Immune dysfunction in MGAT2-CDG: A clinical report and review of the literature.

Glycosylation 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.

Does metabolomic profile differ with regard to birth weight?

BACKGROUND: Macrosomia and child obesity are growing health-care issues worldwide. The purpose of the study was to evaluate how extremely high or low birth weight affects metabolic markers evaluated in newborn screening. METHODS: The study was register-based and included full-term singletons born in Iceland from 2009 to 2012 with newborn screening samples taken 72-96 h after birth. Three groups based on birth weight were compared: low birth weight (<2500 g), appropriate-for-gestational age, and extreme macrosomia (≥5000 g). The comparison was adjusted for possible confounding factors. RESULTS: Compared to appropriate-for-gestational age neonates, both low birth weight and extreme macrosomia were associated with higher levels of glutamic acid. The amino acids alanine and threonine were increased in low birth weight neonates. Free carnitine and some medium- and long-chain acylcarnitines were higher in low birth weight infants. Hydroxybutyrylcarnitine was lower in low birth weight infants, but higher in extremely macrosomic neonates. Acetylcarnitine was higher in low birth weight and extremely macrosomic neonates. Succinylcarnitine was lower and hexadecenoylcarnitine higher in macrosomic newborns. CONCLUSION: Low birth weight and extremely macrosomic neonates show distinctive differences in their metabolomic profile compared to appropriate-for-gestational age newborns. The differences are not explained by gestational age. IMPACT: The key message of this article is that both low birth weight and extremely macrosomic newborns show dissimilar metabolomic profiles compared to appropriate-for-gestational age neonates. The article contributes to knowledge on what affects evaluation of results in newborn screening. The impact of this article is to provide information on metabolism at both ends of the birth weight range after accounting for confounding factors including gestational age.

A new D-galactose treatment monitoring index for PGM1-CDG.

Phosphoglucomutase 1 (PGM1) catalyzes the interconversion of glucose-6-phosphate to glucose-1-phosphate and is a key enzyme of glycolysis, glycogenesis, and glycogenolysis. PGM1 deficiency (OMIM: 614921) was initially defined as a glycogen storage disorder (type XIV), and later re-classified as a PGM1-congenital disorder of glycosylation (PGM1-CDG). Serum transferrin (Tf) glycan isoform analysis by liquid chromatography-mass spectrometry (LC-MS) is used as a primary diagnostic screen tool, and reveals a very unique CDG profile described as a mixture of CDG-type I and CDG-type II patterns. Oral d-galactose supplementation shows significant clinical and metabolic improvements, which are indicated by the Tf glycan isoform normalization over time in patients with PGM1-CDG. Thus, there is a need for biomarkers to guide d-galactose dosage in patients in order to maintain effective and safe drug levels. Here, we present a simplified algorithm called PGM1-CDG Treatment Monitoring Index (PGM1-TMI) for assessing the response of PGM1-CDG patients to d-galactose supplementation. For our single-center cohort of 16 PGM1-CDG patients, the Tf glycan profile analysis provided the biochemical diagnosis in all of them. In addition, the PGM1-TMI was reduced in PGM1-CDG patients under d-galactose supplementation as compared with their corresponding values before treatment, indicating that glycosylation proceeds towards normalization. PGM1-TMI allows tracking Tf glycan isoform normalization over time when the patients are on d-galactose supplementation.

The critical role of psychosine in screening, diagnosis, and monitoring of Krabbe disease.

PURPOSE: Newborn screening (NBS) for Krabbe disease (KD) is performed by measurement of galactocerebrosidase (GALC) activity as the primary test. This revealed that GALC activity has poor specificity for KD. Psychosine (PSY) was proposed as a disease marker useful to reduce the false positive rate for NBS and for disease monitoring. We report a highly sensitive PSY assay that allows identification of KD patients with minimal PSY elevations. METHODS: PSY was extracted from dried blood spots or erythrocytes with methanol containing d5-PSY as internal standard, and measured by liquid chromatography-tandem mass spectrometry. RESULTS: Analysis of PSY in samples from controls (N = 209), GALC pseudodeficiency carriers (N = 55), GALC pathogenic variant carriers (N = 27), patients with infantile KD (N = 26), and patients with late-onset KD (N = 11) allowed for the development of an effective laboratory screening and diagnostic algorithm. Additional longitudinal measurements were used to track therapeutic efficacy of hematopoietic stem cell transplantion (HSCT). CONCLUSION: This study supports PSY quantitation as a critical component of NBS for KD. It helps to differentiate infantile from later onset KD variants, as well as from GALC variant and pseudodeficiency carriers. Additionally, this study provides further data that PSY measurement can be useful to monitor KD progression before and after treatment.

