Molecular analysis of PKU haplotypes in the population of southern Poland

Summary Out of a population of 138598 infants born in southern Poland between 1987 and 1989, and screened for phenylketonuria (PKU), 28 cases were ascertained giving an incidence of 1 in 5000. DNA from 22 of these probands and their parents was isolated and eight polymorphic restriction sites were analyzed within the phenylalanine hydroxylase gene region. Twenty-one different haplotypes (HT) were revealed, five of them representing new categories. The most common haplotypes among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of haplotypes with mutant alleles the most frequent was HT2 (56.8%), whereas the frequency of this haplotype in other European populations, such as French, Danish and German, ranged from 12% to 24%. HT3, being the most common in Danish (38%), and relatively frequent in the other western European (13–14%) populations, appeared to be very rare in our sample (2.3%). The mutation of codon 408 (exon 12, CT, ArgTrp), which has been described to be tightly linked to HT2, was tested on amplified DNA by dot-blot hybridization. This mutation was found in 25 out of 44 proband chromosomes. In one case it was linked to HT5, in the remaining 24 to HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.     

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

Summary DNA 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 Leu⁴⁸Ser amino acid substitution was associated with the mutant haplotype 3 allele and the Glu²²¹Gly amino acid substitution with the mutant haplotype 4 allele of this family. Allele-specific oligonucleotide (ASO) dotblot analysis subsequently detected the Leu⁴⁸Ser 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 Glu²²¹Gly mutation has only been detected within patient E1 and his father.     

Phenylketonuria: detection of a frequent haplotype 4 allele mutation

Summary By sequence analysis of 94 phenylketonuria (PKU) alleles using polymerase chain reaction (PCR) based techniques, we identified a G to A transition in exon 5 of the human phenylalanine hydroxylase gene. This base substitution predicts an Arg¹⁵⁸Glu¹⁵⁸ amino acid exchange and is strongly associated with the mutant haplotype 4 PKU allele.     

Novel PKU mutation on haplotype 2 in French-Canadians.

We analyzed DNA from nine French-Canadian probands from eastern Quebec province; all had hyperphenylalaninemia (phenylketonuria [PKU] or non-PKU forms) caused by mutations at the phenylalanine hydroxylase locus. Analysis of RFLP haplotypes and mutations revealed a novel mutation, an A-to-G transition (met----val) in codon 1 (the translation-initiation codon). It occurred on 5 of the 18 mutant chromosomes and was associated each time with haplotype 2. A proband homozygous for this mutation had the PKU phenotype. In other probands, the codon 1 mutation was inherited once with the splice junction mutation in exon 12 (on haplotype 3), conferring PKU, and was inherited twice with a mutation on haplotype 1, conferring PKU in one proband and non-PKU hyperphenylalaninemia in the other. The other five probands carried mutations, conferring PKU, on the following haplotype combinations: 1/3 (twice), 1/9, 3/4, and 1/1. The mutations on haplotypes 1, 4, and 9 are not yet characterized. This preliminary study reveals a novel PKU mutation and considerable genetic heterogeneity at the phenylalanine hydroxylase locus in French-Canadians.     

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

Summary 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 mutantspecific oligonucleotide probe.     

Geographical distribution gradients of the major PKU mutations and the linked haplotypes

Summary Analysis of 81 phenylketonuria families from Bulgaria, Lithuania and eastern Germany demonstrated a high frequency of haplotype 2 and the associated Arg⁴⁰⁸ Trp⁴⁰⁸ substitution. Haplotype 3 and the splicing mutation in intron 12 are rare or absent in the groups studies. Pooling the data on European populations suggests a Balto-Slavic origin of the defect in codon 408 of the phenylalanine hydroxylase gene and a geographical gradient in the distribution of both major PKU mutations which may contribute to the higher incidence of classic PKU in northern Europeans.     

