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Differential diagnosis of PKU and BH4 deficiency

Differential diagnosis of PKU and BH4 deficiency

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4).1 The diagnosis of BH4 deficiencies is straight forward and should be investigated in all children with HPA.

Following a positive newborn screen for HPA, all infants need to be tested for pterins (neopterin, biopterin and primapterin) from dried blood spots (DBS) or urine and dihydropteridine reductase (DHPR) activity in DBS. These tests should be performed immediately after the first positive report of HPA. Based on the pattern of pterins and DHPR activity (pterins pattern is frequently normal in DHPR-deficient patients), all forms of HPA caused by BH4 deficiency can be identified and differentiated2 (Table 1).

Table 1: Laboratory diagnosis of hyperphenylalaninemia variants

 

PKU

GTPCH deficiency

PTPS deficiency

PCD deficiency

DHPR deficiency

DNAJC12 deficiency

Phe

(DBS, P)

↑*

Phe/Tyr (DBS, P)

Neopterin (DBS, U)

n-↑

n-↑

n

n

Biopterin (DBS, U)

n-↑

↓-n

n-↑

n

Primapterin (DBS, U)

n

n

n

n

n

DHPR (DBS)

n

n

n

n

n

BH4 loading test

+/-

+

+

+

+

+

*may be normal or subnormal in the newborn screening

PKU, phenylketonuria; GTPCH, GTP cyclohydrolase I; PTPS, 6-pyruvoyl-tetrahydropterin synthase; PCD, pterin-4a-carbinolamine dehydratase; DHPR, dihydropteridine reductase; BH4, tetrahydrobiopterin; DBS, dried blood spot; P, plasma; U, urine; n, normal

An oral BH4 challenge (20 mg/kg body weight) is an additional useful test. It is positive in all BH4-deficient patients and in certain mild PKU patients but cannot always differentiate between PKU responders and BH4-deficient patients.3 Mild PAH mutations with a substantial residual enzyme activity are most likely to demonstrate increased activity in the presence of BH4. BH4 appears to be a molecular chaperone to PAH, in that it protects the protein from misfolding during synthesis or promotes reconstitution of the correct 3-dimensional structure in the cytosol.4

Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70 kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH4 by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in cerebrospinal fluid and responded to supplementation with BH4, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism.5 Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH4 are genetically excluded.6

            Since the genotype determines the activity of PAH and thus the metabolic phenotype, there is growing evidence of genotype–phenotype correlation. More than 1,000 PAH variants have been reported to the locus-specific PAH database – available at http://www.biopku.org : 65% are in-frame amino acid substitutions, including changes to nonsense codons, and other common variants, such as deletions (15%), splice variants (12%), indels (4%) and synonymous variants (4%), indicating PKU is a highly heterogeneous disease.7 More recent studies have confirmed a significant relationship between allelic phenotype, enzyme activity and BH4 responsiveness.8 The statistical and analytic power of large databases of mutations has been used to explore the relationship between genotype and phenotype in PKU. The nature of the mutation, or the known effect of the mutation on PAH structure can be used to predict the enzyme activity in a number of cases, but not all. Allelic phenotype value (APV), a number defining the frequency of a phenotype (mild HPA, mild PKU or classic PK) within a cohort of patients with the same genotype, can be used to predict the severity of PKU.9 This information is available for free on the BIOPKU website (http://www.biopku.org). Genotype-based phenotype prediction was found to be almost 100% for classic PKU, almost 50% for mild PKU and almost 90% for mild HPA. It strongly correlates with BH4 responsiveness.

References

[1]        Blau N, Van Spronsen FJ, Levy HL. Phenylketonuria. Lancet 2010;376:1417-27.

[2]        Opladen T. Abu Seda B, Rassi A, et al. Diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots: further development of the method and 5 years experience. J Inherit Metab Dis 2011;34:819-26.

[3]        Feillet F, van Spronsen FJ, MacDonald A, et al. Challenges and pitfalls in the management of phenylketonuria. Pediatrics 2010;126:333-41.

[4]        Muntau AC, Leandro J, Staudigl M, et al. Innovative strategies to treat protein misfolding in inborn errors of metabolism: pharmacological chaperones and proteostasis regulators. J Inherit Metab Dis 2014;37:505-23.

[5]        Anikster Y, Haack TB, Vilboux T, et al. Biallelic mutations in DNAJC12 cause hyperphenylalaninemia, dystonia, and intellectual disability. Am J Hum Genet 2017;100:257-66.

[6]        Blau N, Martinez A, Hoffmann GF, Thony B. DNAJC12 deficiency: A new strategy in the diagnosis of hyperphenylalaninemias. Mol Genet Metab 2018;123:1-5.

[7]        Blau N, Shen N, Carducci C. Molecular genetics and diagnosis of phenylketonuria: state of the art. Expert Rev Mol Diagn 2014;14:655-71.

[8]        Wettstein S, Underhaug J, Perez B, et al. Linking genotypes database with locus-specific database and genotype-phenotype correlation in phenylketonuria. Eur J Hum Genet 2015;23:302-9.

[9]        Garbade SF, Shen N, Himmelreich N, et al. Allelic phenotype values: a model for genotype-based phenotype prediction in phenylketonuria. Genet Med in press (2018).

Nenad Blau

Senior Consultant in Biochemical Genetics
Dietmar-Hopp-Metabolic Center
Division of Clinical Chemistry and Biochemistry
University Children's Hospital
Heidelberg, Germany
PKU
BH4 deficiency
testing
mutations database
BIOPKU