PB0334 - A novel R1162S variant in MYH9 alters the biochemical interactions and dynamics of NMMHC-IIA, induces severe macrothrombocytopenia and perturbs leukocyte function and cellular coherence

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

CL

Clara Llorente-González

MSc
IBMCC,CIC, CSIC-Universidad de Salamanca, Salamanca, Spain.
Salamanca, Castilla y Leon, Spain
MM

María Millán-Salanova

MSc
IBMCC,CIC, CSIC-Universidad de Salamanca, Salamanca, Spain.
Salamanca, Castilla y Leon, Spain

Presenting Author(s)

Ana Marin-Quilez, MSc

Postdoctoral researcher
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, IMIB, CIBERER-U765, Murcia, Spain
Murcia, Murcia, Spain

Co-Author(s)

LD

Lorena Díaz-Ajenjo

Predoctoral researcher
Cancer Research Center - CSIC, University of Salamanca, Instituto de Investigación Biomédica de Salamanca (IBSAL), Spain
Salamanca, Castilla y Leon, Spain
SS

Sandra Santos-Mínguez

MSc
IBSAL, CIC, IBMCC, Universidad de Salamanca-CSIC, Salamanca, Spain
Salamanca, Castilla y Leon, Spain
CM

Cristina Miguel-García

BSc
IBSAL, CIC, IBMCC, Universidad de Salamanca-CSIC, Salamanca, Spain
Salamanca, Castilla y Leon, Spain
RB

Rocío Benito

Postdoctoral researcher
Cancer Research Center - CSIC, University of Salamanca, Instituto de Investigación Biomédica de Salamanca (IBSAL), Spain
Salamanca, Castilla y Leon, Spain
JG

Jose Ramon Gonzalez Porras

Hematologo
Hospital Clínico Universitario Salamanca
salamanca, Castilla y Leon, Spain
José RIVERA Rivera, PhD

PhD
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, Murcia, Spain
Murcia, Murcia, Spain
MV

Miguel Vicente-Manzanares

PhD
IBMCC,CIC, CSIC-Universidad de Salamanca, Salamanca, Spain.
Salamanca, Castilla y Leon, Spain
Jose Mª Bastida, PhD, MD

Hematology Department
Complejo Asistencial Universitario de Salamanca-IBSAL-USAL (Salamanca)
Salamanca, Castilla y Leon, Spain

Background: Patients with MYH9-related disorders (MYH9-RD) show a strong genotype-phenotype correlation based on thrombocytopenia, renal disease, deafness and/or cataracts. Beyond platelets, the cellular effects, and underlying biochemical mechanism, of MYH9 variants which affect to the heavy chain of non-muscle myosin IIA (NMMHCII-A) are poorly characterized. Recurrent infections have not been previously described.

Aims: To characterize the biochemical and dynamic behavior of the MYH9 variant c.3486G>C [p.R1162S] and assess its correlation with alterations observed in leukocytes and recurrent infections.

Methods: MYH9-RD was diagnosed in five patients from the same family with recurrent infections since early age (Table 1). The behavior of neutrophils, activated T cells and monocyte-derived dendritic cells was examined in adhesion and migration assays. NMMHCII-A 1162S mutant was expressed as GFP-coupled protein in a Myh9-deficient cell line for cellular and biochemical characterization.

Results: Leukocyte anomalies consisted of morphology and migratory defects in neutrophils, T lymphoblast and monocyte-derived dendritic cells (mDCs). Upon adhesion to fibronectin, neutrophils showed striking, highly-organized and stack-like NMMHCII-A structures consistent with increased filament stability (Fig.1A). Neutrophils bearing this variant also showed decreased fMLP-dependent migration through Boyden chambers (Fig.1B). T lymphoblasts adhered to fibronectin displayed increased polarization and spreading area (Fig.1C) whereas the adhesion capability of mDCs was impaired (Fig.1D). Further analysis of the migratory ability of mDCs on 3D substrates showed an aberrant CCL19-dependent migration in collagen gels (Fig.1E). Surprisingly, mDCs carrying this variant displayed decreased cellular coherence and increased release of cytoplastic fragments when navigating through collagen fibers (Fig.1F). Expression of GFP-NMMHCIIA R1162S in a MYH9-deficient cell line revealed assembly into abnormally stable mini-filaments dynamically distinct from wild type filaments (Fig.1G).

Conclusion(s): The MYH9 variant p.R1162S led to specific perturbations of the biochemical interactions and dynamics of mutant NMMHCII-A protein. The leukocyte alterations reported here could underlie the recurrent infections observed in patients carrying this variant.