Isolated central form of tetrahydrobiopterin deficiency associated with hemizygosity on chromosome 11q and a mutant allele of PTPS.

6-Pyruvoyl-tetrahydropterin synthase (PTS or PTPS) is involved in tetrahydrobiopterin (BH(4)) biosynthesis, the cofactor for various enzymes including the aromatic amino acid hydroxylases. Inherited PTPS deficiency is a heterogeneous disease with different phenotypes leading to BH(4) depletion. The severe form of PTPS deficiency causes hyperphenylalaninemia and monoamine neurotransmitter deficiency, whereas the mild form gives rise to hyperphenylalaninemia only. From 228 patients with PTPS deficiency at least 32 different mutant alleles have been identified on its corresponding gene, located on chromosome 11q22.3-q23.3. Here we describe a new allele from a child with PTPS deficiency who exhibited a mild but transient form of hyperphenylalaninemia, yet was deficient in CSF monoamines. The patient was found to carry, on her genomic DNA and cDNA, a homozygous A>G transition, leading to PTPS codon alteration Tyr99 to Cys (Y99C). The mother and several members of the maternal family were carriers of the Y99C allele, also verified by the reduced PTPS enzyme activity in erythrocytes. By cytogenetic, molecular, and FISH analyses, a de novo deletion spanning from 11q14 to 11q23.3 on the patient's paternal chromosome was mapped, establishing hemizygosity of the Y99C allele. The PTPS mutation observed in this patient generates a novel phenotype with an apparently isolated central form of BH(4) deficiency.

Dominant negative allele (N47D) in a compound heterozygote for a variant of 6-pyruvoyltetrahydropterin synthase deficiency causing transient hyperphenylalaninemia.

Mutations in the 6-pyruvoyltetrahydropterin synthase (PTPS) gene result in persistent hyperphenylalaninemia and severe catecholamine and serotonin deficiencies. We investigated at the DNA level a family with a PTPS-deficient child presenting with an unusual form of transient hyperphenylalaninemia. The patient exhibited compound heterozygosity for the PTPS-mutant alleles N47D and D116G. Transfection studies with single PTPS alleles in COS-1 cells showed that the N47D allele was inactive, while D116G had around 66% of the wild-type activity. Upon co-transfection of two PTPS alleles into COS-1 cells, the N47D allele had a dominant negative effect on both the wild-type PTPS and the D116G mutant with relative reduction to about 20% of control values. Whereas the mother and the father had reduced enzyme activity in red blood cells (34.7% and 51.7%, respectively) and skin fibroblasts (2.8% and 15.4%, respectively), the clinically normal patient had in these cells activities at the detection limits, although PTPS-cross-reactive material was present in the fibroblasts. The specifically low PTPS activity in the mother's cells corroborated the evidence of a dominant negative effect of the maternal N47D allele on wild-type PTPS.

Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families.

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.

Antenatal diagnosis of tetrahydrobiopterin deficiency by quantification of pterins in amniotic fluid and enzyme activity in fetal and extrafetal tissue.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

Urinary oxalate and glycolate excretion in healthy infants and children.

The molar ratios of oxalate and glycolate over creatinine were determined in single urine samples of 26 infants and 27 children aged 1-5 years. In 135 children aged 5-16 years, two urine specimens were collected, one before breakfast and one at noon. Oxalate was determined by oxalate oxidase, and glycolate was measured by a colorimetric method (improved chromatotropic acid--sulphuric acid assay after prior purification by cation and anion exchanger). Both ratios (expressed in mmol/mol creatinine and analysed on a log-normal basis) were highest in infants 0-6 months old [mean oxalate 147 (95% confidence interval: 60-360), mean glycolate 175 (72-425)]. The mean oxalate ratio was 72 mmol/mol (29-174) at the age of 7-24 months, 44 (19-101) at the age of 2-5 years and 22 (12-40) in adolescents aged 16 years. Molar glycolate ratios were higher, but disclosed the same pattern. Oxalate and glycolate ratios in fasting urines did not differ significantly from those in noon samples (except glycolate in the oldest age group). Oxalate ratios correlated well with glycolate ratios in children up to 5 years of age only. Random urine samples are thus suitable for screening. However, interpretation of data requires use of age-specific reference values that are based on comparable methods.

