Purpose: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). Experimental Design: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1–CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case–control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. Results: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)–like (N = 226). We then examined their association with breast cancer risk in the case–control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35–3.41), 1.57 (95% CI, 1.41–1.75), and 1.19 (95% CI, 1.08–1.31), respectively. The meta-analysis of population-specific datasets showed similar results. Conclusions: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Some patients with IDA are accompanied by thrombocytosis, from which the expression of α-tubulin and β-tubulin within platelets reduced obviously compared with those with normal platelet counts and healthy controls respectively. It is implied that downregulation of tubulin probably is a part of the pathogenesis leading to increased platelet counts in IDA.

Objective: The expression of glucocorticoid receptors within platelets from newly diagnosed Immune Thrombocytopenia (ITP) patients in the adult was investigated. Methods: GR expression in platelets from newly diagnosed ITP patients and healthy controls was measured using flow cytometry. Subsequently, platelets RNA and proteins were isolated and used for confirming the flow cytometry results by using RT-qPCR and ELISA. Results: Flow cytometry showed that the percentages of platelets expressing GRα and GRβ from ITP patients were significantly higher than those from healthy controls (P < 0.05). qPCR and ELISA confirmed that GRα and GRβ were increased at both RNA transcription and protein expression levels within platelets from ITP patients compared with healthy controls. Conclusion: We speculated that the up-regulation of glucocorticoid receptor within platelets may be an important biological feature of platelets in patients with ITP, and may also play an important role in the treatment of ITP, which is worthy of further study.

To observe the differences in proteins between adult patients with chronic immune thrombocytopenic purpura (ITP) and healthy adults. 30 patients with chronic ITP and 30 healthy controls were enrolled into the study. The platelet total protein was extracted from peripheral venous blood of 10 chronic ITP patients and 10 healthy controls respectively, and subjected to two-dimensional electrophoresis (2-DE) to find the differential protein spot between chronic ITP patients and healthy controls, then the differential protein spots were identified by mass spectrometry. Subsequently, platelets RNA and proteins were isolated from the other 20 chronic ITP patients and 20 healthy controls respectively, and used for confirming the 2-DE and mass spectrometry results by using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme linked immunosorbent assay (ELISA). 2-DE combined with mass spectrometry revealed that calreticulin (CRT) expressed normally within platelets from healthy controls, while it reduced within platelets from patients with chronic ITP. qPCR and ELISA confirmed that CRT was decreased at both RNA transcription and protein expression levels within platelets from chronic ITP patients compared with healthy controls. Decreased transcription and expression of CRT within platelets may play an important role in the pathogenesis of chronic ITP, which is worthy of further study. Keywords Chronic immune thrombocytopenic purpura (cITP) Á Platelets Á Calreticulin (CRT) Á Two-dimensional electrophoresis (2-DE) Á Mass spectrometry Á Silver staining Á Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Á Enzyme linked immunosorbent assay (ELISA)

<p>List of all analyzed CHEK2 variants with results of KAP1/CHK2 kinase and localization assays and the results from recent previously published functional analyses of the CHEK2 VUS.</p>