Newborn screening for additional metabolic diseases using second-tier strategies Juergen G. Okun, PhD

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1 Newborn screening for additional metabolic diseases using second-tier strategies Juergen G. Okun, PhD 5th EFLM-UEMS European Joint Congress in Laboratory Medicine October 2018 in Antalya, Turkey

2 Content Newborn screening Outlook Newborn screening in general Usage of second-tier strategies What could be screened next? What is already recommended for the German newborn screening panel?

3 Metabolic Center Heidelberg

4 Newborn screening in Heidelberg Screening of 140,000 newborns / year from the South-West of Germany (Baden-Württemberg, Rheinland-Pfalz and Saarland) Accreditation according to DIN EN ISO for medical laboratories NBS is regulated by the German GBA (Gemeinsamer Bundesausschuss) by a compulsory directive

5 Newborn screening It s not a lab analysis only, it s a programme! Wilson & Jungner criteria Non-invasive gain of material Sensitive and specific lab tests Benefit for the child s health if treated before becoming symptomatic treatable diseases

6 Newborn screening One blood spot only...

7 different treatable disorders. NBS in Germany contains a panel of 16 different disorders recommended by the GBA (Gemeinsamer Bundesausschuss): Metabolic disorders (13): Amino acidopathies (3), organic acidurias (2), fatty oxidation and carnitine cycle defects (6) and enzyme disorders (2) Endocrinopathies (2) Cystic fibrosis (1)

8 Newborn screening in Germany - Prevalences 1. Congenital hypothyroidism (CH) 1 : 3, Congenital adrenal hyperplasia (CAH) 1 : 10, Biotinidase deficiency (BIO) 1 : 60, Galactosemia (classical) (GALT) 1 : 45, Phenylketonuria/hyperphenylalaninemia (incl. cofactor deficiencies) (PKU) 1 : 10, Maple syrup urine disease (MSUD) 1 : 150, Glutaric aciduria type I (GA1) 1 : 120, Isovaleric aciduria (IVA) 1 : 50, Medium-chain acyl-coa dehydrogenase deficiency (MCHAD) 1 : 10, Long-chain-3-OH-acyl-CoA dehydrogenase deficiency (LCHAD) 1 : 80, Very long-chain acyl-coa dehydrogenase deficiency (VLCHAD) 1 : 80, Carnitine palmitoyltransferase I deficiency (CPT-I) 1 : 500, Carnitine palmitoyltransferase II deficiency (CPTII) 1 : 200, Carnitine acylcarnitine translocase deficiency (CACT) 1 : 500, Tyrosinemia type I (since March 2018) (Tyr-I) 1 : 135, Cystic fibrosis (since October 2016) (CF) 1 : 3,300

9 NBS for endocrinopathies, cystic fibrosis and enzyme deficiencies Time-resolved fluoroimmunoassays Congenital hypothyroidism (CH) by TSH Congenital adrenal hyperplasia (CAH) by 17-OH-P Cystic fibrosis (CF): by IRT, PAP (3rd-tier: DNA) Enzyme activity measurements by photo- or fluorimetry Biotinidase deficiency (BIO) Galactosemia (classical) (GALT)

10 NBS for amino acidopathies, organic acidurias, fatty oxidation and carnitine cycle defects by MS/MS Phenylketonuria/hyperphenylalaninemia (PKU) Maple syrup urine disease (MSUD) Glutaric aciduria type I (GA1) Isovaleric aciduria (IVA) Medium-chain acyl-coa dehydrogenase deficiency (MCHAD) Long-chain-3-OH-acyl-CoA dehydrogenase deficiency (LCHAD) Very long-chain acyl-coa dehydrogenase deficiency (VLCHAD) Carnitine palmitoyltransferase I deficiency (CPT-I) Carnitine palmitoyltransferase II deficiency (CPTII) Carnitine acylcarnitine translocase deficiency (CACT) Tyrosinemia type I (since March 2018) (Tyr-I)

11 Tandem-mass spectrometry (MS/MS) MS/MS profiling in DBS provides information about hidden derivatives Many other disorders could be detected by these hidden information Tapping the full potential of the first MS/MS run in combination with second tier tests / strategies on more specific parameters in the DBS

