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Tetrahydrobiopterin responsiveness in phenylketonuria: prediction with the 48-hour loading test and genotype

Karen Anjema1*, Margreet van Rijn1, Floris C Hofstede2, Annet M Bosch3, Carla EM Hollak3, Estela Rubio-Gozalbo4, Maaike C de Vries5, Mirian CH Janssen5, Carolien CA Boelen6, Johannes GM Burgerhof1, Nenad Blau78, M Rebecca Heiner-Fokkema1 and Francjan J van Spronsen1

Author Affiliations

1 Division of Metabolic Diseases, University Medical Center Groningen, Beatrix Children’s Hospital CA33, PO box 30.001, Groningen 9700 RB, The Netherlands

2 University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands

3 Academic Medical Center, University Hospital of Amsterdam, Amsterdam, The Netherlands

4 Maastricht University Medical Center, Maastricht, The Netherlands

5 Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

6 Leiden University Medical Center, Leiden, The Netherlands

7 University Children’s Hospital, Heidelberg, Germany

8 University Children’s Hospital, Zürich, Switzerland

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Orphanet Journal of Rare Diseases 2013, 8:103  doi:10.1186/1750-1172-8-103

Published: 10 July 2013



How to efficiently diagnose tetrahydrobiopterin (BH4) responsiveness in patients with phenylketonuria remains unclear. This study investigated the positive predictive value (PPV) of the 48-hour BH4 loading test and the additional value of genotype.


Data of the 48-hour BH4 loading test (20 mg BH4/kg/day) were collected at six Dutch university hospitals. Patients with ≥30% phenylalanine reduction at ≥1 time points during the 48 hours (potential responders) were invited for the BH4 extension phase, designed to establish true-positive BH4 responsiveness. This is defined as long-term ≥30% reduction in mean phenylalanine concentration and/or ≥4 g/day and/or ≥50% increase of natural protein intake. Genotype was collected if available.


177/183 patients successfully completed the 48-hour BH4 loading test. 80/177 were potential responders and 67/80 completed the BH4 extension phase. In 58/67 true-positive BH4 responsiveness was confirmed (PPV 87%). The genotype was available for 120/177 patients. 41/44 patients with ≥1 mutation associated with long-term BH4 responsiveness showed potential BH4 responsiveness in the 48-hour test and 34/41 completed the BH4 extension phase. In 33/34 true-positive BH4 responsiveness was confirmed. 4/40 patients with two known putative null mutations were potential responders; 2/4 performed the BH4 extension phase but showed no true-positive BH4 responsiveness.


The 48-hour BH4 loading test in combination with a classified genotype is a good parameter in predicting true-positive BH4 responsiveness. We propose assessing genotype first, particularly in the neonatal period. Patients with two known putative null mutations can be excluded from BH4 testing.

Phenylketonuria; PKU; Tetrahydrobiopterin; Sapropterin dihydrochloride; Pharmacological chaperone; Genotype; Loading test