Whole mount EM is useful for the evaluation of suspected PFD due to DGD and detects abnormalities associated with bleeding.
The variability in platelet dense granule ATP release findings amongst patients assessed for diagnostic purposes suggests that the test has limited value for diagnosing platelet disorders.
Background The bleeding risks for nonsyndromic platelet function disorders (PFDs) that impair aggregation responses and/or cause dense granule deficiency (DGD) are uncertain. Objectives Our goal was to quantify bleeding risks for a cohort of consecutive cases with uncharacterized PFD. Methods Sequential cases with uncharacterized PFDs that had reduced maximal aggregation (MA) with multiple agonists and/or nonsyndromic DGD were invited to participate along with additional family members to reduce bias. Index cases were further evaluated by exome sequencing, with analysis of RUNX1 ‐dependent genes for cases with RUNX1 sequence variants. Bleeding assessment tools were used to estimate bleeding scores, with bleeding risks estimated as odds ratios (ORs) relative to general population controls. Relationships between symptoms and laboratory findings were also explored. Results Participants with uncharacterized PFD (n = 37; 23 index cases) had impaired aggregation function (70%), nonsyndromic DGD (19%) or both (11%), unlike unaffected relatives. Probable pathogenic RUNX1 variants were found in 2 (9%) index cases/families, whereas others had PFD of unknown cause. Participants with PFD had increased bleeding scores compared to unaffected family members and general population controls, and increased risks for mucocutaneous (OR, 4‐207) and challenge‐related bleeding (OR, 12‐43), and for receiving transfusions for bleeding (OR, 100). Reduced MA with collagen was associated with wound healing problems and bruising, and more severe DGD was associated with surgical bleeding ( P < .04). Conclusions PFDs that impair MA and/or cause nonsyndromic DGD have significantly increased bleeding risks, and some symptoms are more common in those with more severe DGD or impaired collagen aggregation.
Inherited platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.
Platelet function disorders represent a heterogeneous group of bleeding disorders with diverse molecular causes that are frequently associated with platelet dense granule deficiency and/or impaired aggregation responses. With the exception of Quebec platelet disorder (QPD), bleeding risks for common platelet disorders have not been estimated. This led us to study a Canadian cohort with uncharacterized platelet function disorders and confirmed abnormalities in validated assays for platelet dense granule deficiency and/or light transmission aggregometry (reduced aggregation with ≥2 agonists). Subjects were assessed using: (i) the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) to determine scores and categorize symptom severity, and (ii) CHAT-P, a clinical history assessment tool for assessment of platelet disorders that included questions about general health and bleeding symptoms/problems and questions previously used to assess bleeding risks for QPD. CHAT-P was completed by subjects (or parent in the case of young children) before review by a hematologist. Participants included: 29 individuals with confirmed platelet function disorders of unknown cause (from 7 families, 10 "sporadic" cases), 12 unaffected relatives and 60 general population controls. A one-way ANOVA was used to compare the overall ISTH-BAT scores between the affected individuals, unaffected relatives and healthy controls. Bleeding risks were estimated as odds ratio (OR) with 95% confidence intervals (CI) using CHAT-P data for general population controls as the comparison group. The total number of affected subjects reporting a bleeding problem/symptom from the group of affected individuals was added up and compared with the corresponding numbers of responses for general population controls and unaffected relatives using ANOVA. Summative bleeding scores for CHAT-P items with OR>1 were used to compare the number and range of abnormal bleeding symptoms experienced by subjects. Individuals with confirmed platelet abnormalities had higher ISTH-BAT scores (median: 9, range: 0-18) than unaffected family members (median: 0, range: 0-1) and general population controls (median: 0, range: 0-6) (p < 0.01), and their most severe symptom scores were for: epistaxis, cutaneous bleeding, menorrhagia, bleeding from dental extractions, surgery and a subdural hematoma at birth. Affected individuals had higher risks for bleeding (OR, 95% CI, p value) including: bleeding from minor cuts/wounds lasting >1 hour (56, 3.1-1000, p<0.01); abnormal bruising (15-65, 1.8-140 to 3.7-1200, p<0.01); prolonged nosebleeds (23, 5.9-92, p<0.01) and nosebleeds requiring medical attention (40, 1.5-520, p<0.01), packing (33, 1.8-620, p<0.01) or cautery (27, 1.5-510, p<0.01); wound healing problems (13, 3.4-53, p<0.01); excessive bleeding from injuries/trauma (9.5, 1-87, p=0.03), oral/dental challenges (44, 5.3-370, p<0.01) and surgery (17, 4.1-68, p<0.01). Affected females reported: bleeding interfering with their sex life (6.5, 1.1-38, p=0.04); menses >7 days (11, 2.5-49, p<0.01); flooding/gushing accidents (3.8, 1.2-12, p=0.04 ) and/or clots with menses (13, 2.6-63, p<0.01); menses requiring treatment (7.8, 2.1-29, p<0.01); and excessive bleeding during childbirth (17, 2.7-105, p<0.01), sometimes requiring surgical intervention (41, 2-810, p<0.01). Affected individuals reported more of these bleeding symptoms (median: 15, range: 0-24) than unaffected family members (median: 2, range: 0-6; p<0.01) and general population controls (median: 1, range: 0-14, p<0.01), although there was overlap. Our study illustrates that uncharacterized platelet function disorders are associated with significantly increased bleeding risks and mild rather than severe bleeding problems. It will be important to translate our study findings for patients and healthcare providers to promote evidence-based care of individuals with confirmed dense granule deficiency and/or impaired aggregation responses, which are common amongst individuals tested for bleeding problems. Disclosures No relevant conflicts of interest to declare.
