BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

No abstract

Tyrosinemia type I (TYR I) is caused by autosomal recessive fumarylacetoacetate hydrolase deficiency and is characterized by development of severe liver disease in infancy and neurologic crises. If left untreated, most patients die of liver failure in the first years of life. Intervention with medication is effective when initiated during the first month of life. This improvement in the treatment of TYR I patients influenced the decision to include TYR I in the US Secretary of the Department of Health and Human Services’ (HHS) Recommended Uniform Screening Panel. However, while tyrosine is routinely measured in newborn screening (NBS) by tandem mass spectrometry (MS/MS), elevated tyrosine levels are not specific to TYR I. To improve the specificity of NBS for TYR I, several assays were developed to measure succinylacetone (SUAC) in dried blood spots (DBS). SUAC is a pathognomonic marker of TYR I, and its detection by NBS MS/MS is possible. This review of the current status of NBS for TYR I in the US is the result of discussions at the HHS Secretary’s (Discretionary) Advisory Committee on Heritable Disorders in Newborns and Children about the inconsistent implementation of effective NBS for TYR I in the US. We sought to understand the different TYR I screening practices in US NBS programs. Results indicate that 50 out of 51 NBS programs in the US screen for TYR I, and a successful SUAC performance evaluation scheme is available from the Centers for Disease Control and Prevention. Programmatic and methodological barriers were identified that prevent widespread adoption of SUAC measurements in NBS laboratories. However, since SUAC detection is currently the best approach to NBS for TYR I, a further delay of the addition of SUAC measurement into NBS procedures is discouraged. SUAC measurement should improve both the false positive and false negative rate in NBS for TYR I thereby yielding the desired benefits for affected patients at no expense to the overall population served.

Pallister-Killian syndrome (PKS) is characterized by multiple congenital anomalies including pigmentary skin changes, mental retardation, and the mosaic presence of a tissue-limited isochromosome 12p [i(12p)]. Mechanism(s) of formation and parental origin of the isochromosome are not well understood. In this study, microsatellite DNA markers of chromosome 12p were used to identify the parental origin of the extra chromosome in an 8-year-old previously reported patient with PKS. The i(12p) was found to be maternally inherited. Reported cases of PKS where the parental origin of the i(12p) was determined were also reviewed. In all the cases, with one exception, the errors were found to be maternal in origin. Premeiotic mitotic error may be the most likely mechanism for i(12p) formation in this syndrome.

Newborn screening is the largest genetic testing effort in the United States and is considered one of the ten great public health achievements during the first 10 years of the 21st century. For over 35 years, the Newborn Screening Quality Assurance Program (NSQAP) at the US Centers for Disease Control and Prevention has helped NBS laboratories ensure that their testing does not delay diagnosis, minimizes false-positive reports, and sustains high-quality testing performance. It is a multi-component program that provides comprehensive quality assurance services for dried blood spot testing. The NSQAP, the Biochemical Mass Spectrometry Laboratory (BMSL), the Molecular Quality Improvement Program (MQIP) and the Newborn Screening Translation Research Initiative (NSTRI), aid screening laboratories achieve technical proficiency and maintain confidence in their performance while processing large volumes of specimens daily. The accuracy of screening tests could be the difference between life and death for many babies; in other instances, identifying newborns with a disorder means that they can be treated and thus avoid life-long disability or severe cognitive impairment. Thousands of newborns and their families have benefited from reliable and accurate testing that has been accomplished by a network of screening laboratories and the NSQAP, BMSL, MQIP and NSTRI.

Enzymatic cyclization of homocysteine forms a reactive thiolactone that may play an important role in its cardiovascular toxicity, but reliable quantitation of the free thiolactone metabolite in physiological fluids has not been reported. We have therefore used a highly selective gas chromatography/mass spectrometry (GC/MS) technique combined with the sensitivity of negative chemical ionization (NCI) to develop a quantitative method for the detection of homocysteine thiolactone (HcyTL) in plasma. To improve accuracy the deuterated isomer d(4)-HcyTL was synthesized and added to plasma as internal standard. The plasma was then treated with silica solid-phase extraction and derivatized with heptafluorobutyric anhydride. The derivative was analyzed by GC/MS in NCI mode with methane as the reagent gas and quantified by analyzing for the HcyTL ion [M(-)[bond]HF] and its d(4)-HcyTL counterpart in single-ion monitoring mode. The calibration curve showed a dynamic linear range up to 40 nmol/L. Within-day precision (n = 20, nominal concentration 5.2 nmol/L) was 0.96% and between-day precision was 3.9%, with a detection limit of 1.7 nmol/L and quantification limit of 5.2 nmol/L. Two human plasma samples had HcyTL concentrations of 18 and 25 nmol/L. This facile method for quantitation of homocysteine thiolactone opens the way for more detailed clinical studies of its potential role in homocysteine-induced arteriosclerosis and vaso-occlusive disease.

To induce oxidative stress, HepG2 cells were exposed to a compound known as gramoxone. This compound undergoes a one-electron reduction to form a stable free radical which is capable of generating reactive oxygen species. We demonstrated that exposure of HepG2 cells to gramoxone (0.1 M) resulted in a 2-fold decrease in apoA-I mRNA with no significant change in apoB and apoE mRNA levels. To examine if increased rates of mRNA degradation were responsible for the reduction in apoA-I mRNA levels, mRNA half-lives were measured in the presence of actinomycin D with and without gramoxone treatment. These studies revealed a 4-fold increase in the rate of apoA-I mRNA degradation in cells exposed to gramoxone. In similarly treated cells, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was also increased 2-fold. Consistent with nuclear run-off assays, transient transfection experiments using a series of pGL2-derived luciferase reporter plasmids containing the human apoAI proximal promoter demonstrated that gramoxone treatment increased apoA-I promoter activity 2-fold. We have identified a potential "antioxidant response element" (ARE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gramoxone. Gel mobility shift assays with an ARE oligonucleotide revealed increased levels of a specific protein-DNA complex that formed with nuclear extracts from gramoxone-treated cells. UV cross-linking experiments with the ARE and nuclear extracts from either untreated or gramoxone-treated cells detected proteins of approximately 100 and 115 kDa. When a single copy of the ARE was inserted upstream of the SV40 promoter in a luciferase reporter plasmid, a significant 2-fold induction in luciferase activity was observed in HepG2 cells in the presence of gramoxone. In contrast, a plasmid containing a mutant apoAI-ARE did not confer responsiveness to gramoxone. Furthermore, pGL2 (apoAI-250 mutant ARE), in which point mutations eliminated the ARE in the apoAI promoter, showed no increase in luciferase activity in response to gramoxone. Taken together, the data suggest that gramoxone affects apoA-I mRNA levels by both transcriptional and post-transcriptional mechanisms.

Mechanisms