Class prediction models of thrombocytosis using genetic biomarkers

Abstract

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies.We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V 617 F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V 617 F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy. (Blood. 2010;115:7-14) IntroductionPlatelets mediate the initial first step in hemostasis while simultaneously providing the negatively charged phospholipid surface required for contact phase-mediated propagation of the coagulation cascade. Despite these key functions, molecular defects causally implicated in platelet-associated bleeding or thrombotic risk are largely unknown, best characterized by loss of glycoproteins (GP) IIb/IIIa (␣ IIb ␤ 3 ; Glanzmann thrombasthenia) or the GPIb-IX-V complex (Bernard-Soulier syndrome). 1 Similarly, protein overexpression may favor platelet activation and thrombus formation, providing conceptual support for the presence of biomarkers that may confer enhanced thrombosis susceptibility risk. Thus, polymorphisms within the ITGA2 gene encoding the ␣2 polypeptide of the heterodimeric ␣ 2 ␤ 1 collagen receptor increase receptor surface density and are associated with an increased risk of ischemic heart disease in homozygotes (especially smokers), 2,3 although confirmatory results using meta-analyses for a distinct subset of hemostatic proteins have been somewhat disappointing. 4,5 Similarly, platelet membrane polymorphisms have been linked to stroke in small studies, but the evidence is not strong. [6][7][8][9] Such studies highlight the relevance of identifying additional gene/protein biomarkers that may be causally linked to clinically relevant platelet phenotypes.Platelets retain megakaryocyte-derived mRNA, although the platelet transcriptome is less complex than that of nucleated cells. 10,11 Furthermore, platelets have evolved unique adaptive molecular signals for maintenance of genetic and protein diversity. 12 Quiescent platelets...

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms

“…30 The latter is especially relevant given known platelet functional abnormalities and thrombohemorrhagic consequences in ET. 14 In contrast, no information is available for DAAM1 function in Mk despite the requisite role of actin-based forces on proplatelet formation. Recent data using Daam1-knockout mice have established a crucial role in heart morphogenesis by regulating filamentous actin assembly and cytoskeletal organization.…”

“…12 We have previously shown that distinct platelet phenotypes can be genetically classified using a restricted set of mRNA biomarkers. 14 We have now expanded these observations to further investigate the impact of miRNAs in thrombocytosis, and we have identified a unique fingerprint associated with enhanced megakaryocytopoiesis and/or proplatelet formation. Systematic analyses of miRNA with transcriptomic and proteomic studies using an integrated computational framework identified miR 490 5p and its putative target dishevelled associated activator of morphogenesis 1 (DAAM1) as reciprocally regulated pairs in thrombocythemic platelets, presumably by translational control and not by mRNA target degradation.…”

“…To gain insight into platelet miRNA function and expression patterns in normal and pathologic conditions (thrombocytosis), we microarrayed 79 human platelet samples from a previously described cohort, 14 composed of normal controls (NO; n ϭ 30), patients with RT (n ϭ 22), or patients with ET (n ϭ 27). Absolute platelet miRNA expression patterns ranged from N ϭ 232 (healthy controls) to N ϭ 309 (RT; Figure 1A; supplemental Table 3).…”

“…Based on the unsupervised clustering results, 13 of 19 (68%) JAK2V 617 F and 5 of 8 (63%) JAK2 wild-type ET samples were assigned to the ET-enriched cluster, results consistent with previous genetic classification data using aggregate and condensed mRNA expression profiles. 14 Significance analysis of microarrays identified 21 miRNAs that were most differentially expressed among the cohorts and most pronounced comparing ET to normal ( Figure 1C; Table 1). Aggregate group-wise comparisons demonstrated less pronounced differences between NO and RT, results that were consistent with the phenotypic cosegregation using unsupervised hierarchical clustering.…”

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans

“…All subjects (myeloproliferative neoplasm [MPN; N 5 36], reactive thrombocytosis [RT; N 5 53], or healthy controls [N 5 208]) were enrolled in an institutional review board-approved protocol conducted in accordance with the Declaration of Helsinki, 8 applying well-established clinical criteria for phenotypic classification. 9,10 Informed consent was obtained from all subjects.…”