Apparent higher frequency of phenylketonuria in the Mexican state of Jalisco

The geographic origin of Mexican patients with phenylketonuria (PKU) in Mexico City and in southern California was studied. Compared to patients with other metabolic disorders, patients with PKU were significantly more likely to have originated from the Los Altos region of the state of Jalisco and its environs. The incidence of PKU among mentally retarded students attending special education schools was found to be significantly higher in Jalisco (particularly the Los Altos region) than in the neighboring state of Guanajuato (1.09% vs 0.3%). These results strongly suggest a population of origin effect, the mutant allele(s) having been introduced by the Spanish ancestors of the current population. Our findings also support the addition of PKU to the neonatal screening program for this region of Mexico.     

A missense mutation,S349P,completely inactivates phenylalanine hydroxylase in North African Jews with phenylketonuria

The majority of hyperphenylalaninemias (HPAs) result from mutations at the gene for phenylalanine hydroxylase (PAH). The broad phenotypic variability of these conditions, ranging from phenylketonuria (PKU) to mild benign HPA, is underlain by a wide spectrum of mutations giving rise to various genotypic combinations. Mutant PAH alleles, labeled by specific polymorphic haplotypes and mutations, are becoming useful markers in human population genetics. We report here a mutant PAH allele found in Jews from Morocco and Tunisia, marked by haplotype 4 and a missense mutation, TCA^SerCCA^pro, at codon 349 in exon 10 of the gene. In vitro expression of the mutation showed normal levels of mRNA with virtually no enzymatic activity or protein immunoreactivity, pointing to a highly unstable protein. A homozygote for this mutation showed the most severe (classical) type of PKU, while compound heterozygotes showed two other types of HPA — atypical PKU and high benign HPA — illustrating the interplay between different mutations that gives rise to various HPAs.     

Linkage analysis of neurofibromatosis type 1

Summary In order to investigate the molecular basis of phenylketonuria (PKU) in the Polish population, we screened 44 mutant chromosomes from PKU probands for six known mutations, frequently occurring in western European countries, by polymerase chain reaction amplification of their genomic DNA and hybridization with allele-specific oligonucleotides. Our results show that the majority (66%) of all PKU alleles are characterized by three different mutations: in codon 408 (56.8%), codon 158 (6.8%) and codon 261 (2.27%). Of the mutant haplotype 2 alleles, 96% were linked to the mutation in codon 408. Out of five mutant haplotype 4 alleles, three showed the codon 158 mutation, and out of four mutant haplotype 1 alleles, one had the codon 261 mutation. In two families, MspI digests revealed an additional 13.5-kb band similar in length to that previously reported. However, analysis of exon 9 excluded the presence of the T to C transition originally described, indicating a new MspI variant in the Polish population.     

Familial hypercholesterolemia in South African Afrikaners

Summary Familial hypercholesterolemia (FH), at a prevalence of more than 1 in 100, is at least five times more common in one South African population group than in populations in North America and Europe. Fourteen homozygotic tamilial hypercholesterolemic subjects from this South African group were genotyped for two intragenic DNA restriction fragment length polymorphisms (RFLPs) in the LDL-receptor gene. A Stu I polymorphism is located in exon 8, and a Pvu II polymorphism, in intron 15. Of ten unrelated FH homozygotes genotyped for both RFLPs, nine were homozygous for an S+P- haplotype, and one was heterozygous for an S+P-/S-P+ heplotype. The remaining four were genotyped for Pvu II only and were homozygous for P-. Compared with a previously determined population frequency for the latter, this represents an association (P<0.05) between the frequency for the P- allele and FH in this population, and this finding is consistent with the founder gene effect previously postulated to be present on genealogical and biochemical evidence.     

Two distinct variants of erythrocyte spectrin βIV domain

Summary Thirty-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.     

