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.

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.

Molecular analysis of guanidinoacetate-n-methyltransferase (GAMT) and creatine transporter (SLC6A8) gene by using denaturing high pressure liquid chromatography (DHPLC) as a possible source of human male infertility.

The creatine/phosphocreatine system is essential for cellular phosphate coupled energy storage and production, particularly in tissues subject to high metabolic demands. Male factor infertility is a common condition with unknown etiology in most of the cases. Sperm abnormalities could possibly lead to infertility. As sperm motility depends on intact mitochondrial function and energy levels. Thus reduced intracellular creatine stores may contribute to decreased sperm motility leading to male infertility as creatine /phosphocreatine system plays major role in making and breaking of ATP, thus in energy kinetics. We developed and validated a denaturing high performance liquid chromatograph (DHPLC) method for the molecular analysis of SLC6A8 and GAMT genes involve in creatine biosynthesis and transport as a possible source of human male infertility by analyzing DNA from 64, clinically confirmed, infertile men. No mutation/polymorphism was detected in the exonic regions of both genes in all the patients and in fertile healthy controls indicating that SLC6A8 and GAMT genes may not be directly involved in human male infertility.

The national Austrian newborn screening program - eight years experience with mass spectrometry. past, present, and future goals.

BACKGROUND: the National Austrian Newborn Screening Program for inherited metabolic and endocrinologic disorders was introduced in 1966. The program continuously evolved by expanding the screening panel from phenylketonuria and galactosemia to congenital hypothyroidism, biotinidase deficiency, cystic fibrosis, and congenital adrenal hyperplasia. In 2002, the introduction of tandem mass spectrometry (MS/MS) substantially increased the number of detectable inborn errors of metabolism and now includes disorders of fatty acid oxidation, organic acidurias and various disorders of amino acid metabolism. OBJECTIVE: in this study we report our eight years experience with MS/MS in Austria and give an overview of the incidence of diseases, organization, updates on methods and current development and future aspects. METHODS: a total of 622,489 newborns were screened by MS/MS for more than 20 diseases in Austria between April 2002 and December 2009. Dried blood spot samples were collected and sent to the National Laboratory for Newborn Screening located at the Medical University of Vienna, Vienna, Austria. RESULTS: The resulting overall prevalence of inherited metabolic disorder identified by MS/MS was 1:2855, including 125 newborns with amino acidemias (1:4,980), 46 with organic acidurias (1:13,532), and 47 with fatty acid oxidation disorders (1:13,244). CONCLUSION: the introduction of MS/MS technology in Austria significantly increased the detection of inherited metabolic disorders that were previously not covered. A primary goal is the continuous effort by developing second-tier strategies with the inclusion of more specific markers in order to minimize the risk of false-negatives and to improve the positive predictive value of screening results. Early recognition of these disorders enables diagnosis and treatment before the onset of symptoms.

Transcobalamin II deficiency at birth.

Transcobalamin II deficiency (# MIM 275350) is a rare, recessively inherited disorder of cobalamin transport that leads to intracellular cobalamin depletion with secondary impairment of methionine synthetase and methyl-malonyl CoA mutase activities. Affected individuals may suffer from long-term neurological sequelae if therapy with intramuscular hydroxocobalamin is not initiated promptly. We report two sisters with complete absence of transcobalamin due to homozygosity for a novel mutation (c.insC110) in the TCN2 gene that leads to a premature stop codon and non-functional protein. The older sister, now 4.5 years old, presented at 6 weeks of age with pancytopenia, protein losing enteropathy and a rapidly declining clinical course. Prompt therapy with 1mg hydroxocobalamin/day led to full recovery within days. Her now 1.5 year old sister was diagnosed shortly after birth and was started on hydroxocobalamin prior to onset of clinical symptoms. Interestingly, urinary methylmalonic acid excretion was increased significantly during the first days of life suggesting that functional cobalamin deficiency is present also during fetal life, although not giving rise to clinical symptoms until well after birth.

Long-term stability of amino acids and acylcarnitines in dried blood spots.

BACKGROUND: Dried blood filter cards, collected for newborn screening, are often stored for long periods of time. They may be suitable for the retrospective diagnosis of inborn errors of metabolism, but no data are currently available on the long-term stability of amino acids and acylcarnitine species. METHODS: We analyzed amino acids and acylcarnitines by tandem mass spectrometry in 660 anonymous, randomly selected filter cards from 1989 through 2004. We assessed long-term stability of metabolites by linear regression and estimated annual decrease of concentration for each metabolite. RESULTS: Concentrations of free carnitine increased by 7.6% per year during the first 5 years of storage and decreased by 1.4% per year thereafter. Alanine, arginine, leucine, methionine, and phenylalanine decreased by 6.5%, 3.3%, 3.1%, 7.3%, and 5.7% per year, respectively. Acetylcarnitine, propionylcarnitine, citrulline, glycine, and ornithine decreased by 18.5%, 27.4%, 8.1%, 14.7%, and 16.3% per year during the first 5 years, respectively; thereafter the decline was more gradual. Tyrosine decreased by 1.7% per year during the first 5 years and 7.9% per year thereafter. We could not analyze medium- and long-chain acylcarnitine species because of low physiological concentrations. CONCLUSIONS: Estimation of the annual decrease of metabolites may allow for the retrospective diagnosis of inborn errors of metabolism in filter cards that have been stored for long periods of time.

Influence of hematocrit and localisation of punch in dried blood spots on levels of amino acids and acylcarnitines measured by tandem mass spectrometry.

BACKGROUND: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots. METHODS: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively. RESULTS: Levels of most AA and GAA increased significantly with increasing hematocrit (p<0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p<0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p<0.0001). CONCLUSIONS: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.