Andraz Smon (Presenter)
University Medical Centre Ljubljana
Bio: I am a young researcher working on my PhD on the Faculty of Pharmacy, module Clinical biochemistry and laboratory biomedicine. I have graduated in biochemistry; for my diploma I have worked with GC-MS and GC-FID. Using them I have analysed the volatile compounds in feeding stuff. After the graduation I started working in Unit special laboratory diagnostics (University Children’s Hospital in Ljubljana) and became a PhD student at the Faculty of Pharmacy. My PhD is on the implementation of expanded newborn screening by MS/MS in Slovenia, I am also working on other analyses with GC-MS and GC-FID.
Authorship: Andraz Smon (1), Barbka Repic Lampret (1), Urh Groselj (1) Mojca Zerjav Tansek (1), Tadej Battelino (1,2)
(1) University Medical Centre Ljubljana, (2) University of Ljubljana
| Short Abstract Newborn screening programme in Slovenia currently includes only two diseases, phenylketonuria and congenital hypothyroidism. Last year a pilot study of expanded newborn screening for inborn errors of metabolism using tandem mass spectrometry (MS/MS) started. 10000 dried blood spots from newborns were analysed retrospectively. Newborns with highest elevations of measured analytes were immediately investigated and three newborns with inborn errors of metabolism were found (not counting hyperphenylalaninemia/phenylketonuria); one case of VLCAD, a case of 3-MCC deficiency and a case of GA1. Based on these results the cumulative incidence of inborn errors of metabolism (detected by MS/MS) is high in Slovenia. Follow-up tests on selected newborns to set the cut-off values for chosen disorders are ongoing. |
Long Abstract
Preliminary results from the Slovenian expanded newborn screening pilot study
INTRODUCTION
Newborn screening programme in Slovenia includes only two diseases, phenylketonuria and congenital hypothyroidism. Last year a pilot study of expanded newborn screening for inborn errors of metabolism using tandem mass spectrometry started. 10000 dried blood spots from newborns were analysed retrospectively, as the same filter paper cards from the existing screening programme were used. We tested for the following disorders: medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, glutaric acidemia type 1 (GA 1) and type 2 (GA 2), 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency, maple syrup urine disease (MSUD), very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, isovaleric academia (IVA), propionic academia (PA) /methylmalonic academia (MMA), carnitine uptake disorder (CUD), carnitine palmitoyl transferase 1 (CPT 1) and transferase 2 (CPT 2) deficiency. We also included phenylketonuria, so we could compare our results with the results of the current method for phenylketonuria screening (fluorimetric detection of phenylalanine).
METHODS
Acylcarnitine analysis: Acylcarnitine analysis was done from dried blood spots with Chromsystems MassChrom Amino Acids and Acylcarnitines from Dried Blood kit. Quantification was performed by PerkinElmer 200 HPLC, coupled to AB Sciex 3200 QTRAP.
Organic acid analysis: Urine organic acids were measured with an in-house method [1]. Analysis is done with Agilent 5975C Series GC/MSD (gas chromatography/mass selective detector) system on Agilent Ultra2 25 m x 200 µm x 0.33 µm column.
Samples for gene sequencing and enzyme activity analyses were sent to a laboratory in Netherlands (Academisch Medisch Centrum).
RESULTS
During the study we immediately followed-up every newborn that had a markedly higher concentration of an analyte compared with all other analysed newborns. With this approach we have identified three newborns with inborn errors of metabolism; phenylketonuria was not included in this approach as it is already included in the newborn screening programme.
There was one case of VLCAD deficiency, a case of 3-MCC deficiency and a case of GA 1. For the follow-up testing of VLCAD deficiency we did a repeated acylcarnitine analysis from a new blood spot (normal results), ACADVL gene was sequenced, enzyme activity was measured and palmitate loading test was done. The sequencing identified a heterozygous known pathogenic mutation and two unknown variants with no disease-causing effect found by prediction programs. Enzymatic activity in cultured fibroblasts was reduced, palmitate loading test results were abnormal (elevated C14 and C12 carnitines). Taken together, these results show that the patient suffers from VLCAD deficiency.
Follow-up of the 3-MCC case was done with a repeated acylcarnitine analysis from a new blood spot, which showed significant elevation of C5-OH carnitine and decreased free carnitine. Urine organic acid analysis showed elevations of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine, thus confirming the 3-MCC.
A child with suspected glutaric acidemia type 1 was most recently detected, follow-up tests of acylcarnitine analysis showed elevated C5DC and decreased free carnitine, in urine glutaric acid and 3-hydroxyglutaric acid were strongly elevated, thereby confirming the diagnosis of GA 1. Enzyme activity in lymphocytes was absent.
We also compared our results for phenylketonuria screening with the existing newborn screening in Slovenia. In our cohort we checked 3 children which had the highest phenylalanine concentrations and elevated phenylalanine/tyrosine ratios and checked the results of existing newborn screening to see if they are known patients. All 3 children were known, 2 had phenylketonuria and 1 hyperphenylalaninemia.
CONCLUSION
Based on the preliminary results from the pilot study the cumulative incidence of inborn errors of metabolism is high in Slovenia. We are currently doing follow-up tests on selected newborns with the highest disease possibility to set the cut-off values for the disorders that are planned to be included in Slovenian newborn screening programme. Phenylketonuria screening results with tandem mass spectrometry also seem to be in accordance to the currently used method and will be included in the panel of screened diseases with tandem mass spectrometry.
References & Acknowledgements:
1. Šmon A, Murko S, Repič Lampret B, Battelino T (2014) Pilot research on expanding Slovenian newborn screening programme for inherited metabolic disorders detectable by tandem mass spectrometry. Chem List 108:s183–s186
The study was supported in part by the Slovenian National Research Agency grants J3-2412, J3-9663 and P3-0343.
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