Defining tetrahydrobiopterin responsiveness in phenylketonuria: Survey results from 38 countries.

BACKGROUND: A subset of patients with phenylketonuria benefit from treatment with tetrahydrobiopterin (BH4), although there is no consensus on the definition of BH4 responsiveness. The aim of this study therefore was to gain insight into the definitions of long-term BH4 responsiveness being used around the world. METHODS: We performed a web-based survey targeting healthcare professionals involved in the treatment of PKU patients. Data were analysed according to geographical region (Europe, USA/Canada, other). RESULTS: We analysed 166 responses. Long-term BH4 responsiveness was commonly defined using natural protein tolerance (95.6%), improvement of metabolic control (73.5%) and increase in quality of life (48.2%). When a specific value for a reduction in phenylalanine concentrations was reported (n = 89), 30% and 20% were most frequently used as cut-off values (76% and 19% of respondents, respectively). When a specific relative increase in natural protein tolerance was used to define long-term BH4 responsiveness (n = 71), respondents most commonly reported cut-off values of 30% and 100% (28% of respondents in both cases). Respondents from USA/Canada (n = 50) generally used less strict cut-off values compared to Europe (n = 96). Furthermore, respondents working within the same center answered differently. CONCLUSION: The results of this study suggest a very heterogeneous situation on the topic of defining long-term BH4 responsiveness, not only at a worldwide level but also within centers. Developing a strong evidence- and consensus-based definition would improve the quality of BH4 treatment.

The challenges of managing coexistent disorders with phenylketonuria: 30 cases.

INTRODUCTION: The few published case reports of co-existent disease with phenylketonuria (PKU) are mainly genetic and familial conditions from consanguineous marriages. The clinical and demographic features of 30 subjects with PKU and co-existent conditions were described in this multi-centre, retrospective cohort study. METHODS: Diagnostic age of PKU and co-existent condition, treatment regimen, and impact of co-existent condition on blood phenylalanine (Phe) control and PKU management were reported. RESULTS: 30 patients (11 males and 19 females), with PKU and a co-existent condition, current median age of 14 years (range 0.4 to 40 years) from 13 treatment centres from Europe and Turkey were described. There were 21 co-existent conditions with PKU; 9 were autoimmune; 6 gastrointestinal, 3 chromosomal abnormalities, and 3 inherited conditions. There were only 5 cases of parental consanguinity. Some patients required conflicting diet therapy (n=5), nutritional support (n=7) and 5 children had feeding problems. There was delayed diagnosis of co-existent conditions (n=3); delayed treatment of PKU (n=1) and amenorrhea associated with Grave's disease that masked a PKU pregnancy for 12 weeks. Co-existent conditions adversely affected blood Phe control in 47% (n=14) of patients. Some co-existent conditions increased the complexity of disease management and increased management burden for patients and caregivers. CONCLUSIONS: Occurrence of co-existent disease is not uncommon in patients with PKU and so investigation for co-existent disorders when the clinical history is not completely consistent with PKU is essential. Integrating care of a second condition with PKU management is challenging.

Significance of genotype in tetrahydrobiopterin-responsive phenylketonuria.

BACKGROUND: The value of genotyping to identify tetrahydrobiopterin-responsive (BH(4)-responsive) patients with phenylalanine hydroxylase (PAH) deficiency is a matter of debate. METHODS: We reviewed 250 cases of patients with PAH deficiency, using published data from 198 cases and unpublished data from 52 cases of patients attending our own clinic. Patients underwent analyses for BH(4) load and genetic mutations. Partial and full BH(4) responses were defined as a 10-29% decrease and a >or=30% decrease from baseline in blood phenylalanine levels, respectively. BH(4)-responsive alleles were identified from BH(4)-responsive patients as either homozygous for a specific allele or compound heterozygous for that allele with a null mutation. RESULTS: Most inconsistencies between observed genotype and BH(4) response were associated with mutations in the regulatory domain of PAH (p.R68S, p.I65T, p.L48S and p.F39C), where 20/62 alleles (32.2%) were non-responsive. In the catalytic domain (mutations p.Y414C, p.R261Q, p.E390G, p.A300S, p.R241C, p.A403V and p.V388M), only 8/125 alleles (6.4%) were non-responsive. Seven patients had a genotype with two BH(4)-responsive alleles resulting in no response or only a partial response to BH(4). Ten patients had identical genotypes but inconsistent responses in BH(4) load. CONCLUSIONS: These results show that BH(4) non-responsiveness is associated with genotype. However, patients with mutations in the regulatory domain show inconsistent results. In patients with two responsive alleles, non-responsiveness may be related to negative inter-allelic complementation. In patients with the same genotype and inconsistent results for BH(4) load, external factors such as intestinal absorption of BH(4), catabolic conditions or other genetic factors may be responsible. Further in vitro studies are necessary to clarify the genotype-phenotype correlation in patients with BH(4)-responsive PKU.

