Differential Loss of OAS Genes Indicates Diversification of Antiviral Immunity in Mammals.

One of the main mechanisms of inducing an antiviral response depends on 2'-5'-oligoadenylate synthetases (OAS), which sense double-stranded RNA in the cytoplasm and activate RNase L. Mutations leading to the loss of functional OAS1 and OAS2 genes have been identified as important modifiers of the human immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we performed comparative genomics to search for inactivating mutations of OAS genes in other species of mammals and to establish a model for the diversifying evolution of the OAS gene family. We found that a recombination of the OAS and OAS-like (OASL) loci has led to the loss of OAS2 in camelids, which also lack OAS3. Both paralogs of OASL and OAS3 are absent in Asian pangolins. An evolutionarily ancient OAS paralog, which we tentatively name OAS4, has been lost in pangolins, bats and humans. A previously unknown OAS gene, tentatively named OAS5, is present in Yangochiroptera, a suborder of bats. These differences in the OAS gene repertoire may affect innate immune responses to coronaviruses and other RNA viruses.

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways.

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived protein kinase R (PKR) antagonist RhTRS1 in place of its native PKR antagonists: E3L and K3L (VACVΔEΔK + RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a 'molecular foothold' to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACVΔEΔK + RhTRS1 replication in human cells, mediated by both PKR and ribonuclease L (RNase L). We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage 9. Using our Illumina-based pipeline, we found that some single nucleotide polymorphisms (SNPs) which had evolved during the prior AGM adaptation were rapidly lost, while thirteen single-base substitutions and short indels increased over time, including two SNPs unique to human foreskin fibroblast (HFF)-adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an 'intermediate species' and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

Buccal swab testing with the AmplideX PCR/CE SMN1/2 Plus Kit that assesses copy number and critical mutations for SMA

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that results from mutation of the survival motor neuron 1 gene (SMN1) and the most common genetic cause of infant death. Approximately 95% of SMA cases are caused by a deletion in both alleles of exon 7 in the SMN1 gene. The copy number of the highly homologous SMN2 gene is an important predictor of the severity of SMA as it has been shown to decrease disease severity in a dose-dependent manner. SMN1 and SMN2 only differ by a few nucleotides, presenting a challenge in determining copy numbers. While carriers typically have one copy of SMN1, cis duplication of SMN1 can produce “silent carrier” (2 + 0) genotypes, which are often associated with two SMN1 variants, c.*3 + 80T>G and c.*211_*212del, that can improve the overall carrier detection rate. SMA treatments SPINRAZA®,, Evrysdi™, and ZOLGENSMA® achieve profound benefits on survival and motor milestones by modifying SMN2 splicing or using gene replacement with functional SMN genes. Early detection of SMA (including SMN2 copy number status) and identification of at-risk couples through carrier screening is critical to aid in early intervention and family planning decisions. We developed an accurate and robust single-tube PCR assay and companion software (AmplideX® PCR/CE SMN1/2 Plus Kit*) that uses capillary electrophoresis (CE) to quantify SMN1 and SMN2 copy numbers (0 to ≥4) and determines the presence/absence of the two SMN1 gene duplication “silent carrier” variants, c.*3 + 80T>G and c. *211_*212del, and the SMN2 disease modifier variant c.859G>C. The SMN1/2 Plus Kit has been previously validated for use with DNA isolated from blood. Here, we verify that DNA isolated from buccal swabs can also be used to determine SMN1 and SMN2 copy number and expanded content using this kit. Materials and Methods: A total of 60 DNA samples isolated from buccal swabs, with varying SMN1/2 copies and other positive and negative variants,were tested using the SMN1/2 Plus kit at a single site (Asuragen). Samples were tested in two cohorts: an initial cohort containing 17 samples isolated from buccal swabs with column or magnetic bead-based methods, and a second cohort of 43 samples isolated from matched blood and buccal samples using column-based methods. PCR products were generated using a Veriti thermal cycler and resolved on Applied Biosystems™ 3500xL, 3130xl, 3730xl, and SeqStudio™ Genetic Analyzers. Raw electrophoresis data (.fsa) files were directly imported into an assay-specific analysis module of the AmplideX® Reporter software that automates peak detection and sizebased classification, SMN1 and SMN2 exon 7 copy number quantification, detection of gene duplication and disease modifier variants, and sample- and batch-level quality control checks. Samples were analyzed using the default (kit calibrator) and user-defined calibration (UDC) (buccal DNA) workflows as described in the protocol. Results: For the initial cohort of 17 Buccal swab samples, SMN1 copy number calls were concordant with MLPA reference results (reported as 0, 1, 2, or ≥3) for 16/17 (94.1%) of samples with default calibration and 17/17 (100%) of samples with UDC. Further, concordance for carrier samples (1 SMN1 copy) were 7/7 (100%) using both methods. SMN2 copy numbercallswere concordant with MLPA reference results for 17/17 (100%) of samples with either default calibration or UDC. For the second cohort of 43 buccal swab samples with matched blood samples, SMN1 and SMN2 copy number calls were concordant with the results from the paired whole blood for at least 95% of samples assessed across the four different CE platforms. All variant status calls were concordant between the buccal swab and whole blood results. Conclusions: Here, we demonstrate that buccal swabs are a compatible DNA source for the quantification of 0, 1, 2, 3, and ≥4 gene copies of both SMN1 and SMN2 and the status determination of three clinically significant variants using the single-tube PCR/CE SMN1/2 Plus kit. Although d fault calibration yielded high rates of agreement between copy number results from buccal swabs and reference results, analyzing samples with user-defined calibration (i.e. calibrating to a buccal swab sample) modestly improved concordance. These results suggest that DNA samples isolated from buccal swabs are compatible with this assay and has implications for more facile sample collection and handling, particularly given the strain of COVID-19 on healthcare infrastructure.

2022 Southern Medical Research Conference

The proceedings contain 593 papers. The topics discussed include: association of transthyretin VAL122ILE variant with incident heart failure and mortality among Black Americans: insights from the regards study;vitamin d modulates histone modifications governing the natriuretic peptide receptor-a gene;cell to cell communication through entanglement and superconductivity improving left ventricular function in an uncoupled state;association of serum lipid levels with COVID-19 infection, severity and mortality;impaired glucose tolerance in guanylyl cyclase/natriuretic peptide receptor-a gene-knockout and gene-duplication mutant mice;meta-analysis of randomized vs observational studies of the effects of invasive therapy in patients with non-ST-elevation myocardial infarction and chronic kidney disease;a retrospective analysis of mortality in adult patients with acute coronary syndrome and cardiogenic shock requiring temporary mechanical circulatory support;and mitochondrial myopathy mimicking Guillain-Barre syndrome in a 21-year-old graduate student.