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)     

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.     

Phenylketonuria in U.S. blacks: molecular analysis of the phenylalanine hydroxylase gene

We investigated the frequency, origin, and molecular basis of phenylketonuria (PKU) in U.S. blacks. On the basis of 10 years of Maryland newborn-screening data, we found the frequency to be 1/50,000, or one-third that in whites. We performed haplotype analysis of the phenylalanine hydroxylase (PAH) gene of 36 U.S. blacks, 16 from individuals with classical PKU and 20 from controls. In blacks, 20% of wild-type PAH alleles have a common Caucasian haplotype (i.e., haplotype 1), whereas 80% had a variety of haplotypes, all rare in Caucasians and Asians. One of these, haplotype 15, accounted for a large fraction (30%). Among black mutant PAH alleles, 20% have a haplotype (i.e., either haplotype 1 or haplotype 4) common in Caucasians; 40% have a haplotype rare in Caucasians and Asians, and 40% have one of two previously undescribed haplotypes. Both can be derived from known haplotypes by a single event. One of these haplotypes is characterized by a new MspI restriction site, located in intron 8, which was present in five of 16 black mutant alleles but was not present in 60 U.S. black control, 20 U.S. Caucasian control, or 20 Caucasian mutant PAH alleles. Sequence analysis of DNA from a single individual, homozygous for the new MspI associated haplotype, shows homozygosity for a C----T transition at nucleotide 896 in exon 7 of the PAH cDNA, resulting in the conversion of leucine 255 to serine (L255S).     

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.     

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.     

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.     

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.     

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.     

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)     

Molecular basis for nonphenylketonuria hyperphenylalaninemia.

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygote's ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

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.     

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.     

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.     

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.     

Polymorphic haplotypes and recombination rates at the LDL receptor gene locus in subjects with and without familial hypercholesterolemia who are from different populations.

RFLPs at the low-density lipoprotein (LDL) receptor locus for TaqI, StuI, HincII, AvaII, ApaLI (5' and 3'), PvuII, and NcoI were studied in Swiss and German families with familial hypercholesterolemia (FH). A total of 1,104 LDL receptor alleles were analyzed using Southern blotting and new PCR-based techniques for detection of the TaqI, StuI, HincII, AvaII, NcoI RFLPs. Two hundred fifty-six independent haplotypes from 368 individuals of 61 unrelated Swiss families, as well as 114 independent haplotypes from 184 subjects of 25 unrelated German families, were constructed. In 76 families, clinical diagnosis of FH was confirmed by cosegregation analysis. Of the 43 unique haplotypes consisting of seven RFLPs detected in the Swiss and Germans, only 9 were common in both population samples. Analysis of linkage disequilibrium revealed nonrandom associations between several of the investigated RFLPs. ApaLI (5'), NcoI, PvuII, TaqI, and AvaII or HincII were particularly informative (cumulative informativeness .85). Relative frequencies, heterozygosity indexes, and PICs of the RFLPs from the Swiss and Germans were compared with values calculated from reported haplotype data for Italians, Icelanders, North American Caucasians, South African Caucasians, and Japanese. Pairwise comparisons of population samples by common RFLPs demonstrated unexpected differences even between geographically adjacent populations (e.g., the Swiss and Germans). Furthermore, genetic distances from the Germans to the other Caucasians were larger than to the Japanese. An unexpected lack of correlation between linkage disequilibria and physical distances was detected for the German and Japanese data, possibly because of nonuniform recombination with excessively high rates between exon 13 and intron 15. Hence, the present study revealed a striking variety of polymorphic haplotypes and heterogeneity of RFLP frequencies and recombination rates among the seven population samples.     

Haplotype analysis at the low density lipoprotein receptor locus: application to the study of familial hypercholesterolemia in Israel

Summary Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein (LDL) receptor gene. It has been shown that restriction fragment length polymorphisms (RFLPs) associated with this gene may be used for family and population studies. The present investigation is a population-based study of 19 Jewish families with hypercholesterolemia representing 9 different countries of origin. Ten RFLP sites were used to construct 24 different haplotypes from 112 chromosomes. These haplotypes vary in frequency from 0.9% to 28.6%. Five previously undescribed haplotypes, which comprise 8.1% of the sample, are reported here. The six most common haplotypes account for 70% of the sample. Segregation analysis reveals that, in Israel, distinct LDL receptor haplotypes are associated with hypercholesterolemia in 12 (63%) out of the 19 Jewish families. Five LDL receptor haplotypes co-segregate with hypercholesterolemia. Two of these haplotypes seem to be unique to specific population groups in Israel and may therefore represent founder mutations.     

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.     

Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus.

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.     

<Emphasis Type="Italic">FMR1</Emphasis> haplotype analyses among Indians: a weak founder effect and other findings

This study on allelic/haplotypic fragile X associations evaluated using STR (DXS548, FRAXAC1, FRAXAC2) and SNP (ATL1) markers flanking the (CGG)(n) locus of FMR1is the first report from the large ethnically complex Indian population. Results have been compared with allele/haplotype distributions reported for other major ethnic groups, including White Caucasians, Africans, and Pacific Asians. Though overall allele frequency distributions at the individual loci are more similar to Western Caucasians compared with others, significant differences are observed in haplotypic associations with the mutated X. The striking findings are: (1) high diversity and heterozygosity of haplotypes among fragile X chromosomes ( n=40) and controls ( n=262), including four haplotypes found exclusively in this study sample; (2) weak association of DXS548-FRAXAC1-FRAXAC2 haplotypes, 2-1-3, 6-3-3+ and 7-4-6+ with the disorder, and absence of White Caucasian fragile X haplotypes 6-4-4 and 6-4-5; (3) weak founder effect for the fragile X expansion mutation in the Indians; (4) lack of a continuum of haplotype-based FMR1 alleles between intermediate (CGG)(n) size ranges and expanded alleles; (5) exclusion of ATL1 as a candidate genetic indicator of FMR1 instability. The high STR-based haplotype diversity observed among fragile X lineages, irrespective of ethnic alliances, strongly suggests the inappropriateness of using STR haplotypes to infer predisposition to instability among ethnically separated fragile X pedigrees and may reiterate the need for identifying newer SNPs from this region to not only determine true founder effects for the fragile X mutation, but also decipher possible mechanisms leading to CGG instability.