Tetrahydrobiopterin deficiency without hyperphenylalaninemia –
Detection and characterization of sepiapterin reductase deficiency

Tetrahydrobiopterin (BH4) cofactor is essential for various processes and is present in probably every cell or tissue of higher organisms. BH4 is required for various enzyme activities, and for less defined functions on the cellular level. The de novo biosynthesis pathway of BH4 from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase.

The enzymes that depend on BH4 are the phenylalanine, tyrosine, and tryptophan hydroxylases, the latter two being the rate-limiting steps for catecholamine and serotonin biosynthesis, all NO synthase (NOS) isoforms, and the glyceryl-ether monooxygenase. On a cellular level, BH4 was found to be a growth or proliferation factor for Crithidia fasciculata, hemopoietic cells, and various mammalian cell lines. In the nervous system, BH4 is a self-protecting factor for NO, or a general neuroprotecting factor via the NOS pathway, with a neurotransmitter-releasing function.

In regard to human disease, BH4 deficiency due to autosomal recessive mutations in all enzymes except sepiapterin reductase were described as a cause of hyperphenylalaninemia. Furthermore, several neurological diseases including Dopa-responsive-dystonia (DRD), but also Alzheimer disease, Parkinson disease, autism, and depression were suggested as a consequence of limited cofactor availability.

Patients with autosomal recessive BH4 deficiencies present mostly with progressive neurological deterioration regardless of the different enzyme defects. The clinical manifestation is variable but common symptoms are mental retardation, convulsions, disturbance of tone and posture, abnormal movements, hypersalivation, swallowing difficulties, temperature instability, and oculogyric crises. These patients can be detected through neonatal screening for phenylketonuria (PKU) due to abnormally high levels of phenylalanine in blood.

Recently, we investigated neopterin and biopterin production in cytokine stimulated fibroblasts from patients with different forms of BH4 deficiencies using a newly developed method. Furthermore, we measured the activity of all enzymes involved in BH4 metabolism in normal and stimulated cells. With this method we showed that both, the classical forms of BH4 deficiency and DRD, can be differentiated in fibroblasts. We also investigated fibroblasts from four patients with severe neurotransmitter depletion without hyperphenylalaninemia two of which were initially diagnosed with a “central” form of dihydropteridine reductase deficiency. Pterin metabolites and enzymatic activities revealed SR deficiency, as confirmed by DNA mutation analysis. One patient was homozygous for a TC>CT dinucleotide exchange (354-355TC>CT), predicting a truncated SR protein Q119X. The second patient was a compound heterozygote for a genomic 5 bp deletion (1397-1401delAGAAC) resulting in abolished SR gene expression (N149X), and a A>G transition (448A>G) leading to a R150G amino acid substitution. The third patient was homozygous for the R150G mutation. Recombinant expression of the R150G mutant protein in E. coli revealed a completely inactive SR, whereas the wild type protein was fully active after expression in bacterial cells. We thus describe a new autosomal recessive form of BH4 deficiency with monoamine neurotransmitter depletion and absence of hyperphenylalaninemia, and propose alternative routes for the final step in the BH4 biosynthesis pathway in different tissues.

Clinical features of four patients with SR deficiency include spasticity, dystonia, microcephaly, hypersalivation, hypotonia of the trunk, hypertonia of the limbs, tremor, oculogyric crises, cortical atrophy, and progressive psychomotor retardation. The first two patients were diagnosed at the age of 5 and 10 years and they both responded to L-Dopa/Carbidopa (1-2 mg/kg/d) and 5-hydroxytryptophan (5-6 mg/kg/d). Due to the late diagnosis and thus probably irreversible brain damage a trial with BH4 in one patient was not successful. The third patient was diagnosed as SR-deficient at the age of 25 years, however, the diagnosis at the age of two years was cerebral palsy presenting with diurnal dystonia and hypersomnolence. Although this patient improved on L-Dopa and 5-hydroxytryptophan, initially she also did not tolerate the therapy. The fourth recently diagnosed patient from Heidelberg is like the first two of Turkish origin and diagnosed at the age of 8 years. He is currently on L-Dopa/Carbidopa (6 mg/kg/d).

For many years it was speculated why no patients with SR deficiency had been detected. It has been proposed that either such a deficiency is fatal in utero or possibly compensated by the activity of other reductase(s). As shown here, neither turns out to be true, and alternative reductases replacing absent SR activity at least in peripheral tissues may be responsible for the phenotype (see below). Diagnosis of SR was missed in the past probably due to the fact that these patients present without hyperphenylalaninemia and with normal urinary pterins excretion. Furthermore, normal neopterin and high biopterin and dihydrobiopterin levels in CSF were rather suggestive for the dihydropteridine reductase deficiency. Diagnosis was misleading because both patients with dihydropteridine reductase and SR deficiency present with high biopterin and dihydrobiopterin levels in CSF. Recently, we were able to detect high levels of sepiapterin in the CSF of these patients using new HPLC method.

Based on the current knowledge in patients with SR deficiency peripheral BH4 is synthesized via the salvage pathway catalyzed by the enzymes aldose, carbonyl, and dihydrofolate reductase. In contrast to patients with dihydropteridine reductase deficiency, in these patients dihydrobiopterin is formed from sepiapterin by the action of CR. However, due to the low activity of dihydrofolate reductase in the brain dihydrobiopterin can not be reduced to BH4 and therefore accumulates.

Two hypothetical mechanisms may contribute to the pathogenesis of neurotransmitter deficiency and brain damage in patients with SR deficiency and both are most probably related to the elevated levels of dihydrobiopterin and sepiapterin in CSF. (1) Dihydrobiopterin is a competitive inhibitor of tyrosine and tryptophan hydroxylases and the subnormal concentrations of BH4 production of catecholamines and serotonin is markedly reduced. (2) On the other hand it has been well documented that dihydrobiopterin and sepipaterin compete for BH4 binding to the NOS and that dihydrobiopterin can displace pre-bound BH4 from NOS with >80% efficiency. Accordingly, oxidation of BH4 to dihydrobiopterin, as it occurs in SR-deficient patients, is expected to inhibit NO production, uncouple the reaction, and stimulate superoxide and peroxynitrite production. Peroxynitrite in turn may induce apoptosis of the neuronal cells through the oxidation of lipids, proteins, and DNA. This was documented by the recent finding of low nitrite+nitrate concentrations in CSF of patients with BH4 deficiencies.

References

Bonafé L, Thöny B, Penzien JM, Czarnecki B, Blau N. Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine neurotransmitter deficiency without hyperphenylalaninemia. Am J Hum Genet 69:269-277.2001

Blau N, Bonafé L, Thöny B. Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: Diagnosis and genetics of Dopa-responsive dystonia and sepiapterin reductase deficiency. Mol Genet Metab 74:172-185.2001

Zorzi G, Thöny B, Blau N. Reduced nitric oxide metabolites in CSF of patients with tetrahydrobiopterin deficiency. J Neurochem: 80: in press 2002