OC 45.3 - New α4A-tubulin (TUBA4A) variants associated with familial thrombocytopenia highlight the implication of TUBA4A in thrombopoiesis

Monday, June 24, 2024

15:15 – 15:30 ICT

Room: 110 A-C

Co-Author(s)

AV

Anne Vincenot

Pathologist, PhD
CHU Paris Robert Debré, Paris, France
Paris, Ile-de-France, France
DP

Dominique Pidard

PhD
Université Paris Cité, Inserm UMR-S 1140
Paris, Ile-de-France, France
AM

Aurore Marchelli

Technician
Université Paris Cité, Inserm UMR-S 1140
Paris, Ile-de-France, France
JC

Julie Carré

Pharm
Université Paris Cité, Inserm UMR-S 1140
Paris, Ile-de-France, France
SP

Sonia Poirault-Chassac

Engineer
Université Paris Cité, Inserm UMR-S 1140
Paris, Ile-de-France, France
JB

Jean-Claude Bordet

Researcher
UR4609 Hémostase & Thrombose, Université Claude Bernard Lyon, Lyon, France
Lyon, Rhone-Alpes, France
SB

Sophie Bayart

Physician
CHU Rennes
Rennes, Bretagne, France
MC

Martin Castelle

Physician
Assistance Publique-Hôpitaux de Paris
Paris, Ile-de-France, France
PG

Pascale Gaussem

PharmD, PhD
Université Paris Cité, Inserm UMR-S1140, Hôpital Européen Georges Pompidou
Paris, Ile-de-France, France
MH

Marie-Françoise Hurtaud-Roux

Physician
Assistance Publique-Hôpitaux de Paris
Paris, Ile-de-France, France

Presenting Author(s)

CB

Christilla Bachelot-Loza, PhD

PhD
Université Paris Cité, Inserm UMR-S 1140
Paris, Ile-de-France, France

Background: Tubulins forming microtubules play a major role in proplatelet formation and circulating platelet shape maintenance, as illustrated in patients heterozygous for β1 tubulin (TUBB1) variants associated with hereditary macrothrombocytopenia. Recently, α-tubulin variants, in particular α4A-tubulin (TUBA4A), have been also reported in macrothrombocytopenic subjects.

Aims: To provide data supporting a role of TUBA4A variants in defects of platelet biogenesis and morphology based on study of 5 macrothrombocytopenic patients from two unrelated families carrying two heterozygous variants, respectively (p.(Lys430del) in family A and p.(Asp431Tyr) in family B (CATCH study, ClinicalTrials.gov identifier: NCT04272970).

Methods: Genetic analysis was performed by whole exome sequencing. Platelet structure was analyzed by transmission electron microscopy. The expression of tubulins (TUBB, TUBA and TUBA4A) and their post-translational modification (PTM) was performed by immunoblot; microtubules and marginal band structure were studied by immunofluorescence. Megakaryocyte maturation was carried out in vitro, based on the differentiation of CD34+ progenitor cells in culture, followed by cell morphological analysis and immunofluorescence labeling of microtubules.

Results: The variant TUBA4A allele co-segregated with the platelet phenotype within both families. The most affected patients (family B) showed a drastic decrease of platelet TUBA4A content and an abnormal microtubular marginal band, resulting in largely spherical platelets. In family A, TUBA4A was less reduced and the marginal band and platelet shape were more lightly affected. PTM of α-tubulins were altered, with decreased tyrosination and increased acetylation, having potential impacts on microtubule stability and rigidity. Proplatelet extensions in cultured megakaryocytes showed subtle alteration in family A, but were essentially absent in family B.

Conclusion(s): Our data thus establish the involvement of TUBA4A in platelet biogenesis and structure through proper formation of microtubule networks in megakaryocytes and platelets, and provide new information on pathophysiological mechanisms and effectors underlying macrothrombocytopenia in humans.