Laboratory monitoring of patients with hereditary tyrosinemia type I.

BACKGROUND: The prognosis of patients with Hereditary Tyrosinemia Type 1 (HT-1) has greatly improved with early detection through newborn screening and the introduction of nitisinone (NTBC) therapy. A recent guideline calls for periodic monitoring of biochemical markers and NTBC levels to tailor treatment; however, this is currently only achieved through a combination of clinical laboratory tests. We developed a multiplexed assay measuring relevant amino acids, succinylacetone (SUAC), and NTBC in dried blood spots (DBS) to facilitate treatment monitoring. METHODS: Tyrosine, phenylalanine, methionine, NTBC and SUAC were eluted from DBS with methanol containing internal standards for each analyte and analyzed by liquid chromatography tandem mass spectrometry over 6.5 min in the multiple reaction monitoring positive mode. RESULTS: Pre-analytical and analytical factors were studied and demonstrated a reliable assay. Chromatography resolved an unknown substance that falsely elevates SUAC concentrations and was present in all samples. To establish control and disease ranges, the method was applied to DBS collected from controls (n = 284) and affected patients before (n = 2) and after initiation of treatment (n = 29). In the treated patients SUAC concentrations were within the normal range over a wide range of NTBC levels. CONCLUSIONS: This assay enables combined, accurate measurement of revelevant metabolites and NTBC in order to simplify treatment monitoring of patients with HT-1. In addition, the use of DBS allows for specimen collection at home to facilitate more standardization in relation to drug and dietary treatment.

Multiplex testing for the screening of lysosomal storage disease in urine: Sulfatides and glycosaminoglycan profiles in 40 cases of sulfatiduria.

PURPOSE: To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine. METHODS: Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results. RESULTS: Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders. CONCLUSIONS: Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans.

Correction to: Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Upon publication, it was noted that five of the on-line supplementary figures had incorrect figure: figure legend associations. These were supplementary Figs. 6, 7, 14, 15, and 23.

Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes.

Metabolomic characterization of post-mortem tissues (frontal and parietal cortices, pons, cerebellum, hippocampus, cerebral cortex, liver and kidney) derived from a 37 y.o. male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) was performed in conjunction with four parallel series of control tissues. Amino acids, acylcarnitines, guanidino- species (guanidinoacetic acid, creatine, creatinine) and GABA-related intermediates were quantified using UPLC and mass spectrometric methods that included isotopically labeled internal standards. Amino acid analyses revealed significant elevation of aspartic acid and depletion of glutamine in patient tissues. Evidence for disruption of short-chain fatty acid metabolism, manifest as altered C4OH, C5, C5:1, C5DC (dicarboxylic) and C12OH carnitines, was observed. Creatine and guanidinoacetic acids were decreased and elevated, respectively. GABA-associated metabolites (total GABA, γ-hydroxybutyric acid, succinic semialdehyde, 4-guanidinobutyrate, 4,5-dihydroxyhexanoic acid and homocarnosine) were significantly increased in patient tissues, including liver and kidney. The data support disruption of fat, creatine and amino acid metabolism as a component of the pathophysiology of SSADHD, and underscore the observation that metabolites measured in patient physiological fluids provide an unreliable reflection of brain metabolism.