[Propionic acidemia and sensorineural hearing loss: is there a connection at the molecular genetics level?]. / Propionazidämie und Schallempfindungsschwerhörigkeit: Gibt es eine molekulargenetische Basis?

CURRENT KNOWLEDGE: Propionic acidemia is caused by a gene defect leading to malfunction of the enzyme propionyl-CoA carboxylase (PCC) and in turn to a pathologic accumulation of propionic acid. Many mutations have been found at the molecular genetic level over the past 20 years, and their implications for the limitation of enzyme activity of PCC in propionic acidemia are discussed. SCIENTIFIC QUESTION AND AIMS OF THE STUDY: As an elevated incidence of deafness has been observed in patients with propionic acidemia, the question arises of whether mutations primarily responsible for this disease could also be the underlying cause for a genetic form of deafness. METHODS AND RESULTS: As well as a standard pure tone audiogram, a pedigree was elaborated and DNA isolated for each family concerned. In one family several subjects displayed mutations of both the PCCA and the PCCB -subunits; these included only one girl whose phenotype was affected, however. CONCLUSIONS: Mutation of the PCCB subunit p.R113X has not previously been mentioned in the literature. According to our present knowledge no connection can be assumed between either of the two mutations and the severe sensorineural hearing loss.

Effects of cholesterol and simvastatin treatment in patients with Smith-Lemli-Opitz syndrome (SLOS).

Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome caused by deficiency of 7-dehydrocholesterol reductase catalysing the last step of cholesterol biosynthesis. This results in an accumulation of 7- and 8-dehydrocholesterol (7 + 8-DHC) and, in most patients, a deficiency of cholesterol. Current therapy consists of dietary cholesterol supplementation, which raises plasma cholesterol levels, but clinical effects have been reported in only a few patients. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors were shown to reduce 7 + 8-DHC levels and increase cholesterol concentrations in two small trials with divergent clinical outcome. This retrolective study evaluates the effects of cholesterol only and of cholesterol plus the HMG-CoA reductase inhibitor simvastatin on plasma sterols in 39 SLOS patients and on anthropometric measures in 20 SLOS patients. Cholesterol as well as additional simvastatin decreased the plasma (7 + 8-DHC)/cholesterol ratio. However, the mechanism leading to the decreasing ratio was different. Whereas it was due to an increasing cholesterol concentration in the cholesterol-only cohort, a decreasing 7 + 8-DHC concentration was demonstrated in the cohort receiving additional simvastatin. We could not confirm a positive effect of simvastatin treatment on anthropometric measures or behaviour, as previously reported.

Glutaryl-coenzyme A dehydrogenase deficiency: a distinct encephalopathy.

Clinical course, diagnostic and therapeutic management, and neurodevelopmental outcome were evaluated in 11 patients with glutaryl-coenzyme A dehydrogenase deficiency. In 9 patients macrocephalus was present at or shortly after birth and preceded the neurological disease. In 7 children an acute illness resembling encephalitis appeared after a period of normal development; 2 had developmental delay and progressive "dystonic cerebral palsy." Later, all 9 displayed typical signs of a disorder of the basal ganglia. In 1 patient with macrocephalus the disorder was diagnosed before the onset of neurological disease; in another it was diagnosed prenatally. Computed tomography and magnetic resonance imaging scans revealed severe generalized cerebral atrophy, most striking in the frontal and temporal lobes in 10 patients. Further deterioration was halted after initiation of treatment consisting of low-protein diets, special formulas low in lysine and tryptophan, and supplements of riboflavin and L-carnitine. Only 1 patient showed a slight clinical improvement. Later, dietary therapy was discontinued in 2 older patients and relaxed in a third without observed adverse effects. Two patients in whom treatment could be initiated before the onset of neurological symptoms have developed normally. However, duration of follow-up (6 and 29 months) does not yet allow classification of glutaryl-coenzyme A dehydrogenase deficiency as a treatable disorder. Total body production of glutaric acid, reflected in the daily urinary output, was efficiently reduced by therapeutic measures. Levels of glutaric acid in plasma and cerebrospinal fluid remained unchanged, which may in part explain the overall unsatisfactory outcome. All patients presented with a severe secondary deficiency of carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-year-old girl with partial trisomy 4p and partial monosomy 8p with resemblance to Brachmann-de Lange syndrome--another locus for Brachmann-de Lange syndrome on 4p?

