ACTN1-related thrombocytopenia: identification of novel families for phenotypic characterization

Abstract

Inherited thrombocytopenias (ITs) are a heterogeneous group of syndromic and nonsyndromic diseases caused by mutations affecting different genes. Alterations of ACTN1, the gene encoding for α-actinin 1, have recently been identified in a few families as being responsible for a mild form of IT (ACTN1-related thrombocytopenia; ACTN1-RT). To better characterize this disease, we screened ACTN1 in 128 probands and found 10 (8 novel) missense heterozygous variants in 11 families. Combining bioinformatics, segregation, and functional studies, we demonstrated that all but 1 amino acid substitution had deleterious effects. The clinical and laboratory findings of 31 affected individuals confirmed that ACTN1-RT is a mild macrothrombocytopenia with low risk for bleeding. Low reticulated platelet counts and only slightly increased serum thrombopoietin levels indicated that the latest phases of megakaryopoiesis were affected. Given its relatively high frequency in our cohort (4.2%), ACTN1-RT has to be taken into consideration in the differential diagnosis of ITs.

Figure 1

Functional studies of novel ACTN1 variants. (A) Immunofluorescence analysis in PD220 fibroblast cell line transiently transfected according to standard procedures. Both wild-type (wt) or mutant ACTN1 cDNAs were cloned into the pcDNA3.1-Myc tagged expression vector. The subcellular localization of exogenous α-actinin 1 (green) was examined using c-myc antibodies (9E10; Santa Cruz Biotechnology), whereas the actin filaments were stained with AlexaFluor594 (red) conjugated phalloidin (Invitrogen). Images were obtained with a Nikon C1si confocal microscope, using ×60 Plan Apo objectives. Images were processed for z-projection (maximum intensity), brightness, and contrast regulation, using ImageJ 1.45 (National Institutes of Health). The cells shown are representative of 3 independent experiments. Scale bar = 50 µm. (B) Domain structure of α-actinin and localization of ACTN1 mutations identified in Japanese families (arrowheads) and in this article (arrows). The p.Arg46Gln mutation was also identified in a French family. α-actinin is organized in an actin-binding domain (ABD) at the N terminus, 4 spectrin repeats, and a calmodulin-like domain (CaM) at the C terminus. Antiparallel molecules dimerize in rod-like structures with the ABD at each extremity for cross-linking the actin filaments into bundles.