Molecular phenotype and bleeding risks of an inherited platelet disorder in a family with a RUNX1 frameshift mutation.

INTRODUCTION: Inherited defects in RUNX1 are important causes of platelet function disorders. AIM: Our goals were to evaluate RUNX1-related platelet disorders among individuals evaluated for uncharacterized, inherited platelet function disorders and test a proof of concept that bleeding risks could be quantitatively estimated for typical families with an inherited platelet function disorder. METHODS: Index cases with an uncharacterized inherited platelet function disorder were subjected to exome sequencing with confirmation of RUNX1 mutations by Sanger sequencing. Laboratory findings were obtained from medical records and persistence of platelet non-muscle myosin heavy chain IIB (MYH10), a biomarker of RUNX1 defects, was assessed by Western blotting. Bleeding histories were assessed using standardized assessment tools. Bleeding risks were estimated as odds ratios (OR) using questionnaire data for affected individuals compared to controls. RESULTS: Among 12 index cases who had their exomes sequenced, one individual from a family with eight study participants had a c.583dup in RUNX1 that segregated with the disease and was predicted to cause a frameshift and RUNX1 haploinsufficiency. Unlike unaffected family members (n = 2), affected family members (n = 6) had increased bleeding scores and abnormal platelet aggregation and dense granule release responses to agonists but only some had thrombocytopenia and/or dense granule deficiency. This family's mutation was associated with persistence of MYH10 in platelets and increased risks (OR 11-440) for wound healing problems and mild bleeding symptoms, including bleeding interfering with lifestyle in women. CONCLUSION: Inherited platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.

Human pericytes isolated from adipose tissue have better differentiation abilities than their mesenchymal stem cell counterparts.

Multi-potent mesenchymal stem/progenitor cells are present in almost all organs and tissues, although their identity remains elusive. Several isolation strategies have been pursued to identify these cells prospectively, leading to the isolation of various cell populations endowed with multi-lineage mesodermal potential. Historically, mesenchymal stem cells (MSCs) were the first cell population to be isolated from the stromal fraction of most connective tissues. These cells are able to differentiate towards various mesodermal lineages and are currently the most studied adult mesodermal progenitors. Recently, the isolation of a subpopulation of microvascular pericytes (PCs) endowed with multi-lineage mesodermal potential has led to the identification of mesenchymal progenitors that reside in a defined anatomical location, namely the wall of small blood vessels. To gain insight into these two related cell populations, we performed a detailed analysis of the mesodermal potential of isogenic human MSCs and PCs isolated from white adipose tissue. Although both cell populations expressed known mesodermal markers at similar levels and displayed a comparable growth rate, PCs differentiated towards osteocytes, adipocytes and myocytes more efficiently than their MSC counterparts, as revealed by both histological and molecular assays. Our results show that microvascular PCs are more prone to mesenchymal differentiation than MSCs and therefore represent a preferable source of human adult mesenchymal progenitors when adipose tissue is used as a cell source.

Electron microscopy examination of platelet whole mount preparations to quantitate platelet dense granule numbers: Implications for diagnosing suspected platelet function disorders due to dense granule deficiency.

INTRODUCTION: Dense granule (DG) deficiency (DGD) is a feature of some platelet function disorders (PFD) with a prevalence similar to von Willebrand disease. Most laboratories assess for DGD using whole mount platelet preparations and electron microscopy (EM). We evaluated our experiences with this test and associations between DGD and bleeding. METHODS: Dense granule EM records for 2006-2017 were examined for patients and simultaneously tested controls, and for an overlapping PFD study cohort to evaluate findings and their relationship to bleeding. RESULTS: More patient than control samples had reduced DG counts (6.5% vs 0.3%, P < .01). DG counts showed no relationship to age or mean platelet volume and had acceptable within-subject variability that was higher for DGD than control participants (28% vs 12%). Repeat tests confirmed DGD in all persons with initial DG counts <4.0/platelet, but not in those with less severe reductions (4.0-4.8 DG/platelet) or normal DG counts (≥4.9 DG/platelet). Aggregometry and adenosine triphosphate release tests, respectively, had only ~52% and 70% sensitivity for DGD. Confirmed DGD by EM was associated with higher bleeding scores and a bleeding disorder. CONCLUSION: Whole mount EM is useful for the evaluation of suspected PFD due to DGD and detects abnormalities associated with bleeding.

Variability in platelet dense granule adenosine triphosphate release findings amongst patients tested multiple times as part of an assessment for a bleeding disorder.

