Newborn screening for citrin deficiency and carnitine uptake defect using second-tier molecular tests

Abstract Background: Newborn screening for maple syrup urine disease (MSUD) relies on finding increased concentrations of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine by tandem mass spectrometry (MS/MS). d-Alloisoleucine (allo-Ile) is the only pathognomonic marker of MSUD, but it cannot be identified by existing screening methods because it is not differentiated from isobaric amino acids. Furthermore, newborns receiving total parenteral nutrition often have increased concentrations of BCAAs. To improve the specificity of newborn screening for MSUD and to reduce the number of diet-related false-positive results, we developed a LC-MS/MS method for quantifying allo-Ile. Methods: Allo-Ile and other BCAAs were extracted from a 3/16-inch dried blood spot punch with methanol/H2O, dried under nitrogen, and reconstituted into mobile phase. Quantitative LC-MS/MS analysis of allo-Ile, its isomers, and isotopically labeled internal standards was achieved within 15 min. To determine a reference interval for BCAAs including allo-Ile, we analyzed 541 dried blood spots. We also measured allo-Ile in blinded samples from 16 MSUD patients and 21 controls and compared results to an HPLC method. Results: Intra- and interassay imprecision (mean CVs) for allo-Ile, leucine, isoleucine, and valine ranged from 1.8% to 7.4%, and recovery ranged from 91% to 129%. All 16 MSUD patients were correctly identified. Conclusions: The LC-MS/MS method can reliably measure allo-Ile in dried blood spots for the diagnosis of MSUD. Applied to newborn screening as a second-tier test, it will reduce false-positive results, which produce family anxiety and increase follow-up costs. The assay also appears suitable for use in monitoring treatment of MSUD patients.

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Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses

International Journal of Neonatal Screening ◽

10.3390/ijns6030051 ◽

2020 ◽

Vol 6 (3) ◽

pp. 51 ◽

Cited By ~ 1

Author(s):

Trine Tangeraas ◽

Ingjerd Sæves ◽

Claus Klingenberg ◽

Jens Jørgensen ◽

Erle Kristensen ◽

...

Keyword(s):

Newborn Screening ◽

Screening Program ◽

Good Health ◽

Dried Blood Spots ◽

Case Definitions ◽

Inborn Errors ◽

Disease Spectrum ◽

Expanded Newborn Screening ◽

Second Tier ◽

Biochemical Testing

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies.

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Incorporation of Second-Tier Biomarker Testing Improves the Specificity of Newborn Screening for Mucopolysaccharidosis Type I

International Journal of Neonatal Screening ◽

10.3390/ijns6010010 ◽

2020 ◽

Vol 6 (1) ◽

pp. 10 ◽

Cited By ~ 5

Author(s):

Dawn S. Peck ◽

Jean M. Lacey ◽

Amy L. White ◽

Gisele Pino ◽

April L. Studinski ◽

...

Keyword(s):

Newborn Screening ◽

False Positive ◽

Dermatan Sulfate ◽

False Positive Rate ◽

False Negative ◽

Type I ◽

Mucopolysaccharidosis Type ◽

Mucopolysaccharidosis Type I ◽

Second Tier ◽

Mps I

Enzyme-based newborn screening for Mucopolysaccharidosis type I (MPS I) has a high false-positive rate due to the prevalence of pseudodeficiency alleles, often resulting in unnecessary and costly follow up. The glycosaminoglycans (GAGs), dermatan sulfate (DS) and heparan sulfate (HS) are both substrates for α-l-iduronidase (IDUA). These GAGs are elevated in patients with MPS I and have been shown to be promising biomarkers for both primary and second-tier testing. Since February 2016, we have measured DS and HS in 1213 specimens submitted on infants at risk for MPS I based on newborn screening. Molecular correlation was available for 157 of the tested cases. Samples from infants with MPS I confirmed by IDUA molecular analysis all had significantly elevated levels of DS and HS compared to those with confirmed pseudodeficiency and/or heterozygosity. Analysis of our testing population and correlation with molecular results identified few discrepant outcomes and uncovered no evidence of false-negative cases. We have demonstrated that blood spot GAGs analysis accurately discriminates between patients with confirmed MPS I and false-positive cases due to pseudodeficiency or heterozygosity and increases the specificity of newborn screening for MPS I.

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The Combined Impact of CLIR Post-Analytical Tools and Second Tier Testing on the Performance of Newborn Screening for Disorders of Propionate, Methionine, and Cobalamin Metabolism

International Journal of Neonatal Screening ◽

10.3390/ijns6020033 ◽

2020 ◽

Vol 6 (2) ◽

pp. 33 ◽

Cited By ~ 1

Author(s):

Dimitar K. Gavrilov ◽

Amy L. Piazza ◽

Gisele Pino ◽

Coleman Turgeon ◽

Dietrich Matern ◽

...

Keyword(s):

Newborn Screening ◽

False Positive ◽

Health Care Expenditures ◽

Positive Outcome ◽

Molecular Testing ◽

Second Tier ◽

Multivariate Pattern ◽

Recognition Software ◽

Cobalamin Metabolism ◽

Analytical Tools

The expansion of the recommend uniform screening panel to include more than 50 primary and secondary target conditions has resulted in a substantial increase of false positive results. As an alternative to subjective manipulation of cutoff values and overutilization of molecular testing, here we describe the performance outcome of an algorithm for disorders of methionine, cobalamin, and propionate metabolism that includes: (1) first tier screening inclusive of the broadest available spectrum of markers measured by tandem mass spectrometry; (2) integration of all results into a score of likelihood of disease for each target condition calculated by post-analytical interpretive tools created byCollaborative Laboratory Integrated Reports (CLIR), a multivariate pattern recognition software; and (3) further evaluation of abnormal scores by a second tier test measuring homocysteine, methylmalonic acid, and methylcitric acid. This approach can consistently reduce false positive rates to a <0.01% level, which is the threshold of precision newborn screening. We postulate that broader adoption of this algorithm could lead to substantial savings in health care expenditures. More importantly, it could prevent the stress and anxiety experienced by many families when faced with an abnormal newborn screening result that is later resolved as a false positive outcome.

