1. Phe Tyr PAH BH 4 BH 2 GTP BH 4 -dependent HPA (atypical/malignant PKU) Usually mild PKU/HPA Early CNS symptoms Fe O 2 PAH deficiency Classic PKU Mild PKU Mild HPA (non PKU HPA) Phe level (μmol/l)>600 (1200) 360-600 (600-1200) 120-360 (120- 600) Phe tolerance (mg/day)<400400-800>800 BH 4 responseBH 4 -responsive PKU/HPA Phenylketons FeCl 3 DOPA→DA→NA→A →→→ melanin Norm <120 μmol/l Hyperphenylalaninaemias (HPA) Patomechanism, types

2. ExpectedSzeged Overall1:4500-5000 (20 case/year) 16 PAH deficiency 99% Classic PKU1:9000 (10 case/year) 10 HPA1:9000 (10 case/year) 5 BH 4 deficiency (atypical PKU) 1% 1:250000 (1 case/year) 1 Incidence of hyperphenilalaninamias

3. MS/MS (aminoacid & acylcarnitine) Sampling on 3 rd day of life Primary parameter: blood Phe >102 μmol/l (norm < 120) Secondary parameter: Phe/Tyr > 1.5 Clinical circumstances! Transient hyperphenylalaninaemia  prematurity (Orn, C3 ↑)  parenteral nutrition (Val, Leu, Thr ↑   hepatic disease (sepsis, galactosaemia,…)  drugs 0. day, screening

4. Positive result „normal” newborn Blood Phe 102-360 μmol/l Blood Phe >360 μmol/l Clinical examination week 1-3 Repeat screening Phe ↑ Phe norm Further work-up for suspected HPA newborns

5. Call in the patient – no diet! Detalied clinical history, family anamnesis Physical examination Usually few or no symptoms Mild Phe elevation + feeding difficulty, hypotonia, myoclonus, seizure, salivation → atypical PKU? Laboratory studies Blood Phe, Phe/Tyr (MS/MS) urine FeCl 3, GC-MS BH4 test DNA extraction for mutation analysis EEG Clinical work-up for the suspected PKU newborns Aim: definitve diagnosis, HPA typing, starting treatment

6. BH4 test NEGATIVE BH4 non-responsive Classic PKU / Mild HPA BH4-responsive PKU BH4-dependent (atypical) PKU Hyperhenylalaninemia differential diagnoses POSITIVE BH4 responsive

7. 048121624 BH4 20 mg/kg normal dietdiet blood Phe, Phe/Tyr (MS/MS) BH4 loading Phe > 360 μM POSITIVE (Phe ↓ >30%) → BH4-responsive/atypical PKU? measure pterins, DHPR

8. normal dietdiet POSITIVE (Phe ↓ >30%) → BH4-responsive/atypical PKU? measure pterins, DHPR Combined Phe + BH4 loading Phe < 360 μM 048121624 Phe 100 mg/kg -3 24 -3 1612840 Phe 100 mg/kg BH 4 20 mg/kg blood Phe, Phe/Tyr (MS/MS)

9. Day 1:-3 0 4 8 12 24 hours 1400 1200 1000 800 600 400 200 0 Blood Phe (umol/l) Phe (100 mg/kg) 1193 122 4039 45 Interpreting the BH4 test result - Case 2 888 387 888 812 705 Phe (100 mg/kg) BH4 (20 mg/kg) Day 2: 24 27 31 35 39 51 BH4-dependent

10. Day 1:-3 0 4 8 12 24 hours Blood Phe (umol/l) Phe (100 mg/kg) 412 137 65 42 35 Interpreting the BH4 test result - Case 2 96 140 BH4 (20 mg/kg) Day 2: 24 27 31 35 39 51 BH4-responsive 1400 1200 1000 800 600 400 200 0 96 670 254 533 440

11. Send DNA sample for mutation analysis to Semmelweis University, 2 nd Dept. Pediatrics Screen for 6 most frequent PAH mutation (R408W, R158Q, R261Q, R252W, IVS 10nt546, IVS12 splice-site) Whole gene sequencing Molecular genetical diagnoses

12. Classic PKU treament, advices Treat immediately after diagnosis Gold standard: Phe-free medical food (enriched with vitamins, trace elements, additional energy) Breastfeeding is encouraged, ⅓ of daily protein intake Diet overshoot: Phe-deficiency: lethargy, feeding problem, diarrhea, anaemia, anorexia In PKU: target Phe-level: 120-360 μmol/l In HPA (120-360 μmol/l): no treament is necessary (except pregnancy)

13. BH4-deficiency treament, advices BH4 5-10 mg/kg/day Neurotransmitter precursors: L-DOPA (Madopar 1-3, 4-7, 8-12 mg/kg/day) 5-hidroxi triptophan (Tript-OH 6-9 mg/kg/day) MAO-B blocker selegiline (0.25 mg/kg/day) Low-Phe diet if necessary

14. Follow-up Blood sample via mail regularly Control check-ups: 0-3 years:every 3 months 3-6 years: every 6 months > 6 years:yearly Physical examination, laboratory studies

15. Patient education