1. Genotype-phenotype correlation In PKU Szeged, December 12, 2011.

2. Introduction HPA – BH4-responsive – classic atypical (BH4-dependent) Severity depends partially on the mutation of PAH enzyme Genotype correlates with the metabolic phenotype in most of the cases – homozygous, severe null mutations … or „mild” mutations Most of the patients are compound heterozygous (or – in Hungary - R408W homozygous) There are significant genotype-phenotype inconsistencies between identical PAH mutation bearing patients So the PKU/HPA phenotype manifestation is more complex than it could be predictable based on the Mendelian inheritance of the mutated alleles

3. N-terminal regulatory domain: AA 1–142 Catalytic domain: AA 143–410 C-terminal domain: AA 411–452 → tetramerization PAH enzyme

4. www.pahdb.mcgill.ca 12q22-q24.1, 13 exon 564 mutation PAH gene mutations

5. Metabolic phenotypeBH4-responsivenessMutation type mildMostly responsive (80%)mild/mild moderateMay be responsive, depending of the „mild” mutation type mild/null severe (classic)Non-responsivenull/null

6. BH4-responsive PAH mutations ~70 known mutation Mostly point-mutation (never splice, insertion or deletion) Mostly in the catalytic domain, but not in the cofactor-binding site

7. BH4-responsive PAH mutations

8. mutációallél R408W45null A403V5may be responsive IVS12+1G>A5splice, null R261Q5 may be responsive L48S4 responsive IVS10-11G>A3splice, null IVS3-22C>T3silent R158Q3 IVS7-4delT3unknown, splice? c.15delCT2 P281L2null IVS11+1G>A2splice, null c.39delC2 IVS4+5G>T2splice mutációallél E390G1responive T380M1responive Y414C1 may be responsive R252W1null IVS4+47C>T1 P225T1 IVS10+94G>A1silent IVS7+1G>A1splice I174V1 V177L1 IVS4-5C>G1splice S350Y1 Q172X1null G148D1unknown M276T1 I95F1 R243X1null V245A1HPA F302V1unknown

9. Our cases: null mutations Since 2006 we could not have the patients' DNA sequenced 52 patients' DNA sequence and mutations are known Classic PKU: null mutations Allél 1Allél 2esetszámmutáció típus R408W 14missense IVS12+1G> A IVS11+1G>A2splice R408WIVS10-11G>A1missense/splice R408WIVS12+1G>A1missense/splice

10. MutationBH4-response (allele %) In vitro residual enzyme activity (%) L48S8339 Regulatory domain A403V10032 Catalytic domain E390G10072 Catalytic domain, enzyme surface Y414C9336 Tetramerization R261Q8638 Active site secondary structure, tetramerization R158Q5610 Catalytic domain Trefz et al. J Inherit Metab Dis (2009) 32:22–26 Non-null mutations

11. BH 4 loading with 20 mg/kg bw in 3 patients, all genotype Y414C/R408W. Lindner M et al, Mol Genet Metab 2001; 73:104-106. Y414C (mild)/R408W (null) → moderate PKU? Same genotype – different BH4-responsiveness Y414CBH4-resp: 93%Residual enzyme activity: 36%

12. HPA cases Allele 1Allele 2 Phe level (  mol/l) BH4 loading test? T380Mwt130not performed E390GA403V180 not performed no diet S350YA403V240 not performed on diet

13. Interesting cases Allele 1Allele 2Phe levelRemark KAR408WR261Q600-1200on diet Matalon 2005BH4-responsive Desviat 2004non-responzive Erlandsen 2004partially responsive DGR252W?300-1200

14. Tesztelt BH4-reszponzív esetek Allél 1Allél 2Phe szint Megjegyzés SZB & SZFIVS7-4delTA403V360 BH4-reszponzív NÁR408WA403V240 BH4-reszponzív UIAV245AR158Q240 BH4-reszponzív

15. Pathogenic mutation phenotype 1 phenotype 2 Genetically determined factors: other intragenic differences, polymorphisms modifying genes: aminoacid transport, competition, metabolism, excretion Non-genetically determined factors phenotype 3 Genotype-phenotype in PKU

16. Confirming the genotype may help in the judgement and predicition of the severity of the disease There are significant genotype-phenotype inconsistencies between similar PAH mutation bearing patients: mainly the regulatory domain mutations lead to unpredictable phenotype, in the cases of compound heterozygosy BH4-loading test result may vary in a patient - intracellular environment, metabolic status influence the mutated enzyme activity The standard(ized) Phe+BH4 loading test is the gold standard for planning the treatment! Conclusion