Novel homozygous frameshift mutation of ITGB3 in the Glanzmann thrombasthenia patient with abnormal bone metabolism and congenital bone defects

Abstract

Background: Glanzmann thrombasthenia (GT) is a rare inherited bleeding disorder caused by dysfunction of the integrin αIIbβ3 in platelets. The subunit β3, encoded by ITGB3 also plays a significant role in bone metabolism. Whether GT patients with β3 deficiency also suffer from bone pathology remains unclear.

Method: The 21-year-old female patient presenting with bleeding diathesis and multiple congenital bone defects in her right hand, and her seven family members were included in the study. Whole exome sequencing as well as Sanger sequencing were conducted to identify GT-associated mutations within the family. The platelet function of the family was detected by the platelet aggregation test and thromboelastography (TEG). The expression levels of CD41 (αIIb) and CD61 (β3) on the platelet surface and total in platelet were detected by flow cytometry and Western blot. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. X-ray imaging, bone densitometry and bone metabolism index were performed to evaluate bone development and metabolism.

Result: A novel homozygous frameshift mutation c.2143_2158delinsCT (p.Lys715Leufs*36) of ITGB3 was found in the proband. Platelet aggregation by ADP, collagen, epinephrine, and arachidonic acid was absent, TEG showed hypocoagulability and decreased platelet function, and the expression levels of αIIb and β3 on the platelet surface and total in platelet were significantly reduced (< 5%) in the proband. The parents, second elder sister and grandmother of proband were heterozygous carriers without bleeding symptoms and had normal platelet aggregation function and αIIb/β3 protein expression. Structural modeling strongly suggested that the mutation creates a truncation in cytoplasmic domains of β3, resulting in the mutant β3/αIIbβ3 inactivated and low expression. The proband was born with partial absence of phalanges in digits 2-4 and the deformity of fingers 1 and 5 in her right hand, bone densitometry indicated significant osteopenia and increased risk of fracture in her right radius, and no other gene mutations related to bone pathology were identified.

Conclusion: A novel mutation of ITGB3 which results in GT was identified. This is the third reported case of GT combined with bone defect. Our work expands ITGB3 mutation spectrum and provide further insights into the potential association between GT and bone development and metabolism.

Keywords: ITGB3; Bone defects; Bone metabolism; GPIIb/IIIa; Glanzmann thrombasthenia; Novel mutation.

Fig. 1

X-ray imaging, BMD of the proband, and molecular modeling of the mutant β3. (A) X-ray imagine of the proband’s right hand. The X - ray image of the proband’s right hand reveals that the proband was born with partial absence of the phalanges in digits 2–4, as well as deformities in digits 1 and 5. (B) Radial BMD Scan of the Proband’s Right Forearm. Schematic representation of the radial BMD scan of the right forearm of the proband indicated a significantly lower radial BMD, suggesting significant osteopenia and increased risk of fracture. (C) Molecular modeling analysis of the mutant β3. The mutation results in the substitution of lysine with leucine at the 715th amino acid position of β3, followed by premature termination at the 750th amino acid. This truncation leads to the loss of the cytoplasmic domains (residues 742–788), ultimately causing inactivation and reduced expression of the β3/αIIbβ3

Fig. 2

Family Pedigree, sanger sequencing, platelet aggregation curves, thrombelastogram and expression of αIIb and β3. (A) Pedigree of the GT family and sanger sequencing validation. NM_000212.3 is the reference transcript. III3 (proband) had a homozygous frameshift mutation c.2143_2158delinsCT (16 nucleotides were deleted, and CT were inserted in their place) in exon 14 of ITGB3 gene. I4 (grandmother), II2 (father), II3 (mother), abd III2 (the second older sister) were found to be heterozygous carriers. II4 and II5 passed away at the ages of 7 years and 20 days, respectively. (B) Platelet aggregation in the proband. Platelet aggregation by ADP, Col, EPI, and AA was absent in the proband. (C) TEG analysis. TEG results indicated hypocoagulability, as well as reduced platelet function and fibrinogen activity. (D) Expression of CD41 and CD61 on platelet surface. The expression of CD41 and CD61 on the platelet surface in the proband was significantly reduced (< 5%). In contrast, the expression levels were normal in the heterozygous carriers (for simplcity, only the parents of the proband are presented in the figure). (E) Expressions of total αIIb and β3 in platelets. The expressions of total αIIb and β3 in platelets of the proband were significantly reduced (< 5%) but were normal in the heterozygous carriers. 1: normal control, 2: proband, 3–6: the parents, second older sister, and grandmother of proband, respectively