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.     

Haplotype distribution and mutations at the PAH locus in Croatia

Summary Restriction 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.     

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.     

Phenylketonuria in Spain: RFLP haplotypes and linked mutations

In order to investigate the molecular basis of phenylketonuria (PKU) in Spain, we analyzed the restriction fragment length polymorphism (RFLP) haplotypes and common mutations in the phenylalanine hydroxylase (PAH) gene in 32 unrelated Spanish PKU families. The distribution of RFLP haplotypes differs from that of northern Europe. Mutant haplotypes 2 and 3 were completely absent in our sample. Approximately 65% of the mutant alleles are confined to three RFLP haplotypes, namely haplotypes 1, 6 and 9, also frequently found in other Mediterranean populations. We screened for previously described PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides, and found IVS10,165T, E280K and P281L as the major mutations, representing 41% of the PKU alleles. Other mutations found were Y414C, and a new one, P244L. Mutations R408W and IVS12, prevalent in northern Europe, as well as others present in southern European populations (R252W, R261Q, L249F) were not detected in our sample. Our results reveal the genetic heterogeneity present in the Spanish PKU population, which shows similarities to others of Mediterranean origin.     

A termination mutant prevalent in Norwegian haplotype 7 phenylketonuria genes.

RFLPs in the phenylalanine hydroxylase (PAH) gene locus were determined in 47 Norwegian nuclear families that had at least one child with phenylketonuria (PKU). The PKU haplotype distribution differed somewhat from that of other European populations. Mutant haplotype 7 is relatively rare in other populations but constituted 20% of all mutant haplotypes in Norway. In 14 of the 17 mutant haplotypes 7, a previously unreported deletion of the BamHI restriction site in exon 7 of the PAH gene was observed. The abrogation of the BamHI site was shown to be due to a G-to-T transversion, changing Gly 272 to Ter 272 in exon 7 of the gene, thus directly identifying the PKU mutation. Unlike the families of the other PKU patients, the families with this mutation clustered along the southeastern coast of Norway, suggesting a founder effect for this mutation.     

Polymorphic DNA haplotypes at the phenylalanine hydroxylase locus and their relation to phenotype in Swedish phenylketonuria families

Summary The genetic heterogeneity at the phenylalanine hydroxylase (PAH) locus was studied in 88 families including 93 of the 105 children with phenylketonuria (PKU) or hyperphenylalaninemia (HPA) detected through the Swedish neonatal screening program from 1966 to the end of 1986. Haplotypes based on eight restriction fragment length polymorphisms (RFLPs) at the PAH locus could be constructed for 132 normal and 136 mutant alleles. The normal alleles were of 27 different RFLP haplotypes, 9 of which have not been described previously, but there was a dominance of a few haplotypes common to many European populations. The distribution of mutant alleles was significantly different from that in neighboring countries, even though over 90% of all mutant alleles were confined to six RFLP haplotypes, also prevalent in other European populations. Allele-specific oligonucleotide hybridization analysis for the Arg⁴⁰⁸ to Trp⁴⁰⁸ mutation and for the G to A splicing mutation in intron 12 showed exceptions to the previously reported linkage of these mutations to mutant haplotypes 2 and 3, respectively. Correlation of mutant alleles with clinical phenotypes pointed to the presence of at least two different mutations associated with each of six haplotypes. We argue that PKU/HPA in the Swedish population may be caused by at least 13 different mutations in addition to the 4 already identified. The theoretical informativity of RFLP analysis in heterozygote detection and prenatal diagnosis in PKU/HPA families was estimated at approximately 85%. Carrier detection could, in effect, be accomplished for 88% of the 56 healthy siblings in the families studied.     

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.     

