Rare, but in severe cases symptomatic at an early stage: disorders of BH4 metabolism
The little patient is only a few days old when an increased concentration of phenylalanine is detected in her blood as part of newborn screening. She is one of around 5,000 newborns in whom hyperphenylalaninemia (HPA) is detectable. Hyperphenylalaninaemia includes "mild HPA", which does not require treatment, as well as "classic" and "atypical" phenylketonuria, each of which requires different therapies.
Phenylalanine hydroxylase deficiency (PAH)
In classic phenylketonuria (incidence 1:10,000), there is a deficiency of phenylalanine hydroxylase (PAH) due to a PAH gene defect. Without PAH, phenylalanine cannot be metabolized to tyrosine and accumulates in the body. At concentrations of over 600 μmol/L in the blood, phenylalanine has a neurotoxic effect. A low-phenylalanine diet initiated early in infancy can prevent severe neurological damage.
Deficiency of tetrahydrobiopterin (BH4)
In only around 1-2% of all HPA cases, the tetrahydrobiopterin (BH4) metabolism is impaired. BH4 serves as a cofactor for PAH, but also for enzymes involved in neurotransmitter synthesis (tyrosine hydroxylase and tryptophan hydroxylase) and NO synthase. If BH4 is missing, phenylalanine cannot be broken down by PAH and the starting molecules for dopamine and serotonin synthesis are missing. The clinical severity of congenital BH4 deficiency can vary greatly, with the main symptoms reflecting the lack of neurotransmitters.
During the immediate confirmatory diagnostics, it becomes clear that the young patient has BH4 deficiency. Due to the severe symptoms, treatment is started immediately. This consists of the administration of synthetic BH4 (sapropterin) and supplementation with neurotransmitter precursors (L-dopa in combination with carbidopa and 5-hydroxytryptophan) as well as adherence to a phenylalanine-balanced diet.
PTS deficiency identified, but genetic cause initially puzzling.
A number of enzymes are involved in the biosynthesis of BH4 and the dihydrobiopterin/tetrahydrobiopterin redox system. Disorders of the redox system can be detected or ruled out by testing the activity of dihydropteridine reductase (DHPR) as the central enzyme of the system. By determining the concentration of intermediates or metabolites along the biosynthetic pathway, the enzyme that is deficient can be identified. In the case of the infant, this is 6-pyruvoyltetrahydropterin synthase (PTS).
PTS-related BH4 deficiency follows an autosomal recessive pattern of inheritance, i.e. both PTS alleles must have a pathogenic alteration. Despite intensive efforts using Sanger sequencing and, in a second step, exome sequencing, only one heterozygous missense variant (affecting only one allele) can be identified in the PTS gene. This leads to an exchange of histidine for proline at amino acid position 24. His24 is one of the binding sites for the PTS cofactor zinc.
In-depth trace search with genome and RNA sequencing
We suspect the alteration of the second allele in previously unanalyzed deep-intronic or regulatory regions of the PTS gene. However, not only the detection, but also the assessment of changes found in these regions is a challenge. In the course of a reanalysis, we decide to get to the bottom of the underlying genetics by means of genome and RNA sequencing. RNA sequencing makes it possible to visualize the influence of genetic changes at the gene expression level - which provides us with the decisive clue in the present case.
On the allele not affected by the missense variant, we find a deep-intronic insertion of 90 base pairs. Since this sequence also has new splicing sites (splice junctions), a new exon is created that is detectable in the patient's transcripts but absent in control samples. As a consequence, the reading frame shifts, resulting in a lack of functional PTS enzyme.
Success for therapy and beyond
With the missense variant on one allele and the deep-intronic insertion on the second allele, we were able to detect biallelic changes in the gene (on both parental gene copies) in the sense of autosomal recessive inheritance. The underlying genetics explain the clinical picture, support the biochemical diagnosis and confirm the therapeutic approach chosen for the young patient.
This is essential not only for clinical care, but also for various other aspects, such as reimbursement of the therapy by the health insurance company. Parents of affected children can be advised and supported if they wish to have children: Carrier testing can be used to assess the risk of recurrence as part of family planning and targeted prenatal diagnostics can be carried out during a further pregnancy.