12 Second-tier Strategy - Definition

13 Project NBS 2020 using second-tier LC-MS/MS Project NBS 2020: Feasibility study for inclusion of 25 additional diseases in regular NBS (91,000 newborns/y) Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders Peter Monostori 1 *, Glynis Klinke 1, Sylvia Richter 1, Akos Barath 2, Ralph Fingerhut 3, Matthias R. Baumgartner 3, Stefan Koelker 1, Georg F. Hoffmann 1, Gwendolyn Gramer 1, Juergen G. Okun 1 1 Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, Center for Pediatric and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, Germany, 2 Department of Pediatrics, University of Szeged, Szeged, Hungary, 3 Children's Research Center, Division of Metabolism, University Children's Hospital Zurich, Zurich, Switzerland These authors contributed equally to this work. PLOS ONE September 15, 2017

14 Propionate metabolism PA = Propionic acidemia MMA= Methylmalonic acidemia Cbl = Cobalamin (Vit. B12) deficency PA MMA CblC CblB CblA CblF CblD CblE CblG Adaptation from Coelho et al. (2008)

15 Propionic, methylmalonic acidemias & combined remethylation disorders Clinical symptoms and complications Diagnosis Severe metabolic decompensation with metabolic acidosis and hyperammonemia, potentially life threatening, Catastrophic illness Freqencies: 1: (MMA), 1:100,000 to 150,000 (for PA &Cbls) MS/MS (DBS): acylcarnitine profile: propionyl carnitine OA (urine): 3-OH-propionic, methymalonic, methylcitric acids by GCMS

16 Second-tier Strategy - 1 st tier: C3 & 2 nd -tier MMA, 3-OH- PA and MCA by LC-MS/MS Increased propionylcarnitine (C3) levels and corresponding ratios (C3/C2, C3/C16, C3/Met) in newborn screening are indicative for this group of fatal disorders. This alteration is relatively non-specific, but it is a spin-off finding of the regular performed acylcarnitine profiling (1 st -tier run) in dried blood (DBS). This results in the necessity of a 2 nd -tier run using LC-MS/MS in the same DBS sample: MMA, 3-OH-PA and MCA

17 Development of methods

18 Material and Methods Sample preparation technique Underivatized Derivatised with 3.0N hydrochloric acid in n-butanol Column Waters Acquity BEH Amide 150x2.1 mm, 1.7 μm Waters Acquity BEH C18 100x2.1 mm, 1.7 μm Waters Atlantis HILIC Silica 50x2.1 mm, 3 μm Phenomenex Luna Omega Polar C18 100x2.1 mm, 1.6 μm Phenomenex Gemini C6-Phenyl 150x2.0 mm, 3 μm Red underlined configurations were selected for subsequent method validation Extraction solvent ACN/water 20/80 (v/v) ACN/water 40/60 (v/v) ACN/water 60/40 (v/v) ACN/MeOH/water 20/20/60 (v/v) ACN/MeOH/water 20/40/40 (v/v) ACN/water 20/80 (v/v) +0.4% formic Eluents acid ACN/water 20/80 (v/v) +0.1% A: Water acetic +0.1% acid formic acid A: Water +0.4% formic acid A: Water +0.1% acetic acid B: ACN/MeOH 50/50 (v/v) LC method type B: ACN/MeOH 50/50 (v/v) +0.4% formic acid Isocratic: 85% Eluent B: ACN/water A (water +0.4% 80/20 formic (v/v) +0.1% acid) formic and acid 15% Eluent B (ACN/MeOH B: ACN 50/ % (v/v)); formic flow acid rate 200 μl/min. Gradient: Eluents A and B and flow rate are the same as above, but %B is changed as follows: min: 10%; min: 10% to 30%; min: 30%; min: 30% to 10%; min: 10%.

