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A novel platform for the electronic detection of nucleic acids on microarrays is introduced and shown to perform well as a selective detection system for applications in molecular diagnostics. A gold electrode in a printed circuit board is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe, containing ferrocene-modified nucleotides and complementary to the target in the region adjoining the capture probe binding site, is held in close proximity to the SAM in a sandwich complex. The SAM allows electron transfer between the immobilized ferrocenes and the gold, while insulating the electrode from soluble redox species, including unbound signaling probes. Here, we demonstrate sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. In addition, single nucleotide polymorphism discrimination is shown. A genotyping chip for the C282Y single nucleotide polymorphism associated with hereditary hemochromatosis is used to confirm the genotype of six patients' DNA. In addition, a gene expression-monitoring chip is described that surveys five genes that are differentially regulated in the cellular apoptosis response. Finally, custom modification of individual electrodes through sequence-specific hybridization demonstrates the potential of this system for infectious disease diagnostics. The versatility of the electronic detection platform makes it suitable for multiple applications in diagnostics and pharmacogenetics.

ObjectiveA novel algorithm to identify fetal microdeletion events in maternal plasma has been developed and used in clinical laboratory‐based noninvasive prenatal testing. We used this approach to identify the subchromosomal events 5pdel, 22q11del, 15qdel, 1p36del, 4pdel, 11qdel, and 8qdel in routine testing. We describe the clinical outcomes of those samples identified with these subchromosomal events.MethodsBlood samples from high‐risk pregnant women submitted for noninvasive prenatal testing were analyzed using low coverage whole genome massively parallel sequencing. Sequencing data were analyzed using a novel algorithm to detect trisomies and microdeletions.ResultsIn testing 175 393 samples, 55 subchromosomal deletions were reported. The overall positive predictive value for each subchromosomal aberration ranged from 60% to 100% for cases with diagnostic and clinical follow‐up information. The total false positive rate was 0.0017% for confirmed false positives results; false negative rate and sensitivity were not conclusively determined.ConclusionNoninvasive testing can be expanded into the detection of subchromosomal copy number variations, while maintaining overall high test specificity. In the current setting, our results demonstrate high positive predictive values for testing of rare subchromosomal deletions. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research. Many laboratories are testing for pharmacogenetic (PGx) markers, common genetic variants that are usually considered only when a patient is likely to be exposed to a Accepted for publication June 21, 2010. R.B., A.E.-B., C.S., A.V., and M.Z. are employees of AutoGenomics (manufacturer of several pharmacogenetic assays used in this study); M.B., A.B., and K.M. are employees of Quest Diagnostics Inc.; J.M. is an employee of Idaho Technology (manufacturer of the reagents used to genotype CYP2C9 and VKORC1 loci for this project);

Renal cell carcinoma with (angio) leiomyomatous stroma (RCCLMS) is included as a provisional entity in the 2016 World Health Organization (WHO) classification of renal epithelial neoplasia; however, debate remains whether it represents a distinct entity or a heterogenous group of renal cell carcinomas (RCCs) with overlapping morphology. Also, its relationship to similar tumors occurring in the setting of tuberous sclerosis complex (TSC) is not fully addressed. We analyzed the clinicopathologic, immunohistochemical, and molecular characteristics of 23 sporadic RCCs associated with smooth muscle stroma and classified them into 2 groups, independent of molecular results: (1) RCCLMS (n=18) and (2) clear cell renal cell carcinoma (CCRCC) (n=5). The classification of a case as “RCCLMS” was based on morphologic comparison with 5 “index” RCCs from 3 patients with TSC showing similar features and the presence of diffuse CK7 expression. To investigate mutational and copy number alterations, a 170-gene solid tumor panel was utilized to sequence 14 RCCLMSs and control of 5 CCRCCs. Also, 4 RCCLMSs, suspicious for chromosome 8 monosomy, were further evaluated by a broader 479 gene sequencing panel that included ELOC (also referred to as TCEB1). Clinical information and follow-up data were obtained from electronic medical records. The mean age of patients with RCCLMS was 52 years (range, 33 to 69) with male:female ratio of 1:2. Macroscopically, all tumors were solitary and predominantly (82%) tan/red, circumscribed, and solid. The average tumor size was 2.3 cm (range, 1.1 to 4.5). Microscopically, the distinctive feature included tumor nodules of elongated and frequently branching tubules lined by cells with voluminous clear to mildly eosinophilic cytoplasm (100%), separated by focal to prominent smooth muscle stroma. Additional frequently identified features included: biphasic pattern of collapsed acini surrounding tubules with voluminous cytoplasm (50%), focal papillary architecture (39%), peritumoral lymphoid aggregates (39%), and hemosiderin-laden macrophages (33%). All 11 (100%) RCCLMSs with available staging information were pT1; 78% were WHO/International Society of Urologic Pathology (ISUP) grade 2 and 22% grade 3. Immunophenotypically, RCCLMSs were characterized by diffuse CK7, CAM5.2 and CD10 reactivity (100%). All patients with available follow-up (n=10) were alive and without disease progression after a mean and median follow-up of 25.2 (range: 1 to 58) and 25 months, respectively. The molecular results showed recurrent mutations in all RCCLMS: TSC1 (4), TSC2 (4), MTOR (6), and/or ELOC (2). Five control CCRCCs demonstrated primary alterations in VHL gene, while all 14 RCCLMS cases tested had intact VHL gene. Of 2 RCCLMSs with confirmed monosomy 8, 1 showed a hotspot ELOC mutation without TSC/MTOR mutations, and 1 showed a previously undescribed 3-bp in-frame ELOC deletion, along with a truncating TSC1 mutation. In conclusion, RCCLMS, as defined herein, harbors recurrent mutations of TSC1/TSC2, MTOR, and/or ELOC, consistent with hyperactive MTOR complex. Our findings argue that these tumors represent the sporadic counterpart to morphologically identical tumors occurring in TSC patients. Finally, the data support that RCCLMS is a novel subtype of RCC with unique morphologic, immunohistochemical, and molecular characteristics that is distinct from CCRCC and clear cell-papillary RCC.

Background Four SARS-CoV-2 variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. Method Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March, 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. Results 2779 patients identified with either alpha (N = 1153), gamma (N = 122), delta (N = 808) or omicron variants (N = 696) were selected for analysis. No difference in frequency of hospitalization, ICU admission, and death were found among alpha, gamma, and delta variants. However, patients with omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (X2 = 12.8 p < 0.001, X2 = 21.6 p < 0.002, X2 = 9.6 p = 0.01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with delta or omicron variants (218/808 [26.9%] and 513/696 [73.7%], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between omicron sub-variants BA.1 and BA.2. Conclusions Disease severity associated with alpha, gamma, and delta variants is comparable while omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with omicron infection.