David S. Konecki

Molecular basis of phenotypic heterogeneity in phenylketonuria.

BACKGROUND Phenylketonuria is a metabolic disorder that results from a deficiency of the hepatic enzyme phenylalanine hydroxylase. Its clinical phenotype varies widely, and to date more than 10 mutations in the phenylalanine hydroxylase gene have been identified in persons with the disorder. We attempted to relate the clinical phenotype of patients to their genotype. METHODS We studied 258 patients with phenylketonuria from Denmark and Germany for the presence of eight mutations previously found in patients from these countries. The in vitro activity of the enzymes associated with these mutations was determined by expression analysis in heterologous mammalian cells. The level of activity was then used to predict the in vivo level of phenylalanine hydroxylase activity in patients with various combinations of mutant phenylalanine hydroxylase alleles. RESULTS The eight mutations involved 64 percent of all mutant phenylalanine hydroxylase alleles in the patients. Expression analysis showed that these mutant enzymes produced from 0 to 50 percent of normal enzyme activity. The predicted level of phenylalanine hydroxylase activity correlated strongly with the pretreatment serum level of phenylalanine (r = 0.91, P less than 0.001 in the Danish patients and r = 0.74, P less than 0.001 in the German patients), phenylalanine tolerance in the Danish patients (r = 0.84, P less than 0.001), and the serum phenylalanine level measured after standardized oral protein loading in the German patients (r = 0.84, P less than 0.001). CONCLUSIONS Our results strongly support the hypothesis that there is a molecular basis for phenotypic heterogeneity in phenylketonuria. The establishment of genotype will therefore aid in the prediction of biochemical and clinical phenotypes in patients with this disease.

Allelic association of the cystic fibrosis locus and two DNA markers, XV2c and KM19, in 55 German families

SummaryAllelic association between cystic fibrosis and two linked markers is demonstrated in a sample of 55 German families. It is shown by example how these observations can be used for genetic risk calculation.

The phenylketonuria locus: current knowledge about alleles and mutations of the phenylalanine hydroxylase gene in various populations

SummaryThe hyperphenylalaninemic disorders of classic phenylketonuria (PKU), mild phenylketonuria, and hyperphenylalaninemia (HPA), result from a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) or its cofactor (tetrahydrobiopterin). Use of the complementary DNA of this enzyme has allowed the establishment of a restriction fragment length polymorphism (RFLP) haplotype-analysis system. This haplotype analysis system provides the means for determination of mutant PAH alleles in most affected families and is the basis for mutational analysis of the PKU locus. This review is focused on two major areas of current PKU research: (1) the use of DNA haplotype analysis in the study of the population genetics of PAH deficiency, and (2) the study of genotypes, and their various combinations, as a means of explaining and predicting the phenotypic variability observed for the disorders of PAH deficiency.

Linkage disequilibrium between mutation and RFLP haplotype at the phenylalanine hydroxylase locus in the German population

SummaryRestriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus have been determined in 60 German families with PAH deficiency. Similar to the Danish population, about 90% of the mutant alleles are confined to four distinct haplotypes. There are however, differences in the frequency distributiion of these haplotypes among the mutant alleles between the two populations. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 in the Danish population, a tight association between the mutation and the RFLP haplotype has also been observed in Germany. The results provide strong evidence that the splicing mutation occurred on a haplotype 3 chromosome and that the mutant allele has spread into different populations smong Caucasians.

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)

DNA haplotype analysis at the phenylalanine hydroxylase locus in the Turkish population

SummaryThirty-nine Turkish phenylketonuria (PKU) families were investigated for their DNA haplotypes at the phenylalanine hydroxylase (PAH) locus. There was a threefold higher incidence of consanguinity in the population studied compared with the general Turkish population. The PAH DNA haplotype 6 was found to be almost exclusively associated not only with the mutant PAH genes but also with the classic phenotype in 39% of the Turkish patients. This haplotype was of no importance in northern European populations. The two DNA haplotypes (1 and 4) that were almost equally frequent among the normal and the mutant PAH genes in northern European populations show virtually the same distribution in Turkish individuals. In all populations studied, these haplotypes are associated with different phenotypes.

The identification of two mis-sense mutations at the PAH gene locus in a Turkish patient with phenylketonuria

SummaryDNA sequence analysis of the 13 exons and intron/exon boundaries of the phenylalanine hydroxylase (PAH) gene has detected two base transitions, resulting in mis-sense mutations, in the genomic DNA of a Turkish patient (E1) with phenylketonuria (PKU). The Leu48→Ser amino acid substitution was associated with the mutant haplotype 3 allele and the Glu221→Gly amino acid substitution with the mutant haplotype 4 allele of this family. Allele-specific oligonucleotide (ASO) dotblot analysis subsequently detected the Leu48→Ser mutation in the haplotype 4 PKU alleles of nine (18.8%) of the 48 unrelated Caucasian PKU families investigated. This mutation results in mild PKU in the homozygous state. The Glu221→Gly mutation has only been detected within patient E1 and his father.

DNA sequence polymorphisms in exonic and intronic regions of the human phenylalanine hydroxylase gene aid in the identification of alleles

Five sequence polymorphisms at the phenylalanine hydroxylase (PAH) gene locus were observed to be in tight association with specific alleles of this locus. Since these polymorphisms can be detected using polymerase chain reaction (PCR) methodology, application of a combination of these polymorphisms reduces the effort involved in PAH DNA haplotype analysis, which is needed for population genetic analysis or diagnosis of the disease status. In addition our results indicate the evolution of haplotype 3, 4 and 7 PAH alleles from a common ancestor, whereas PAH haplotypes 5, 6, and 11 arose form another common ancestor allele. These data reveal that two of the polymorphisms investigated originated before the separation of races.

RFLP-patterns in japanese PKU families : new polymorphisms for the mutant phenylalanine hydroxylase gene

SummaryNew RFLP patterns are present in Japanese families with members suffering from phenylketonuria indicating a deletion at the 3′ end of the PAH-gene.

Haplotype distribution and mutations at the PAH locus in Croatia.

SummaryRestriction fragment length polymorphism (RFLP) haplotypes and mutations at the phenylalanine hydroxylase (PAH) locus have been studied in 25 unrelated families from Croatia. The results of RFLP analysis demonstrated that 80% of the mutant alleles were associated with three haplotypes (1, 2 and 4). Eight mutations were detected on the background of six mutant haplotypes, comprising 68% of phenylketonuria (PKU) alleles in Croatia. The mutation in codon 408 was most frequent, as was the haplotype 2 allele with which it was associated. These data are in accordance with formerly published population genetic analyses at the PAH locus, and with studies revealing the molecular basis of the phenotypic heterogeneity of PKU. The codon 281 mutation was more frequent in Croatia than previously observed in other populations.