Recurrent mutation in the human phenylalanine hydroxylase gene

Am. J. Hum. Genet. 46:919-924, 1990

Recurrent Mutation in the Human Phenylalanine HydroxylaseGeneYoshiyuki Okano,* Tao Wang,* Randy C. Eisensmith,* Fleming Guttlert and Savio L. C. Woo*

*Howard Hughes Medical Institute, Department of Cell Biology and Institute of Molecular Genetics, Baylor College of Medicine, Houston; andtjohn F. Kennedy Institutiet, Glostrup, Denmark

Summary

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 1of the PAH gene in this population. This mutation has previously been found in North Africa, where it isin linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have beentransferred from one haplotype background to another by a double crossover or gene conversion event, thefact that the mutation is exclusively associated with the two different haplotypes in the two distinct popu-lations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the hu-man PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may representhot spots for mutation in the human PAH locus.

Introduction

The study of phenylketonuria (PKU) at the molecularlevel began with the isolation of several phenylalaninehydroxylase (PAH) cDNA clones from a human livercDNA library (Robson et al. 1982; Kwok et al. 1985).The use of these clones as probes in Southern analysesrevealed the presence of eight RFLPs in or near the hu-man PAH gene (Woo et al. 1983; Lidsky et al. 1985b;DiLella et al. 1986a). The presence of these RFLPs per-mitted prenatal diagnosis and carrier screening in PKUfamilies (Lidsky et al. 1985a; Ledley et al. 1988). Thusfar, over 46 different haplotypes have been observedat the PAH locus (Woo 1988; Daiger et al. 1989a).An examination of the frequency and distribution

ofPAH haplotypes among normal and mutant individ-uals in several populations demonstrated a strong as-sociation between specific haplotypes and PKU alleles(DiLella et al. 1986a, 1987a; Chakraborty et al. 1987;Aulehla-Scholz et al. 1988; Herrmann et al. 1988;

Received October 10, 1989; revision received December 11, 1989.Address for correspondence and reprints: Savio L. C. Woo, Howard

Hughes Medical Institute, Baylor College of Medicine, One BaylorPlaza, Houston, TX 77030.C) 1990 by The American Society of Human Genetics. All rights reserved.0002-9297/90/4605-0009$02.00

Lichter-Konecki et al. 1988b, 1989; Rey et al. 1988;Riess et al. 1988; Chen et al. 1989; Daiger et al. 1989a,1989b; Hertzberg et al. 1989; Sullivan et al. 1989). Thissituation is similar to that seen in the human 13-globinlocus, where P-thalassemia mutations are tightly linkedto specific haplotypes present in different ethnic popu-lations (Orkin and Kazazian 1984). The subsequentcharacterization of the molecular defects associated withseveral common mutant PAH alleles confirms the pres-ence of linkage disequilibrium between specific PKUmutations and PAH haplotypes (DiLella et al. 1986b,1987b; Marvit et al. 1987). The results suggest thatthese PKU mutations have occurred fairly recently, sincethey have not yet had time to spread to other haplo-types by crossover or gene conversion events.The presence of linkage disequilibrium has hereto-

fore been observed in every PKU mutation examined(DiLella et al. 1986b, 1987b; Lichter-Konecki et al.1988a; Lyonnet et al. 1989; Wang et al. 1989, 1990;Okano et al. 1990). These findings further suggest thateach PKU mutation has been caused by an indepen-dent mutational event which has occurred on a specifichaplotype background. In 1-thalassemia, where over40 mutations have been observed, several mutationswere shown to be present in several different haplotypes,either because of recurrent mutational events or throughcrossovers or gene conversions (Wong et al. 1986). Lyon-

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Okano et al.

net et al (1989) have previously reported in codon 280a PKU mutation which results in the substitution oflysine for glutamate. This mutation was observed tobe in linkage disequilibrium with haplotype 38 in theAlgerian population. In the present paper, we reportthe presence of the same codon 280 mutation in a haplo-type 1 allele of a Danish PKU patient. This is the firstinstance of the same mutation appearing on differentchromosomal haplotype backgrounds in PKU.