INTRODUCTION: Lumi-aggregometry quantification of platelet dense granule adenosine triphosphate (ATP) release is commonly used for diagnosing platelet function disorders. As the test findings show considerable variability for healthy controls, we postulated that patient findings might also be variable and investigated patients who were assessed for dense granule ATP release defects more than once. METHODS: Analyses were performed on prospectively collected data for first and second tests for subjects tested for dense granule ATP release defects more than once by the Hamilton Regional Laboratory Program (HRLMP) between January 2007 and June 2013 (cohort I). Similar analyses were performed for subjects who were recruited to a platelet disorder study (cohort II) and were assessed for ATP release defects more than once before October 2015. RESULTS: A total of 150 unique subjects had multiple ATP release tests. Results with individual agonists were variable for many subjects. While normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), impaired release with multiple agonists was confirmed in only some subjects (cohort I: 34%; cohort II: 54%). Inconsistent findings were common (cohort I: 36%; cohort II: 39%). ISTH bleeding scores showed no relationship to the test findings. The finding of impaired ATP release with 2 or more agonists on both tests was not associated with an increased likelihood of a definite bleeding disorder. CONCLUSION: 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.

Dietary fat, genes, and human health.

These studies show that a macronutrient like dietary fat plays an important role in gene expression. In the cases presented here, dietary fat regulates gene expression leading to changes in carbohydrate and lipid metabolism. The interesting outcome of these studies is the finding that the molecular targets for dietary fat action did not converge with the principal targets for hormonal regulation of gene transcription, like hormone receptors. Instead, PUFA-RF targets elements that play key ancillary roles in gene transcription. This is important because it shows how PUFA can interfere with hormone regulation of a specific gene without having generalized effect on overall hormonal control, i.e. PUFA effects are promoter-specific. How PUFA-RF interferes with gene transcription will require the isolation and characterization of PUFA-RF along with the tissue-specific factors targeted by PUFA-RF. A different story emerges when fatty acids activate PPAR. Based on the studies presented here and elsewhere, long chain-highly unsaturated fatty acids (like 20:5,n-3 and 22:6, n-3) or high levels of fat activate PPAR. PPAR directly activates genes like AOX, but also inhibits transcription of genes like S14, FAS, apolipoprotein CIII, transferrin. For S14, the mechanism of inhibition involves sequestration of RXR, a critical factor for T3 receptor binding to DNA. Thus, PPAR can have generalized effects on T3 action or on other nuclear receptors, like vit. D (VDR) and retinoic acid (RAR) receptors, that require RXR for action. For apolipoprotein CIII and transferrin, PPAR/RXR heterodimers compete for HNF-4 binding sites (DR + 1). In addition to HNF-4, COUP-TF, ARP-1 and RXR all bind the DR + 1 type motif. These factors are important for tissue-specific regulation of gene transcription. PPAR can potentially interfere with the transcription of multiple genes through disruption of nuclear receptor signaling leading to changes in phenotype. Clearly, more studies are required to assess the role PPAR plays in the fatty acid regulation of gene transcription and its contribution to chronic disease. Finally, it is clear that dietary fat has the potential to affect gene expression through multiple pathways. Depending on the gene examined, PUFA might augment or abrogate gene transcription which leads to specific phenotypic changes altering metabolism, differentiation or cell growth. These effects can be beneficial to the organism, such as the n-3 PUFA-mediated suppression of serum triglycerides or detrimental, like the saturated and n-6 PUFA-mediated promotion of insulin resistance. How such effects contribute to the onset or progression of specific neoplasia is unclear. However, studies in metabolism might provide important clues for this connection.

The CCAAT box binding factor, NF-Y, is required for thyroid hormone regulation of rat liver S14 gene transcription.

Triiodothyronine (T3) activates rat liver S14 gene transcription through T3 receptors (TRbeta) binding distal thyroid hormone response elements located between -2.8 and -2.5 kilobase pairs upstream from the transcription start site. Previous studies suggested that proximal promoter elements located between -220 to -80 base pairs upstream from the 5' end of the S14 gene were involved in hormone activation of the S14 gene. This report identifies an inverted CCAAT box (or Y box) at -104ATTGG-100 as a core cis-regulatory element. Gel shift studies using rat liver nuclear proteins show that at least three CCAAT-binding factors interact with this region as follows: NF-Y and c/EBP-related proteins formed major complexes, whereas NF-1/CTF forms a minor complex in gel shift assay. Mutation of the Y box indicated that loss of NF-Y binding, but not c/EBP or NF-1, correlated closely with a decline in basal activity and a loss of T3-mediated transactivation. Substitution of the S14 Y box in reporter genes with elements binding only NF-Y elevated basal activity and T3-mediated transactivation, whereas substitution with elements binding c/EBP-related proteins or SP1 displayed low basal activity and T3-mediated transactivation. These studies indicate that NF-Y and TRbeta functionally interact to confer T3 control to the S14 gene.