Incidental findings in a series of 2500 gene panel tests for a genetic predisposition to cancer: Results and impact on patients.

With next generation sequencing, physicians are faced with more complex and uncertain data, particularly incidental findings (IF). Guidelines for the return of IF have been published by learned societies. However, little is known about how patients are affected by these results in a context of oncogenetic testing. Over 4 years, 2500 patients with an indication for genetic testing underwent a gene cancer panel. If an IF was detected, patients were contacted by a physician/genetic counsellor and invited to take part in a semi-structured interview to assess their understanding of the result, the change in medical care, the psychological impact, and the transmission of results to the family. Fourteen patients (0.56%) were delivered an IF in a cancer predisposition gene (RAD51C, PMS2, SDHC, RET, BRCA2, CHEK2, CDKN2A, CDH1, SUFU). Two patients did not collect the results and another two died before the return of results. Within the 10 patients recontacted, most of them reported surprise at the delivery of IF, but not anxiety. The majority felt they had chosen to obtain the result and enough information to understand it. They all initiated the recommended follow-up and did not regret the procedure. Information regarding the IF was transmitted to their offspring but siblings or second-degree relatives were not consistently informed. No major adverse psychological events were found in our experience. IF will be inherent to the development of sequencing, even for restricted gene panels, so it is important to increase our knowledge on the impact of such results in different contexts.

Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist.

The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.

Key role of the P2Y(1) receptor in tissue factor-induced thrombin-dependent acute thromboembolism: studies in P2Y(1)-knockout mice and mice treated with a P2Y(1) antagonist.

BACKGROUND: ADP plays a key role in hemostasis, acting through 2 platelet receptors: the P2Y(1) receptor and an unidentified P2 receptor, called P2cyc, coupled to adenylyl cyclase inhibition, which is the target of the antiplatelet drug clopidogrel. We showed that the P2Y(1) receptor is an essential cofactor in thrombotic states induced by intravenous infusion of collagen and epinephrine. The aim of the present study was to assess the role of this receptor in thrombin-dependent tissue factor-induced thromboembolism. METHODS AND RESULTS: Human thromboplastin was injected intravenously into wild-type or P2Y(1)-deficient mice, and the effects on platelet count and mortality were determined and plasma thrombin-antithrombin III (TAT) complexes were quantified. P2Y(1)-deficient mice were resistant to the thromboembolism induced by injection of thromboplastin. Whereas the platelet count decreased sharply in wild-type mice, there was no significant drop in platelets in P2Y(1)-knockout mice. The platelet consumption in wild-type mice was probably due to thrombin generation, because it was abolished by hirudin. Thromboplastin also led to a rise in TAT complexes in plasma, again reflecting thrombin formation. This effect, however, was less important in P2Y(1)-knockout mice than in wild-type mice, indicating that less thrombin was generated in the absence of P2Y(1). Similar results were obtained after intravenous administration of N:(6)-methyl-2'-deoxyadenosine-3':5'-bisphosphate, a selective antagonist of the P2Y(1) receptor, to wild-type mice. CONCLUSIONS: Our results demonstrate a role of the P2Y(1) receptor in thrombotic states involving thrombin generation and provide further evidence for the potential relevance of this receptor as a target for antithrombotic drugs.

Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors.

Platelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y(1) receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADPbetaS. Platelets incubated for 1 h at 37 degrees C with 1 mM ADPbetaS and resuspended in Tyrode's buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADPbetaS treated platelets loaded with fura-2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT(2A) receptor while adrenaline had no such effect. These results suggested that the refractory state of ADPbetaS treated platelets was entirely due to desensitization of the P2Y(1) receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y(1) and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y(1) and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y(1) sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y(1) receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y(1) receptor, where ADPbetaS treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y(1) and P2cyc receptors are differently regulated during platelet activation.