A frameshift mutation in exon 2 of the phenylalanine hydroxylase gene linked to RFLP haplotype 1

Summary A deletion of a single base in codon 55 (exon 2) of the phenylalanine hydroxylase (PAH) gene has been identified by direct DNA sequencing of 94 phenyl-ketonuria (PKU) chromosomes. This mutation alters the reading frame so that a stop signal (TAA) is generated in codon 60 of the PAH gene. Haplotype analysis revealed that all PKU alleles showing the codon 55 frameshift mutation exhibited haplotype 1. In our panel of DNA probes 13% of all mutant haplotype 1 alleles carry this particular mutation. Patients who were compound heterozygotes for this deletion and R408W in exon 12, or the splice mutation in intron 12, were affected by severe PKU. Thus, the clinical data provide additional evidence that haplotype 1 PKU alleles carry molecular defects which confer a null phenotype. In addition, we were able to show that the newly detected mutation occurs on alleles of different ethnic background.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase (PAH) locus in Asian families with phenylketonuria (PKU)

DNA polymorphisms at the phenylalanine hydroxylase (PAH) locus have proved highly effective in linkage diagnosis of phenylketonuria (PKU) in Caucasian families. More than 10 RFLP sites have been reported within the PAH structural locus in Caucasians. With information from affected and unaffected offspring in PKU families it is often possible to reconstruct complete RFLP haplotypes in parents and to use these haplotypes to follow the segregation of PKU within families and to determine the distribution of PKU chromosomes within populations. To establish the utility of these RFLPs in characterizing Asian families with PKU, we typed eight DNA sites in 21 Chinese families and 12 Japanese families with classical PKU. The eight RFLPs were chosen for their informativeness in Caucasians. From these families we reconstructed a total of 91 complete PAH haplotypes, 44 from non-PKU chromosomes and 47 from PKU-bearing chromosomes. Although all eight marker sites are polymorphic in both Chinese and Japanese, there is much less haplotypic variation in Asians than in Caucasians. In particular, one haplotype alone, haplotype 4, accounts for more than 77% of non-PKU chromosomes and for more than 80% of PKU-bearing chromosomes. Haplotype 4 is also relatively common in Caucasians. The next most common Asian haplotype is 10 times less frequent than haplotype 4. By contrast, in many Caucasian populations the sum of the frequencies of the five most common haplotypes is still less than 80%, and several of the most common haplotypes are equally frequent. Even though the extent of haplotypic variation in Asians is severely limited, the few haplotypes that are found often differ at a number of RFLP sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Missense mutations associated with RFLP haplotypes 1 and 4 of the human phenylalanine hydroxylase gene.

We report missense mutations associated with haplotype 1 and haplotype 4 alleles of the human phenylalanine hydroxylase (PAH) gene. Individual exon-containing regions were amplified by polymerase chain reaction from genomic DNA of a PKU patient who was a haplotype 1/4 compound heterozygote. The amplified DNA fragments were subcloned into M13 for sequence analysis. Missense mutations were observed in exons 5 and 7, resulting in the substitution of Arg by Gln at residues 158 and 261 of the enzyme, respectively. Expression analysis in heterozygous mammalian cells after site-directed mutagenesis demonstrated that the Arg158-to-Gln158 mutation is a PKU mutation, whereas the Arg261-to-Gln261 mutation is apparently silent in the assay system. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the Arg158-to-Gln158 mutation is present in two of six mutant haplotype 4 alleles among the Swiss and constitutes about 40% of all mutant haplotype 4 alleles in the European population. The mutation is not present in normal alleles or in any mutant alleles of other haplotypes. The results provide conclusive evidence that there is linkage disequilibrium between mutation and haplotype in the PAH gene and that multiple mutations have occurred in the PAH gene of a prevalent haplotype among Caucasians.     

