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.     

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.     

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.     

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.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

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.     

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)     

DNA haplotype analyses of patients with hyperphenylalaninemia.

Linkage analysis of phenylketonurics has shown a strong association between the DNA haplotype at the phenylalanine hydroxylase (PAH) locus and phenylketonuria (PKU). Similarly, a genetic linkage between less severe forms of hyperphenylalaninemia (HPA) and the PAH locus has been suggested. In the present study we analyzed this linkage in more detail. Haplotypes at the PAH locus were determined for 19 individuals with moderately elevated plasma phenylalanine and normal urinary neopterin/biopterin ratios. Fourteen of these individuals had plasma phenylalanine levels of 4-10 mg/dl (mild HPA), and the other five had plasma phenylalanine levels of 10-19 mg/dl (atypical PKU). Thirteen of the 15 HPA families consisted of an affected child and at least one other sibling. Elevated plasma phenylalanine was seen to genetically segregate with specific PAH alleles in each family. Summation of the LOD scores for both categories of moderate plasma phenylalanine elevation gave a maximum value of 3.556 at theta = 0. At theta = 0 this gives a probability of linkage between the PAH locus and the locus for moderate phenylalanine elevations that is approximately 3,600:1. None of the alleles segregating with either mild HPA or atypical PKU were of haplotype 2 or 3, and 13/20 were of types 1 or 4. This is in agreement with the most deleterious mutations being on haplotypes 2 and 3 and with the less severe mutations being on haplotypes 1 and 4. chi 2 Analyses indicated no statistically significant correlation between HPA and a particular haplotype or restriction-enzyme site.     

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

Summary Restriction 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.     

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.     

Compound heterozygosity in nonphenylketonuria hyperphenylalanemia: the contribution of mutations for classical phenylketonuria

Hyperphenylalaninemia (HPA) results from defective hydroxylation of phenylalanine in the liver, in most cases because of defective phenylalanine hydroxylase. HPA is highly variable, ranging from moderate elevation of plasma phenylalanine with no clinical consequences to a severe disease, classical phenylketonuria (PKU). Non-PKU HPA was found in excess of PKU in Israel, while the opposite is true in Europe. To study the genetic basis of non-PKU HPA, we performed haplotype analysis at the phenylalanine hydroxylase locus in 27 families with non-PKU HPA. All individuals with this condition were compound heterozygotes. In six of these families, in which both PKU and non-PKU HPA were segregating, haplotype analysis showed that non-PKU HPA resulted from compound heterozygosity for a PKU mutation and a second mutation, with milder effect, which is probably expressed only when it interacts with the severe mutation. The involvement of PKU mutations in non-PKU HPA was further demonstrated in Jewish Yemenite families with non-PKU HPA, in which the individuals with this condition were carriers of the single PKU allele which exists in this community. In addition, two previously known PKU point mutations (R261Q and R408W) were found in individuals with non-PKU HPA. These mutations are associated, in our population, with the same haplotypes as those with which it is associated in Europe. Based on the above-mentioned genetic model for non-PKU HPA, successful prenatal diagnosis of this condition was performed in one family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haplotype distribution and molecular defects at the phenylalanine hydroxylase locus in Italy

Summary In order to investigate the molecular basis of phenylketonuria (PKU) in Italy, we characterized the RFLP haplotypes at the phenylalanine hydroxylase gene in 38 unrelated Italian PKU families. The distribution of haplotypes associated with PKU alleles differs from that of other European populations. In particular, haplotypes 1 and 6 are present in 39.7% and 17.6% of the PKU chromosomes, whereas the frequencies of haplotypes 2 and 3 are 5.9% and 2.9%, respectively. The characterization of PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides shows that 1 out of 2 haplotypes 3 carries the splicing mutation and that 2 out of 4 haplotypes 2 carry the missense mutation associated with these haplotypes in North European populations. Our results indicate that the two molecular defects most frequent in Northern Europe represent a minority of PKU mutations in Italy.     

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.     

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.     

RFLP haplotyping and mutation analysis of the phenylalanine hydroxylase gene in Dutch phenylketonuria families

Restriction fragment length polymorphism haplotyping of mutated and normal phenylalanine hydroxylase (PAH) alleles in 49 Dutch phenylketonuria (PKU) families was performed. All mutant PAH chromosomes identified by haplotyping (n = 98) were screened for eight of the most predominant mutations. Compound heterozygosity was proven in 40 kindreds. Homozygosity was found for the IVS12nt1 mutation in 5 families, and for the R158Q and IVS10nt546 mutations in one family each. All patients from these families suffer from severe PKU, providing additional proof that these mutations are deleterious for the PAH gene. Genotypical heterogeneity was evident for mutant haplotype 1 (n = 27) carrying the mutations R261Q (n = 12), E280K (n = 4), P281L (n = 1) and unknown (n = 10), and likewise for mutant haplotype 4 (n = 30) carrying the mutations R158Q (n = 13), Y414C (n = 1) and unknown (n = 16). Mutant haplotype 3 (n = 20), in tight association with mutation IVS12nt1, appeared to be in strong linkage disequilibrium (LDE) with its normal counterpart allele (n = 4). Mutant haplotype 6 (n = 4), in tight association with the IVS10nt546 mutation, showed moderate LDE with its counterpart allele (n = I). The distribution of the mutant PAH haplotypes 1, 3 and 4 among the Dutch PKU population resembles that in other Northern and Western European countries, but it is striking that mutant haplotype 2 and its associated mutation R408W is nearly absent in The Netherlands, in strong contrast to its neighbouring countries.     

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.     

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.     

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria

Summary Eight polymorphic restriction enzyme sites at the phenylalanine hydroxylase (PAH) locus were analyzed from the parental chromosomes in 33 Danish nuclear families with at least one phenylketonuric (PKU) child. Determination of haplotypes of 66 normal chromosomes and 66 chromosomes bearing mutant allele (S) demonstrated that there are at least two haplotypes which occur predominantly on PKU chromosomes and rarely otherwise. Overall, the relative frequencies of the various haplotypes are significantly different on PKU-and normal-allele bearing chromosomes, even though there is no predominantly occurring unique haplotype which can characterize the PKU chromosomes. In addition, no significant association (linkage disequilibrium) between any single polymorphic site and the mutant allele (s) was observed. The results suggest that either the phenylketonuric mutation was very ancient so that the polymorphic sites and the mutation have reached linkage equilibrium or the mutant allele (s) are the results of multiple mutations in the phenylalanine hydroxylase gene in man. Furthermore, a crude relationship between standardized linkage disequilibria and physical map distances of the polymorphic sites indicates that there is no apparent recombination hot-spot in the human phenylalanine hydroxylase gene, since the recombination rate within the locus apears to be uniform and likely to be occurring at a rate similar to that within the HLA gene cluster. The limitations of this later analysis are discussed in view of the sampling errors of disequilibrium measure used, and the potential untility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.     

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene.

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiple of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between these alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations.     

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)