MPV17-related hepatocerebral mitochondrial DNA depletion syndrome, previously known as Navajo Neurohepatopathy (NNH), is a rare genetic disease affecting Navajo children of the American Southwest. These children can suffer from several severe symptoms like brain damage and liver disease, and a diagnosis leads to death by age 10, on average. The…
MPV17-related hepatocerebral mitochondrial DNA depletion syndrome, previously known as Navajo Neurohepatopathy (NNH), is a rare genetic disease affecting Navajo children of the American Southwest. These children can suffer from several severe symptoms like brain damage and liver disease, and a diagnosis leads to death by age 10, on average. The only known effective therapy for NNH is a liver transplant. Currently, the disease is diagnosed through a lengthy and expensive process of gene sequencing, but oftentimes patients with the most severe forms of NNH deteriorate quickly; thus a rapid diagnostic would be beneficial to beginning the transplant process as early as possible. Here, Tentacle Probes, a novel technology to detect genetic mutations, were proposed to rapidly and accurately diagnose NNH. Because of Tentacle Probes' double binding site kinetics, they can detect mutations more accurately than other types of genetic probes. Probes specific to the NNH mutation were designed for use with a real-time polymerase chain reaction (PCR) detection platform. Initial synthetic DNA testing of Tentacle Trobes showed capable differentiation between mutated and non-mutated samples. However, experiments to validate those results at Phoenix Children's Hospital before moving to patient samples showed that test viability decreased over time. Efforts to diagnose the issues that led to decreased viability suggested four possible explanations that are as follows (in order of decreasing likelihood): first, undesired products from improper PCR primer design was supported by double bands in DNA gel electrophoresis; second, DNA may have degraded over time or due to repeated cycles of freezing and thawing stock solutions, and this was supported by smeared DNA gel electrophoresis; third, probe degradation, specifically of the fluorescent reporter, is possible; finally, contaminants that inhibit the PCR reaction may have been introduced. A combination of these factors may also have caused the change in assay viability. As a result of these most likely possibilities, new primers were designed and steps suggested to return viability to the assay. Thus, the various limitations and requirements for this Tentacle Probe diagnostic have been identified, and as assay development continues following the promising initial results achieved, we are confident that a rapid method if diagnosing NNH is on its way to help the children afflicted with this devastating disease receive timely access to treatment.
Background: Cysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move…
Background: Cysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move a step beyond detection and into the relative quantification of Cys-SOH within specific proteins found in a complex biological setting--namely, human plasma.
Results: This report describes the possibilities and limitations of performing such analyses based on the use of thionitrobenzoic acid and dimedone-based probes which are commonly employed to trap Cys-SOH. Results obtained by electrospray ionization-based mass spectrometric immunoassay reveal the optimal type of probe for such analyses as well as the reproducible relative quantification of Cys-SOH within albumin and transthyretin extracted from human plasma--the latter as a protein previously unknown to be modified by Cys-SOH.
Conclusions: The relative quantification of Cys-SOH within specific proteins in a complex biological setting can be accomplished, but several analytical precautions related to trapping, detecting, and quantifying Cys-SOH must be taken into account prior to pursuing its study in such matrices.
