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Glanzmann thrombasthenia (GT) is a bleeding disorder characterized by
impaired platelet aggregation in response to most physiological agonists and
caused by either a complete lack or dysfunction of the platelet integrin αIIbβ3
(glycoprotein IIb/IIIa). In this study we identified a novel homozygous
non-synonymous variant (c.995A>T, p.Asp332Val) in exon 11 of ITGA2B leading to replacement of
Aspartic Acid by Valine. According to the ACMG (American College of Medical
Genetics and Genomics) variant classification guidelines, this variant is
classified as likely pathogenic. This amino acid change affects the second
Calcium-binding domain of αIIb integrin. The Index patient’s platelet
aggregation induced by all agonist except Ristocetin is absent, and the
expression of the GPIIb/IIIa-complex is severely decreased.
Keywords: Glanzmann
thrombasthenia, ITGA2B,
Calcium-binding domain, Novel likely pathogenic variant
INTRODUCTION
Glanzmann thrombasthenia (GT) is the most
common inherited platelet disorder characterized by a deficiency or functional
defect of platelet integrin αIIbβ3. Most often GT is inherited autosomal
recessively, a few patients with an autosomal dominant inheritance of GT have
been identified. GT was first described by Glanzmann in 1918 as a platelet
abnormality presenting with haemorrhage and thrombasthenia (weak) platelets.
Characteristical for this disease is severely impaired or absent platelet
aggregation in response to multiple physiological agonists. Platelets lacking
integrin αIIbβ3 may adhere to the subendothelium after injury, however,
platelet spreading on the exposed surface is defective as well as thrombus
formation [1]. Patients suffer from a lifelong moderate to severe haemorrhagic
syndrome which can manifest rapidly after birth. Bleeding symptoms comprise
haematomas, petechiae, gastrointestinal and mucocutaneous bleeding (i.e., epistaxis).
GT cannot be clinically distinguished from other platelet disorders; therefore,
comprehensive diagnostic investigation is important to elucidate the underlying
cause of the haemorrhagic diathesis [2-4].
This study describes the clinical and biochemical phenotype of a
patient with GT caused by a novel homozygous likely pathogenic variant which
results in an amino acid change located in the second Calcium-binding domain of
αIIb integrin.
PATIENT, MATERIALS AND METHODS
The index patient is a 6 year old boy who came for counseling before a
planned dental restoration. He and his
family came originally from Iran. The parents are first cousins. He suffers
from increased bleeding symptoms since early childhood, including petechiae on
the entire skin and easy bruising (Figure
1A). He reported prolonged epistaxis: 2-3 nose bleeding episodes during the
year lasted as minor bleeding even for a couple of days. After minor injuries
or blood drawing the bleeding symptoms persist for 2-3 h. His parents reported
major bleeding and prolonged wound healing after circumcision as an infant. In
Iran he successfully received repeated platelet concentrates after sustained
bleeding episodes. After Glanzmann Thrombasthenia was diagnosed here, the index
patient underwent tooth extraction (5 teeth) under treatment with activated
recombinant factor VII (rFVIIa) without any major bleeding. He did not need any
platelet concentrate.
The mother reported that she had minor bleeding symptoms as a child
like epistaxis and easy bruising. She had no increased bleeding symptoms giving
birth to her children. The father and the 4 year old brother are unaffected.
One brother underwent an adenoidectomy without bleeding complications.
Platelet aggregometry analyses
Platelet-rich plasma (PRP) was prepared from citrated blood. Platelet
aggregometry (APACT4) was performed using 2.0 and 10.0 μg/ml collagen (Takeda,
Linz, Austria), 4.0 and 10.0 μmo/l/l adenosine diphosphate (ADP)
(Sigma-Aldrich, St. Louis, MO, USA), 8.0 μmol/l epinephrine (Sanofi-Aventis,
Frankfurt, Germany), 0.3 and 0.5 mg/ml arachidonic acid (MöLab GmBH,
Langenfeld, Germany) and 1.2 mg/ml ristocetin (American Biochemical and
Pharmaceutical LTD, Frankfurt, Germany).