Material and Methods

1. Amplification of Genomic DNA

Genomic DNA was isolated from white blood cellsof a Danish PKU patient bearing haplotype 1 and 2mutant alleles. The 13 exon-containing regions of the90-kb PAH gene were amplified by polymerase chainreaction (PCR) for 35 cycles, according to a methoddescribed elsewhere (Wang et al. 1990). Phosphorylatedoligonucleotides intronic to the gene were purchasedfrom Genetic Designs Inc. (Houston) and were usedas amplification primers.

2. Sequencing of PCR ProductsAmplified exonic DNAs were purified by elution from

agarose gels onto Gene-Clean (Bio 101) according tomanufacturer's instructions. The purified double-strandDNAs were sequenced directly using SequenaseT (USB)and dimethyl sulphoxide, according to a method de-scribed elsewhere (Wang et al. 1990).

Normal MutantGAT CG AT C

G --ow. *- G+ A

(Exon -7)

GIU280Normal 5'-CCCCGAACCGT-3'

Mutant 5 ----AAA---- 3'

Lys280Figure I Identification of missense mutation in exon 7 of thehuman PAH gene. The amplified exon 7-containing regions froma normal individual and from the patient were sequenced with theprimer (5'-AGATGACGCTCAGTGTG-3'). The patient shows G andA bands, while the normal individual shows a G band. The G-to-Atransition in exon 7 results in the substitution of the Glu8'3 codonby the Lys28'1 codon.

normal DNA sequence (fig. 1). This result demonstratesthe presence of two alleles within the patient's DNA,one of which bears a G-to-A transition at the first baseof codon 280. This missense mutation causes the sub-stitution of lysine for glutamate at amino acid 280 ofPAH.

3. Dot Hybridization

Amplified samples were applied directly onto Zeta-probe membranes (BioRad) by using a dot-blot mani-fold (Schleicher and Schuell) and were analyzed by hy-bridization with allele-specific oligonucleotide probes(17-mer) for the normal and mutant alleles separately,according to a method described elsewhere (DiLella etal. 1987a, 1988).

Results

Identification of a Missense Mutation in the PAH Gene

Exon 7 plus flanking intronic regions of the PAH geneof the Danish PKU patient were obtained by PCR am-plification, as elsewhere reported by Okano et al. (1990).The amplified product was subjected to direct DNAsequence analysis. The sequence gel revealed two bands(G and A) at the same position in exon 7 of the pa-tient's DNA, while only a G band was present in the

Linkage Disequilibrium between the Missense Mutationand Haplotype I

Genomic DNA samples from the Danish PKU fam-ily were independently amplified by PCR and were ana-lyzed by dot-blot hybridization using allele-specificoligonucleotide probes. The normal 280 probe is com-plementary to the antisense strand of the normal genesequence. It hybridized with DNA of all three mem-bers of the family, indicating successful amplification(fig. 2). The mutant 280 probe is complementary tothe sense strand of the normal exon sequence exceptfor a G-A mismatch at the mutation site. It hybridizedwith the proband sample, demonstrating that the mis-sense mutation is present in the patient's genomicDNAand is not an artifact of PCR. It also hybridized withthe paternal sample, which bears a mutant haplotype1 allele, but not with the maternal sample, which bearsa mutant haplotype 2 allele. The maternal haplotype2 allele previously has been shown, by dot-blot hybrid-

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Mutation in Phenylalanine Hydroxylase Gene