We describe a 3-year-old girl with partial trisomy 4p and partial monosomy 8p who had prenatal and postnatal growth retardation, mental retardation, no speech development, mild synophrys, hirsutism, apparently low-set ears, dysphonic hoarse voice, hyperactivity, and small hands with proximal placement of the thumbs. She had recurrent lung infections, due to earlier aspiration and immune deficiency (chronic granulomatous disease). Cytogenetic findings in this and other cases with suggestive phenotype may point to an additional locus for Brachmann-de Lange phenotype.

Ventricular septal defect closure in a neonate with combined methylmalonic aciduria/homocystinuria.

Methylmalonic acidemia with associated homocystinuria is a rare inborn error of amino acid metabolism affecting energy supply on the cellular level. Its effects on recovery from surgically induced organ ischemia are largely unknown. We report the successful closure of a nonrestrictive ventricular septal defect by following a normothermic strategy combined with ample metabolic substrate supply.

Sensitive in vivo assay of the phenylalanine hydroxylating system with a small intravenous dose of heptadeutero L-phenylalanine using high pressure liquid chromatography and capillary gas chromatography/mass fragmentography.

A method is described which allows the in vivo determination of the phenylalanine hydroxylating system in atypical and classical phenylketonuria. Phenylalanine-d7 is administered i.v. (0.030 g/kg body weight) within 10 min. Tyrosine-d6 in plasma is measured from 30 to 240 min post load by using a computerized capillary gas chromatography/mass fragmentography system. In two patients with hyperphenylalaninemia, the residual activity of the phenylalanine hydroxylating system was 15.7 and 3.7% of the normal, in two phenylketonurics 1.5 and 0.3% respectively. The in vivo figures correspond well to the in vitro assay of the residual activity of the phenylalanine hydroxylase in needle liver biopsy material.

Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry.

A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.

Genotype-phenotype correlations in phenylketonuria.

Genotyping of the phenylalanine hydroxylating system offers a new way of characterizing patients with phenylalanine hydroxylase (PAH) deficiency. This paper investigates the power of genotyping as a parameter for differential diagnosis and as a measure of the risk factor of brain damage in well-treated patients with phenylketonuria (PKU). Thirty-three PKU patients were followed up over 9 years and the quality of dietary treatment, plasma phenylalanine (phe) in the newborn period before treatment and intellectual outcome at the age of 9 years were measured and correlated with the predicted residual activity (PRA) of the phe hydroxylase system as estimated from mutation analysis of the PAH gene. Patients were grouped in group Ia (PRA = 0%), group Ib (PRA = 5-15%) and group II (PRA > or = 25% of the normal activity). Mean plasma phe levels in the newborn in group Ia were 37.9 +/- 6.5 (2296 +/- 394), in group Ib 40.8 +/- 15.9 (2472 +/- 963) and in group II 16.2 +/- 4.2 (981 +/- 254) mg/dl (mumol/l). Difference in mean plasma values of groups Ia and Ib on the one hand and group II on the other were highly significant (P < 0.0001). No difference could be seen between groups Ia and Ib. There was a higher mean IQ at the age of 9 years in group II (97.4 +/- 5.4) in comparison with groups Ia (92.7 +/- 12.8) and Ib (85.0 +/- 14.4). The difference between group Ib and group II was significant (P < 0.040).(ABSTRACT TRUNCATED AT 250 WORDS)

Facts and artefacts in mevalonic aciduria: development of a stable isotope dilution GCMS assay for mevalonic acid and its application to physiological fluids, tissue samples, prenatal diagnosis and carrier detection.