Flow cytometry analyses
Flow cytometry analyses were performed according to Lahav et al. [12]
using FACSCalibur (Becton Dickinson, Heidelberg, Germany). Aliquots of diluted
PRP (5 × 107 to 5 × 107 platelets/ml) were fixed and
stained with FITC-labelled monoclonal surface antibody against CD41 (fibrinogen
receptor GPIIb/IIIa-complex), CD42a (von Willebrand factor (VWF) receptor
GPIb/IX) and CD42b (VWF receptor GPIb) (Coulter, Immunotech, Marseille, France),
respectively.
For VWF-binding analyses, diluted PRP (5 × 107 to 5 × 107
platelets/ml) was stimulated with different concentrations of ristocetin (0-1
mg/ml) and ADP (0-2 µmol/l) for 3 min at RT, respectively. Platelets were
stained with FITC-labeled anti-VWF (Bio-Rad AbD Serotec, Puchheim, Germany) and
Alexa Fluor 488-labelled anti-fibrinogen (Invitrogen, Waltham, MA, USA).
For secretion analyses, diluted PRP (5 × 107 platelets/ml)
was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2,
0.5 and 1 U/ml; Siemens Healthineers, Marburg, Germany) in the presence of 1.25
mM fibrinolysis inhibiting factor Gly-Pro-Arg-Pro (Bachem, Bubendorf,
Switzerland). Platelets were stained with monoclonal FITC-labelled anti-CD62
antibody (α-granule secretion) and anti-CD63 antibody (δ-granule secretion)
(both Immunotech, Marseille, France).
Molecular genetic analysis
Sanger sequencing was performed for the coding region and splice sites
of ITGA2B (NM_000419.4) and ITGB3 (NM_000212.2) as described
previously [13] and aligned using CodonCode® software. Variants were
evaluated using mutation analyzing software Alamut®, in silico pathogenicity prediction and
occurrence in Glanzmann database (Sinai central), NCBI ClinVar and HGMD®
[Access 11/2018].
RESULTS
Molecular genetic analysis of the index patient revealed a novel
homozygous variant (c.995A>T, p.Asp332Val) in exon 11 of ITGA2B. The highly conserved (up to C. elegans) Aspartic Acid at position
p.332 (301 in mature protein) is exchanged by the physiochemical different
Valine (Grantham dist.:152 [0-215]). The variant is neither listed in
population databases (dbSNP, gnomAD, ESP) nor in disease databases (Sinai
central, HGMD and ClinVar) [Access 11/2018]. In silico pathogenicity prediction was concordant pathogenic using
SIFT, Polyphen2 and MutPred).
Family genotyping revealed that the consanguine parents and the brother
of the index patient are heterozygous carriers of this variant (Figure 2).
DISCUSSION AND CONCLUSION
In this study we describe the severe bleeding phenotype of a young boy
carrying a novel likely pathogenic (Class4) non-synonymous variant in the ITGA2B-gene. Wild type Aspartic Acid
(Asp; D) at position p.301 in the mature protein (p.332 with signal peptide) is
replaced by Valine. This change affects the second Calcium-binding domain of
αIIb integrin, which is a sequence of 9 amino acids: p. 297-DVNGDGRHD-
Biochemical and molecular genetic analysis of our patient revealed the
diagnosis of Glanzmann Thrombasthenia and identified most likely the disease
causing mutation. Therefore, successful treatment in case of bleeding symptoms
or surgery could be successfully performed. Identifying the disease causing
mutation in GT enables to understand the biochemical and molecular genetic
mechanisms responsible for the bleeding phenotype.
ACKNOWLEDGEMENT
We thank Simone Rosenfelder and Eileen Lerner for excellent technical
assistance.
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