Temporal metabolomics in dried bloodspots suggests multipathway disruptions in aldh5a1-/- mice, a model of succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD; OMIM 271980) is a rare disorder featuring accumulation of neuroactive 4-aminobutyric acid (GABA; γ-aminobutyric acid, derived from glutamic acid) and 4-hydroxybutyric acid (γ-hydroxybutyric acid; GHB, a short-chain fatty acid analogue of GABA). Elevated GABA is predicted to disrupt the GABA shunt linking GABA transamination to the Krebs cycle and maintaining the balance of excitatory:inhibitory neurotransmitters. Similarly, GHB (or a metabolite) is predicted to impact ß-oxidation flux. We explored these possibilities employing temporal metabolomics of dried bloodspots (DBS), quantifying amino acids, acylcarnitines, and guanidino- metabolites, derived from aldh5a1+/+, aldh5a1+/- and aldh5a1-/- mice (aldehyde dehydrogenase 5a1 = SSADH) at day of life (DOL) 20 and 42 days. At DOL 20, aldh5a1-/- mice had elevated C6 dicarboxylic (adipic acid) and C14 carnitines and threonine, combined with a significantly elevated ratio of threonine/[aspartic acid + alanine], in comparison to aldh5a1+/+ mice. Conversely, at DOL 42 aldh5a1-/- mice manifested decreased short chain carnitines (C0-C6), valine and glutamine, in comparison to aldh5a1+/+ mice. Guanidino species, including creatinine, creatine and guanidinoacetic acid, evolved from normal levels (DOL 20) to significantly decreased values at DOL 42 in aldh5a1-/- as compared to aldh5a1+/+ mice. Our results provide a novel temporal snapshot of the evolving metabolic profile of aldh5a1-/- mice while highlighting new pathomechanisms in SSADHD.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Moonlighting newborn screening markers: the incidental discovery of a second-tier test for Pompe disease.

PURPOSE: To describe a novel biochemical marker in dried blood spots suitable to improve the specificity of newborn screening for Pompe disease. METHODS: The new marker is a ratio calculated between the creatine/creatinine (Cre/Crn) ratio as the numerator and the activity of acid α-glucosidase (GAA) as the denominator. Using Collaborative Laboratory Integrated Reports (CLIR), the new marker was incorporated in a dual scatter plot that can achieve almost complete segregation between Pompe disease and false-positive cases. RESULTS: The (Cre/Crn)/GAA ratio was measured in residual dried blood spots of five Pompe cases and was found to be elevated (range 4.41-13.26; 99%ile of neonatal controls: 1.10). Verification was by analysis of 39 blinded specimens that included 10 controls, 24 samples with a definitive classification (16 Pompe, 8 false positives), and 5 with genotypes of uncertain significance. The CLIR tool showed 100% concordance of classification for the 24 known cases. Of the remaining five cases, three p.V222M homozygotes, a benign variant, were classified by CLIR as false positives; two with genotypes of unknown significance, one likely informative, were categorized as Pompe disease. CONCLUSION: The CLIR tool inclusive of the new ratio could have prevented at least 12 of 13 (92%) false-positive outcomes.

Precision newborn screening for lysosomal disorders.

PURPOSE: The implementation of newborn screening for lysosomal disorders has uncovered overall poor specificity, psychosocial harm experienced by caregivers, and costly follow-up testing of false-positive cases. We report an informatics solution proven to minimize these issues. METHODS: The Kentucky Department for Public Health outsourced testing for mucopolysaccharidosis type I (MPS I) and Pompe disease, conditions recently added to the recommended uniform screening panel, plus Krabbe disease, which was added by legislative mandate. A total of 55,161 specimens were collected from infants born over 1 year starting from February 2016. Testing by tandem mass spectrometry was integrated with multivariate pattern recognition software (Collaborative Laboratory Integrated Reports), which is freely available to newborn screening programs for selection of cases for which a biochemical second-tier test is needed. RESULTS: Of five presumptive positive cases, one was affected with infantile Krabbe disease, two with Pompe disease, and one with MPS I. The remaining case was a heterozygote for the latter condition. The false-positive rate was 0.0018% and the positive predictive value was 80%. CONCLUSION: Postanalytical interpretive tools can drastically reduce false-positive outcomes, with preliminary evidence of no greater risk of false-negative events, still to be verified by long-term surveillance.

Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy.

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 <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.

Chronic Phenotype Characterization of a Large-Animal Model of Hereditary Tyrosinemia Type 1.

