Abstrakta

1. Population genetics of cystic fibrosis and the EuroCareCF patient registry project: Implications for neonatal screening (review)

Macek M. Jr. on behalf of the Euro CareCF Workpackage 2

Department of Biology and Medical Genetics – Cystic Fibrosis Centre, Charles University Prague – 2nd Medical School and Faculty Hospital Motol; Prague, Czech Republic

Cystic Fibrosis (CF) is a severe life-shortening, autosomal recessive disorder that is affecting in Europe alone about 35,000 individuals. Mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF and a spectrum of CFTR-related disorders. In our presentation we will demonstrate their population distribution and relevance of these findings to neonatal screening. Despite impressive advances in understanding the disease, patient life expectancy and quality of life remain limited because resources are scattered in Europe. CF highlights the complex challenges of genetic diseases in the post-genomic era, pioneering explanations for pathological mechanisms and rational approaches to therapy. Moreover, CF is a model monogenic disorder for molecular medicine, with its complex pathogenesis serving as a paradigm for the study of important multifactorial diseases. Within the EuroCareCF project demographic data from over 29,000 CF patients were collected and illustrate the disparities in care between Western and Eastern European countries. One of the ways to overcome this problem is uniform and early diagnosis of the disease by neonatal screening.

Acknowledgements: Supported by the

EuroCareCF and MSM0064203.

2. From suspicion to diagnosis in children with inborn errors of metabolism

Zeman J.

Charles University, First Faculty of Medicine, Department of Pediatrics and Institute for Inherited Metabolic Diseases, Prague, Czech Republic

In addition to common cardiovascular and infectious disorders, more than 5,800 rare diseases including metabolic, endocrinologic and genetic disturbances enrolled in the Orphanet database are also an important threat to the health. Early diagnostics of life-threatening or chronically debilitating rare diseases more or less well treatable if recognized early after birth in still asymptomatic neonates represent together with the proper genetic counseling and eventual prenatal diagnostics in affected families one of the most effective preventive programmes in medicine. Although in many regions also the economic impact of new laboratory technologies of newborn screening becomes relevant, expanded newborn screening programs on the national level may represent the chance not only to improve quality of life in affected individuals but also to decrease the cost estimates for treatment and follow-up. Especially in centers using screening methods including electrospray-ionization mass spectrometry (ESI-MS), capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) and/or other sophisticated methods, the confirmation of the diagnosis on biochemical and/or molecular level in the child with positive screening is easier and usually cheaper than in critically ill neonates in the intensive care unit with broad spectrum of possible diagnoses. Despite the logistic problems and economic constraints, expanded neonatal screening is gradually introduced in a number of countries. In most centers for neonatal screening, the high quality research information on the cost and effectiveness from other countries are of importance due to low incidence of individual diseases.

Acknowledgements: Supported by

project VZ 64165.

3. Quality assurance program for MS/MS analysis in newborn screening and metabolic diagnosis

Lukacs Z.

Hamburg University Medical Center, Dept. of Pediatrics and Institute of Clinical Chemistry, Germany

Tandem mass spectrometry for the analysis of acylcarnitines and amino acids using dried blood samples has more commonly been introduced since 2000. It instantly provided a challenge to quality assurance (QA) in laboratories as more than 30 parameters were simultaneously determined in one run. We started to offer our QA program in 2004 and have seen participation rise from 21 laboratories to more than 45 laboratories in 2008. This also reflects the increasing awareness for QA issues. Participants include laboratories from Europe, North America, Australia and Asia. The program is free of charge and further participants are welcome. For all diagnostic laboratories the most important focus is on the correct diagnosis. Thus, each batch of QA samples contains specimens from real patients and/or healthy controls. Laboratories are asked to report the supposed diagnosis and their quantitative results. The latter can be compared to all other participants. In addition, results were also referred to individual cut-off thresholds or popular analyte ratios were compared. Especially, the first failed to ameliorate the problem of comparability because of highly variable philosophies in choosing individual cut-offs (e.g. purely statistical vs patient values). Generally, most problems were encountered with very rare disorders like LCHADD and glutaric aciduria I. In addition, the concentrations of key analytes in those diseases may be only marginally elevated. Further harmonization of methods seems to be useful to allow better comparability of results. This becomes even more necessary as more and more doctors rely on acylcarnitine and amino acid concentrations in dried blood for patient follow-up.

4. Spectrum of Medium Chain Acyl CoA Dehydrogenase (MCAD) mutations identified from newborn screening of 1.14 million ethnically diverse infants

Oerton J.¹, Andresen B. S.², Leonard J.³, Khalid J. M.¹, Dezateux C.¹

¹UCL Institute of Child Health, MRC Centre of Epidemiology for Child Health, London, England

²University of Southern Denmark, Department of Biochemistry and Molecular Biology, Odense, Denmark

