Thomas F. Metz

Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria

BACKGROUND The interest in neonatal screening for lysosomal storage disorders has increased substantially because of newly developed enzyme replacement therapies, the need for early diagnosis, and technical advances. We tested for Gauchers disease, Pompes disease, Fabrys disease, and Niemann-Pick disease types A and B in an anonymous prospective nationwide screening study that included genetic mutation analysis to assess the practicality and appropriateness of including these disorders in neonatal screening panels. METHODS Specimens from dried blood spots of 34,736 newborn babies were collected consecutively from January, 2010 to July, 2010, as part of the national routine Austrian newborn screening programme. Anonymised samples were analysed for enzyme activities of acid β-glucocerebrosidase, α-galactosidase, α-glucosidase, and acid sphingomyelinase by electrospray ionisation tandem mass spectrometry. Genetic mutation analyses were done in samples with suspected enzyme deficiency. FINDINGS All 34,736 samples were analysed successfully by the multiplex screening assay. Low enzyme activities were detected in 38 babies. Mutation analysis confirmed lysosomal storage disorders in 15 of them. The most frequent mutations were found for Fabrys disease (1 per 3859 births), followed by Pompes disease (1 per 8684), and Gauchers disease (1 per 17,368). The positive predictive values were 32% (95% CI 16-52), 80% (28-99), and 50% (7-93), respectively. Mutational analysis detected predominantly missense mutations associated with a late-onset phenotype. INTERPRETATION The combined overall proportion of infants carrying a mutation for lysosomal storage disorders was higher than expected. Neonatal screening for lysosomal storage disorders is likely to raise challenges for primary health-care providers. Furthermore, the high frequency of late-onset mutations makes lysosomal storage disorders a broad health problem beyond childhood. FUNDING Austrian Ministry of Health, Family, and Women.

Simplified Newborn Screening Protocol for Lysosomal Storage Disorders

BACKGROUND Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays. METHODS After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid-liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate. RESULTS Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory. CONCLUSIONS Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.

The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots

Lysozomal storage disorders are just beginning to be routinely screened using enzyme activity assays involving dried blood spots and tandem mass spectrometry (MS/MS). This paper discusses some of the analytical challenges associated with published assays including complex sample preparation and potential interference from excess residual substrate. Solutions to these challenges are presented in the form of on-line two-dimensional chromatography to eliminate off-line liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the use of ultra-high-performance liquid chromatography (UHPLC) to separate excess substrate from all other analytes and multiplexed sample introduction for higher throughput required of a population screening assay. High sensitivity, specificity and throughput were demonstrated using this novel method.

Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening

The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within 4h including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3h revealed in intra-day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography-ultra high performance liquid chromatography-tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day.

Evaluation of a novel, commercially available mass spectrometry kit for newborn screening including succinylacetone without hydrazine.

Newborn screening for tyrosinemia type I (Tyr-I) is mandatory to identify infants at risk before life-threatening symptoms occur. The analysis of tyrosine alone is limited, and might lead to false-negative results. Consequently, the analysis of succinylacetone (SUAC) is needed. Current protocols are time-consuming, and above all, include hazardous reagents such hydrazine. We evaluated a novel, commercial kit to analyze amino acids, acylcarnitines and SUAC with a significantly less harmful hydrazine derivative in a newborn screening laboratory. Dried blood spot specimens from 4683 newborns and samples from known patients with inborn errors of metabolism (IEM) were analyzed by a novel protocol and compared to an in-house screening assay. All samples were derivatized with butanol-HCl after extraction from 1/8-inch DBS punches. For the novel protocol, the residual blood spots were extracted separately for SUAC, converted into hydrazone, combined with amino acids and acylcarnitines, and subsequently analyzed by mass spectrometry using internal isotope-labeled standards. All newborns were successfully tested, and 74 patients with IEMs including three with Tyr-I (SUAC 1.50, 4.80 and 6.49; tyrosine levels 93.10, 172.40 and 317.73, respectively) were detected accurately. The mean SUAC level in non-affected newborns was 0.68 μmol/l (cut-off 1.29 μmol/l). The novel assay was demonstrated to be accurate in the detection of newborns with IEM, robust, and above all, without the risk of the exposure to highly toxic reagents and requirement of additional equipment for toxic fume evacuation.

