Clinical utility of regions of homozygosity (ROH) identified in exome sequencing: when to pursue confirmatory uniparental disomy testing for imprinting disorders?

OBJECTIVES: Regions of homozygosity (ROH) could implicate uniparental disomy (UPD) on specific chromosomes associated with imprinting disorders. Though the algorithms for ROH detection in exome sequencing (ES) have been developed, optimal reporting thresholds and when to pursue confirmatory UPD testing for imprinting disorders remain in ambiguity. This study used a data-driven approach to assess optimal reporting thresholds of ROH in clinical practice. METHODS: ROH analysis was performed using Automap in a retrospective cohort of 8,219 patients and a prospective cohort of 1,964 patients with ES data. Cases with ROH on imprinting-disorders related chromosomes were selected for additional methylation-specific confirmatory testing. The diagnostic yield, the ROH pattern of eventually diagnosed cases and optimal thresholds for confirmatory testing were analyzed. RESULTS: In the retrospective analysis, 15 true UPD cases of imprinting disorders were confirmed among 51 suspected cases by ROH detection. Pattern of ROH differed between confirmed UPD and non-UPD cases. Maximized yield and minimized false discovery rate of confirmatory UPD testing was achieved at the thresholds of >20 Mb or >25 % chromosomal coverage for interstitial ROH, and >5 Mb for terminal ROH. Current recommendation by ACMG was nearly optimal, though refined thresholds as proposed in this study could reduce the workload by 31 % without losing any true UPD diagnosis. Our refined thresholds remained optimal after independent evaluation in a prospective cohort. CONCLUSIONS: ROH identified in ES could implicate the presence of clinically relevant UPD. This study recommended size and coverage thresholds for confirmatory UPD testing after ROH detection in ES, contributing to the development of evidence-based reporting guidelines.

Development and validation of a cost-effective virtual reality educational tool to reduce anxiety and improve set-up accuracy in radiotherapy patients.

PURPOSE: This study proposes a cost-effective method for educating radiotherapy patients through an immersive virtual reality (VR) system. METHODS: The VR educational tool comprises VR glasses, a handheld controller, the scientific knowledge of radiotherapy, radiotherapy demonstration, and an audio introduction. To verify its efficacy, 120 radiotherapy patients with tumors were prospectively enrolled and divided into the control group or VR intervention group. After the first treatment, set-up errors, including three translation errors and three rotation errors, were recorded in six directions. In addition, participants were required to complete a questionnaire before radiotherapy to assess anxiety and understanding degrees. The questionnaire was scored using a five-point Likert Scale. Finally, Spearman's rank correlation test was used to evaluate set-up errors and questionnaire scores. RESULTS: The set-up errors are significantly reduced in AP, SI, total translation, Roll and total rotation in the intervention group compared with the control group (p < 0.05). The scores are higher in the intervention group than in the control group in question 1 (2.1 ± 0.58 vs. 3.3 ± 0.55), question 2 (1.3 ± 0.44 vs. 2.5 ± 0.65), question 4 (2.2 ± 0.65 vs. 3.2 ± 0.82), question 5 (1.8 ± 0.59 vs. 3.1 ± 0.79), and all subscales (5.5 ± 1.2 vs. 8.9 ± 1.3 and 6.4 ± 1.3 vs. 9.2 ± 1.5). The scores of high, moderate, and low correlation are 47 (74%), 15 (23%), and 2 (3%) for the control group and 44 (69%), 17 (26%), and 3 (5%) for the intervention group, respectively. CONCLUSION: The VR educational tool can significantly improve comprehension and reduce anxiety. There is a strong correlation between set-up errors and questionnaire scores. The VR educational tool may help reduce set-up errors for radiotherapy patients.