³UCL Institute of Child Health, London, England

In those of European descent, on average 55% of children diagnosed as MCAD deficiency (MCADD) through newborn screening are c.985A>G homozygotes (HMZs), compared with 80% presenting clinically. We have previously shown that, in England, c.985A>G homozygosity is not found in Black or Asian ethnic groups. We report the spectrum of MCAD mutations identified through newborn screening of an ethnically diverse English population.1.14 million babies (79.5% White, 10.2% Asian [from Indian sub-continent], 4.7% Black and 5.5% Mixed/other ethnic origin), born in a three year period, were screened at age 5–10 days using electrospray tandem mass spectrometry analysis of underivatised blood spot samples to quantitate octanoylcarnitine (C8). All presumptive positives (average C8 >0.5 µmol/L) were investigated with repeat biochemistry and tested for c.985A>G, with extended mutation screening for those not c.985A>G HMZs. New mutations were identified, the clinical significance of which was unclear. An independent expert panel assigned cases to MCADD of “definite” or “uncertain” phenotype. Of 126 children confirmed as MCADD (1.1 per 10,000), 87 (69%) were considered definite (79 White, 3 Asian, 2 Mixed/other and 3 unknown). Of these, 66 (52%) were c.985A>G HMZs (64 White, 2 Mixed/other), 4 (5%) 799G>A HMZs (1 Asian), 3 HMZ of other mutations (157C>T, 244InsT, 727C>T; 1 Asian) and 14 heteroallelic (985A>G/799A>G in 7). A further 39 (31%) children (26 mutations) were diagnosed with MCADD of “uncertain” phenotype (27 White, 11 Asian, 1 Black), 7 Asian babies were IVS10-6T>G HMZs, and 2 were 1237C>T HMZs. The overall prevalence of MCADD in Asian infants was 1.2 per 10,000. The spectrum of MCAD mutations differs in ethnic groups. MCADD is as prevalent amongst Asian infants as in general population but is more likely to be of an uncertain phenotype. Extended mutation screening provides useful diagnostic information when investigating screen-positive infants in a multi-ethnic population.

5. Screening for defects of peroxisomal ß-oxidation

Janzen N.¹, Sander S.², Sass O.³, Gokcay G.⁴, Peter M.², Sander J.²

¹Medical School Hannover, Childrens Hospital, Hannover, Germany

²Screening-Labor Hannover, Laboratory, Hannover, Germany

³University, Childrens Hospital, Freiburg, Germany

⁴University, Childrens Hospital, Istambul, Turkey

For detection of defects of peroxisomal ß-oxidation in newborns and infants we included butylated hexacosanoyl carnitine (C26 carnitine) into the MS/MS measurement of acylcarnitines and aminoacids from dried blood spots. For identification of C26 carnitine we used m/z 596.6 Da with a fragment of 85.0 Da. Trideuterated octadecanoyl carnitine (dC18) served as internal standard (m/z 487.4) for C26 carnitine as well as for C18 acylcarnitines of the standard screening program. Since C26 carnitine is not commercially available direct quantitation was not possible. Therefore we calculated relative units (RU) related to the concentration (7.8 µmol/L) of dC18. Analysis of 27,000 blood samples on filterpaper for newborn or selective screening showed concentrations above 0.02 RU in 20 cases (0.07%). Among these there were 5 samples of 4 patients for whom peroxisomal biogenesis defects were clinically confirmed. As a second tier analysis from the same sample for immediate confirmation we used MS/MS measurement of the C27 bile acid precursors dihydroxycholestanoic acid (DHCA) and trihydroxycholestanoic acid (THCA) after chromatographic separation on a Luna 3µ C18(2) 50 x 2 mm column (Phenomenex) (m/z 433.25 and 449.25 Da). In Zellweger syndrom patients DHCA and THCA concentrations ranged from 0.08 to 4.03 and 0.23 to 6.63 µmol/L respectively. Among those samples showing unspecific elevation of C26 carnitine concentrations of both C27 bile acid precursors were within the normal range below 0.06 µmol/L for DHCA and 0.02 µmol/L for THCA. Three of these samples were from galactosemic patients and 2 from infants presenting with acute liver disease. We conclude that specific early detection of at least an important part of the defects of peroxisomal ß-oxidation can be achieved just by focussing on an additional mass in a standard metabolic screening program provided it is combined to immediate second tier analysis of di- and trihydroxycholestanoic acid.

6. The impact of a second tier blood-spot methylmalonic acid (MMA) using tandem mass spectrometry (MS/MS) on routine newborn screening for IEM

Ranieri E., Gerace R., Bartlett B., Harrison J. R., Fletcher J.

SAPathology, Women’s & Children’s Hospital Campus, Genetic Medicine, Adelaide, South Australia, Australia

Background: In 1999 the South Australian Neonatal Screening Centre introduced MS/MS into routine newborn screening for IEM. A 2nd tier test for methylmalonic acid (MMA) was added as a specific diagnostic marker for a group of inherited disorders collectively known as methylmalonic acidemias.

Methods: A non-derivatised blood-spot LC-MS/MS MMA method was developed using a 5 x 100 mm Phenomenex C6-phenyl column with isocratic buffer acetonitrile:water:formic acid) at a flow rate of 150 µL/min directly into an API4000 MS/MS (MDS-SCIEX) operated in negative ion mode. MMA was eluted from a 3 mm blood-spot with methanol and levels determined against a(d3)-MMA by monitoring the MRM pairs of 117.1/73.1 and 120.1/76.1 in a 5 minute LC run.

Results: In over 154,000 consecutive newborn blood spot specimens tested there were 207 (0.13%) that required a repeat sample due to an elevated propionyl-carnitine (C3; 99th centile >5.4 µmol/L whole blood) in addition to its associated ratios C3/C2, C3/C16 and C3/methionine. Of the 207 requiring a recollection, 12 babies had a sustained elevation of C3 and were recalled for a plasma and urine MMA and B12 determination at an average infant age of 21 days. Of these, 9 had an elevated MMA and shown to be significantly B12 deficient. Retrospective analysis of the 207 blood spot samples showed an elevated MMA (>1.1 µmol/L whole blood) in all 9 confirmed B12 deficient cases. Since introducing the LC-MS/MS MMA blood spot method we have identified a mother, as a result of an elevated C3 and MMA in her baby, with pernicious anaemia due to antibodies against intrinsic factor.

Conclusion: The incorporation of a 2nd tier LC-MS/MS MMA blood spot method into a routine newborn screening programme has had a significant impact on reducing the false positive rate associated with the measurement of C3 carnitine.

