BERNARD SOULIER SYNDROME IN PREGNANCY

Bernard-Soulier syndrome is an inherited platelet disorder, transmitted in an autosomal recessive pattern. Thrombocytopenia and large defective platelets are characteristics, often presents early with bleeding symptoms, such as epistaxis, ecchymosis, menometrorrhagia, and gingival or gastrointestinal bleeding. Diagnosis can be conrmed by platelet aggregation studies and ow cytometry. Differential diagnosis includes other inherited giant platelet disorders, as well as von Willebrand disease and immune thrombocytopenic purpura. During pregnancy, it can present as recurrent rst trimester miscarriages, antepartum, intrapartum and postpartum haemorrhage. Treatment remains generally supportive with platelet transfusions and recombinant factor VII has also been described in literature.

Mapping TRPM7 Function by NS8593

The transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a ubiquitously expressed membrane protein, which forms a channel linked to a cytosolic protein kinase. Genetic inactivation of TRPM7 in animal models uncovered the critical role of TRPM7 in early embryonic development, immune responses, and the organismal balance of Zn2+, Mg2+, and Ca2+. TRPM7 emerged as a new therapeutic target because malfunctions of TRPM7 have been associated with anoxic neuronal death, tissue fibrosis, tumour progression, and giant platelet disorder. Recently, several laboratories have identified pharmacological compounds allowing to modulate either channel or kinase activity of TRPM7. Among other small molecules, NS8593 has been defined as a potent negative gating regulator of the TRPM7 channel. Consequently, several groups applied NS8593 to investigate cellular pathways regulated by TRPM7. Here, we summarize the progress in this research area. In particular, two notable milestones have been reached in the assessment of TRPM7 druggability. Firstly, several laboratories demonstrated that NS8593 treatment reliably mirrors prominent phenotypes of cells manipulated by genetic inactivation of TRPM7. Secondly, it has been shown that NS8593 allows us to probe the therapeutic potential of TRPM7 in animal models of human diseases. Collectively, these studies employing NS8593 may serve as a blueprint for the preclinical assessment of TRPM7-targeting drugs.

Study of Bernard–Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells

Bernard–Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.

Inherited Giant Platelet Disorder, Kashmiri Thrombocytopenia, a Common Syndrome Found in Srinagar, India

The causative genetic mutations of inherited giant platelet disorders (IGPD) encompass genes coding for the platelet glycoprotein Ib-IX complex (Bernard Soulier syndrome and its variants), myosin heavy chain 9 (MYH9 gene mutated in May-Hegglin anomaly and other IGPDs), GATA-01 (GATA-related thrombocytopenia), TUBB-1, ITGA2, ITGAB3, FLNA and some others. IGPDs are frequently associated with other disorders including renal disease, sensorineural deafness, and leukocyte inclusion bodies. Most are accompanied with variable degrees of bleeding diathesis, while others, like TUBB1 IGPD, do not have any bleeding manifestations. Harris platelet syndrome (HPS), previously called asymptomatic constitutional macrothrombocytopenia, is an autosomal dominant disorder characterized by low-normal to severe thrombocytopenia IGPD and absence of bleeding. HPS has also been observed in healthy blood donors from the northeastern part of India (Bengal) and some areas of Bangladesh, Bhutan and Nepal. We describe a high prevalence of an autosomal dominantly inherited form of IGPD with mild to severe thrombocytopenia in the Muslim population in Kashmir Valley in the northern Indian subcontinent. 830 voluntary, healthy, male blood donors from Kashmir Valley were included in the study. They were aged 15-55 years (median 31 years) and underwent ancillary screening as follows; CBC, peripheral smear, HBV, HCV, HIV, ANA and Anti-H pylori antibodies. 15% of the donors had thrombocytopenia (mean platelet count 109.6 compared to 189.9 in controls; p=<0.0001). No differences were noted in age between the 2 groups. The mean platelet volume (MPV) in thrombocytopenic subjects was higher (12.53 + 0.78 vs 9.52 + 1.03 fl). The red cell distribution width (RDW) in thrombocytopenic subjects was higher than in those with normal counts (15.6 + 1.61 Vs 13. 22 + 1.36, p=<0.001). Hematocrit and other red cell indices were not different in the 2 groups. None of the participants had a history of bleeding, renal disease, sensorineural deafness, or leukocyte inclusion bodies. Peripheral blood platelet morphology revealed large platelets in all subjects. In a pilot study of 7 families, Kashmiri thrombocytopenia was compatible with autosomal dominant inheritance affecting both genders. The congenital nature of Kashmiri thrombocytopenia was demonstrated by analyses of 34 consecutive neonates born in Sher-i-Kashmir Institute Hospital; among 20 girls and 19 boys, we found 18% (2 male and 5 female) to have low platelet count, the mean platelet count of the affected group when compared to unaffected group were 102.6 vs 234 (p=<0.001) respectively. We then searched for a causative mutation using the following approaches. We sequenced the MYH9 gene and no mutation was found. We then employed SNP array analyses using Shared Genome Segment (SGS) and Whole Genome Association Study (WGAS). We were able to exclude all previously reported IGPD-causing genes. SGS that overlapped with WGAS narrowed the target into 3 chromosome regions in Chr. 5 (rs6872531-rs100072476), Chr. 9 (rs11999541-rs12682912) and Chr. 10 (rs11013452-rs7083349). The performed SNP analyses included large genomic segments as candidates for a Kashmiri thrombocytopenia-causative gene. To further narrow down the cause of this disorder, we recruited 3 TRIO families (an affected parent and a child) for stronger linkage analysis and next-generation sequencing to continue the search for the cause of the Kashmiri thrombocytopenia. Disclosures No relevant conflicts of interest to declare.