Precision medicine identifies a pathogenic variant of the ITGA2B gene responsible for Glanzmann's thrombasthenia in a cat

Abstract

Background: A nonpedigreed male cat presented with epistaxis, severe bladder hemorrhage, and secondary urethral obstruction after cystocentesis.

Objectives: To characterize the phenotype of a cat with bleeding diathesis and use a precision medicine approach to identify the molecular genetic defect by whole genome sequencing.

Methods: Adenosine diphosphate (ADP) and arachidonic acid (AA)-induced whole blood platelet aggregometry was performed in the affected cat and a healthy cat. Platelet activation, measured by P-selectin expression, and surface integrin subunit β3 expression were evaluated by flow cytometry in the affected cat and healthy control. Total integrin subunit αIIb expression was assessed by western blot. Whole genome sequencing at 30× coverage was used to identify genetic variants that segregated in the affected cat compared to 194 cats from the 99 Lives Sequencing Consortium.

Results: Platelet aggregometry identified significant impairment in platelet aggregation in response to ADP and AA compared to the control cat. Targeted protein expression analyses by flow cytometry and immunoblot analysis determined that the surface expression and total expression of the integrin, αIIbβ3, was absent. Whole genome sequencing identified a homozygous c.1986delC frameshift variant in the integrin subunit αIIb (ITGA2B) gene that was not detected in the control population. The p.Pro662fs (ITGA2B P662X) variant terminates translation of the protein at the extracellular domain of the integrin prematurely, which is predicted to affect expression of the β3 unit.

Conclusions and clinical importance: This novel ITGA2B variant and the associated phenotype closely resemble Glanzmann's thrombasthenia, which has never been reported in cats.

Keywords: Glanzmann's thrombasthenia; ITGA2B; integrin αIIbβ3; whole genome sequencing.

FIGURE 1

Representative tracings of adenosine diphosphate (ADP) and arachidonic acid (AA)‐induced whole blood platelet aggregometry in a cat with bleeding diathesis and a healthy control cat. No aggregation was detected upon activation by ADP or AA in the cat. Aggregation of platelets on the paired electrodes (red and blue curves) in response to ADP or AA resulted in gradual increase in electrical impedance, measured as aggregation units (AU) in the control cat

FIGURE 2

Representative scatter plot diagrams and histograms of flow cytometric in the cat with bleeding diathesis and a healthy control cat. A, Platelets were identified based on their forward‐(FS) and side‐scatter (SC) properties in unstimulated state (resting). Cat platelets underwent shape change in response to thrombin. Note the shift in FS and SC properties in thrombin‐activated platelets (red) compared to resting platelets (blue). B, Scatter dot plots demonstrating the co‐expression of P‐selectin (CD62P+) and integrin subunit β3 (CD61+) in adenosine diphosphate (ADP)‐activated platelets in the control cat (red box). C, A histogram illustrating the number of platelets expressing integrin subunit β3 (CD61+). Platelets from the cat (gray) failed to express integrin subunit β3 on the plasma membrane compared to platelets in a healthy cat (white). D, In contrast, despite the cat's ability to respond to ADP by expression P‐selectin (blue box) (CD62P+), no platelets co‐expressed P‐selectin and integrin subunit β3 (CD61−)

FIGURE 3

Representative immunoblot analysis of platelet integrin subunit αIIb in platelet lysates from a healthy control and the cat with bleeding diathesis. Molecular weight of integrin subunit αIIb is approximately 110 kDa. Immunoblot demonstrates that the cat's platelets did not express integrin subunit αIIb when compared to those in the healthy control. Beta actin (~42 kDa) was used as loading control. The analysis was repeated twice to ensure consistent results

FIGURE 4

Illustration of the predicted wildtype protein structure (A) for integrin subunit αIIb, encoded by ITGA2B and truncated protein structure (B) for a cat that is homozygous for the ITGA2B P662X frameshift variant (C). Note how the truncated protein (B) has altered beta‐propeller and thigh domain and lacks the extracellular calf and transmembrane domains that are otherwise found in the wildtype protein structure. Additionally, note that in the chromatogram the affected cat has a deletion of a cytosine compared to the reference genome and unaffected cat