Matern, Dietrich

AbstractGlycosylation is a critical post/peri‐translational modification required for the appropriate development and function of the immune system. As an example, abnormalities in glycosylation can cause antibody deficiency and reduced lymphocyte signaling, although the phenotype can be complex given the diverse roles of glycosylation. Human MGAT2 encodes N‐acetylglucosaminyltransferase II, which is a critical enzyme in the processing of oligomannose to complex N‐glycans. Complex N‐glycans are essential for immune system functionality, but only one individual with MGAT2‐CDG has been described to have an abnormal immunologic evaluation. MGAT2‐CDG (CDG‐IIa) is a congenital disorder of glycosylation (CDG) associated with profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations. Here, we report a 4‐year old female with MGAT2‐CDG due to a novel homozygous pathogenic variant in MGAT2, a 4‐base pair deletion, c.1006_1009delGACA. In addition to clinical features previously described in MGAT2‐CDG, she experienced episodic asystole, persistent hypogammaglobulinemia, and defective ex vivo mitogen and antigen proliferative responses, but intact specific vaccine antibody titers. Her infection history has been mild despite the testing abnormalities. We compare this patient to the 15 previously reported patients in the literature, thus expanding both the genotypic and phenotypic spectrum for MGAT2‐CDG.

Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.

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.

Measurement of the absolute concentration of the biomarker psychosine in dried blood spots (DBS) is useful for diagnosis and prognosis of Krabbe disease and to support newborn screening of this leukodystrophy. As for assays for more common diseases, it is important to achieve congruence when multiple clinical laboratories provide testing. Four clinical laboratories, one research laboratory, and a commercial vendor collaborated with the goal to achieve congruence in quantitative psychosine measurement in DBS. A set of DBS calibrators was prepared by a single vendor and used in each reference laboratory to make a standard curve of measured psychosine in DBS versus the stated calibrator psychosine level. Congruence between the participating five laboratories was achieved using the psychosine DBS calibrators. This allowed application of disease-specific reference ranges obtained by the reference laboratory with the most extensive data by the other reference laboratories. Congruence between multiple reference laboratories in the measurement of the absolute concentration of biomarkers in DBS (and by extension other samples) is possible by the use of a common set of DBS calibrators.

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.

Abstract Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B12 deficiency. Methods: We added 1 nmol of the internal standard MMA-d3 to 500 μL of plasma or 100 μL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 °C for 15 min. For separation, we used a Supelcosil LC-18, 33 × 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d3, respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. Results: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 μmol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030x − 0.032 (Sy|x = 1.03 μmol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3.8–8.5% and 1.3–3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 μmol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 μmol/L), 96.0% (0.60 μmol/L), and 94.8% (2.02 μmol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100–150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. Conclusions: This method is well suited for large-scale MMA testing (≥100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.

Abstract Medical nutrition therapy for long‐chain fatty acid oxidation disorders (LC‐FAODs) currently emphasizes fasting avoidance, restricted dietary long‐chain fatty acid intake, supplementation with medium chain triglycerides, and increased carbohydrate intake. We hypothesize that increasing dietary protein intake relative to carbohydrate intake would preserve metabolic control yet induce physical benefits including reduced hepatic lipogenesis. Therefore, we compared two dietary approaches with similar fat intake but different carbohydrate to protein ratios in participants diagnosed with LC‐FAODs. Thirteen participants were enrolled and randomized into either a high‐protein (PRO) or a high‐carbohydrate (CHO) diet for 4 months. Baseline and 4‐month assessments included body composition, ectopic lipid deposition, and resting energy expenditure. End of study assessments also included total energy expenditure, metabolic responses to oral feedings, and whole‐body fatty acid oxidation capacity. At the end of the dietary intervention, both groups had similar energy expenditure, fat and glucose oxidation rates, and glucolipid responses to mixed meal and oral glucose loads. Neither dietary group experienced worsening symptoms related to their LC‐FAOD. Compared to the CHO group, the PRO group exhibited increased blood levels of short‐chain acylcarnitines, reduced intrahepatic lipid content, and maintained lean body mass while the CHO group lost lean mass. In patients with LC‐FAODs, increasing protein intake maintained metabolic control, reduced liver fat without risk of metabolic decompensation, and helped preserve lean body mass. We propose that a modest increase in dietary protein along with fasting avoidance and fat restriction may improve body composition and energy expenditure in patients with LC‐FAODs.

Abstract BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were &lt;7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

Homocystinuria (HCU) due to cystathionine-β-synthase deficiency is generally regarded as a rare disease, but within the Qatari population has an incidence of 1 in 1800 live births. Most newborn screening methods for HCU using dried blood spots (DBS) rely on the detection of an elevated methionine level or a rapid screen for total homocysteine (tHCY). However, screening based on methionine levels alone lacks specificity and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for tHCY exhibit variable results with high false positive rates. This report describes a LC-MS/MS method for detection of tHCY on DBS, with improved specificity. tHCY was extracted from DBS with a solution containing dithiothreitol and subsequently butylated with hydrochloric acid in n-butanol. The butyl esters were separated by liquid chromatography on a reverse-phase column and the homocysteine (HCY), detected by tandem mass spectrometry. The butyl ester of HCY eluted at 1.8 min. Total analysis time was 6.1 min per sample, including column flush and equilibration. This method allows for the quantification of tHCY over a linear range from 0.3 to 200 µM. Intraassay and interassay imprecision and recoveries were acceptable. Good concordance was observed with another LC-MS/MS method. Application of this method improves specificity and reduces false positive rates in screening for HCU.