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The Role of Extended CFTR Gene Sequencing in Newborn Screening for Cystic Fibrosis

International Journal of Neonatal Screening ◽

10.3390/ijns6010023 ◽

2020 ◽

Vol 6 (1) ◽

pp. 23 ◽

Cited By ~ 2

Author(s):

Anne Bergougnoux ◽

Maureen Lopez ◽

Emmanuelle Girodon

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Dna Analysis ◽

Genetic Diseases ◽

Major Drawback ◽

Coding Regions ◽

Sequencing Technologies ◽

Second Tier ◽

Real Challenge

There has been considerable progress in the implementation of newborn screening (NBS) programs for cystic fibrosis (CF), with DNA analysis being part of an increasing number of strategies. Thanks to advances in genomic sequencing technologies, CFTR-extended genetic analysis (EGA) by sequencing its coding regions has become affordable and has already been included as part of a limited number of core NBS programs, to the benefit of admixed populations. Based on results analysis of existing programs, the values and challenges of EGA are reviewed in the perspective of its implementation on a larger scale. Sensitivity would be increased at best by using EGA as a second tier, but this could be at the expense of positive predictive value, which improves, however, if EGA is applied after testing a variant panel. The increased detection of babies with an inconclusive diagnosis has proved to be a major drawback in programs using EGA. The lack of knowledge on pathogenicity and penetrance associated with numerous variants hinders the introduction of EGA as a second tier, but EGA with filtering for all known CF variants with full penetrance could be a solution. The issue of incomplete knowledge is a real challenge in terms of the implemention of NBS extended to many genetic diseases.

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Performance of the Four-Plex Tandem Mass Spectrometry Lysosomal Storage Disease Newborn Screening Test: The Necessity of Adding a 2nd Tier Test for Pompe Disease

International Journal of Neonatal Screening ◽

10.3390/ijns4040041 ◽

2018 ◽

Vol 4 (4) ◽

pp. 41 ◽

Cited By ~ 6

Author(s):

Shu-Chuan Chiang ◽

Pin-Wen Chen ◽

Wuh-Liang Hwu ◽

An-Ju Lee ◽

Li-Chu Chen ◽

...

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Newborn Screening ◽

Pompe Disease ◽

Screening Test ◽

Disease Screening ◽

Confirmatory Test ◽

Tandem Mass ◽

Lysosomal Storage ◽

Second Tier

Early diagnosis of lysosomal storage diseases (LSDs) through newborn screening (NBS) has been adapted widely. The National Taiwan University Hospital Newborn Screening Center launched the four-plex tandem mass spectrometry LSD newborn screening test in 2015. The test determined activities of acid α-glucosidase (GAA; Pompe), acid α-galactosidase (GLA; Fabry), acid β-glucocerebrosidase (ABG; Gaucher), and acid α-l-iduronidase (IDUA; MPS-I) in dried blood spots (DBS). Through 2017, 64,148 newborns were screened for these four LSDs. The screening algorithm includes enzyme activity/ratio as the cutoffs for the first screening test and a second-tier test for Pompe disease screening. The second-tier Pompe disease screening test measured activity inhibition by acarbose. Twenty-nine newborns required a confirmatory test; six were confirmed to have Pompe disease, and nine were confirmed to have Fabry disease. The screen-positive rate for Pompe disease was 0.031%. Therefore, in Pompe disease newborn screening, a validated 2nd tier test is necessary to decrease false positives.

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Newborn Screening for Cystic Fibrosis by Use of a Multiplex Immunoassay

Clinical Chemistry ◽

10.1373/clinchem.2009.132480 ◽

2010 ◽

Vol 56 (3) ◽

pp. 445-450 ◽

Cited By ~ 16

Author(s):

Barbara A Lindau-Shepard ◽

Kenneth A Pass

Keyword(s):

Cystic Fibrosis ◽

Newborn Screening ◽

Screening Program ◽

Pancreatic Disease ◽

High Rate ◽

Dna Testing ◽

Second Tier ◽

Sample Set ◽

Positive Results ◽

Immunoreactive Trypsinogen

Abstract Background: Since its beginnings, newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high rate of false-positive results (screen positive, diagnosis negative), despite attempts to reduce this rate by use of altered cutoffs and second-tier DNA testing. IRT exists as 2 isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Assay standardization between programs is a continuing problem because the IRT assays currently in use variously recognize either 1 or both isoforms. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. Methods: Using 2 different Luminex bead sets, we developed assays for each IRT isoform separately and then combined them. Using the sum of IRT1 and IRT2 values (IRT1+IRT2), we compared the results with a CF kit currently in use. Results: In a sample set consisting of 16 cases confirmed positive for CF, we established a cutoff at >97 μg/L total IRT. Seven of 8 carriers with 1 CF mutation screen-positive by the standard method were also screen-positive by IRT1+IRT2. Of 32 cases screen-positive by standard IRT, 11 were screen-negative by IRT1+IRT2. None of these 11 cases had CF mutations identified by the screening program. Conclusions: These data indicate that the multiplex method with specificity for 2 isoforms of IRT has performance comparable to that of a standard IRT method and the advantage of improved standardization by detection of the 2 isoforms.

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