Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.

BACKGROUND: Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques. METHODS: Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA). RESULTS: NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel. CONCLUSION: This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Structural modeling of ion channels using AlphaFold2, RoseTTAFold2, and ESMFold.

Ion channels play key roles in human physiology and are important targets in drug discovery. The atomic-scale structures of ion channels provide invaluable insights into a fundamental understanding of the molecular mechanisms of channel gating and modulation. Recent breakthroughs in deep learning-based computational methods, such as AlphaFold, RoseTTAFold, and ESMFold have transformed research in protein structure prediction and design. We review the application of AlphaFold, RoseTTAFold, and ESMFold to structural modeling of ion channels using representative voltage-gated ion channels, including human voltage-gated sodium (NaV) channel - NaV1.8, human voltage-gated calcium (CaV) channel - CaV1.1, and human voltage-gated potassium (KV) channel - KV1.3. We compared AlphaFold, RoseTTAFold, and ESMFold structural models of NaV1.8, CaV1.1, and KV1.3 with corresponding cryo-EM structures to assess details of their similarities and differences. Our findings shed light on the strengths and limitations of the current state-of-the-art deep learning-based computational methods for modeling ion channel structures, offering valuable insights to guide their future applications for ion channel research.

Is There a Need for Abdominal CT Scan in Trauma Patients With a Low-Risk Mechanism of Injury and Normal Vital Signs?

Background Clinically significant injuries are often missed in trauma patients with low-risk mechanisms of injury and lack of "red flags," such as abnormal vital signs. The purpose of this retrospective analysis was to evaluate the efficacy of computed axial tomography (CT) for identifying occult injuries in a high-volume trauma center. Methods Records from our institutional trauma registry were retrospectively extracted, examining encounters from January 2015 to October 2019. Those patients between the ages of 18 and 65 who were referred to the trauma team with a CT scan of the abdomen and had low-risk mechanisms of injury, a Glasgow Coma Scale (GCS) score of 15, and normal vital signs at presentation were included. Patients in the lowest trauma categorization (Level Three, Consult) met the study definition for the low-risk mechanism of injury. Demographic and clinical data were abstracted for all patients. For this analysis, patients were divided into two groups based on age (18 - 40 years or 40 - 65 years). Injuries found on CT, their clinical significance, and the likelihood of being missed without CT were determined. Results Of 2,103 blunt trauma patients that received a CT scan of the abdomen from January 2015 to October 2019, 134/2,103 (6.4%) met the inclusion criteria (mean age: 44.6 years; 72.3% male). Patients between the ages of 40 and 65 years comprised 61.2% (82/134) of the study population. Of the included patients, 17.2% (23/134) had at least one acute traumatic injury identified after CT imaging of the torso. Occult injuries found on CT included rib fracture with associated lung injuries (10/23, 43.5%), splenic laceration (4/23, 17.4%), liver laceration (3/23, 13.0%), gluteal hematoma with active bleeding (1/23, 4.3%), sternal fractures (3/23, 13.0%), and thoracic or lumbar spine fractures (2/23, 8.7%). An independent review of the medical records determined that 9.0% (12/134) of these patients had traumatic injuries that would have been missed based on clinical examination without CT. Conclusions Based on our experience, utilizing CT imaging of at least the abdomen as a routine screening measure for all trauma consults - even low-risk patients with normal vital signs - can rapidly and accurately identify clinically significant injuries that would have been otherwise missed in a notable portion of the population.

Design and validation of an oligonucleotide microarray for the detection of genomic rearrangements associated with common hereditary cancer syndromes.

BACKGROUND: Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders. METHODS: We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs. RESULTS: Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing. CONCLUSION: The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.