7-Substituted pterins. A new class of mammalian pteridines.

Three novel pteridines have been isolated from the urine of patients with a new variant of 6-(L-erythro-1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) deficiency, showing hyperphenylalaninemia. From the results of high performance liquid chromatography, oxidative degradation, and gas chromatography-electron impact mass spectrometry, their structures were identified as 7-(D-erythro-1',2',3'-trihydroxypropyl)-pterin (7-neopterin), 7-(L-erythro-1',2'-dihydroxypropyl)-pterin (7-biopterin), and 6-oxo-7-(L-erythro-1',2'-dihydroxypropyl)-pterin (6-oxo-7-biopterin). The ratio of biopterin to 7-biopterin in the patients' urines was 1:1, and after oral loading with tetrahydrobiopterin, 7-biopterin excretion rose parallel to biopterin. This finding suggests that 7-substituted pterins may be formed endogenously by a yet unknown isomerization reaction. The cause of hyperphenylalaninemia is still unclear. The activities of the enzymes involved in tetrahydrobiopterin biosynthesis and regeneration were found to be normal in the patients, and no effect of 7-biopterin on these enzymes was observed in vitro. However, compared with the normal cofactor, tetrahydrobiopterin, the Km values of tetrahydro-7-biopterin for phenylalanine hydroxylase and dihydropteridine reductase are 20 and 5 times higher, respectively.

Primapterin, anapterin, and 6-oxo-primapterin, three new 7-substituted pterins identified in a patient with hyperphenylalaninemia.

Three unknown compounds present in the urine of a patient with mild hyperphenylalaninemia were identified to be L-erythro-7-iso-biopterin, D-erythro-7-iso-neopterin, and L-erythro-6-oxo-7-iso-biopterin. The newly identified pterins were named primapterin, anapterin, and 6-oxo-primapterin, respectively. Primapterin and anapterin are present in very low concentrations in every human urine, as well as in the liver of man and mouse, whereas 6-oxo-primapterin was detected in the patient's urine only. Substantial amounts of primapterin were excreted in the patient described. The metabolic origin of primapterin and anapterin is still obscure.

Application of gas chromatography-mass spectrometry to the study of biopterin metabolism in man. Detection of biolumazine and 2'-deoxysepialumazine.

The separation characteristics of the trimethylsilyl ether derivatives of various naturally occurring and synthetic pteridines on a apolar glass capillary column, together with their mass spectra, permit their identification and quantitation in biological samples. Examples are given of the determination of the ratio of monapterin to neopterin in urine, of monitoring excreted pterin metabolites after a loading test with 6-methyltetrahydropterin in urine and of structure elucidation of lumazines , previously unknown in man. 6- Methylisoxanthopterin was shown to be the main metabolite in urine after administration of 6-methyl-5,6,7,8-tetrahydropterin. Biolumazine and 2'- deoxysepialumazine were found in human faeces after administration of ( 6R ,S)-5,6,7,8-tetrahydro-L-erythro-biopterin.

Glycolic acid in urine. A colorimetric method with values in normal adult controls and in patients with primary hyperoxaluria.