Linkage disequilibrium between RFLP haplotype 2 and the affected PAH allele in PKU families from the Berlin area of the German Democratic Republic

Summary Probands from 26 PKU-affected families of the Berlin area were analyzed with respect to the allele frequency distribution of six RFLPs in linkage with the normal and the PKU alleles of the phenylalanine hydroxylase gene. These investigations confirm most of the RFLP haplotypes observed by Güttler and colleagues in the Danish population and describe two additional ones. They detect no significant differences in the single RFLP or RFLP haplotype distribution on the normal chromosomes in comparison with the Danish families and confirm a prevalent association of the RFLP haplotypes 1,4 and 7 with the normal PAH allele. In contrast to the Danish investigations, in our study the PKU allele is found most frequently linked to haplotype 2, rather then to haplotype 3. In one of our patients we found a substitution of the normal 19-kb MspI fragment by a 13.5- and a 5.5-kb fragment, reported up to now only in one other German family.     

Phenylalanine hydroxylase gene: novel missense mutation in exon 7 causing severe phenylketonuria.

By direct sequence analysis of 94 mutant phenylalanine hydroxylase alleles using polymerase chain reaction-based techniques, we identified a C to T transition in exon 7 of the human phenylalanine hydroxylase gene that is associated with RFLP haplotypes 1 and 4. A leucine for proline substitution at position 281 can be predicted from the nucleotide sequence of the mutant codon. Expression analysis in cultured mammalian cells after site-directed mutagenesis proved that the base substitution is a disease causing gene lesion. Dot-blot hybridization analysis using allele-specific oligonucleotides revealed that 25% of all mutant haplotype 1 alleles in the German population bear this mutation. In addition, this mutation could be detected on one mutant haplotype 4 allele. The fact that this mutation is associated with only 25% of all mutant haplotype 1 alleles suggests that multiple mutations may be associated with this haplotype. The occurrence of several different mutations would be in agreement with the clinical heterogeneity observed in the group of patients whose PKU alleles belong to haplotype 1.     

Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic und mutational analysis at the SCN4A locus, which encodes the -subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.This paper is dedicated to Professor Dr. Dr. Peter Emil Becker on the occasion of this 85th birthday     

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

Summary The 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.     

Phenylketonuria mutations in Germany

We report the spectrum of mutations and associated modified haplotypes in patients with phenylketonuria living in Germany. A total of 546 independent alleles was investigated, including 411 of German and 65 of Turkish descent. Mutations were identified for 535 PKU alleles (98%) and there were 91 different mutations. The most common mutation was R408W on 22% of alleles. Two mutations, IVS12+1G→A and IVS10–11G→A accounted for just under 10% of alleles, whereas the remaining mutations were found at relative frequencies of 6% or less; 43 mutations were observed once only. IVS10–11G→A was the most common mutation (38% of alleles) in the subgroup of patients of Turkish descent. Modified haplotypes were determined from the analysis of four silent mutations, three diallelic restriction fragment length polymorphisms, a variable number of tandem repeats minisatellite and a short tandem repeat microsatellite in the phenylalanine hydroxylase gene, showing that a considerable proportion of mutations must have recurred in independent founders; other mutations may have changed chromosomal haplotype backgrounds by gene conversion. The spectrum of PKU mutations in Germany reflects the history of a heterogenous Central European population living at the crossroads of migration throughout the centuries. Received: 11 January 1999 / Accepted: 11 March 1999     

PKU locus: Genetic linkage with human amylase (Amy) loci and assignment to linkage group I

Summary The linked alpha-amylase loci Amy 1 and Amy 2 were evaluated for their linkage relationship to the PKU locus using data collected from two (one Czech and one Polish) groups of families. The five sibships informative for Amy 1: PKU give a score of 1.505 at =0.00 and the eight sibships informative for Amy 2: PKU give a score of 2.709 at =0.00. Due to the tandem position of Amy 1 and Amy 2 loci, these data could be combined, and linkage between Amy and PKU loci established with a score 4.214 at =0.00. The practical significance of the linkage, especially for identifying PKU allele carriers, is emphasized.