Background
Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity ...of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants.
Objectives
To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology.
Patients/Methods
Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting.
Results
Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels.
Conclusions
This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.
Abstract
Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of ...glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.
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 preventative treatments. Until recently, this diagnosis has usually been performed
Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the 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 platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in
(n=2) and
(n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.
Platelet transfusions can fail to prevent bleeding in patients with inherited platelet function disorders (IPDs), such as Glanzmann's thrombasthenia (GT; integrin αIIbβ3 dysfunction), Bernard-Soulier ...syndrome BSS; glycoprotein (GP) Ib/V/IX dysfunction, and the more recently identified nonsyndromic
variants. Here, we used IPD mouse models and real-time imaging of hemostatic plug formation to investigate whether dysfunctional platelets impair the hemostatic function of healthy donor wild-type (WT) platelets. In
mice or mice with platelet-specific deficiency in the integrin adaptor protein TALIN1 ("GT-like"), WT platelet transfusion was ineffective unless the ratio between mutant and WT platelets was ~2:1. In contrast, thrombocytopenic mice or mice lacking the extracellular domain of GPIbα ("BSS-like") required very few transfused WT platelets to normalize hemostasis. Both
and GT-like, but not BSS-like, platelets effectively localized to the injury site. Mechanistic studies identified at least two mechanisms of interference by dysfunctional platelets in IPDs: (i) delayed adhesion of WT donor platelets due to reduced access to GPIbα ligands exposed at sites of vascular injury and (ii) impaired consolidation of the hemostatic plug. We also investigated the hemostatic activity of transfused platelets in the setting of dual antiplatelet therapy (DAPT), an acquired platelet function disorder (APD). "DAPT" platelets did not prolong the time to initial hemostasis, but plugs were unstable and frequent rebleeding was observed. Thus, we propose that the endogenous platelet count and the ratio of transfused versus endogenous platelets should be considered when treating select IPD and APD patients with platelet transfusions.
This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type ...bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbβ3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2.
Germline mutations in RUNX1 can cause a familial platelet disorder that may lead to acute myeloid leukemia, an autosomal dominant disorder characterized by moderate thrombocytopenia, platelet ...dysfunction, and a high risk of developing acute myeloid leukemia or myelodysplastic syndrome. Discerning the pathogenicity of novel RUNX1 variants is critical for patient management.
To extend the characterization of RUNX1 variants and evaluate their effects by transcriptome analysis.
Three unrelated patients with long-standing thrombocytopenia carrying heterozygous RUNX1 variants were included: P1, who is a subject with recent development of myelodysplastic syndrome, with c.802 C>Tp.Gln268∗ de novo; P2 with c.586A>Gp.Thr196Ala, a variant that segregates with thrombocytopenia and myeloid neoplasia in the family; and P3 with c.476A>Gp.Asn159Ser, which did not segregate with thrombocytopenia or neoplasia. Baseline platelet evaluations were performed. Ultrapure platelets were prepared for platelet transcriptome analysis.
In P1 and P2, but not in P3, transcriptome analysis confirmed aberrant expression of genes recognized as RUNX1 targets. Data allowed grouping patients by distinct gene expression profiles, which were partitioned with clinical parameters. Functional studies and platelet mRNA expression identified alterations in the actin cytoskeleton, downregulation of GFI1B, defective GPVI downstream signaling, and reduction of alpha granule proteins, such as thrombospondin-1, as features likely implicated in thrombocytopenia and platelet dysfunction.
Platelet phenotype, familial segregation, and platelet transcriptomics support the pathogenicity of RUNX1 variants p.Gln268∗ and p.Thr196Ala, but not p.Asn159Ser. This study is an additional proof of concept that platelet RNA analysis could be a tool to help classify pathogenic RUNX1 variants and identify novel RUNX1 targets.
•Germinal RUNX1 variants may cause hematological malignancies at a young age.•Discerning the pathogenicity of novel RUNX1 variants is critical for patient management.•Gene expression analysis demonstrates the deleterious effects of Gln268∗ and Thr196Ala variants.•Platelet transcriptomics is a useful tool for pathogenicity assessment of novel RUNX1 variants.
Abstract
RUNX1
-related disorder (
RUNX1
-RD) is caused by germline variants affecting the
RUNX1
gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making ...genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in
RUNX1
-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain
RUNX1
variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a
knock-in
murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1
WT/WT
, RUNX1
WT/L43S
, and RUNX1
L43S/L43S
). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1
L43S/L43S
and RUNX1
WT/L43S
mice had a significantly longer tail-bleeding time than RUNX1
WT/WT
mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1
L43S/L43S
and RUNX1
WT/L43S
displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin α
IIb
β
3
activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1
L43S/L43S
and RUNX1
WT/L43S
using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1
L43S/L43S
and RUNX1
WT/L43S
suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin α
IIb
β
3
activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with
RUNX1
variants in the clinical setting.
Inherited Platelet Disorders: An Updated Overview Palma-Barqueros, Verónica; Revilla, Nuria; Sánchez, Ana ...
International journal of molecular sciences,
04/2021, Letnik:
22, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 10
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.
PDF