Ii1 2 1

Normal Probe

Mutant Probe

Figure 2 Transmission of the missense mutant alleles in a Dan-ish PKU family. Genomic DNA was isolated from leukocytes. Theexon 7-containing region (291 bp) of PAH was amplified by PCR,dot-blotted onto z-probe membrane, and hybridized with allele-specificoligonucleotide probes. The following probes were used to detectthe substitution at codon 280 in exon 7: the normal probe (5'-TCACGGTTCGGGGGTAT-3') is the antisense DNA strand, and themutant probe (5'-ATACCCCCAAACCGTGA-3') is the sense DNAstrand. Blackened portions of square and diamond denote mutant

haplotype 1 allele; cross-hatched portions of circle and diamond de-note mutant haplotype 2 allele; white portions of square and circledenote normal haplotype alleles.

ization analysis using allele-specific oligonucleotides,to bear the Arg408-to-Trp408 missense mutation (DiLellaet al. 1987b). Thus the Glu280-to-Lys280 mutation inexon 7 is associated with a haplotype 1 allele in thisfamily.

Allele-specific oligonucleotide hybridization analy-sis was then performed on the panel of Danish PKUfamilies that had previously been haplotyped. Amonga total of 67 mutant alleles analyzed, three bear thecodon 280 mutation and all are haplotype 1 alleles (ta-ble 1). This mutation is not present in either 50 normalalleles or 49 non-haplotype 1 mutant alleles.A second patient is a compound heterozygote of

haplotype 1 and 4 alleles, of which the haplotype 4allele is an unknown mutant. A third patient is a com-

Table I

Association of the Glu2SO-to-LysZW Mutation with MutantHaplotype I Alleles in Denmark

No. OF HYBRIDIZING ALLELES/TOTAL

No. OF GENES ANALYZED

HAPLOTYPE Normal Alleles Mutant Alleles

1 ......0/18 3/182 0...... /1 0/113 0/...... /1 0/224 0...... /18 0/11Other 0...... 0/12 0/5

pound heterozygote of haplotype 1 and 3 alleles, ofwhich the haplotype 3 allele was shown, by dot-blothybridization analysis, to bear the splicing mutationat the exon 12/intron 12 boundary (DiLella et al.1986b). All three patients show a classical PKU pheno-type, which is defined as neonatal blood phenylalaninelevels of >1,200 jmol/liters and a phenylalanine toler-ance at 5 years of age of 10-20 mg/kg of body weight/d(Guttler et al. 1987).

Discussion

Lyonnet et al. (1989) have recently reported a Glu280-to-Lys280 mutation which is present in about 10% ofall PKU alleles in North Africa. The same mutationhas now been observed in three Danish PKU patientsbut has not been found in the Swiss, Hungarian, andCzechoslovakian populations (data not shown). Mostimportant, the identical mutations are present on twodifferent haplotype backgrounds. In North Africa themutation is in linkage disequilibrium with haplotype38, and in Denmark it is associated with haplotype 1.This discrepancy can be resolved by either of two hy-potheses: (1) it represents two independent mutationalevents recurring at the same nucleotide of the 90 kbPAH gene, or (2) it is the result of a crossover or geneconversion event between the two haplotypes duringmeiosis. Although haplotypes 1 and 38 differ from eachother at four individual RFLP sites (BglII, PvuIIa,PvuIIb, and HindIII), a simple double crossover or geneconversion event including exon 7 and flanking intronsof the PAH gene could have transferred the mutationsite from one haplotype background to another (fig.3). If this was the underlying basis for the mutationto be present in the two different haplotype back-grounds, it might be expected that the mutation willbe associated with both haplotypes in at least one ofthe two populations. The fact that linkage disequi-

921

922 Okano et al.

230 230Glu -Lys

4 9101112Exons 1 2 3 4 5 678>7 r13

t t at ftHaptotype Bgtll Pvult(e) Pvull(b) EcoRi Mspl Xmnl HindIli EcoRV

1 - + - - + - -

38 + - + - + - -

HypotheticalCrossover EventsHypotheticalGene Conversion

Figure 3 RFLP haplotypes 1 and 38 at the human PAH locus.The molecular structure of the human PAH gene is shown schemati-cally with its 13 exons and about 90 kb of DNA. The arrows cor-respond to the polymorphic restriction sites. A plus sign (+) anda minus sign (-) are used to designate, respectively, the presence andabsence of a polymorphic restriction site. An equals sign (=) is usedto designate a 4.4-kb HindIII allele. The large X's (IX ) and the two-headed horizontal arrow (a) indicate the hypothetical crossover-eventssite and the gene conversion part, respectively.