7. Italian guidelines for expanded newborn screening using tandem mass spectrometry

Antonozzi I.¹, Carducci C.¹, Burlina A.², Caruso U.³, Cerone R.³, Corbetta C.⁴, Giordano G.², La Marca G.⁵, Pasquini E.⁵

¹Sapienza Università di Roma, Dip Medicina Sperimentale, Rome, Italy

²Università di Padova, Dipartimento di Pediatria, Padova, Italy

³Università di Genova, Dipartimento di Pediatria, Genova, Italy

⁴Istituti Clinici di Perfezionamento, Laboratorio Screening Neonatale e Biochimica, Milano, Italy

⁵Università di Firenze, Dipartimento di Pediatria, Firenze, Italy

The Italian Society for Newborn Screening (SISN) and the Italian Society for the Study of Inborn Errors of Metabolism (SISMME) have coordinated an evaluation of Italian pilot studies on expanded newborn screening and the compilation of evidence-oriented guidelines to obtain a uniform organization of the nationwide service. In these guidelines the components of newborn screening system that are critical to achieving the objectives of screening expansion using MS/MS, have been considered. The reported evaluation were also based on the Italian multi centric (Firenze, Roma, Padova, Genova) pilot study for the nationwide based implementation of extended screening. Until November 2007, 182,323 newborns entered the study. A panel of 39 diseases was screened and 98 true positives were found. The global recall rate was 1.04%, ranging from 0.105 to 1.99%. The average detection rate was 1:1,901 and the positive predictive value was 5.08. The evaluated panel showed an optimal cost benefit ratio provided that the dimension of the screening centers was >50,000 newborns. In this case, the estimated costs of the traditional + expanded screening were calculated as 55.35 €/sample. A strong recommendation was made on the strict correlation between screening and laboratory confirmation, on the participation to the international Quality and Proficiency control programs, on the importance of a close cooperation between the Paediatrician specialist and the Screening Center. The Guidelines are available at www.sismme.it.

8. Expanded newborn screening for metabolic disorders by tandem mass spectrometry in Italy: current status

Cassanello M.¹, La Marca G.², Burlina A. B.³, Carducci C.⁴, Ruoppolo M.⁵, Caruso U.⁶, Giordano G.⁷, Pasquini E.⁸, Malvagia S.², Donati A.⁸, Antonozzi I.⁴, Cerone R.⁹

¹G. Gaslini Institute, Labsiem, Department of Pediatrics, Genoa, Italy

²Meyer Institute, Clinical Biochemistry Lab, Florence, Italy

³Padua University, Department of Pediatrics, Padua, Italy

⁴Sapienza University, Genetic and Metabolic Diseases Service, Rome, Italy

⁵Federico II University, CEINGE, Naples, Italy

⁶Genoa University, Labsiem, Department of Pediatrics, Genova, Italy

⁷Padua University, MS Lab- Depth of Pediatrics, Padua, Italy

⁸Meyer Institute, Department of Pediatrics, Florence, Italy

⁹Genoa University, Department of Pediatrics, Genoa, Italy

Neonatal screening has been performed in Italy for more than 35 years. Since 1992 neonatal screening for PKU/HPA, congenital hypothyroidism (CH) and cystic fibrosis (CF) is mandatory by law with a full coverage for PKU/HPA and CH and about 70% for CF. Expanded newborn screening (NBS) by tandem mass spectrometry was introduced as pilot program in Padua, Tuscany, Liguria, Rome and Naples in 1999, 2001, 2003, 2004 and 2007 respectively. Nowadays only in Tuscany region expanded NBS screening is mandatory by low since November 2004, while the pilot programs proceed in the other above mentioned Centres. At 2008, October, a total of 270,609 newborns have been tested with an average of recall rate of 1.14%. 25 organic acidosis, 22 ß-oxidation defects and 71 aminoacidopathies (including 62 PKU/HPA) have been detected, all confirmed by biochemical and or molecular testing. The overall incidence of metabolic diseases in the examined population results to be 1:4,832 (1:2,293 including PKU/HPA). The most frequent disorders (not PKU/HPA) are: MCAD (N=10), MMA (6) and 3-methylcrotonylglycinuria (6). Further, ten newborns showed steadily high C4 and C4/C3 ratio. Additionally metabolic diseases have been diagnosed in six relatives of newborns showing abnormalities at NBS testing. Although the coverage of expanded NBS in Italy was only of 10.4% in 2007 (the newborn population consisted of about 565,000 individuals), some other Italian Regions are strongly interested in the topic and activation of new pilot programs is in progress. The Italian Society of Neonatal Screening (SISN) and the Italian Society for the Study of Inborn Metabolic Diseases (SISMME) have developed in May 2008 national guidelines for the implementation and management of expanded newborn screening, aimed to harmonize all screening programs, already working or in progress, and to stimulate the support of the Central Health Authority as well (available, in Italian, on www.sismme.it).

9. Improving newborn screening performance: the Mayo Clinic experience (2004–2008)

Matern D., Raymond K., Tortorelli S., Gavrilov D., Oglesbee D., Rinaldo P.

Mayo Clinic College of Medicine, Biochemical Genetics Laboratory, Rochester, MN, United States

The continued expansion of newborn screening (NBS) programs to include additional conditions increases the accountability of NBS laboratories to provide testing with the highest possible sensitivity and specificity to allow for identification of affected patients while minimizing the false positive rate. Some assays and analytes are particularly problematic. Over recent years, our laboratory tried to improve this situation by developing second tier tests to reduce false positive results in the screening for congenital adrenal hyperplasia (CAH), Methylmalonic acidemias, Homocystinuria, and Maple Syrup Urine Disease (MSUD). In addition, succinylacetone was included into the primary screen for inborn errors of amino acid, fatty acid, and organic acid metabolism by tandem mass spectrometry, virtually eliminating false positive results for tyrosinemia. Beginning in 2004, this approach was applied to Mayo’s NBS program resulting in a significant improvement of performance metrics (see below). With the ongoing expansion of NBS programs to include conditions such as Lysosomal Storage Disorders, efficient and effective second tier strategies are indispensable because false positive results for these disorders will likely have more serious implications than for the currently screened conditions. Our laboratory is prepared to assist others in the implementation of currently employed strategies to improve NBS performance.