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by deficiency in fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. In this study, we investigated whether fumarylacetoacetate hydrolase deficient (FAH-/-) pigs, a novel large-animal model of HT1, develop fibrosis and cirrhosis characteristic of the human disease. FAH-/- pigs were treated with the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3 cyclohexandione (NTBC) at a dose of 1 mg/kg per day initially after birth. After 30 days, they were assigned to one of three groups based on dosing of NTBC. Group 1 received ≥0.2 mg/kg per day, group 2 cycled on/off NTBC (0.05 mg/kg per day × 1 week/0 mg/kg per day × 3 weeks), and group 3 received no NTBC thereafter. Pigs were monitored for features of liver disease. Animals in group 1 continued to have weight gain and biochemical analyses comparable to wild-type pigs. Animals in group 2 had significant cessation of weight gain, abnormal biochemical test results, and various grades of fibrosis and cirrhosis. No evidence of hepatocellular carcinoma was detected. Group 3 animals declined rapidly, with acute liver failure. In conclusion, the FAH-/- pig is a large-animal model of HT1 with clinical characteristics that resemble the human phenotype. Under conditions of low-dose NTBC, FAH-/- pigs developed liver fibrosis and portal hypertension, and thus may serve as a large-animal model of chronic liver disease.

Newborn screening for proximal urea cycle disorders: Current evidence supporting recommendations for newborn screening.

Current newborn screening (NBS) for urea cycle disorders (UCD) is incomplete as only distal UCDs are included in most NBS programs by measuring elevated amino acid concentrations. NBS for the proximal UCDs involves the detection in NBS spots of low citrulline values, a finding which is often overlooked because it is considered to be inadequate. We retrospectively analyzed NBS blood spots from known UCD patients comparing the utility of the Region 4 Stork (R4S) interpretive tools to conventional cutoff based interpretation. This study shows the utility of R4S tools in detecting all UCDs, and provides evidence to support the nomination to add proximal UCDs to the recommended uniform screening panel.

Outcomes of four patients with homocysteine remethylation disorders detected by newborn screening.

PURPOSE: We evaluated the clinical outcome in homocysteine remethylation disorders following newborn screening (NBS) and initiation of early specific treatment. METHODS: Five patients with remethylation disorders were included in this study. RESULTS: Two asymptomatic patients (one with cblG and one with cblE) were identified by NBS using an approach that combines a postanalytical interpretive tool (available on the Region 4 Stork (R4S) collaborative project website, http://www.clir-r4s.org) and a second-tier test for total homocysteine determination. Both the initial screening and the second-tier test are performed on the same blood spot, with no additional patient contact, resulting in no false-positive outcomes. Two additional patients with methylenetetrahydrofolate reductase deficiency were detected by NBS using low methionine as a marker. Although already symptomatic despite the early diagnosis, the latter two patients greatly improved with treatment and their outcomes are compared with that of another patient with methylenetetrahydrofolate reductase deficiency and significant morbidity who was diagnosed clinically at 3 months of age. CONCLUSION: Early detection by NBS and timely and specific treatment considerably improve at least short-term outcomes of homocysteine remethylation disorders. When a remethylation disorder is suspected, group-specific treatment could be started prior to the completion of in vitro confirmatory testing because all disorders from this group require similar intervention.

Simultaneous Testing for 6 Lysosomal Storage Disorders and X-Adrenoleukodystrophy in Dried Blood Spots by Tandem Mass Spectrometry.

BACKGROUND: Newborn screening for lysosomal storage disorders (LSD) has revealed that late-onset variants of these conditions are unexpectedly frequent and therefore may evade diagnosis. We developed an efficient and cost-effective multiplex assay to diagnose six LSDs and several peroxisomal disorders in patients presenting with diverse phenotypes at any age. METHODS: Three 3-mm dried blood spot (DBS) punches were placed into individual microtiter plates. One disc was treated with a cocktail containing acid sphingomyelinase-specific substrate and internal standard (IS). To the second DBS we added a cocktail containing substrate and IS for ß-glucosidase, acid α-glucosidase, α-galactosidase A, galactocerebrosidase, and α-L-iduronidase. The third DBS was extracted with methanol containing d4-C26 lysophosphatidylcholine as IS and stored until the enzyme plates were combined and purified by liquid-liquid and solid-phase extraction. The extracts were evaporated, reconstituted with the extract from the lysophosphatidylcholine plate, and analyzed by flow injection tandem mass spectrometry. RESULTS: Reference intervals were determined by analysis of 550 samples from healthy controls. DBS from confirmed patients with 1 of the 6 LSDs (n = 33), X-adrenoleukodystrophy (n = 9), or a peroxisomal biogenesis disorder (n = 5), as well as carriers for Fabry disease (n = 17) and X-adrenoleukodystrophy (n = 5), were analyzed for assay validation. Prospective clinical testing of 578 samples revealed 25 patients affected with 1 of the detectable conditions. CONCLUSIONS: Our flow injection tandem mass spectrometry approach is amenable to high-throughput population screening for Hurler disease, Gaucher disease, Niemann-Pick A/B disease, Pompe disease, Krabbe disease, Fabry disease, X-adrenoleukodystrophy, and peroxisomal biogenesis disorder in DBS.