19 Sample preparation & LC-MS/MS run 2x 3.2 mm DBS In filter plate units + 120uL IS (MeOH/ Water) + 10uL 50mM ZnSO 4 (in MeOH) 5 min vortex 10 min at 5000g RT Autosampler 5µL PHenomenex Gemini C6-Phenyl (150 x 2,0 mm, 3 µm) + Security Guard Column A: 0,4% FA B: MeOH/ACN (50:50) 200µL/min; Gradient LC-MS/MS (LC-ESI-MS/MS)

20 LC-MS/MS chromatograms N MMACbl PA N MMACbl PA N MMACbl PA! Succinic acid is isobar to Methylmalonic acid Lactic acid is isobar to 3- hydroxypropionic acid 3-OHPA MMA MCA

21 Method verification - Retrospective analysis Initial 1 st tier Biomarker C3 C3/C2 3OHPA MMA MCA NBS Controls (n=250) NBS CblB (n=1) NBS CblC (n=9) n (2/9) - NBS PA (n=4) n (2/4)- ERNDIMNo.2 MMACbl / / ERNDIMNo.4 MMACbl / / ERNDIMNo.5 MMACbl / / n/ ERNDIMNo.7 MMACbl / / ERNDIM No.1 PA (n=1) / / ( ) ERNDIM No.3 PA (n=1) / / ERNDIM No.6 PA (n=1) / / ERDNIM: Summary Phenotypical difference reflected in biochemical profile Effect of long term sample storage => Preanalytical issue for retrospective samples? 2 nd tier (HD) ERDNIM: = concentration above Cut off = concentration under Cut off n= in normal range () = moderate / = not measured Monostori et al. (2017)

22 Method verification Treated patients Initial 1 st tier Biomarker C3 C3/C2 3OHPA MMA MCA Treated CblB (n=17) n (1/17) - n (1/17) - n (8/17)- n (3/17) - Treated CblC (n=10) n (2/10)- n (2/10)- n (2/10)- Treated CblA (n=2) n (1/2)- Treated MMAmut (-) (n=11) n (1/11)- Treated MMAmut (0) (n=4) n (1/4)- Treated PA (n=21) n (7/21)- MMACbl Biomarker: MMA > MCA PA Biomarker: MCA > 3OHPA 2 nd tier (HD) Monostori et al. (2017) Summary Therapy effect: Renal elimination due to carnitine supplementation Effect of long term sample storage => Preanalytical issue for retrospective samples?

23 Method verification Symptomatically diagnosed patients 1 st tier 2 nd tier C3 C3/C2 3OHPA MMA MCA S1 Cbl C, D, F or J S6 Cbl C ( ) S5 MMA S7 MMA S2 PA Gramer et al. (2018)

24 Conclusion Development & verification of the 2 nd tier method for PA, MMA, and Cbl disorders Effect of long term sample storage but this is not a problem for fresh newborn screening samples MMACbl Biomarker: MMA > MCA PA Biomarker: MCA > 3OHPA

25 Outlook I What could be done next? Diseases which should be tested in a study design: Urea cycle defects by the hidden information in the MS/MS based amino acid profiling (already included in NBS 2020) Lysosomal storage diseases by an LC-MS/MS based attempt (Multiplex 7 or 6, Liu et al. 2017, Elliot et al 2016)

26 List of lysosomal storage disorders including Neuronal ceroid lipofuscinoses Multiplex Enzyme Disease Reference Iduronidase MPS I Iduronatsulfatase MPS II alpha-n-acetylglucosaminidase MPS IIIB 7 Galaktose-6-Sulfat-Sulfatase MPS IVA Liu et al Arylsulfatase B MPS VI Glucuronidase MPS VII TPP1 (Tripeptidyl Peptidase) NCL2 Iduronidase MPS I alpha-galactosidase A Fabry 6 beta-glucosidase acidic Sphingomyelinase Gaucher NPAB Elliot et al 2016 Galactocerebrosidase Krabbe alpha-glucosidase Pompe

27 Outlook II What is already recommended? Recommended by the GBA for Germany: Screening for severe combined immuno deficiencies (SCID) by TREC as the first genetical approach (expected start in 2019) Screening for sickle cell disease (SCD) using capillary electrophosis or a MS/MS based attempt (still under consideration, possible start 2020?)

28 Newborn screening It s not a lab analysis only, it s a programme... that has to be developed continiously by looking for better screening techniques or strategies. that has to be extended continiously by including new treatable disorders. that has to be evaluated continiously with regard to the benefit of an early treatment of the screened children.

29 Acknoledgement This work and the pilot study "Newborn screening 2020º at the Newborn Screening Center of the University Hospital Heidelberg are generously supported by the Dietmar Hopp Foundation, St. Leon Rot ( We acknowledge the financial support of the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding programme Open Access Publishing. Thank you for your attention!