librium is exclusively maintained in both populationswould suggest that these represent two independentmutational events recurring at the same site of the PAHgene on different haplotype backgrounds in the twopopulations. Second, the Glu280-to-Lys280 mutation in-volves a CpG dinucleotide (fig. 1). CpG is the most com-mon site of methylation in mammalian DNA, anddeamination of 5-methylcytosine will lead to a C-to-Ttransition. Such a deamination event at the first nucleo-tide of codon 280 on the antisense strand would haveresulted in the G-to-A transition on the sense strand.Since the frequency of this transition is 42-fold higherthan that of random mutation (Cooper and Youssoufian1988; Silva and White 1988), it is likely that this muta-tion has occurred independently in the two distinctpopulations. This mutation thus represents the first ob-served instance of recurrent mutation in the PAH genethat causes PKU.The Glu280-to-Lys280 mutation is the second muta-

tion found to be in linkage disequilibrium with haplo-type 1 in the Caucasian population, the first being thepreviously reported Arg261-to-Gln261 mutation (Okanoet al. 1990). This observation provides unambiguoussupport to our previously suggested hypothesis thatthere are multiple mutations associated with mutanthaplotype 1 alleles in the Danish population. Thisphenomenon reflects the fact that haplotype 1 is preva-lent among normal PAH alleles and has a higher prob-ability of sustaining multiple mutational events. In con-trast, haplotypes 2 and 3 are relatively rare amongnormal PAH alleles, and only one mutation was found

to be associated with each of these two mutant haplo-types in the northern European population. This in-clusive association between mutation and haplotype formutant haplotypes 2 and 3, however, is not maintainedin some other populations. For example, in Italy notall mutant haplotype 2 and 3 alleles are linked, re-spectively, to the Arg408-to-Trp408 and the splicing mu-tations (authors' unpublished data). In addition, amutant haplotype 2 allele associated with anothermutation, Mett-to-ValP, has been observed in a French-Canadian family (John et al. 1989). Thus, the associa-tion between mutations and haplotypes must be con-sidered independently for individual populations.

Clinically, PKU is a heterogeneous metabolic disor-der (Guttler 1980), reflecting the presence of multiplemutations in the PAH gene of various severity. Lyonnetet al. (1989) have reported that patients homozygousfor the Glu280-to-Lys280 mutation in haplotype 38 ex-hibit a mild phenotype, while patients who are geneticcompound heterozygotes for both the Glu280-to-Lys280mutation and other unknown mutations show a classi-cal PKU phenotype. A similar correlation between geno-type and phenotype has been observed in two of theDanish patients examined in the present study. Thesetwo patients are compound heterozygotes for theGlu280-to-Lys280 mutation and for either the Arg408-to-Trp408 or the splicing mutation, both of which areassociated with a severe clinical phenotype in theirhomozygous state. Since both patients in the presentstudy exhibited a severe clinical phenotype, the datasuggest that the Glu280-to-Lys280 mutation results inmarginal enzymatic activity in the patient liver, so thatthese patients exhibit the milder hyperphenylalaninemicphenotype at the homozygous state but exhibit the se-vere classical phenotype at the hemizygous-equivalentstate. This interpretation is consistent with our hypoth-esis that mutant genotypes can be correlated with bio-chemical and clinical phenotypes in patients with PKU(Guttler et al. 1987).

AcknowledgmentThis work was supported in part by NIH grant HD-17711

to S.L.C.W., who is also an Investigator with the HowardHughes Medical Institute.

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