Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventive treatments. Until recently, this has been performed by Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of disorder. The remaining had disease with no specific manifestations. Overall, high-throughput sequencing test attained a molecular diagnosis in 70% of the patients. This sensitivity increased to 90% among patients suspected to have a defined platelet disorder. We found 58 different candidate variants in 29 genes, among which 70% were not previously described. Following consensus guidelines, we qualified 68% and 27% of the candidate variants as pathogenic and likely pathogenic, respectively. Apart from establishing definitive diagnosis of well-known inherited platelet disorders in most patients, in few others high-throughput sequencing provided molecular results suggestive of less recognized disorders, such as sitosterolemia, filamin and actinin deficiencies and abnormalities at the level of G-protein coupled receptors and effector enzymes. Noteworthy, high-throughput sequencing diagnosed novel patients with very rare molecular pathology in DIAPH1 (n=2) and RASGRP2 (n=3). Overall, our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream of genetic diagnosis practice in inherited platelet disorders.

Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 10¹¹ platelet per day as well as appropriate survival and functions are life essential events. Inherited platelet disorders (IPDs), affecting either platelet count or platelet functions, comprise a heterogenous group of about sixty rare diseases caused by molecular anomalies in many culprit genes. Their clinical relevance is highly variable according to the specific disease and even within the same type, ranging from almost negligible to life-threatening. Mucocutaneous bleeding diathesis (epistaxis, gum bleeding, purpura, menorrhagia), but also multisystemic disorders and/or malignancy comprise the clinical spectrum of IPDs. The early and accurate diagnosis of IPDs and a close patient medical follow-up is of great importance. A genotype–phenotype relationship in many IPDs makes a molecular diagnosis especially relevant to proper clinical management. Genetic diagnosis of IPDs has been greatly facilitated by the introduction of high throughput sequencing (HTS) techniques into mainstream investigation practice in these diseases. However, there are still unsolved ethical concerns on general genetic investigations. Patients should be informed and comprehend the potential implications of their genetic analysis. Unlike the progress in diagnosis, there have been no major advances in the clinical management of IPDs. Educational and preventive measures, few hemostatic drugs, platelet transfusions, thrombopoietin receptor agonists, and in life-threatening IPDs, allogeneic hematopoietic stem cell transplantation are therapeutic possibilities. Gene therapy may be a future option. Regular follow-up by a specialized hematology service with multidisciplinary support especially for syndromic IPDs is mandatory.

Predominantly antibody deficiencies (PADs) are the most prevalent primary immunodeficiencies, but their B-cell defects and underlying genetic alterations remain largely unknown. We investigated patients with PADs for the distribution of 41 blood B-cell and plasma cell (PC) subsets, including subsets defined by expression of distinct immunoglobulin heavy chain subclasses. Blood samples from 139 patients with PADs, 61 patients with common variable immunodeficiency (CVID), 68 patients with selective IgA deficiency (IgAdef), 10 patients with IgG subclass deficiency with IgA deficiency, and 223 age-matched control subjects were studied by using flow cytometry with EuroFlow immunoglobulin isotype staining. Patients were classified according to their B-cell and PC immune profile, and the obtained patient clusters were correlated with clinical manifestations of PADs. Decreased counts of blood PCs, memory B cells (MBCs), or both expressing distinct IgA and IgG subclasses were identified in all patients with PADs. In patients with IgAdef, B-cell defects were mainly restricted to surface membrane (sm)IgA+ PCs and MBCs, with 2 clear subgroups showing strongly decreased numbers of smIgA+ PCs with mild versus severe smIgA+ MBC defects and higher frequencies of nonrespiratory tract infections, autoimmunity, and affected family members. Patients with IgG subclass deficiency with IgA deficiency and those with CVID showed defects in both smIgA+ and smIgG+ MBCs and PCs. Reduced numbers of switched PCs were systematically found in patients with CVID (absent in 98%), with 6 different defective MBC (and clinical) profiles: (1) profound decrease in MBC numbers; (2) defective CD27+ MBCs with almost normal IgG3+ MBCs; (3) absence of switched MBCs; and (4) presence of both unswitched and switched MBCs without and; (5) with IgG2+ MBCs; and (6) with IgA1+ MBCs. Distinct PAD defective B-cell patterns were identified that are associated with unique clinical profiles.

In addition to mutations in ITG2B or ITGB3 genes that cause defective α_IIbβ₃ expression and/or function in Glanzmann’s thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of α_IIbβ₃. The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2. CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in α_IIbβ₃ integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients’ neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation. Publisher9s Note: There is an Inside Blood Commentary on this article in this issue. Key Points New function-disrupting mutations in CalDAG-GEFI, p.S381F and p.R113X, were identified in 2 unrelated families of distinct ethnic origin. Homozygous carriers of these mutations displayed relevant bleeding diathesis and moderate impairment and/or delay in platelet aggregation.

Immune thrombocytopenia (ITP) is an autoimmune disorder that induces a decrease in the number of circulating platelets due to spleen destruction and inability of megakaryocytes to restore normal counts. Immunosuppressive therapy with glucocorticoid drugs constitutes the first line of treatment. However, lack of response to these agents is not uncommon, and the management of refractory patients is a matter of controversy. In fact, day-to-day clinical practice shows that, in spite of the current guidelines, splenectomy, which is currently considered a suitable second-choice therapy, is being replaced by treatment with thrombopoietin receptor agonists. These boost platelet production by megakaryocytes. The use of one of these, namely eltrombopag, has been permitted for ITP patients refractory to first-line drugs or splenectomy, for the last 10 years. This review summarizes the experience reported using eltrombopag in ITP, paying attention to efficacy and safety. Results from clinical trials will be discussed, and studies performed in the course of daily clinical practice will also be reviewed, as these are useful to assess the potential of the drug in real-world settings. The management of adverse events and the use of eltrombopag in particular situations will also be covered. The experience reported so far permits us to suggest that eltrombopag efficiently induces recovery of platelet counts. Furthermore, recent papers have demonstrated that a sustained response after discontinuation, initially thought to be problematic, may be possible in a nonnegligible number of cases. The safety profile is satisfactory, although patients presenting with thromboembolism risk factors should be treated with caution until the eltrombopag-associated prothrombotic risk is fully established. In summary, although larger studies are still needed to clarify some issues, eltrombopag may be a useful alternative tool for ITP patients refractory to conventional medical management or splenectomy.