2004 – False Positive Rate: 0.12%;

Positive Predictive Value: 27%

2005 – False Positive Rate: 0.08%;

Positive Predictive Value: 37%

2006 – False Positive Rate: 0.09%;

Positive Predictive Value: 47%

2007 – False Positive Rate: 0.06%;

Positive Predictive Value: 47%

2008 – False Positive Rate: 0.07%;

Positive Predictive Value: 53%

10. Diet therapy of galactosemia

Mitish M., Beljaeva I., Yatsyk G., Odinaeva N.

Scientific Center for Children’s Health, Department of Neonatology, Moscow, Russia

To estimate the influence of early diagnostics and therapy on the development of infants with galactosemia. Neonatal screening, determination of galactose in blood by two-dimensional chromatography, DNA-diagnostics, diet therapy. The introduction of neonatal screening for galactosemia (in Russia since 2007) gives the possibility of early diagnostics of this pathology. During the last 2 years we observed 6 infants with galactosemia: in 4 cases the diagnosis was made during the 1st week of life (in the course of neonatal screening programme), in other 2 cases the diagnosis was made on the 34–40th day of life when infants were admitted to the Department of Neonatology of the Scientific Center for Children’s Health. At the admission these infants had hepatosplenomegaly, vomiting and a diarrhea, jaundice, intoxication, and ascites (1 infant). They also had a low level of spontaneous movements and hyporeflexia. Molecular-genetic analysis showed the following mutations: K285N (homozygote boy), Q188R – 4 infants (heterozygotes) and Q188R + L385 (heterozygote boy). The diet therapy was started before the genetic confirmation of the diagnosis – infants were fed with Nutrilon SL (2 infants) or Humana SL (4 infants). This feeding continued after discharge from the Department of neonatology (we observed these patients up to the age of 1 year); with age the diet broadened under control of galactose level in blood. All infants developed properly; their physical, psychological and motor development did not differ from the same in infants without galactosemia. Early diagnostics and timely beginning of treatment by special diet provides normal development of infants with galactosemia.

11. Development and validation of a fast quantitative method for plasma, serum and urine dimethylarginines analysis using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)

Di Gangi I. M.¹, Gucciardi A.¹, Chiandetti L.², Giordano G.³

¹Universita’ di Padova, Pediatria, Padua, Italy

²Azienda Ospedaliera, Pediatria, Padua, Italy

³JRC, IHCP, PCE, Commissione Europea, Ispra (VA), Italy

Introduction: N^G,N^G-dimethyl-L-arginine, or asymmetric-dimethylarginine (ADMA) is a naturally occurring amino acid that circulates in plasma. Free ADMA, and related amino acids, N^G-monomethyl-L-arginine (MMA), N^G,N’^G-dimethyl-L-arginine (SDMA), are producted normally by all cells from hydrolysis of proteins containing methylated L-arginine residues. They are excreted in urine and they are detectable in plasma, tissues and in cells cytosol. ADMA acts as endogenous competitive inhibitor of endothelial nitric oxide synthase (eNOS) and, blocking NO production, it initiates and promotes processes involved in cardiovascular disorders such as atherogenesis and plaque progression, pre-eclampsia, renal failure, hypertension and hypercholesterolemia. Recently, high concentration of ADMA was correlated with inborn errors of metabolism among which urea cycle defects including two enzymes: argininosuccinate-synthase (ASS) and argininosuccinate-lyase (ASL).

Design and methods: We developed and validated a UPLC-MS/MS method for separation and quantification of arginine, ADMA, SDMA, MMA, homo-arginine and citrulline with a short run time (2 min) using a small volume of sample (0.01 mL), necessary condition to make analysis in neonatal field.

Results: Correlation coefficients (r2) of calibration curves ranged from 0.9954 to 0.9993. Intra-day assay CVs (n=30) for ADMA, SDMA and MMA were 6.6%, 7.7% and 7.9% and inter-day assay CVs (n=9) were 9.8%, 12.8% and 14.1%, respectively. Recovery ranged from 99.8% to 100.6%. Limit of detection (LOD) of method was resulted of 0.05 µM (S/N ratio=3). Accuracy medium value was 3% (for ADMA 1.7%).

Conclusions: This study present simple and fast LC-tandem MS-based method for the simultaneous determination and quantification of ADMA and methylated arginines, in biological fluids using a small volume of sample, in a run time suitable for routine analysis. Data from calibration curves and quality controls reveal accuracy and precision of method.

12. Egypt experience implementing a pilot newborn screening program using tandem mass spectrometry

Hassan F.¹, El Mougy F.¹, Hady S. A.², Selim L.², Sharaf S.¹, Mandour I.¹, Morgan M.¹, Mehaney D.¹, Monem  M. A.¹, Salem F.², Oraby A.², El Nekhely I.³

¹Faculty of Medicine, Cairo University, Clinical & Chemical Pathology, Cairo, Egypt

²Faculty of Medicine, Cairo University, Pediatrics, Cairo, Egypt

³Ministry of Health & Population, Children with special needs programs, Cairo, Egypt

Problem statement: It is suspected that metabolic disorder rates among newborn in Egypt has high incidence due to the prevalence of consanguinity in Egypt (38%), where NBS is neither mandatory nor available. This pilot study is the first attempt to elicit prevalence of metabolic disorders in Egypt by tandem mass spectrometry (MS/MS).

