Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

CONTEXT.­: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. OBJECTIVE.­: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. DESIGN.­: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. RESULTS.­: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. CONCLUSIONS.­: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

Recommendations for Interpreting and Reporting Silent Carrier and Disease-Modifying Variants in SMA Testing Workflows.

Genetic testing for SMA diagnosis, newborn screening, and carrier screening has become a significant public health interest worldwide, driven largely by the development of novel and effective molecular therapies for the treatment of spinal muscular atrophy (SMA) and the corresponding updates to testing guidelines. Concurrently, understanding of the underlying genetics of SMA and their correlation with a broad range of phenotypes and risk factors has also advanced, particularly with respect to variants that modulate disease severity or impact residual carrier risks. While testing guidelines are beginning to emphasize the importance of these variants, there are no clear guidelines on how to utilize them in a real-world setting. Given the need for clarity in practice, this review summarizes several clinically relevant variants in the SMN1 and SMN2 genes, including how they inform outcomes for spinal muscular atrophy carrier risk and disease prognosis.

Multisite Evaluation and Validation of a Sensitive Diagnostic and Screening System for Spinal Muscular Atrophy that Reports SMN1 and SMN2 Copy Number, along with Disease Modifier and Gene Duplication Variants.

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.∗3+80T>G and c.∗211_∗212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a single-tube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were ≥98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results.

Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication.

Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.

Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.

DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a small-volume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons.

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.

Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly.

Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.

Identification and characterization of a Mef2 transcriptional activator in schistosome parasites.

Myocyte enhancer factor 2 protein (Mef2) is an evolutionarily conserved activator of transcription that is critical to induce and control complex processes in myogenesis and neurogenesis in vertebrates and insects, and osteogenesis in vertebrates. In Drosophila, Mef2 null mutants are unable to produce differentiated muscle cells, and in vertebrates, Mef2 mutants are embryonic lethal. Schistosome worms are responsible for over 200 million cases of schistosomiasis globally, but little is known about early development of schistosome parasites after infecting a vertebrate host. Understanding basic schistosome development could be crucial to delineating potential drug targets. Here, we identify and characterize Mef2 from the schistosome worm Schistosoma mansoni (SmMef2). We initially identified SmMef2 as a homolog to the yeast Mef2 homolog, Resistance to Lethality of MKK1P386 overexpression (Rlm1), and we show that SmMef2 is homologous to conserved Mef2 family proteins. Using a genetics approach, we demonstrate that SmMef2 is a transactivator that can induce transcription of four separate heterologous reporter genes by yeast one-hybrid analysis. We also show that Mef2 is expressed during several stages of schistosome development by quantitative PCR and that it can bind to conserved Mef2 DNA consensus binding sequences.

Cercarial transformation and in vitro cultivation of Schistosoma mansoni schistosomules.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.