Background: Platelet function disorders are a common cause of a bleeding problem. While many rare and severe forms of platelet function disorders are well studied, many common platelet function defects are uncharacterized. In a family with uncharacterized platelet secretion defects, we identified a single base pair insertion in the gene RUNX1 by exome sequencing. We report on the clinical and laboratory phenotype of the defect in this family. Methods: Bleeding histories of affected and unaffected family members, obtained using a standardized questionnaire, were scored using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). Laboratory data evaluated included blood counts, platelet aggregation responses, dense granule ATP release findings (lumiaggregometry) and platelet dense granule counts, evauated by whole mount preparations and electron microscopy. Affection status was based on the recorded opinion of the subject's hematologist, which concurred with diagnostic laboratory findings. Exome sequencing was performed on the index case, followed by evaluation of all family members for the candidate mutation by polymerase chain reaction (PCR) amplification and Sanger sequencing. Results: Family members investigated (median age: 25.5, range 1-69] included 5 males (affected: n=4, unaffected: n=1) and 3 females (affected: n=2, unaffected: n=1). ISTH BAT bleeding scores for affected members were elevated (median: 10.5, range 4-20), unlike unaffected family members (n=2) and healthy controls (n=40) (ranges: 0-1 and 0-2). Platelet counts were low in 2/6 affected individuals (109 platelets/L : affecteds: median: 164, range: 125-169). While unaffected family members had unremarkable platelet function findings, the affected individuals had absent or reduced dense granule secretion with all agonists tested (including thrombin, collagen, epinephrine, U46619 and arachidonic acid), and in aggregation tests, they had absent secondary aggregation with epinephrine, reduced maximal aggregation with collagen (1.25 and 5.0 μg/mL) and thromboxane analogue U46619, variable responses to arachidonic acid (reduced in 4/5 affecteds), and normal responses to ADP and ristocetin. Only some (3/6) affected family members had dense granule deficiency (lower reference interval limit: 4.9/platelet; affecteds: median: 5.2, range: 4-6). A single base pair insertion (A) in exon 6 of the RUNX1 gene (NM_001754.4:c.583dup) was identified in the index case using exome sequencing. The mutation results in a reading frame shift at codon Ile195, that is expected to end in a premature termination codon 17 positions downstream. Sanger sequencing further confirmed this mutation was present in 6/6 affected family members and it was absent in both unaffected family members. Further evaluation of the family history indicated that only one family member (deceased aunt of the index case) had developed leukemia or myelodysplasia although her mutation status is unknown. Conclusion: We identified a novel, frameshift RUNX1 mutation in a family with an inherited platelet function disorder associated with increased bleeding symptoms. This variant has not been previously reported in exome sequencing of over 60 000 individuals as documented by the ExAC consortium. The variable platelet count and dense granule numbers among affected individuals in this family emphasize the importance of phenotyping multiple family members. RUNX1 defects should be considered as a potential cause of inherited abnormalities in dense granule secretion, even in individuals without dense granule deficiency or a striking family history of leukemia/myelodysplasia. Disclosures No relevant conflicts of interest to declare.
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