Methods: During the period between January 2008 & November 2008 of this pilot program (25,000) neonates (3–7 days) from 3 governorates and 850 patients (3 months to 15 y) presenting to the neurometabolic outpatient (OP) clinic at Cairo University Children Hospitals (CUCH) were offered expanded metabolic screening by MS/MS. Dried blood spots samples obtained from Egyptian Ministry of Health & Population (MOHP) national screening program for congenital hypothyroidism (CH) and from OP were derivatized by an in-house method for sample preparation. Method validation was achieved through participation in CDC NBSQA program and our own QC samples.

Results: From the 25,000 screened neonates, 19 cases were detected approximately (1/1300). They were 5 PKU (1/5000), 1 case MSUD (1/25000), 13 cases organic acid and fatty acid oxidation disorders approximately (1/1900). First Egyptian reference values for expanded screening by MS/MS were calculated from this sample. From 850 patients presenting to neurometabolic clinic, 46 patients with metabolic disorders were detected approximately (1/18). They were 30 PKU cases (1/28), 3 MSUD (1/280), 2 patients with citullinemia, 2 homocystinuria (1/422) and 10 cases with abnormal acylcarnitine profile (1/85).

Conclusion: Evaluation of Egypt MS/MS screening pilot program demonstrated that this technology was effective in identifying several metabolic disorders. Overall incidence is very high particularly among disabled children, who could have been saved if expanded NBS was offered. The study provided first Egyptian reference values by MS/MS for newborns.

13. NeoBase non-derivatized MS/MS assay for amino acids, free carnitine, acylcarnitines and succinylacetone: analytical and clinical performance

Huusko J.¹, Pitts R.², Cherkasskiy A.², Gurnani P.², Ostrinskaya A.², Trometer J.², Wang A.², Cerda B.²

¹PerkinElmer, Genetic Screening, Turku, Finland

²PerkinElmer, Genetic Screening, Waltham, United States

Background: Currently amino acids (AA) and acylcarnitines (AC) are widely measured by MS/MS based methods to detect inborn errors of metabolism (IEMs). However, succinylacetone (SUAC), as a primary marker for Tyrosinemia type 1, is an important addition to MS/MS newborn screening.

Objectives: 1) To develop a non-derivatized MS/MS assay for the simultaneous extraction and measurement of 11 amino acids, free carnitine, 30 acylcarnitines and succinylacetone. 2) To compare the analytical and clinical performance of the new NeoBase non-derivatized assay to the NeoGram derivatized AAAC assay.

Method: The analytical and clinical performances of the methods were studied across multiple newborn screening facilities. Identical dried blood spot (DBS) specimens (9416) that included 104 true positive samples and 320 artificially-enriched samples were analyzed by both methods.

Results: The two methods had equivalent analytical performance and the clinical correlation was excellent. Importantly, four true positive tyrosinemia type 1 samples which had normal Tyr levels with the NeoGram assay were all detected with the new NeoBase assay and SUAC screening.

Conclusion: The new non-derivatized NeoBase method is effective in detecting IEMs in neonatal DBS and the measurement of SUAC allows for improved Tyrosinemia type 1 screening.

14. Characterization of newborns with transient elevation of C3-Carnitine

Chang H.-Y.¹, Chen P.-W.¹, Chien Y.-H.², Wang S.-F.¹, Chen P.-H.³, Hsieh S.-H.³, Chen H.-Ch.¹, Hsu W.-L.¹, Hwu W.-L.²

¹National Taiwan University Hospital, Medical Genetics, Taipei, Taiwan

²National Taiwan University Hospital, Medical Genetics and Pediatrics, Taipei, Taiwan

³National Taiwan University, Computer Science and Information Engineering, Taipei, Taiwan

Propionylcarnitine (C3-carnitine) is the biomarker used in the tandem mass spectrometry (MS/MS) screening for methylmalonic acidemia (MMA) and propionic acidemia (PA). False-positive rate of C3-carnitine screening is relative high in view of the rarity of MMA and PA. Also cofactor-deficiency forms of these diseases may not be detected by the screening. This retrospective data mining aimed at characterizing newborns with transient elevation of C3-carnitine (or false-positive C3-carnitine screening), to see if they would alternatively be patients with cofactor-deficiency. 489,298 newborns were screened between January 1, 2002 and November 11, 2008. When C3-carnitine levels were higher than the cut-off value (4.74 μmol/L), a second dried blood spot (DBS) were requested to repeat C3-carnitine measurements. Those negative for the second DBS or normal at the confirmatory step were defined as transient elevation. DBS acylcarnitine profiles and methionine levels were analyzed retrospectively. Methionine level may decrease in Cbl C, D, F and B12 deficiency. 7,232 (1.48% of the total screened population) newborns were positive in the first DBS, while only 23 were also positive in the second DBS. Finally 6 cases were confirmed as MMA. Data mining revealed 8 of the 7,232 (0.11%) with elevated C3-carnitine/methionine ratio (>0.7), including one MMA, 4 cases having normal C3-carnitine at 2nd DBS and 3 cases having normal C3-carnitine at confirmation stage. The first DBS data from the 7 cases with transient elevation of C3-carnitine also revealed elevated free carnitine, C2-carnitine, C4DC-carnitine, and C16-Carnitine, but low methionine as compared to the total population (p <0.05). This study discovered a specific group of cases who might have problems other than classical MMA or PA. This data is important in developing new strategies to identify more inborn errors leading to C3-carnitine fluctuation.

