Last data update: Jun 17, 2024. (Total: 47034 publications since 2009)
Records 1-5 (of 5 Records) |
Query Trace: Cuthbert CD [original query] |
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Can incorporating molecular testing improve the accuracy of newborn screening for congenital adrenal hyperplasia?
Sarafoglou K , Gaviglio A , Wolf C , Lorentz CP , Lteif A , Kyllo J , Radloff G , Detwiler Z , Cuthbert CD , Hodges JS , Grosse SD , Greene CN , Cordovado S . J Clin Endocrinol Metab 2024 ![]() BACKGROUND: Single-tier newborn screening (NBS) for CAH using 17-hydroxyprogesterone (17OHP) measured by fluoroimmunoassay (FIA) in samples collected at 24-48 hours produces a high false-positive rate (FPR). 2nd tier steroid testing can reduce the FPR and has been widely implemented. We investigated the accuracy of an alternative multi-tier CAH NBS protocol that incorporates molecular testing of the CYP21A2 gene and reduces the 1st tier 17OHP cutoff to minimize missed cases. METHODS: Created a Minnesota-specific CYP21A2 pathogenic variants panel; develop a rapid, high-throughput multiplex, allele-specific-primer-extension assay; perform 1-year retrospective analysis of Minnesota NBS results comparing metrics between a conventional steroid-based two-tier protocol and a molecular-based multi-tier NBS protocol, applied post-hoc. RESULTS: CYP21A2 gene sequencing of 103 Minnesota families resulted in a Minnesota-specific panel of 21 pathogenic variants. Centers for Disease Control and Prevention (CDC) created a molecular assay with 100% accuracy and reproducibility. Two-tier steroid-based screening of 68,659 live births during 2015 resulted in 2 false negatives (FNs), 91 FPs, and 1 true positive (TP). A three-tier protocol with a lower 1st-tier steroid cutoff, 2nd-tier 21-variant CYP21A2 panel and 3rd-tier CYP21A2 sequencing would have resulted in 0 FNs, 52 FPs and 3 TPs. CONCLUSIONS: Incorporation of molecular testing could improve the accuracy of CAH NBS, although some distinct challenges of molecular testing may need to be considered before implementation by NBS programs. |
Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing.
Hendrix MM , Cuthbert CD , Cordovado SK . Int J Neonatal Screen 2020 6 (2) 1-15 ![]() ![]() An increasing number of newborn screening laboratories in the United States and abroad are moving towards incorporating next-generation sequencing technology, or NGS, into routine screening, particularly for cystic fibrosis. As more programs utilize this technology for both cystic fibrosis and beyond, it is critical to identify appropriate DNA extraction methods that can be used with dried blood spots that will result in consistent, high-quality sequencing results. To provide comprehensive quality assurance and technical assistance to newborn screening laboratories wishing to incorporate NGS assays, CDC's Newborn Screening and Molecular Biology Branch designed a study to evaluate the performance of nine commercial or laboratory-developed DNA extraction methods that range from a highly purified column extraction to a crude detergent-based no-wash boil prep. The DNA from these nine methods was used in two NGS library preparations that interrogate the CFTR gene. All DNA extraction methods including the cruder preps performed reasonably well with both library preps. One lower-concentration, older sample was excluded from one of the assay evaluations due to poor performance across all DNA extraction methods. When 84 samples, versus eight, were run on a flow cell, the DNA quality and quantity were more significant variables. |
The Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention: thirty-five year experience assuring newborn screening laboratory quality
De Jesus VR , Mei JV , Cordovado SK , Cuthbert CD . Int J Neonatal Screen 2015 1 (1) 13-26 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. |
Succinylacetone as primary marker to detect tyrosinemia type I in newborns and its measurement by newborn screening programs.
De Jesus VR , Adam BW , Mandel D , Cuthbert CD , Matern D . Mol Genet Metab 2014 113 67-75 ![]() 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. |
Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry
Turgeon CT , Magera MJ , Cuthbert CD , Loken PR , Gavrilov DK , Tortorelli S , Raymond KM , Oglesbee D , Rinaldo P , Matern D . Clin Chem 2010 56 (11) 1686-95 ![]() 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). METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d(3)-MMA, d(3)-MCA, d(8)-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 mumol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations. |
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