Rapid detection of twenty-nine common Chinese glucose-6-phosphate dehydrogenase variants using a matrix-assisted laser desorption/ionization-time of flight mass spectrometry assay on dried blood spots.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease. Current neonatal screening methods for G6PD deficiency primarily rely on the use of biochemical tests. However, only 15%-20% of female carriers were estimated to have been detected using these tests. As a better alternative, DNA-based tests could be used for G6PD deficiency screening. We aimed to develop a matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assay for G6PD variant detection. METHODS: A MALDI-TOF MS assay with multiprimer extension (multi-PEX) was developed to rapidly and accurately detect the 29 common G6PD variants in the Chinese population using a dried blood spot as a template. A parallel study screening 571 unrelated neonatal samples using the MALDI-TOF MS and fluorescence quantitative enzymatic assays was performed. All results were confirmed by Sanger sequencing in a blind study. RESULTS: In 571 unrelated neonatal samples, 34 positive samples, including 26 samples from hemizygous males and eight samples from heterozygous females, were correctly identified, yielding a clinical sensitivity of 100%. The results were validated using Sanger sequencing with 100% concordance. In contrast, the fluorescence quantitative enzymatic assay had a 75% false negative and 88.8% false positive rate for the detection of heterozygous G6PD deficient females. CONCLUSIONS: We established a reliable MALDI-TOF MS assay for G6PD deficiency screening in the Chinese population maximizing the chance of detection of heterozygous G6PD deficient females and reducing the false negative and false positive rates associated with routinely used newborn screening procedures.

Diagnostic yield of additional exome sequencing after the detection of long continuous stretches of homozygosity (LCSH) in SNP arrays.

Long continuous stretches of homozygosity (LCSH) are associated with risk of recessive disorders. Though LCSH can be detected by SNP microarrays, additional testing is necessary to clarify the clinical significance. This study is to assess the yield of additional exome sequencing (ES) after LCSH detection and inform the likelihood of eventual diagnosis. In 2226 patients referred to SNP microarrays, 35 patients met the criteria of indicative LCSH. These patients were recruited and went through additional ES. The diagnostic yield was analyzed, and the LCSH pattern was compared between eventually diagnosed cases and those undiagnosed. The results showed additional ES attained a diagnostic yield of 31.4% (11/35), but only one-third of the yield (11.4%, 4/35) was relevant to LCSH. In contrast, two-thirds of the diagnostic variants (20%, 7/35) were de novo or dominantly inherited, irrelevant to the original LCSH finding. No particular LCSH pattern, including the chromosomal coverage or LCSH size, was found to associate with the diagnostic outcome. We concluded that additional ES after LCSH detection could reveal diagnostic variants, but it is strongly recommended to consider all possible inheritance mode, as the diagnostic variants may be irrelevant to the original LCSH finding.

Genistein decreases A549 cell viability via inhibition of the PI3K/AKT/HIF­1α/VEGF and NF­κB/COX­2 signaling pathways.

Genistein is an important chemopreventive agent against atherosclerosis and cancer. However, whether genistein is effective in the treatment of lung cancer, and its underlying mechanism, remains to be determined. The present study demonstrated that genistein treatment of A549 lung cancer cells decreased viability in a dose­ and time­dependent manner, and induced apoptosis. Additionally, A549 cells exhibited significantly increased reactive oxygen species formation and cytochrome­c leakage, and activated caspase­3, B­cell lymphoma 2­associated X protein and apoptosis inducing factor expression levels, which are involved in the mitochondrial apoptosis pathway. Furthermore, the phosphatidylinositol­4,5­biphosphate 3­kinase (PI3K)/protein kinase B (AKT)/hypoxia­inducible factor­1α (HIF­1α) and nuclear factor­κB (NF­κB)/cyclooxygenase­2 (COX­2) signaling pathways were significantly downregulated by genistein treatment. In conclusion, reduced proliferation and increased apoptosis in A549 lung cancer cells was associated with inhibition of the PI3K/AKT/HIF­1α/ and NF­κB/COX­2 signaling pathways, which implicates genistein as a potential chemotherapeutic agent for the treatment of lung cancer.

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells.

Lung cancer is the most common type of malignant tumor, but the molecular mechanisms for lung cancer progression remains to be elusive. Here, we demonstrated that FBP1 (Fructose-1, 6-bisphosphatase) was frequently down-regulated in lung cancer tissues and cells, and FBP1 down-regulation was associated with poor prognosis in lung cancer patients. Restored FBP1 expression inhibited glucose uptake and lactate production, but induced oxygen consumption. Restored FBP1 expression also inhibited lung cancer cells proliferation and invasion under hypoxia in vitro, and inhibited lung cancer growth in vivo. Moreover, we confirmed DNA methylation in the promoter contributed to the decrease of FBP1 expression in lung cancer cells. We identified Zinc finger E-box-binding homeobox 1 (ZEB1) bond to FBP1 promoter to enhance DNA methylation in lung cancer cells. Our findings indicate that the down-regulation of FBP1 is a critical oncogenic event in lung cancer progression.