15. LCHAD deficiency – the most frequent fatty acid oxidation disorder in newborn screening in the Czech Republic

Chrastina P., Bartl J., Hornik P., Hladikova J., Paulova M., Stastna S., Elleder M., Zeman J.

General Faculty Hospital and 1st Medical Faculty of Charles University, Institute of Inherited Metabolic Disorders, Prague, Czech Republic

Objectives: To prepare conditions for national-wide neonatal screening of inherited metabolic disorders (IMDs) in Czech Republic by tandem mass spectrometry (MS/MS) we analysed 83,820 samples from neonates born between 2000 and 2007 from our catchment area.

Methods: The metabolites were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS.

Results: We detected 21 patients with IMD (14x PKU/HPA, 3x LCHAD deficiency, 1x MCAD deficiency, 1x 3-methylcrotonyl-CoA carboxylase deficiency, 1x propionic acidemia and 1x methylmalonic acidemia). The frequency of screened IMD in our catchment area is 1:3,991. The frequency of phenylketonuria and LCHAD deficiency are 1:5,988 and 1:27,940, respectively.

Conclusions: Routine newborn screening for IMD by MS/MS is useful for diagnoses of IMD. Early detection of IMD enables counselling of families, close monitoring of child and the opportunity to provide appropriate medical treatment. LCHAD deficiency is the most frequent fatty acid oxidation disorder in newborn screening by tandem mass spectrometry in comparison with high incidence of MCAD deficiency in Western Europe.

Acknowledgements: Supported by the Grant MZOVFN2005 by Ministry of Health of the Czech Republic.

16. Frequency of homocystinuria due to cystathionine beta-synthase deficiency in the Czech Republic: implications for neonatal screening

Kozich V., Janosik M., Sokolova J., Janosikova B., Krijt J., Klatovska V.

Institute of Inherited Metabolic Disorders, Charles University, Prague, Czech Republic

Homocystinuria due to cystathionine beta-synthase deficiency is considered a rare disease with frequency between 1:65,000 and 1:900,000. Neonatal screening programs based on detecting elevated blood methionine levels are mostly ascertaining patients with the severe form of disease. However, molecular epidemiological studies in several North European countries suggested that frequency of homocystinuria – including also the milder pyridoxine responsive variants – may be much higher ranging between 1:6,400 and 1:20,500. The aim of our study was to determine the expected frequency of homocystinuria in a typical Central European population. Mutations c.833T>C (p.I278T), c.1105C>T (p.R369C) and c. 1224-2A>C (p.W409_G453del) were analyzed in 600 anonymous newborn blood spots by PCR-RFLP. Population frequencies of the c.833T>C, c.1105C>T and c. 1224-2A>C mutations were 0.0019 (95% CI 0.00063–0.0045), 0.005 (95% CI 0.0018–0.011) and 0, respectively. In a model which combines data on diagnosed patients and frequency of homozygotes calculated from Hardy Weinberg equilibrium the birth prevalence of homocystinuria in the Czech Republic is estimated to be 1:15,500 (95% CI 1:7,500–1:38,000). Our study demonstrates that the population frequency of heterozygotes for pathogenic mutations in the cystathionine beta-synthase gene in Czech newborns is similar to Northern Europe, and suggests that the majority of patients with homocystinuria- most likely with pyridoxine responsive forms that manifest only by thromboembolia in adulthood – may remain undiagnosed. Since determination of methionine in blood spots usually fails to detect patients with the milder forms of disease, ascertainment of these patients by neonatal screening will most likely require a change in methodology. Development of methods for routine determination of total homocysteine and/or of the methionine-to-cystathionine ratio is desirable in light of the presented epidemiological evidence.

17. Screening test for enzyme defects in the second part of purine de novo synthesis

Kratschmerova H., Vyskocilova P., Adam T.

Palacky University in Olomouc, Laboratory for Inherited Metabolic Disorders, Olomouc, Czech Republic

Objectives: Enzyme defects of purine de novo synthesis (PDNS) (adenylosuccinate lyase deficiency, AICA-ribosiduria) known to date are characterized by abnormal concentrations of substrates in cells and their excretion into body fluids in the dephosphorylated form (SAICAr, SAdo, AICAr). The aim of this study was to develop a screening photometric method for determination of all ribosides related to the second part of PDNS.

Methods: The dephosphorylated intermediates (ribosides) of the second part of PDNS and sulphonamides (potential interferences) were used in this study. Because all ribosides (except FAICAr) have aromatic primary amino group, Bratton-Marshall reaction was used for their detection (diazotation and copulation with N-1-naphtylethylenediamine dihydrochloride). After acidic hydrolysis (1 hour in 1,5 M-HCl; FAICAr converted to AICAr) samples were neutralized and then acetylated by acetanhydride for 30 minutes (prevention of sulphonamides interference). The azo-compounds were analysed spectrophotometrically at 530 nm on microplate using multimode microplate reader (Infinite 200, Tecan, Switzerland).

Results: The method allows determination of all the ribosides in the range of 40–500 μmol/l with limit of detection lower than 20 μmol/l. No interfering compounds were observed in three hundred urine samples analyzed by this method. In case of positive result a full spectrum in the range of 480–580 nm allow distinguishing individual ribosides.

Conclusions: All the ribosides related to the second part of PDNS can be analysed by the presented method. The method can be fully robotized, has analytical throughput of 4 samples/min and is applicable in neonatal screening.

