A limited spectrum of phenylalanine hydroxylase mutations is observed in phenylketonuria patients in western Poland and implications for treatment with 6R tetrahydrobiopterin.

Phenylketonuria (PKU) is an autosomal recessive defect in hepatic metabolism of phenylalanine, which is secondary to mutations in the phenylalanine hydroxylase (PAH) gene. Sixty-seven ethnically Polish PKU patients, followed at the Outpatient Department of Pediatrics and Developmental Medicine in Poznan, Poland, were assessed for mutations in the PAH gene. Two mutations were identified in 61 of 67 patients and a single mutation was identified in the remaining six patients. The four most prevalent mutations (p.R408W, 68%; c.1066-11G>A, 6%; c.1315+1G>A, 5.2%; c.822-832delGCCCATGTATA, 3.7%) accounted for 83% of the mutant alleles. Fifteen additional mutations were identified of which most (13/15) were observed in an individual patient. Before knowledge of PAH genotypes, 19 patients were challenged with a 20 mg kg(-1) dose of 6R tetrahydrobiopterin (BH(4)) and serum phenylalanine concentration was monitored in hospital over 24 h. Two patients responded to the BH(4) challenge with a reduction of serum phenylalanine concentration >30% from baseline. PAH genotypes of the two responsive patients would have been predicted, as they contained mutations recognized as BH(4) responsive, whereas the 17 patients who were unresponsive would have been predicted as their mutations were either recognized as non-responsive or were highly deleterious frame-shift mutations. Overall, only 7.5% (5/ 67) of patients had PAH mutations recognized as responsive to co-factor therapy. Among the PKU patients from western Poland, PAH mutations responsive to BH(4) therapy are poorly represented; therefore, genotyping may be useful for identifying candidate patients likely to respond to BH(4) before physiological challenge.

Inhibition of endogenous pancreatic enzyme secretion by oral pancreatic enzyme treatment.

BACKGROUND: The existence of a feedback mechanism for exocrine pancreatic secretion in humans is controversial. Exclusion of proteases from the duodenum stimulates exocrine pancreatic secretion. Conversely, addition of exogenous enzymes could reduce the enzyme secretion. Further investigation of the feedback mechanism should be performed under the most physiological conditions. In the present study we investigated exocrine pancreatic function by measuring fecal enzyme output in healthy volunteers consuming a normal diet, before and during a time course of exogenous pancreatic enzyme supplementation. MATERIAL AND METHODS: Twenty-five healthy subjects (HS) were given two different doses (30 and 60 FIP proteases kg(-1) d(-1)) divided by the number of meals. In all subjects, fecal elastase-1 (E1) concentrations and chymotrypsin (ChT) activities were measured without and with enzyme supplements after 7 days of treatment. In eight subjects, E1 concentrations and ChT activities were measured daily for 10 consecutive days. The subjects were given a dose regimen of 100 FIP proteases kg(-1) d(-1)(divided by the number of meals) for the first 7 days. RESULTS: Oral pancreatic treatment dose-dependently inhibited endogenous pancreatic secretion measured with the use of E1 concentrations. In both regimen groups, the differences were statistically significant. The exogenous enzymes, which interfere with colorimetric method for ChT, dose-dependently increased ChT output. However, only the higher dose resulted in a statistically significant difference. In the subgroup of eight HS, time-dependent changes of fecal enzyme output occurred with a decrease of E1 concentrations and an increase of ChT activity from the second up to eighth or ninth day of the experiment. CONCLUSION: Exogenous applied pancreatic enzymes, dose- and time-dependently inhibited endogenous pancreatic secretion. The obtained results strongly support the existence of a protease mediated feedback mechanism in humans.

A simplified method for detection of the mutations predominantly causing cystic fibrosis and phenylketonuria in Polish families.

Genomic DNA was isolated from dried blood specimens and subsequently used as a template in simplified PCR-based detection assays of delta F508 mutation of CFTR gene and of R408W mutation of PAH gene in families with cystic fibrosis and phenylketonuria, respectively. Products of amplification of CFTR gene were analyzed in NuSieve agarose gel. The amplification-created restriction site with TaqI digestion was used for detection of the PAH gene mutation.

Genetic background of clinical homogeneity of phenylketonuria in Poland.

In order to elucidate the clinical homogeneity and severity of the hyperphenylalaninaemias in Poland, a total of 71 children with typical phenylketonuria (PKU) originating from western and northern Poland were screened for 13 mutations in the phenylalanine hydroxylase (PAH) gene. Eighty percent of all PKU alleles tested were found to carry an identified mutation. One mutation, namely the R408W mutation, accounted for more than 63% of mutant PAH alleles in Poland, the other 27% being accounted for by six mutations: IVS12nt1 (5%), IVSnt546 (5%), Y414C (4%), R252W (1.5%), R261Q (< 1%), and G272ter (< 1%). The predominance of the R408W mutation resulted in a high rate of homozygotes (35.2%) and compound heterozygotes for this mutation in children from western and northern Poland. The frequency and deleterious nature of this mutation probably accounts for the clinical homogeneity and severity of the hyperphenylalaninaemias in Poland. In addition, the high rate of the R408W mutation and its association with mutant haplotype 2 at the PAH locus in Poland give additional support to the Balto-Slavic origin of this mutant gene.

The codon 408 mutation associated with haplotype 2 is predominant in Polish families with phenylketonuria.

The incidence of phenylketonuria (PKU) in the western part of Poland is 1 in 5000 live births. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase locus have been analysed in 46 Polish families with PKU. Among 43 fully-informative families 16 RFLP haplotypes were identified. Haplotype 2 is the most frequently (62%) associated with Polish PKU alleles, and the codon 408 mutation is in complete linkage disequilibrium with this haplotype in Poland. This finding is in agreement with observations in other eastern European countries (German Democratic Republic, Czechoslovakia, and Hungary) and in contrast to the genotype distribution observed in western European countries. The present observation suggests the spread of classical PKU, due to the codon 408 mutation associated with haplotype 2, from east to west in European populations. Perhaps more important for genetic counselling, 62% of all PKU chromosomes in the Polish population can now be detected using only one mutant-specific oligonucleotide probe.

Haplotype analysis of phenylalanine hydroxylase alleles in polish families with phenylketonuria.

Eight polymorphic restriction enzyme sites at phenylalanine hydroxylase locus from the parental chromosomes in Polish families with phenylketonuria were analyzed. Among 28 chromosomes studied, we identified haplotypes found within the Danish population. Haplotype 2 has been found in 25% of affected alleles. One of the patients studied is homozygous for this haplotype.

Hematoxylin and thionin techniques for staining myelin and cells: variations and critical steps.

The action of the delipidization step and the type of differentiation fluid was assessed for two stains, Harris' hematoxylin and thionin, in frozen sections of rat brains. For thionin, all procedural variations used produced a blue or purple cell staining. Myelin stained red when sections were delipidized and differentiated in acetic acid, acid-formalin, or acid-alcohol. Myelin remained white when the delipidization step was omitted or when differentiation was performed in alcohol. Myelin staining was achieved with Harris' hematoxylin when sections were delipidized and were differentiated in either acid-formalin or borax-ferricyanide. The former produced red fibers against a red-purple or light purple cell background with some cell staining. The latter produced purple fibers contrasting sharply with a buff background and minimal cell staining. Cells were further accentuated in the hematoxylin techniques by a thionin counterstain.