A stable isotope dilution assay using D3-mevalonic acid was developed and applied to the study of mevalonic aciduria. The method also appears to be suitable for the evaluation of different therapeutic regimens in patients with hypercholesterolemia. Mevalonic acid was isolated by liquid partition chromatography and quantified as the underivatized lactone by means of ammonia chemical ionization selected ion monitoring capillary gas chromatography-mass spectrometry. In heterozygotes there was significantly greater urinary excretion of mevalonic acid, while the range of enzymatic activity of mevalonate kinase showed an overlap with that of controls. The analysis of amniotic fluids of two pregnancies at risk for mevalonic aciduria showed a 3277-fold elevation as compared to controls in the first case, diagnostic of an affected fetus, and a normal value in the second one. Mevalonic acid concentration was much increased in tissues of the affected and aborted fetus. Concentrations ranged from 840 to 1120 mumol/kg in various tissues and were as high as 1810 mumol/kg in brain. Concentrations in control fetal tissues were approximately 1 mumol/kg.

[Selective screening for amino and organic acid inborn errors]. / Selektives Screening auf Amino- und Organoazidopathien.

Aminoacidopathies and organoacidopathies are the most common acute life-threatening inborn errors of metabolism in the neonatal period. In the Federal Republic of Germany approximately 1 out of 5000 newborns is currently diagnosed as having an aminoacidopathy and approximately 1 out of 9000 newborns an organoacidopathy. Especially in the case of organoacidopathies there is substantial evidence that this number represents an underestimation. Many cases of amino- and organoacidopathies are still likely to remain undiagnosed. The incidence figures would warrant neonatal population screening for these disorders; however, the complexity and expense of the current methods prohibit this approach. Instead specialized investigations are carried out in children who develop symptoms indicative of an inborn error of metabolism. This approach is called selective screening. Early diagnosis, therefore, rests on a high degree of suspicion. In this paper clinical and laboratory findings of amino- and organoacidopathies are summarized. They can be nonspecific and misinterpreted. In the neonate and infant the presentation is commonly that of an acute overwhelming disease, whereas in the older child unexplained mental and/or neurological problems are often the leading symptom. We present an algorithm for the quick and comprehensive diagnosis of acutely presenting inborn errors of metabolism using commonly available parameters. However, in many cases the definitive diagnosis is not reached by selective metabolic screening of a single urine specimen of a patient, but requires close cooperation between the referring physician and the metabolic specialist. Multiple analyses, sometimes of different physiological fluids, or even in vivo and in vitro loading tests may be necessary.(ABSTRACT TRUNCATED AT 250 WORDS)

Sapropterin dihydrochloride: a new drug and a new concept in the management of phenylketonuria.

Phenylketonuria (PKU) is characterized by persistent hyperphenylalaninemia, due to mutations in the gene coding for phenylalanine hydroxylase (PAH). If untreated, patients develop profound mental retardation. The principal treatment for PKU is lifelong dietary phenylalanine restriction, requiring the administration of special phenylalanine-free protein supplements. Adhering to the diet is burdensome, and poor compliance and control of blood phenylalanine are common, especially in adolescents and adults. A subset of patients, particularly those with milder forms of PKU, shows a clinically significant reduction in blood phenylalanine when treated with pharmacological doses of tetrahydrobiopterin, the cofactor of PAH. A tablet formulation of sapropterin dihydrochloride is approved for therapeutic use in Europe and the USA. Clinical trials have demonstrated durable reductions in blood phenylalanine, and/or increased dietary phenylalanine tolerance, in some patients with hyperphenylalaninemia due to PKU. Although further data are needed, especially with regard to long-term neuropsychological outcomes or possible use in pregnancy, sapropterin appears to represent a useful addition to the management of PKU.

Final intelligence in late treated patients with phenylketonuria.

UNLABELLED: Despite neonatal screening programmes, there is still a number of patients with phenylketonuria who are not diagnosed and start treatment late. The question in this study was to evaluate which factors will contribute, other than the quality and duration of dietary treatment, to final outcome in late treated patients with phenylketonuria. We retrospectively analysed the data of 40 patients with phenylketonuria, of whom 2 patients at 35 and 24 years of age had a normal IQ despite never being treated. In 38 patients starting dietary treatment between 0.7 and 7 years of age, mean IQ/DQ at diagnosis was 52.7 (SD = 16) (mean age 2.5 years), final IQ (mean age 33.5 years) was 79.0 (SD = 16), the difference was highly significant (P < 0.0001). Important factors for the final intelligence in adult late treated patients with phenylketonuria were onset (r = -0.46, P < 0.009) and DQ/IQ (r = 0.51, P < 0.002) when dietary treatment was started. Thus, in late treated patients with phenylketonuria, in addition to the quality and duration of treatment, the outcome is mainly influenced by the age of starting treatment and also by the intellectual status of the patient. In one of the two patients with normal intelligence, nuclear magnetic resonance spectroscopy showed that brain phenylalanine was undetectable even though blood phenylalanine was 30 mg/dl. A second metabolic disorder may protect these patients from severe brain damage. CONCLUSION: These data indicate that brain damage in untreated or late treated patients with phenylketonuria is influenced by various genetic factors.

