Background: Rare, monoallelic missense variants in ACTN1 (encoding α-actinin-1, ACTN1) are associated with the non-syndromic disorder ACTN1-related thrombocytopenia (A-RT). Variants affecting the rod domain of ACTN1 may be associated with a milder platelet phenotype. ACTN1 is an ISTH Tier 1 thrombocytopenia gene included in diagnostic next generation sequencing (NGS) panels for thrombocytopenia. As a result, many variants of unknown significance (VUS) are identified that can be difficult to resolve.

Aims: 1. To report a novel de novo ACTN1 rod domain variant in a child with isolated macrothrombocytopenia.
2. To use computational techniques to predict the pathogenicity of this novel variant and previously reported missense variants affecting the rod domain.

Methods: Extended evaluation of the pedigree was completed at the Hospital for Sick Children, Toronto with research ethics board approval (REB number 1000007948). A systematic search of the literature, HGMD Professional and the ClinVar database was performed to identify previous reports of rare missense variants in the rod domain of ACTN1 associated with thrombocytopenia. Structural modelling of variants was performed using Chimera.

Results: Clinical and laboratory results are provided in Figure 1. NGS of a 31-gene thrombocytopenia panel revealed a heterozygous ACTN1 variant (c.1861G>A, p.(Glu621Lys)) classified by the reporting laboratory as a VUS. No other credible variants were identified. Sanger sequencing of parental DNA was consistent with a de novo variant. Computational tools produced conflicting predictions of pathogenicity for this variant and several previously reported variants affecting the rod domain of ACTN1 (Figure 2). Despite additional investigations, the variant remains a VUS.

Figure 1. Clinical and laboratory features. A) Pedigree diagram. The proband is shown with an arrow. Black symbol indicates thrombocytopenia. White symbol indicates normal platelet count. G, reference allele. A, variant allele. B) Key clinical and laboratory data. NR, local normal range. C) Immunofluorescence structured illumination microscopy images of a typical normal donor platelet (left) and a large patient platelet (right) with >4X normal cell volume (maximum intensity renders; scale same for both images).

Figure 2. Variants affecting the rod domain of ACTN1 associated with thrombocytopenia. Diagrammatic representation of the rod domain of ACTN1 and position of the 26 missense variants associated with thrombocytopenia. The novel variant reported here is shown in red. The predicted amino acid substitution is given for each variant with reference to the canonical transcript ENST00000193403.10. The Revel score ranges from 0-1, with higher scores predicting more deleterious variants. The effect on hydrogen bond formation was predicted using Chimera. The current classification of the variant was taken from depositions in ClinVar as at 3rd March 2021. ACMG, American College of Medical Genetics and Genomics. LPV, likely pathogenic variant. SLR, spectrin-like repeat. VUS, variant of unknown significance.

Conclusions: Prediction of pathogenicity is particularly challenging where pedigrees are small or uninformative, where conditions lack pathognomonic features and where computational methods produce conflicting results. Development of experimental and computational tools alongside continued case reporting should be encouraged in order to improve the clinical utility of NGS.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/assigning-pathogenicity-to-a-novel-de-novo-actn1-variant-in-a-child-with-macrothrombocytopenia-a-case-description-and-review-of-previously-identified-variants/