative or qualitative defect of GPIb-IX-V complex, deriving from a
homozygous defect of GPIb␣, GPIb␤, or GPIX gene, is responsible
for BSS, an illness characterized by a bleeding diathesis, thrombocytopenia, and large platelets that fail to interact with vWF.9
On the basis of linkage analysis, we hypothesized that autosomal dominant macrothrombocytopenia could be a mild form of
BSS deriving from mutations of GPIb␣. In 6 of 12 families, a
heterozygous C ⬎ T transition at position 515 was identified. This
mutation was previously reported as the Bolzano variant in 2 Italian
patients with BSS patients—one was homozygous19 and the other
was compound heterozygous.24 In the homozygous patient, the
GPIb-IX-V complex was found to be expressed on the platelet
surface, albeit with a reduced number of copies, but GPIb␣ was
unable to bind vWF, probably because of an abnormal conformation of the expressed molecule deriving from the Ala156-Val
substitution. It is unclear how this point mutation in the extracytoplasmic, leucine-rich repeat region leads to a defect in the
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
mechanism of thrombopoiesis. However, it has been hypothesized
that the altered conformation of this domain may be responsible for
an abnormal interaction of the cytoplasmic tail of GPIb␣ with the
cytoskeleton, resulting in abnormalities of platelet number and
morphology.19 As expected, quantitative and qualitative defects of
the GPIb/IX/V complex were also observed in our patients.
Because of the heterozygous condition, the residual amount of
normal GPIb␣ was sufficient to support vWF-mediated platelet
aggregation after ristocetin stimulation (data not shown).
Among the patients without the Bolzano variant, flow cytometry studies identified a defect of the GPIb/IX/V complex in 4 of 6.
In these 4 patients, the clinical findings, the hereditary pattern, and
the reduction of the GPIb/IX/V complex were consistent with a
heterozygous form of BSS. The screening for mutations of GPIb␣,
GPIb␤, GPIV, and GPV is in progress. However, preliminary
results in some patients indicate the absence of variations, at
least in the coding region of these genes, suggesting that other
genes might be responsible for this disease (Savoia, personal
communication).
In conclusion, 10 of 12 patients, originally diagnosed as
affected by autosomal dominant macrothrombocytopenia, had a
heterozygous BSS phenotype, as determined by platelet GP
analysis. In 6 patients, the diagnosis was supported by the presence
of a mutation in the GPIb␣ gene. The analysis excluded a
molecular defect of the GPIb/IX/V complex in the remaining 2
patients, who therefore represent the only individuals affected by
“true” autosomal dominant macrothrombocytopenia.
Because BSS is classically described as a recessive disorder,
heterozygous subjects are expected to be asymptomatic. Although
little attention has so far been devoted to these subjects, a careful
search in the literature allowed us to collect data on 38 heterozygous relatives of BSS patients.27-37 Some were indeed asymptomatic, whereas others had from mild to moderate bleeding diatheses.
Platelet count was reduced (lowest value 80 ⫻ 109/L) in 9 of 23
subjects, MPV was increased in 17 of 23, and a defect of
GPIb-IX-V complex was observed in 25 of 36. Moreover, one
white family has been reported in whom a mild form of BSS was
transmitted with an autosomal dominant mechanism.14 Literature
data and our experience suggest that most patients with heterozygous BSS have a reduced platelet count, recognizable defect of
GPIb-IX-V complex, or increased MPV—or any combination of
these. In some patients these abnormalities are severe enough to
elicit a bleeding diathesis.
There are 2 possible explanations for the high prevalence of the
Bolzano variant in our sample. First, the mutation has such a
detrimental action on platelet production that its effect is detectable
in the heterozygous state. Second, this allele has a relatively high
frequency in the Italian population. However, because qualitative
and quantitative platelet abnormalities were previously described
in heterozygous carriers for other BSS mutations27-37 and 3 of 6
Italian BSS chromosomes (all those characterized for mutations)
carried the Bolzano variant,19,23,25 we favor the second hypothesis.
On this basis, the results of our investigation are most relevant to
Italian populations, and we cannot exclude that different results
would be obtained in other countries.
Heterozygous patients with BSS patients, like homozygous
patients,9 show a wide spectrum of clinical and platelet findings.
The heterogeneity is likely due to several factors, such as the effect
of different mutations and the genetic background, which influence
variability not only among families but also among members of the
same family. Therefore, the severity of BSS ranges from irrelevant
to moderate and from moderate to severe in heterozygous and
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
HETEROZYGOUS BERNARD-SOULIER SYNDROME
homozygous patients, respectively. The current classification of
BSS as a recessive disorder hampers the diagnosis of those
symptomatic, heterozygous patients whose illness is transmitted as
a dominant trait.
Based on data reported concerning European, North American,
and Japanese populations, the frequency of homozygous BSS has
been estimated to be approximately 1 in 1 million,9 and, according
to the Hardy-Weinberg law, the frequency of heterozygotes is 1 in
500. However, the proportion of heterozygous subjects with low
platelet count, bleeding tendency, or both is unknown. In fact, the
diagnosis is almost always missed when there are no homozygous
1335
BSS patients in the family. Therefore, a heterozygous BSS
condition must be suspected in patients with autosomal dominant
thrombocytopenia or platelet macrocytosis.
Acknowledgments
We thank Prof Mario Cazzola (Institute of Haematology, IRCCS
San Matteo-University of Pavia) for referring to us one of the
investigated families and Dr Maurizio Margaglione (IRCCS Hospital CSS) for providing some of the GPIb␣ primers.
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2001 97: 1330-1335
doi:10.1182/blood.V97.5.1330
Autosomal dominant macrothrombocytopenia in Italy is most frequently
a type of heterozygous Bernard-Soulier syndrome
Anna Savoia, Carlo L. Balduini, Maria Savino, Patrizia Noris, Maria Del Vecchio, Silverio Perrotta,
Simona Belletti, Vincenzo Poggi and Achille Iolascon
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