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

DNA haplotype data from the phenylalanine hydroxylase (PAH) locus are available from a number of European populations as a result of RFLP testing for genetic counseling in families with phenylketonuria (PKU). We have analyzed data from Hungary and Czechoslovakia together with published data from five additional countries--Denmark, Switzerland, Scotland, Germany, and France--representing a broad geographic and ethnographic range. The data include 686 complete chromosomal haplotypes for eight RFLP sites assayed in 202 unrelated Caucasian families with PKU. Forty-six distinct RFLP haplotypes have been observed to date, 10 unique to PKU-bearing chromosomes, 12 unique to non-PKU chromosomes, and the remainder found in association with both types. Despite the large number of haplotypes observed (still much less than the theoretical maximum of 384), five haplotypes alone account for more than 76% of normal European chromosomes and four haplotypes alone account for more than 80% of PKU-bearing chromosomes. We evaluated the distribution of haplotypes and alleles within these populations and calculated pairwise disequilibrium values between RFLP sites and between these sites and a hypothetical PKU "locus." These are statistically significant differences between European populations in the frequencies of non-PKU chromosomal haplotypes (P = .025) and PKU chromosomal haplotypes (P much less than .001). Haplotype frequencies of the PKU and non-PKU chromosomes also differ significantly (P much less than .001. Disequilibrium values are consistent with the PAH physical map and support the molecular evidence for multiple, independent PKU mutations in Caucasians. However, the data do not support a single geographic origin for these mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Phenylketonuria mutations and their relation to RFLP haplotypes at the PAH locus in Czech PKU families

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism /variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and I65T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.     

PKU in Slovakia: mutation screening and haplotype analysis

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.     

Recurrent mutation in the human phenylalanine hydroxylase gene.

We report the identification of a missense mutation of Glu280 to Lys280 in the phenylalanine hydroxylase (PAH) gene of a phenylketonuria (PKU) patient in Denmark. The mutation is associated with haplotype 1 of the PAH gene in this population. This mutation has previously been found in North Africa, where it is in linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have been transferred from one haplotype background to another by a double crossover or gene conversion event, the fact that the mutation is exclusively associated with the two different haplotypes in the two distinct populations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the human PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may represent hot spots for mutation in the human PAH locus.     

Phenylketonuria mutation in Chinese haplotype 44 identical with haplotype 2 mutation in northern-European Caucasians

Summary DNA amplification with the polymerase chain reaction was employed to identify the phenylketonuria (PKU) mutation in Chinese PKU families. The amplified DNA was hybridized with oligonucleotides corresponding to the two most common mutant alleles, i.e., mutations associated with PKU haplotype 2 and 3 among Caucasians of northern-European ancestry. The results of analysis demonstrate that the mutation in Chinese haplotype 44 is a single-base substitution corresponding to the mutation associated with haplotype 2 in Caucasians, whereas the mutations of the phenylalanine hydroxylase gene in haplotypes 4, 7, 11 and 28 among Chinese do not correlate with either of the two mutations identified in northern-European Caucasians.     

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.     

Analysis of the phenylalanine hydroxylase gene in the Spanish population: mutation profile and association with intragenic polymorphic markers.

The aim of this study was to characterize the phenylketonuria (PKU) alleles in the Spanish population, by both identifying the causative mutations and analyzing the RFLP haplotypes and the VNTR and short-tandem-repeat alleles associated with the phenylalanine hydroxylase (PAH) gene. We have investigated 129 independent mutant chromosomes, using denaturing gradient gel electrophoresis (DGGE) and direct sequencing. Ninety percent of the alleles were identified, and a total of 40 different mutations were detected. The mutational spectrum includes seven previously unreported mutations: P122Q, D129G, P147S, D151G, A165T, S196fs, and P407S. Seven mutations represent 43% of the Spanish PKU alleles, the most common being IVS10nt-11g-->a (14.7%), I65T (8.5%), and V388M (6.2%). The remaining 33 mutations are rare. The mutation profile and relative frequencies are markedly different from those in northern Europe, also showing unique features compared with those in other, southern European populations. The association analysis with polymorphic markers in the PAH gene provides valuable information for population-genetic studies and investigation of the origins of the mutations. This study may serve as reference in the analysis of the contemporary distributions and frequencies of the PKU mutations in related populations, with particular relevance in Latin American countries.     