Continuous age- and sex-adjusted reference intervals of urinary markers for cerebral creatine deficiency syndromes: a novel approach to the definition of reference intervals.

BACKGROUND: Urinary concentrations of creatine and guanidinoacetic acid divided by creatinine are informative markers for cerebral creatine deficiency syndromes (CDSs). The renal excretion of these substances varies substantially with age and sex, challenging the sensitivity and specificity of postanalytical interpretation. METHODS: Results from 155 patients with CDS and 12 507 reference individuals were contributed by 5 diagnostic laboratories. They were binned into 104 adjacent age intervals and renormalized with Box-Cox transforms (Ξ). Estimates for central tendency (µ) and dispersion (σ) of Ξ were obtained for each bin. Polynomial regression analysis was used to establish the age dependence of both µ[log(age)] and σ[log(age)]. The regression residuals were then calculated as z-scores = {Ξ - µ[log(age)]}/σ[log(age)]. The process was iterated until all z-scores outside Tukey fences ±3.372 were identified and removed. Continuous percentile charts were then calculated and plotted by retransformation. RESULTS: Statistically significant and biologically relevant subgroups of z-scores were identified. Significantly higher marker values were seen in females than males, necessitating separate reference intervals in both adolescents and adults. Comparison between our reconstructed reference percentiles and current standard age-matched reference intervals highlights an underlying risk of false-positive and false-negative events at certain ages. CONCLUSIONS: Disease markers depending strongly on covariates such as age and sex require large numbers of reference individuals to establish peripheral percentiles with sufficient precision. This is feasible only through collaborative data sharing and the use of appropriate statistical methods. Broad application of this approach can be implemented through freely available Web-based software.

Streamlined determination of lysophosphatidylcholines in dried blood spots for newborn screening of X-linked adrenoleukodystrophy.

BACKGROUND: Pre-symptomatic hematopoietic stem cell transplantation is essential to achieve best possible outcomes for patients with the childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD). We describe a high-throughput method for measurement of C20-C26 lysophosphatidylcholines (LPCs) and biochemical diagnosis of X-ALD using the same dried blood spots (DBS) routinely used for newborn screening. METHODS: LPCs are extracted from 3-mm DBS punch with methanol containing an isotopically labeled LPC as internal standard. This extract is transferred to a 96-well plate, evaporated and then reconstituted in mobile phase for flow injection analysis tandem mass spectrometry (FIA-MS/MS) in selected reaction monitoring mode for measurement of four different LPCs (C20, C22, C24, C26) and the internal standard (d4-C26-LPC). Analysis time is 1.5min per sample. RESULTS: The mean CVs from the intra- and inter-assay experiments for LPCs were 6.3-15.1% for C20-LPC, 4.4-18.6% for C22-LPC and 4.5-14.3% for C24-LPC. Limits of detection were determined for C20-LPC (LOD=0.03µg/mL), C22-LPC (0.03µg/mL), C24-LPC (0.03µg/mL) and C26-LPC (0.01µg/mL). Reference ranges were established from DBS of 130 newborns and 20 adults. Samples of patients with X-ALD (n=16), peroxisomal biogenesis disorders (n=8), and X-ALD carriers (n=12) were analyzed blindly and all were correctly identified. CONCLUSION: Analysis of LPC species by FIA-MS/MS is a fast, simple and reliable method to screen for X-ALD and other peroxisomal disorders in DBS. To maximize specificity, abnormal results can be verified by a 2nd tier assay using LC-MS/MS.

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines.

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.