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1. Novel Pooling Strategies for Genetic Testing, with Application to Newborn Screening

   https://doi.org/10.1287/mnsc.2021.4289  

   El Hajj, Hussein ; Bish, Douglas R. ; Bish, Ebru K. ; Kay, Denise M. ( March 2022 , Management Science)

   Newborn screening (NBS) is a state-level initiative that detects life-threatening genetic disorders for which early treatment can substantially improve health outcomes. Cystic fibrosis (CF) is among the most prevalent disorders in NBS. CF can be caused by a large number of mutation variants to the CFTR gene. Most states use a multitest CF screening process that includes a genetic test (DNA). However, due to cost concerns, DNA is used only on a small subset of newborns (based on a low-cost biomarker test with low classification accuracy), and only for a small subset of CF-causing variants. To overcome the cost barriers of expanded genetic testing, we explore a novel approach, of multipanel pooled DNA testing. This approach leads not only to a novel optimization problem (variant selection for screening, variant partition into multipanels, and pool size determination for each panel), but also to novel CF NBS processes. We establish key structural properties of optimal multipanel pooled DNA designs; develop a methodology that generates a family of optimal designs at different costs; and characterize the conditions under which a 1-panel versus a multipanel design is optimal. This methodology can assist decision-makers to design a screening process, considering the cost versus accuracy trade-off. Our case study, based on published CF NBS data from the state of New York, indicates that the multipanel and pooling aspects of genetic testing work synergistically, and the proposed NBS processes have the potential to substantially improve both the efficiency and accuracy of current practices. This paper was accepted by Stefan Scholtes, healthcare management. 

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2. Equity in genetic newborn screening

   https://doi.org/10.1002/nav.21882  

   El Hajj, Hussein ; Bish, Douglas R. ; Bish, Ebru K. ( December 2019 , Naval Research Logistics (NRL))

   State‐level newborn screening allows for early treatment of genetic disorders, which can substantially improve health outcomes for newborns. As the cost of genetic testing decreases, it is becoming an essential part of newborn screening. A genetic disorder can be caused by many mutation variants; therefore, an important decision is to determine which variants to search for (ie, thepaneldesign), under a testing budget. The frequency of variants that cause a disorder and the incidence of the disorder vary by racial/ethnic group. Consequently, it is important to consider equity issues in panel design, so as to reduce disparities among different groups. We study the panel design problem using cystic fibrosis (CF) as a model disorder, considering the trade‐offs between equity and accuracy, under a limited budget. Most states use a genetic test in their CF screening protocol, but panel designs vary, and, due to cost, no state's panel includes all CF‐causing variants. We develop models that design equitable genetic testing panels, and compare them with panels that maximize sensitivity in the general population. Our case study, based on realistic CF data, highlights the value of equitable panels and provides important insight for newborn screening practices.

    

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3. ClinVar and HGMD genomic variant classification accuracy has improved over time, as measured by implied disease burden

   https://doi.org/10.1186/s13073-023-01199-y  

   Sharo, Andrew G. ; Zou, Yangyun ; Adhikari, Aashish N. ; Brenner, Steven E. ( July 2023 , Genome Medicine)

   Curated databases of genetic variants assist clinicians and researchers in interpreting genetic variation. Yet, these databases contain some misclassified variants. It is unclear whether variant misclassification is abating as these databases rapidly grow and implement new guidelines.

   Using archives of ClinVar and HGMD, we investigated how variant misclassification has changed over 6 years, across different ancestry groups. We considered inborn errors of metabolism (IEMs) screened in newborns as a model system because these disorders are often highly penetrant with neonatal phenotypes. We used samples from the 1000 Genomes Project (1KGP) to identify individuals with genotypes that were classified by the databases as pathogenic. Due to the rarity of IEMs, nearly all such classified pathogenic genotypes indicate likely variant misclassification in ClinVar or HGMD.

   While the false-positive rates of both ClinVar and HGMD have improved over time, HGMD variants currently imply two orders of magnitude more affected individuals in 1KGP than ClinVar variants. We observed that African ancestry individuals have a significantly increased chance of being incorrectly indicated to be affected by a screened IEM when HGMD variants are used. However, this bias affecting genomes of African ancestry was no longer significant once common variants were removed in accordance with recent variant classification guidelines. We discovered that ClinVar variants classified as Pathogenic or Likely Pathogenic are reclassified sixfold more often than DM or DM? variants in HGMD, which has likely resulted in ClinVar’s lower false-positive rate.

   Considering misclassified variants that have since been reclassified reveals our increasing understanding of rare genetic variation. We found that variant classification guidelines and allele frequency databases comprising genetically diverse samples are important factors in reclassification. We also discovered that ClinVar variants common in European and South Asian individuals were more likely to be reclassified to a lower confidence category, perhaps due to an increased chance of these variants being classified by multiple submitters. We discuss features for variant classification databases that would support their continued improvement.

    

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4. Identifying SARS-CoV-2 Variants of Concern through Saliva-Based RT-qPCR by Targeting Recurrent Mutation Sites

   https://doi.org/10.1128/spectrum.00797-22  

   Ham, Rachel E. ; Smothers, Austin R. ; Che, Rui ; Sell, Keegan J. ; Peng, Congyue Annie ; Dean, Delphine ( June 2022 , Microbiology Spectrum)

   Mostafa, Heba H. (Ed.)

   ABSTRACT SARS-CoV-2 variants of concern (VOCs) continue to pose a public health threat which necessitates a real-time monitoring strategy to complement whole genome sequencing. Thus, we investigated the efficacy of competitive probe RT-qPCR assays for six mutation sites identified in SARS-CoV-2 VOCs and, after validating the assays with synthetic RNA, performed these assays on positive saliva samples. When compared with whole genome sequence results, the SΔ69-70 and ORF1aΔ3675-3677 assays demonstrated 93.60 and 68.00% accuracy, respectively. The SNP assays (K417T, E484K, E484Q, L452R) demonstrated 99.20, 96.40, 99.60, and 96.80% accuracies, respectively. Lastly, we screened 345 positive saliva samples from 7 to 22 December 2021 using Omicron-specific mutation assays and were able to quickly identify rapid spread of Omicron in Upstate South Carolina. Our workflow demonstrates a novel approach for low-cost, real-time population screening of VOCs. IMPORTANCE SARS-CoV-2 variants of concern and their many sublineages can be characterized by mutations present within their genetic sequences. These mutations can provide selective advantages such as increased transmissibility and antibody evasion, which influences public health recommendations such as mask mandates, quarantine requirements, and treatment regimens. Our RT-qPCR workflow allows for strain identification of SARS-CoV-2 positive saliva samples by targeting common mutation sites shared between variants of concern and detecting single nucleotides present at the targeted location. This differential diagnostic system can quickly and effectively identify a wide array of SARS-CoV-2 strains, which can provide more informed public health surveillance strategies in the future. 

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5. High-Surety Isothermal Amplification and Detection of SARS-CoV-2

   https://doi.org/10.1128/mSphere.00911-20  

   Bhadra, Sanchita ; Riedel, Timothy E. ; Lakhotia, Simren ; Tran, Nicholas D. ; Ellington, Andrew D. ( June 2021 , mSphere)

   Rasmussen, Angela L. (Ed.)

   ABSTRACT Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes. 

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