[Endocarditis lenta caused by Lactobacillus salivarius subsp. salicinicus (author's transl)]. / Endocarditis lenta durch Lactobacillus salivarius subsp. salicinicus

After two tooth extractions performed without antibiotic cover endocarditis lenta occurred in a ten-year-old girl. The causative organism isolated was Lactobacillus salivarius subsp. salicinicus, the first such reported case. The child has a small, haemodynamically insignificant, ventricular septal defect. A cure was achieved after long-term administration of penicillin G in high doses, at first combined with ampicillin. There were no complications.

[Point of micropreparative high pressure liquid chromatography and thin-layer chromatography for the identification of indole compounds in human plasma (author's transl)]. / Kombinierte Anwendung der mikropräparativen Hochdruck-Flüssigkeitschromatographie und der Dünnschichtchromatographie beim Nachweis von Indolmetaboliten im menschlichen Plasma

Results are presented on the analysis of indolic metabolites of tryptophan in human plasma, using high-pressure liquid chromatography and thin-layer chromatography. Dichloromethane/ethanol extracts of denaturated plasma were analysed. Thin-layer chromatography proved to be more advantageous for the analysis of this class compounds because it is possible to use a specific staining reagent (4-dimethylaminobenzaldehyde). The advantage of high-pressure liquid chromatography lies in the rapid isolation and purification of unknown compounds for identification in an off-line method. This application is demonstrated with the isolation of N-acetyltryptophan from human plasma. Preliminary results are presented on the plasma concentration of indole-3-lactic acid, indole-3-acetic acid and N-acetyltryptophan in healthy persons, phenylketonurics, and uremic patients.

Quantitative determination of cortisol in human plasma by high-pressure liquid chromatography.

A method is described for the determination of cortisol in human plasma by high-pressure liquid chromatography. The simplified extraction procedure makes the method applicable to routine clinical assays. Partition chromatography is carried out on a Zorbax-Sil column with the eluent system dichloromethane-ethanol-water. A 78% recovery was obtained for cortisol. The detection limit is 1 mug per 100 ml in 1 ml of plasma. Cortisol values were determined in samples from a random selection of patients.

Direct detection of a major mutation responsible for phenylketonuria in the population of the Federal Republic of Germany.

Forty-six individuals having phenylketonuria (PKU) alleles at the phenylalanine hydroxylase (PAH) locus were tested for the haplotype 2 PKU mutation by allele-specific hybridization following in vitro DNA amplification. Patients and carriers previously shown to have a mutant haplotype 2 PAH allele demonstrated conservation of this mutation. In vitro DNA amplification greatly facilitated this analysis and provides the possibility of population screening for 37% of the mutant German PAH alleles.

[Pre- and postnatal diagnosis of organoacidopathies]. / Prä- und postnatale Diagnostik der Organoazidopathien.

Organoacidopathies are the most common life-threatening inborn errors of metabolism presenting acutely in the neonatal period. Early diagnosis rests on a high degree of suspicion. Clinical and laboratory findings are often nonspecific and can be misinterpreted. We present an algorithm for a quick and comprehensive diagnosis of these disorders using commonly available parameters. Different methods for the prenatal diagnosis of organoacidopathies are discussed and our experience with over 150 cases presented. The method of choice is the precise quantification of elevated levels of metabolites in amniotic fluid obtained by amniocentesis at 12-18 weeks of pregnancy. Quantification is best done by stable isotope dilution analysis with the addition of the labelled metabolite to the amniotic fluid. A positive prenatal diagnosis allows a decision of the family for a termination of pregnancy or the immediate institution of therapy after birth. The conduction of a prenatal diagnosis requires the knowledge of the exact diagnosis of a previously affected child.