Matrix-assisted laser desorption/ionization for simultaneous quantitation of (acyl-)carnitines and organic acids in dried blood spots

RATIONALE Screening for inborn errors of metabolism using mass spectrometry is part of nationwide newborn screening programs and involves the detection of disease relevant (acyl-)carnitines and organic acids from dried blood spots. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) is a well-established tool for proteomics approaches. In recent years, this technique has become more and more integrated in analysis and identification of small metabolites and disease biomarkers in daily clinical laboratories. METHODS We used a combination of both MALDI and high-resolution accurate mass (HR/AM) mass spectrometry using a linear ion trap-Orbitrap for the identification of small molecules from dried blood spots that serve as biomarkers for inborn errors of metabolism. The levels of detected metabolite species were compared between healthy newborns and affected patients with various inborn errors of metabolism using isotopically labeled internal standards and new bioinformatics software, respectively. RESULTS (Acyl-)carnitine levels from normal and affected patients could be quantified and differentiated. Additionally, using the high resolving power of full scan Orbitrap mass spectrometry and novel software tools we demonstrated the identification and quantification of disease-specific organic acids. CONCLUSIONS MALDI-HR/AM and full scan spectra to obtain information for the metabolic status of patients is a promising complementary approach to electrospray ionization mass spectrometry by simplified sample preparation, facilitating the screening of hundreds of metabolites from small sample volumes.

Demethylation of the promoter region of GPX3 in a newborn with classical phenylketonuria

OBJECTIVES Phenylketonuria (PKU) is characterized by a high phenylalanine (phe) in plasma and oxidative stress. However, the monitoring of oxidative stress in newborns with PKU using the activity levels of antioxidant enzymes is not optimal. We investigated the possibility of monitoring an increased reactive oxygen species (ROS) production using DNA methylation changes of an oxidative stress response element in the promoter region of an enzymatic antioxidant gene. DESIGN AND METHODS Using DNA extracted from blood leukocytes, the cytosine phosphodiester bond guanine positions of an overlapping CCAAT box/metal response element (CGATTGGCTG) of the glutathione peroxidase 3 promoter activated by oxidative stimuli and expressed in plasma were analysed for methylation changes in 20 newborns with hyperphenylalaninemia and 20 healthy controls. RESULTS A demethylated allele was detected in a PKU patient at a phe level of 465μmol/L on day 2 after birth, but not in other patients (phe<465μmol/L, ≥day 2 after birth; phe>465μmol/L, ≥day 3 after birth) and healthy controls (phe<465μmol/L, ≥day 2 after birth). CONCLUSIONS The detection of the demethylated allele could be time and phe concentration dependent. The demethylated allele is suggested as an early epigenetic marker for an extracellular monitoring of an increased ROS production in newborns with PKU.

Aberrant DNA methylation of calcitonin receptor in Fabry patients treated with enzyme replacement therapy

Fabry disease is as a result of a deficiency of the enzyme alphagalactosidase A which leads to a gradual lysosomal accumulation of globotriaosylceramide (GB3) in cells of the body. Neuropathic pain is one of the prominent symptoms of Fabry disease in the initial stages of the disease [1,2,3], and a possible mechanism involves the release of inflammatory molecules such as calcitonin gene related peptide (CGRP) which activates both calcitonin receptor-like receptor (CRLR) and calcitonin receptor (CALCR) on cell membranes in target tissues, generating signals that regulate various functions within the cell such as neuropathic pain. The involvement of CGRP and CGRP receptors in pain transmission and modulation in central and peripheral nervous systems has been demonstrated in several studies and they appear to play an essential role in peripheral inflammation and development of neuropathic pain [4,5]. While the mechanism of symptom reduction through enzyme replacement therapy (ERT) is not known [6,7], an emerging evidence suggests that DNA methylation changes can affect important CpG (cytosine phosphodiester bond guanine) sites for pain hypersensitivity in neuropathic pain [8]. We therefore carried out a retrospective investigation of the DNA methylation status of the promoter region of the calcitonin receptor in Fabry patients (6 non-ERT treated and 3 ERT treated) and 6 healthy controls respectively, using bisulphite modified DNA isolated from blood leukocytes, methylation specific PCR, high resolution melting, and sequencing as previously described [9]. The inclusion criteria for Fabry patients were: the presence of acute unexplained periods of pain or chronic pain in the limbs, hypohidrosis, angiokeratomas, and anenzyme activity of GLA b2.8 μmol/l/h [1]. An ethic approval for this study was available (EK478/2009). At position −78504 CpG in the promoter region of CALCR, 3 ERT treated Fabry patients showed a total methylation (GGAGAAGTGCGTA) on both alleles (Fig. 1, Panel B), 3 non-ERT treated Fabry patients and 5 healthy controls respectively showed a demethylation on both alleles (GGAGAAGTGTGTA) (Fig. 1, Panel A), 3 non-ERT treated Fabry patients and 1 healthy control respectively showed a partial methylation on one allele (GGAGAAGTGC/TGTA) (Fig. 1, Panel C). The specific methylation affected a DNA sequence (GTGCG) that is similar to a consensus binding sequence (R-CGTG) for a hypoxia inducible factor 1α protein (HIF-1α). HIF-1α is important in nerve inflammation and is involved in the development of neuropathies [10,11]. The methylation at−78504 CpG on both alleles of the promoter region of CACLR in ERT treated Fabry patients is predicted to prevent the transcription factor HIF-1 from binding to its responsive element