Search

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (dohle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of ca

Background: Inherited macrothrombocytopenias (IMT) are rare and often misdiagnosed as immune thrombocytopenia (ITP) leading to unnecessary treatments including splenectomy. Giant platelets are suggestive of IMT rather than ITP, but are not detected by automated cell counters, which can underestimate mean platelet volume (MPV). Aim(s): MYH9 disorders are characterised by macrothrombocytopenia and leukocyte inclusions which are often missed on blood films. They may also cause nephropathy, sensorineural hearing loss or cataract, with a strong correlation between genotype and phenotype, hence the importance of correct diagnosis. Method(s): We used Next-Generation Sequencing (NGS) to investigate a large cohort (N=121) of patients with lifelong thrombocytopenia not previously diagnosed with IMT. Result(s): Variants of the MYH9 gene, which encodes the non-muscle myosin heavy chain IIA (NMMIIA) were by far the most common with 10 rare benign variants (N=14), 3 variants of uncertain significance (VUS) (N=4) and 7 pathogenic variants (N=15) identified. NMMIIA immunofluorescent (IF) staining on blood film is the current diagnostic gold standard but not widely available. MPV values for subjects with MYH9 variants were not consistently elevated. In contrast, mean platelet diameter (MPD) derived from scanned blood films was clearly increased in IMT compared to ITP. When available, the blood films of patients with pathogenic mutations were evaluated. All demonstrated macrothrombocytopenia; however, inclusion bodies were not easily appreciated. NMMIIA-IF was performed in 4 cases; all demonstrated abnormal NMMIIA clustering, including 2 cases with a VUS. Conclusion(s): From our cohort, MYH9 disorders are the commonest cause of IMT. MPDs are markedly and consistently increased in MYH9 disorders and useful for differential diagnosis. As neutrophil inclusions are often missed on stained blood films, NMMIIA-IF is critical for diagnosis which may also include MYH9 genotyping.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Abnormalities of platelet size are one of the distinguishing features of inherited thrombocytopenias (ITs), and evaluation of blood films is recommended as an essential step for differential diagnosis of these disorders. Nevertheless, what we presently know about this subject is derived mainly from anecdotal evidence. To improve knowledge in this field, we evaluated platelet size on blood films obtained from 376 patients with all 19 forms of IT identified so far and found that these conditions differ not only in mean platelet diameter, but also in platelet diameter distribution width and the percentage of platelets with increased or reduced diameters. On the basis of these findings, we propose a new classification of ITs according to platelet size. It distinguishes forms with giant platelets, with large platelets, with normal or slightly increased platelet size, and with normal or slightly decreased platelet size. We also measured platelet diameters in 87 patients with immune thrombocytopenia and identified cutoff values for mean platelet diameter and the percentage of platelets with increased or reduced size that have good diagnostic accuracy in differentiating ITs with giant platelets and with normal or slightly decreased platelet size from immune thrombocytopenia and all other forms of IT