18. Identification of a novel FAH large deletion mutation in a Korean neonate with hereditary tyrosinemia type 1

Lee Y.-W.¹, Park H.-D.², Lee D.-H.³, Choi T.-Y.⁴, Lee Y.-K.¹, Ki Ch.-S.²

¹Soonchunhyang University Bucheon Hospital and Soonchunhyang University College of Medicine, Laboratory Medicine and Genetics, Bucheon, South Korea

²Samsung Medical Center, Sungkyunkwan University School of Medicine, Laboratory Medicine and Genetics, Seoul, South Korea

³Soonchunhyang University College of Medicine, Pediatrics, Seoul, South Korea

⁴Soonchunhyang University Hospital and Soonchunhyang University College of Medicine, Laboratory Medicine, Seoul, South Korea

Hereditary tyrosinemia type I (HT1; MIM 276700), caused by decreased activity of fumarylacetoacetate hydrolase (FAH), is the most severe disorder linked to the tyrosine catabolic pathway. Patients with HT1 and their associated mutations in the FAH gene have been reported around the world, to date no genetically confirmed case of HT1 has been reported in Korea. We present a female Korean neonate in whom HT1 was associated with a novel large-deletion mutation in the FAH gene which has never before been reported. The patient’s newborn screening test revealed increased concentrations of methionine and tyrosine. Subsequently, urine organic acid analysis showed increased urinary excretion of 4-hydroxyphenyllactate, 4-hydroxyphenylpyruvate, succinate, and succinylacetone. Long-range PCR and sequence analysis of the FAH gene revealed a large deletion mutation, covering exon 13 to exon 15 (c.960+1130_*1260+10539del18036). The proband’s parents are both heterozygous for the mutation. We report the diagnosis of a Korean patient with HT1 by molecular analysis and the discovery of a novel large-deletion mutation in the FAH gene. The long-range PCR method developed in the present study may be useful for identification of HT1 patients with deletions of exons 13–15.

19. New biotinidase assay for plate readers

Lerch A.¹, Reynolds J.², Adams J.²

¹Astoria-Pacific, Marketing, Portland, OR, United States

²Astoria-Pacific, Chemistry, Portland, OR, United States

Astoria-Pacific International has developed a new microtiter plate kit which has 510 (k) status for the semi-quantitative determination of biotinidase activity in dried whole blood spots. The kit is used in conjuction with commercially available micropipettes, incubator/shakers and microplate readers. The SPOTCHECK^® Biotinidase Microplate Reagent Kit is intended for in vitro screening of decreased biotinidase activity, primarily for the diagnosis and treatment of biotinidase deficiency in newborns. Patient samples of whole blood collected on standardized filter paper are eluted in a standard 96-well plate. The plate is incubated with substrate in a buffer at 37 degrees Celsius for 240 minutes on a combination incubator/shaker. Following incubation, interfering proteins in the reaction mixture are removed through precipitation and vacuum filtration. Reaction products in the filtrate generated from biotinidase activity are subsequently mixed with reagents to form a pink dye. Biotinidase activity in the sample is proportional to the dye’s absorbance, which is measured at 550 nm using a microplate reader. Performance evaluation using normal and deficient neonatal dried blood spots as well as deficient controls, demonstrated the kit’s safety and effectiveness when compared against Astoria-Pacific’s approved-for-market segmented flow analyzer kit.

20. Improved newborn population screening for classical homocystinuria

Lindner M.¹, Gan-Schreier H.¹, Kebbewar M.², Fang-Hoffmann J.¹, Abdoh G.³, Ben-Omran T.⁴, Bener A.⁵, Al-Rifai H.⁶, Al-Khal A. L.⁷, Zschocke J.², Wilcken B.⁸, Hoffmann G. F.⁹

¹Univ.-Children’s Hospital, Div. Metabolic Diseases, Heidelberg, Germany

²Institute of Human Genetics, Heidelberg, Germany

³Hamad Medical Corporation, NSU, Doha, Qatar

⁴Hamad Medical Corporation, Weill Cornell Medical College, Dept Metabol Diseases, Doha, Qatar

⁵Hamad Medical Corporation, Weill Cornell Medical College, Doha, Qatar

⁶Hamad Medical Corporation, Neonatology, Doha, Qatar

⁷Hamad Medical Corporation, Doha, Qatar

⁸The Children’s Hospital at West-Mead, Westmead, Australia

⁹Univ.-Children’s Hospital, General Pediatrics, Heidelberg, Germany

Classical homocystinuria (cystathionine β-synthase deficiency, CBS) was shown to have a high incidence in the State of Qatar. This disease is poorly detectable by current newborn screening strategies using methionine as the primary indicator. Total homocysteine (t-Hcy) is expected to be a better biochemical parameter. To facilitate early recognition, novel biochemical and molecular testing strategies for newborn screening of CBS deficiency were developed. t-Hcy is determined in DBS with high performance liquid chromatography (HPLC) with tandem mass spectrometry detection which quantifies t-Hcy over a linear working range up to 50 µmol/L. DNA was extracted from dried blood spots and tested for two prevalent Qatari mutations. Both methods were performed in parallel and proved to be suitable for high throughput screening. Over two years we screened 29,466 Qatari newborns, 12,603 of native Qatari origin. Biochemical screening proved superior to genetic screening even in this highly consanguineous population. From June to December 2008 another 8,740 samples were screened exclusively with t-Hcy quantification. CBS deficiency was detected in 10 neonates. All showed highly elevated t-Hcy in DBS, whilst methionine was elevated above the 99,5th quantile (65 µmol/l) in only 4. With a methionine cut-off at the 99th quantile (40 µmol/l) all patients could have been detected but a false positive rate of 1% seems inacceptable. Molecular screening would have missed one patient homozygous for a mutation not previously identified in the Qatari population. Our study shows that a reliable NBS for CBS deficiency may be possible with a second tier strategy with methionine as the first screening and a second tier analysis of t-Hcy for >99th quantile of methionine. Whether this strategy proves to be specific and sensitive and probably able to detect even milder forms of CBS deficiency is currently being tested in a population of mainly Caucasian ancestry.

