High-resolution melting analysis of single nucleotide polymorphisms.

The technology of Single Nucleotide Polymorphism (SNP) detection has evolved steadily through mobility shift studies, mass cleavage product evaluations, heterodimer differences in chemical, conformational, and enzymatic properties, mass spectroscopy and sequencing, to allele-specific hybridization probe methods. Each method presented challenges of labor intensity, unreliable efficiencies, complicated optimizations, and issues of sample quantity and quality. Concurrently the value of SNP detection in basic research and personalized medicine has continued to grow. Accessing the secrets of genetic individuality is the next frontier in moving medicine from the description of very low frequency and highly deleterious nucleotide changes to the study of very low frequency polymorphisms, lower penetrance polymorphisms, and polymorphisms with public health importance. High-Resolution Melting (HRM) analysis of SNP status became an option for high throughput settings with the development of double-stranded dyes that do not interfere with PCR amplification in saturation, eliminate dye jumping, and nearest neighbor sequence changes influence melt temperature via amplicon strand locking chemistry. This method is able to distinguish transitions, transversions, and identify novel changes at or near the SNP of interest rapidly, inexpensively, and without post-amplification assay techniques or extensive technical interpretation of data. For probe or solid matrix based assays, the investigator initially defines a set of target sequences for binding. These assays are not only difficult due to the optimization of binding conditions but are unable to detect sequences that were not included in the design, often have marginalized binding due to a "one size fits all" reaction, and are not distinct in the case of heterozygotes.

H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice.

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe(2+) from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe(2+) to catalytically inactive Fe(3+) inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p < 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.

Enhanced primer selection and synthetic amplicon templates optimize high-resolution melting analysis of single-nucleotide polymorphisms in a large population.

AIMS: High-resolution melting (HRM) screening and scanning for single-nucleotide polymorphisms (SNPs) afford the advantages of a quicker, less expensive, and less demanding option compared to other methods for sequence analysis. The evaluation of large populations of patients for multiple SNPs in a high-throughput manner is the next phase in individualized medicine. RESULTS: We demonstrated that Tm profiles can be generated from gDNA samples that clearly differentiate homozygous ancestral, homozygous SNP, and heterozygous genotypes, while identifying samples of unique outcome without the cumbersome processes of normalization, temperature shifting, and difference plot generation. CONCLUSIONS: Through expanded primer selection criterion and inclusion of a cloning fragment length double-stranded DNA sequence-specific control template, we are now able to generate additional data via HRM melt domains that are greatly simplified, while considering both the peak melt temperature and profile.

Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease.

Increases in heme oxygenase-1 (HO-1) and administration of heme degradation products CO and biliverdin inhibit vascular inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin (alb) promoter-driven Sleeping Beauty (SB) transposase plasmid with a wild-type rat hmox-1 (wt-HO-1) transposable element was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had three- to five-fold increases in HO-1 activity and protein expression in liver, similar to hemin-treated mice. Immunohistochemistry demonstrated increased perinuclear HO-1 staining in hepatocytes. Messenger RNA transcription of the hmox-1 transgene in liver was confirmed by quantitative real-time polymerase chain reaction restriction fragment length polymorphism (qRT-PCR RFLP) with no detectible transgene expression in other organs. The livers of all HO-1 overexpressing mice had activation of nuclear phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-Akt, decreased nuclear expression of nuclear factor-kappa B (NF-kappaB) p65, and decreased soluble vascular cell adhesion molecule-1 (sVCAM-1) in serum. Hypoxia-induced stasis, a characteristic of SCD, but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice despite the absence of hmox-1 transgene expression in the skin suggesting distal effects of HO activity on the vasculature. No protective effects were seen in SCD mice injected with nonsense (ns-) rat hmox-1 that encodes carboxy-truncated HO-1 with little or no enzyme activity. We speculate that HO-1 gene delivery to the liver is beneficial in SCD mice by degrading pro-oxidative heme, releasing anti-inflammatory heme degradation products CO and biliverdin/bilirubin into circulation, activating cytoprotective pathways and inhibiting vascular stasis at sites distal to transgene expression.

Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy.

Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors.