**1. Introduction**

Platelets are essential for clot formation after tissue trauma. Initiation of the platelet plug occurs by adhesion of platelets to the damaged vascular endothelium mediated by interactions of glycoprotein Ib/IX/V complexes with von Willebrand factor (vWF), and GPVI and integrin

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α2β1 with collagen [1]. Extension of the platelet plug requires activation of αIIbβ3 through an "inside-out" signaling cascade which enables receptor cross-linking with fibrinogen and vWF and activation of "outside-in" signaling events [1, 2].

Primary hemostasis relies on both adequate function and number of platelets. Abnormalities in platelet function and/ or number may be acquired (liver disease, chronic kidney disease) or inherited (inherited platelet function disorders, IPFDs or inherited platelet number disorders, IPNDs). The group of inherited macrothrombocytopenias is included in the heterogeneous IPNDs and are characterized by large platelets, thrombocytopenia and bleeding tendencies in affected individuals (Figure 1A, Figure 1B, Figure 1C and Figure 1D) [3].

**Figure 1.** A normal blood film and three blood films demonstrating macrothrombocytopenia associated with muta‐ tions in different genes (*MYH9, NBEAL2* and *GFI1B*, respectively). (A) A blood film with platelets of normal appear‐ ance (black arrows). (B) *MYH9*-related disorder with characteristic inclusion bodies in the neutrophils (small black arrow) and large platelets (red arrow). Normal-sized platelets are also seen (long black arrow). (C) Gray platelet syn‐ drome showing distinctive pale or gray platelets (black arrows). (D) *GFI1B*-related thrombocytopenia (c.880-881insC mutation) resulting in red cells with atypical shapes and sizes (red arrow) and thrombocytopenia with platelets that appear large with normal granulation (long black arrow) as well as hypogranular or gray (short black arrows).

Unfortunately, inherited macrothrombocytopenia is under-recognized with the presence of large platelets on blood film examination often leading to a misdiagnosis of immune throm‐ bocytopenic purpura (ITP), resulting in subsequent inappropriate treatment with steroids or in some cases removal of the spleen [4]. Diagnostic algorithms have traditionally been based around biological laboratory tests examining functional properties and activation pathways in isolated platelets [3, 5–7]. This phenotypic approach is poorly standardized, technically difficult and not easily reproducible [6–11]. In addition, numerous pre-analytical variables may affect phenotypic test results. These variables include the effect of food (garlic), alcohol, drugs (herbal remedies, non-steroidal anti-inflammatory drugs, anti-platelet medications) and stimulants (smoking and caffeine) on platelet function, activation of platelet samples during venipuncture and transport necessitating careful sample handling as well as the relatively large volume of blood needed (which becomes a major problem when assessing pediatric samples) [12–14]. Despite these complex phenotypic tests, many cases remain without a definitive diagnosis.

Genetic technology may overcome many of the problems surrounding phenotypic testing for thrombocytopenia as DNA is stable, can easily be transported long distances and is not affected by diet or drugs. Moreover, genetic-based tests have provided opportunities to reduce redundancy and heterogeneity of diagnostic algorithms and have shifted our ability to describe inherited platelet disorders from a level of the defective platelet pathway involved, to a molecular level.

The Sanger sequencing method [15] has long been considered the "gold standard" technology to rapidly analyze small regions across a limited number of samples, but it is not suited to screening large numbers of genes in multiple patients [16]. The emergence of next-generation sequencing (NGS) technologies as a diagnostic approach has been able to generate more test sequence increasing the number of gene targets and decreasing the costs [17, 18]. Human whole genome sequencing (WGS) or whole exome sequencing (WES) [19, 20] have proven to be clinically appropriate and practical modalities in describing new genetic mutations in families and identifying known pathogenic mutations in individuals formerly without a diagnosis [17].

Testing approaches may vary depending on whether a novel genetic mutation is likely. WGS and WES are powerful platforms in discovering novel causal variants in individuals with rare penetrant monogenic disorders [21], whilst a candidate gene approach allows assessment of known mutations in genes causing clinical phenotypes.

Whole genome approaches incorporating NGS have recently reported novel mutations in an essential platelet transcription factor GFI1B [22, 23], and a WES approach followed by targeted Sanger sequencing was used successfully to describe mutations in *ACTN1* causing macro‐ thrombocytopenia [24, 25]. Acknowledging these advancements, we employed a targeted candidate gene approach to explore cases of suspected inherited macrothrombocytopenia that remained uncharacterized despite phenotypic testing and hypothesized this to be an effective approach to diagnose inherited macrothrombocytopenia.