21. Biotinidase assay for neonatal biotinidase deficiency screening

Mattsson P.¹, Ukonaho T.¹, Mäkinen M.-L.¹, Karvinen J.¹, Karvonen H.¹, Singer D.², McHugh W.², Seppälä J.¹

¹PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland

²Bureau of Public Health Laboratories, State of Ohio Department of Health, Ohio, United States

Biotinidase (BTD) deficiency is an inherited autosomal recessive disorder (1:60,000) affecting the vitamin biotin metabolism. The symptoms of untreated disorder include neurological and cutaneous findings, seizures, ataxia, hair loss, development delay and a potential fatal outcome. Early treatment with biotin prevents the onset of the symptoms. The objective was to develop a precise, quick and easy-to-use assay to screen newborns with biotinidase deficiency by determining biotinidase enzyme activity in dried blood spot (DBS) specimens. The biotinidase assay is based on the enzyme’s ability to utilize biotin 6-aminoquinoline (BAQ) as a substrate. Lyophilized BAQ substrate is reconstituted and added to a well containing a punched dried blood spot (DBS) specimen. BTD enzyme, dissolved from the DBS to the reaction buffer, cleaves the BAQ to a fluorescent end-product 6-aminoquinoline (6-AQ). After 3 hours incubation at 50°C, the reaction is stopped by the addition of absolute ethanol. The 6-AQ is measured with VICTOR^® fluorometer or equivalent. The biotinidase activity is reported as enzyme units (U): 1 U = 1 nmol of the 6-AQ formed during one minute and in one deciliter of blood (1 nmol/min/dL). A biotinidase assay with a 5–6 h run time was developed. The measuring range of the assay was from 16 to 390 U and the limit of detection and quantitation was 16 U. Within plate and total variation near the medical decision level (80 U) was 7.1% and 12%, respectively. A median of 162 U was obtained in a study where 1498 routine neonatal DBS specimens were assayed. All 20 deficient specimens included in the study were detected as positive. In conclusion, a fast (5–6 h), easy-to-use (only three pipetting steps) assay for a 96-well microplate format with a low assay variation has been developed for the determination of biotinidase activity in neonatal DBS specimens.

22. Selective screening of urine’s organic acids

Novikova I., Fadeeva A., Grechanina J., Maksimova V., Kanyuka M., Delevskaya V.

Kharkiv Specialized Medical Genetic Centre, Biochemical Laboratory, Kharkiv, Ukraine

Determination of urine’s organic acids is the necessary technique for diagnostics of metabolic diseases. Organic acids (OA) are the key metabolites of practically all ways of intermediate metabolism. There were accumulating carboxylic acids in the body. Kharkov Specialized Medical Genetic Centre (KhSMGC) started selective screening of urine’s organic acids with GS-MS Agilent equipment (GS 6890, MS 5975C) last year. We use sample preparation procedure via sylylation and modified it. Our sample preparation method include: two steps extraction of urine’s OA with ethylacetate or dietylethers; removing of residual water from samples with magnesium sulfate; derivatization with BSTFA 1% TMCS. We use isopropilmalonic acid (IMA) as internal standard. For most of the compounds individual 50 mmol/l aqueous stock solutions were prepared from the acid or its salt. Ten calibration solutions, used successively during this study were prepared by combining aliquots of stock solutions of 25 OA. Individual calibration curves for all metabolites are established and integrated using the Agilent ChemStantion Program. We also use AMDIS Program for metabolites identification. Metabolite identities are confirmed by using NIST library. There were identified 82 compounds and analyzed 55 patient’s urines. Selective screening on OA disorders had never been provided in Ukraine until GS-MS technology was introduced in our establishment. The diagnostic of organic acidurias achieved at the close collaboration of doctor-geneticist and laboratory staff in KhSMGC.

23. Phenylketonuria in the era of neonatal screening in the Czech Republic, physician’s view

Pazdirkova R., Zatloukalova R.

3rd Faculty of Medicine, Charles University, Prague, Department of Pediatric, Prague, Czech Republic

Since national screening was introduced in 1975 in the Czech Republic infants with phenylketonuria (PKU) are diagnosed within the first two weeks of life. Approximately 1 in 7,984 people are born with hyperphenylalaninaemia. It represents approximately 12 neonates with PKU each year. This autosomal recessive genetic disorder is characterised by a deficiency in hepatic enzyme phenylalanine hydroxylase (PAH). History changed during the 1960´s and 1970´s, when treatment using a low-protein diet started and the era of neonatal screening began. Untreated patients had microcephaly, severe mental retardation, epilepsy, sometimes progressive supranuclear motor failure and cutaneous problems. More recently born patients who benefited from the early treatment has had almost normal cognitive and psychomotor development. The early treated patients enjoy great opportunity of living “normal” life. However, there still remains a lot of problems to be solved by the patients themselves as well as by their doctors and nutritionists. Our aim is to describe complications and difficulties caused by treating PKU with diet. Children find it difficult to adhere to the strict low-protein diet and to drink untasteful synthetic amino acids mixtures. Other problems are high cost of the specific nutrition, regular fasting system and need of frequent blood and clinical checks. In case compliance is not good children are not only threatened in their psychomotor development but also some nutritional deficiencies can occur. The increased level of phenylalanine in maternal blood causes spontaneous abortion or has toxic effect on the development of foetus. Syndrome of maternal PKU can be prevented by using a strict diet 3 months before the conception and during the whole pregnancy. Historically treatment was terminated at the end of childhood when brain development was considered complete. Now we recommend life-long diet, because adult people off diet frequently show subtle neurological signs.