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As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we use a data set of 256 SARS-CoV-2-positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. During this time frame, two targets in the N gene (N2 and N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3'-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk, 20.02; 95% CI, 11.36 to 35.72; P 0.0001). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3'-mismatch to the N2 probe PBS and increases target dropout risk (relative risk, 11.92; 95% CI, 8.17 to 14.06; P 0.0001). These findings highlight the robust capability of MassARRAY SARS-CoV-2 Panel target results to reveal circulating virus diversity, and they underscore the power of multitarget design to capture variants of concern.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

To develop a novel pharmacogenetic genotyping panel, a multidisciplinary team evaluated available evidence and selected 29 genes implicated in interindividual drug response variability, including 130 sequence variants and additional copy number variants (CNVs). Of the 29 genes, 11 had guidelines published by the Clinical Pharmacogenetics Implementation Consortium. Targeted genotyping and CNV interrogation were accomplished by multiplex single‐base extension using the MassARRAY platform (Agena Biosciences) and multiplex ligation‐dependent probe amplification (MRC Holland), respectively. Analytical validation of the panel was accomplished by a strategic combination of > 500 independent tests performed on 170 unique reference material DNA samples, which included sequence variant and CNV accuracy, reproducibility, and specimen (blood, saliva, and buccal swab) controls. Among the accuracy controls were 32 samples from the 1000 Genomes Project that were selected based on their enrichment of sequence variants included in the pharmacogenetic panel (VarCover.org). Coupled with publicly available samples from the Genetic Testing Reference Materials Coordination Program (GeT‐RM), accuracy validation material was available for the majority (77%) of interrogated sequence variants (100% with average allele frequencies > 0.1%), as well as additional structural alleles with unique copy number signatures (e.g., CYP2D6*5, *13, *36, *68; CYP2B6*29; and CYP2C19*36). Accuracy and reproducibility for both genotyping and copy number were > 99.9%, indicating that the optimized panel platforms were precise and robust. Importantly, multi‐ethnic allele frequencies of the interrogated variants indicate that the vast majority of the general population carries at least one of these clinically relevant pharmacogenetic variants, supporting the implementation of this panel for pharmacogenetic research and/or clinical implementation programs.

Congenital heart defects (CHDs) are caused by a disruption in heart morphogenesis, which is dependent, in part, on a network of transcription factors (TFs) that regulate myocardial development. Heterozygous sequence variants in the basic helix-loop-helix TF gene heart and neural crest derivatives expressed 2 (HAND2) have been reported among some patients with CHDs; however, HAND2 has not yet been established as a Mendelian disease gene. We report a 31-month-old male with unicommissural unicuspid aortic valve, moderate aortic stenosis, and mild pulmonic stenosis. Chromosome analysis revealed a normal 46,XY karyotype, and a CHD sequencing panel was negative for pathogenic variants in NKX2.5, GATA4, TBX5, and CHD7. However, chromosomal microarray (CMA) testing identified a heterozygous 546.0-kb deletion on chromosome 4q34.1 (174364195_174910239[GRCh37/hg19]) that included exons 1 and 2 of SCRG1, HAND2, and HAND2-AS1. Familial CMA testing determined that the deletion was paternally inherited, which supported a likely pathogenic classification as the proband's father had previously undergone surgery for Tetralogy of Fallot. The family history was also notable for a paternal uncle who had previously died from complications related to an unknown heart defect. Taken together, this first report of a HAND2 and HAND2-AS1 deletion in a family with CHDs strongly supports haploinsufficiency of HAND2 as an autosomal dominant cause of CHD.

BackgroundSaliva is an optimal specimen for detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and non-invasive collection. However, together with intrinsic enzymes and oral microbiota, children’s unique dietary habits may introduce substances that interfere with diagnostic testing.MethodsTo determine whether children’s dietary choices impact SARS-CoV-2 detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n=5) who self-collected saliva at home before and at 0, 20, and 60 minutes after eating from 20 foods they selected. Each of seventy-two specimens was split into two volumes and spiked with SARS-CoV-2-negative or -positive standards prior to side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay.ResultsDetection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 minutes after eating 11 of 20 foods. Interference resolved at 20 and 60 minutes after eating all foods except hot dog in one participant. This represented a significant improvement in detection of nucleic acids compared to saliva collected at 0 minutes after eating (P=0.0005).ConclusionsWe demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting 20 minutes after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

The COVID-19 pandemic sparked rapid development of SARS-CoV-2 diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines RT-PCR and MALDI-TOF mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly-specific for the alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Exposure to a high fat diet (HFD) during gestation stimulates neurogenesis and expression of hypothalamic orexigenic neuropeptides that affect consummatory and emotional behaviors. With recent studies showing a HFD to increase inflammation, this report investigated the neuroinflammatory chemokine, CXCL12, and compared the effects of prenatal CXCL12 injection to those of prenatal HFD exposure, first, by testing whether the HFD affects circulating CXCL12 in the dam and the CXCL12 system in the offspring brain, and then by examining whether prenatal exposure to CXCL12 itself mimics the effects of a HFD on hypothalamic neuropeptides and emotional behaviors. Our results showed that prenatal exposure to a HFD significantly increased circulating levels of CXCL12 in the dam, and that daily injections of CXCL12 induced a similar increase in CXCL12 levels as the HFD. In addition, prenatal HFD exposure significantly increased the expression of CXCL12 and its receptors, CXCR4 and CXCR7, in the hypothalamic paraventricular nucleus (PVN) of the offspring. Finally, the results revealed strong similarities in the effects of prenatal HFD and CXCL12 administration, which both stimulated neurogenesis and enkephalin (ENK) expression in the PVN, while having inconsistent or no effect in other regions of the hypothalamus, and also increased anxiety as measured by several behavioral tests. These results focus attention specifically on the CXCL12 chemokine system in the PVN of the offspring as being possibly involved in the stimulatory effects of prenatal HFD exposure on ENK-expressing neurons in the PVN and their associated changes in emotional behavior.

The preservation and stability mechanisms of soil organic carbon (SOC) are the important factors to evaluate the capacity of soil carbon pool and the potential of sustainable utilization. To understand the preservation time and mechanisms of SOC under burial conditions, in the present study, the distributions of total organic carbon (TOC) and stable organic carbon (StOC), and their correlations with the contents of clays and clay minerals and different forms of iron oxides were investigated in the Baoji and Luochuan loess–paleosol profiles. Four facts were observed as the followings. (1) The labile SOC almost was decomposed and the mostly stable SOC was preserved in the loess and paleosol after 375 kyr since their formation. StOC could be preserved at least 762 kyr in both loess and paleosol under burial condition. (2) The TOC was positively correlated with clay contents, with correlation coefficients of 0.72 (Baoji) and 0.63 (Luochuan). (3) The TOC, StOC, mineral-protected organic carbon (MOC), and recalcitrant organic carbon (ROC) were positively correlated with kaolinite, with correlation coefficients of 0.93, 0.72, 0.52, 0.81 (Baoji) and 0.78, 0.58, 0.50, 0.49 (Luochuan), respectively, both with neither illite nor vermiculite. (4) The TOC was highly correlated with complex iron (Fe p ) with correlation coefficients of 0.90 (Baoji) and 0.82 (Luochuan), so with amorphous oxides of iron (Fe o ) as well. Among them, Fe o mainly affected by sorption and Fe p by complexation on SOC preservation, whereas kaolinite had both chemical and physical effects. The values of coefficients further highlight that the contributions of inorganic matters to the fixation of organic carbon were ranked to an order of kaolinite > Fe p > Fe o .