An improved Chromotropic acid-sulfuric acid assay for urinary glycolic acid is described. The sample (0.5 ml or less) is precleaned by filtering through strongly acidic and strongly basic ion-exchangers and compared with a standard made up in normal urine. From the many compounds tested, only glyceric acid in pathologically high concentrations interfers partially; this interference can be recognized at 776 nm and eliminated by a second determination. Per man-day, 8--12 samples can be analyzed. Recovery of 0.8 mmol/l glycolic acid added to the urines of 12 persons was 100.6 +/- 4.7% (1 S.D., n=12) without, and 101.9 +/- 5.3% (1 S.D., n=12) with correction for isotope dilution of [1-14C]glycolic acid, respectively, using 0.5-ml sample volumes. The variation coefficients of a single determination were 1.5 and 2.1% without and with correction for isotope dilution, respectively (n=7). The method was checked by mass fragmentography. The following normal values were found in adults (n=15; x +/- 1 S.D. (range)): 47.3 +/- 10.1 (24.4--63.7) mmol/mol creatinine and 0.60 +/- 0.15 (0.29--0.91) mmol/day or 45.8 +/- 11.3 (22.2--69.0) mg/day. Two patients (F.G. and A.S.) with primary hyperoxaluria type I excreted glycolic acid between 112 and 379 mmol/mol creatinine and 1.21--5.64 mmol/day or 92--429 mg/day. Under vitamin B-6 treatment, urinary excretion decreased in one patient (F.G.) to 71--131 mmol/mol creatinine and 0.92--2.0 mmol/day or 70--152 mg/day.

New tyrosine metabolites in humans: hawkinsin and cis- and trans-4-hydroxycyclohexylacetic acids. Unusual adsorption of deuterated and non-deuterated hawkinsin during gas chromatography.

In a new inborn error of metabolism, where obviously a defect of 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) exists, hawkinsin [(2-cystein-S-yl-1,4-dihydroxycyclohex-5-en-1-yl) acetic acid] and cis- and trans-hydroxycyclohexylacetic acids were found in the urine. A partially reversible adsorption of deuterated and non-deuterated hawkinsin (as the penta-trimethylsilyl derivative) in gas chromatography--mass spectrometry has inhibited a mass fragmentographic quantitation of this compound to date. However, quantitation seems to be possible using mass framentography of 1,4-dihydroxycyclohexylacetic acid, formed by desulfuration of the sample with active nickel.

A new sulfur amino acid, named hawkinsin, identified in a baby with transient tyrosinemia and her mother.

An unknown compound present in the urine of a girl with prolonged transient tyrosinemia and her mother was isolated and identified as (2-L-cystein-S-yl-1,4-dihydroxycyclohex-5-en-1-yl)-acetic acid (IVa). The new amino acid was named hawkinsin (Haw) and characterized by gas chromatography-mass spectrometry (GC-MS) of its penta-trimethylsilyl (TMS) derivative and of its desulfuration components. Haw was compared with the synthetic reference compound using GC-MS, IR, TLC, PC, ion-exchange chromatogrpahy and high-voltage electrophoresis. IVa and (2,6-bis-L-cystein-S-yl-1,4-dihydroxycyclohexyl-1)-acetic acid were synthesized from 4-quinolacetic acid, the latter was prepared in two different ways. It is postulated that Haw originates from an intermediate in the 4-hydroxy-phenylpuruvate hydroxylase reaction (EC 1.14.2.2), and that mother and child are heterozygous for an inborn error of metabolism characterized by a defect in this hydroxylase system, which is unable to rearrange the intermediate to homogentisic acid.

Hydantoin-5-propionic aciduria in folic acid nondependent formiminoglutamic aciduria observed in two siblings.

Hydantion-5-propionic acid was detected in massive amounts in the urine of two previously described sisters with folic acid nondependent formiminoglutamic aciduria. HPA was identified rigorously, e.g. by gas chromatography-mass spectrometry, and was measured quantitatively by selected ion monitoring (mass fragmentography) using deuterated HPA as internal standard. Before histidine loading, both girls with the postulated formiminotransferase deficiency excreted an amount of HPA more than 50 times greater than the control subjects. After histidine ingestion, HPA excretion was still doubled or tripled. With the exception of the father, the values for the other members of the family also markedly exceeded the normal range and were of the same order of magnitude as in folate deficiency. Measurement of HPA excretion in urine seems to be a valuable supplement or alternative to the enzymatic FIGu test for the detection of general or functional folate deficiency or impaired folate utilization and it will be indispensable for the detection of (as yet unknown) 4-imidazolone-5-propionic acid hydrolase deficiency.