Recurrence of the R408W Mutation in the Phenylalanine Hydroxylase Locus in Europeans

The relative frequency of the common phenylalanine hydroxylase (PAH) mutation R408W and its associations with polymorphic RFLP, VNTR, and short-tandem-repeat (STR) sites in the PAH gene were examined in many European populations and one representative North American population of defined European descent. This mutation was found to cluster in two regions: in northwest Europe among Irish and Scottish peoples, and in eastern Europe, including the Commonwealth of Independent States. This allele was significantly less frequent in intervening populations. In eastern European populations, the R408W mutation is strongly associated with RFLP haplotype 2, the three-copy VNTR allele (VNTR 3), and the 240-bp STR allele. In northwestern European populations, it is strongly associated with RFLP haplotype 1, the VNTR allele containing eight repeats (VNTR 8), and the 244-bp STR allele. An examination of the linkage between the R408W mutation and highly polymorphic RFLP, VNTR, and STR haplotypes suggests that recurrence is the most likely mechanism to account for the two different major haplotype associations of R408W in Europe.     

Phenylalanine hydroxylase gene haplotypes in Polynesians: evolutionary origins and absence of alleles associated with severe phenylketonuria.

A total of 630 haplotypes for the phenylalanine hydroxylase (PAH) gene locus were established in five groups of Polynesians comprising Samoans, Tongans, Cook Islanders, Maori, and Niueans. Considerable genetic continuity was demonstrated between these widely dispersed populations, since three common haplotypes (4, 1, and 7) constituted over 95% of alleles. A control group of individuals from Southeast Asia shared the same major haplotypes, 4, 1, and 7, with Polynesians. These data provide further support for the theories of genetic homogeneity and of Asian affinities of the Polynesian precursor populations. The absence of severe phenylketonuria (PKU) in both Polynesians and Southeast Asians is consistent with the lack of PAH haplotypes 2 and 3, on which the severe PKU mutants have arisen among Caucasians.     

Linkage disequilibrium between the four most common cystic fibrosis mutations and microsatellite haplotypes in the Celtic population of Brittany

Microsatellite haplotypes were determined for 117 chromosomes carrying the four most frequent mutations in the cystic fibrosis (CF) gene identified in the Breton population of Celtic origin, as well as for 83 normal chromosomes (noncarriers of a CF mutation). Each of the three non-ΔF508 mutations was associated with a single haplotype: 1078delT with 16-31-13, G551D with 16-7-17, and W846X with 16-32-13. Although these results suggest identity-by-descent for each mutation, recurrent mutations, although unlikely, could not be completely ruled out. The four most frequent haplotypes on normal chromosomes and the three most frequent haplotypes on ΔF508 chromosomes are the same as those found in Ireland, Spain, and Italy. This suggests that some haplotypes, associated or not with the ΔF508 mutation, were present in an ancestral population from which all four populations descended. Received: 27 November 1995 / Revised: 1 February 1996     

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.     

Haplotype distribution of the human phenylalanine hydroxylase locus in Scotland and Switzerland.

RFLP haplotypes at the phenylalanine hydroxylase (PAH) locus were determined in 45 nuclear Caucasian families from Switzerland and Scotland. The RFLPs at the PAH locus are highly informative, and prenatal diagnosis is possible in 85% of the families studied. The data were combined with the profiles previously observed in the Danish population, in order to study the variation in RFLP haplotype distribution among European populations. A total of 22 different haplotypes were observed in Denmark, Switzerland, and Scotland. Fifteen and 19 haplotypes are associated with the normal (non-PKU) and with the mutant chromosomes, respectively. The haplotype distribution and the allele frequency of normal chromosomes remain constant between Denmark, Switzerland, and Scotland. However, both the haplotype distribution and allele frequencies of mutant chromosomes show significant variation between the three countries. Our results suggest there may be additional mutations in the PAH gene that cause PKU.