sequence with equal intensity to the corresponding G band in the normal ... from the proband and both parents of three PKU families were PCR-amplified for the.
Proc. Nati. Acad. Sci. USA Vol. 88, pp. 2146-2150, March 1991
Genetics
Founder effect of a prevalent phenylketonuria mutation in the Oriental population (population genetics of phenylketonuria in Asia/founder and drift hypothesis/missense mutation)
TAO WANG*t, YOSHIYUKI OKANO*, RANDY C. EISENSMITH*, MICHELE L. HARVEY*, WILSON H. Y. Lot, SHU-ZHEN HUANGt, YI-TAO ZENGt, LI-FANG YUANt, JUN-ICHI FURUYAMA§, TOSHIAKI OURAI, STEVE S. SOMMERII, AND SAVIo L. C. Woo* *Howard Hughes Medical Institute, Department of Cell Biology and Institute of Molecular Genetics, Baylor College of Medicine, Houston, TX 77030; tDepartment of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100730, People's Republic of China; 1Laboratory of Medical Genetics, Shanghai Children's Hospital, Shanghai, People's Republic of China; §Department of Genetics, Hyogo
College of Medicine, Nishinomiya 663, Japan; ITraining Division, Osaka Municipal Rehabilitation Center for the Disabled, Osaka 547, Japan; and I'Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905
Communicated by C. C. Tan, December 17, 1990
screening has been implemented in China and a PKU frequency of :1 in 16,000 births has been observed (8). An examination of the PAH locus in normal and phenylketonuric individuals by Southern blot analysis reveals the presence of more than 43 haplotypes (9). It is interesting to note that the distribution of restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus shows significant variations in different populations (10-13). In Caucasians, four haplotypes (haplotypes 1-4) account for >90%o of all PKU chromosomes and 86% of all PKU families are heterozygous at one or more RFLP sites (12). In contrast, haplotype 4 alone accounts for >80%o of all normal and mutant haplotypes among Orientals, with only 32% of families showing heterozygosity (13, 14). This observation indicates that the PAH locus is much less polymorphic in Orientals and suggests that they might exhibit a different genetic background of PKU. Although the molecular defects responsible for PKU have been studied extensively in Caucasians, relatively little is known about the molecular basis and evolution of PKU among Orientals. Twelve PKU mutations reported thus far account for >75% of mutant alleles in the Caucasian population and most are in linkage disequilibrium with their respective haplotype. However, none of these mutations has yet been found in non-Caucasians (15-24). The only Oriental PKU mutation yet reported is a C-to-T transition in exon 3 of the PAH gene (Rl11X), which is not present among Caucasians (25). This study reports a second Oriental PKU mutation found in a classic PKU patient from northern China. This mutation, tightly linked to RFLP haplotype 4, is prevalent in both Chinese and Japanese PKU patients. These results support the hypothesis that the "northern Mongoloid" group represents a founding population for PKU in eastern Asia.
A missense mutation has been identified in ABSTRACT the human phenylalanine hydroxylase [PAH; phenylalanine 4-monooxygenase; L-phenylalanine,tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] gene in a Chinese patient with classic phenylketonuria (PKU). A G-to-C transition at the second base of codon 413 in exon 12 of the gene results in the substitution of Pro413 for Arg"'3 in the mutant protein. This mutation (R413P) results in negligible enzymatic activity when expressed in heterologous mammalian cells and is compatible with a classic PKU phenotype in the patient. Population genetic studies reveal that this mutation is tightly linked to restriction fragment length polymorphism haplotype 4, which is the predominant haplotype of the PAH locus in the Oriental population. It accounts for 13.8% of northern Chinese and 27% of Japanese PKU alleles, but it is rare in southern Chinese (2.2%) and is absent in the Caucasian population. The data demonstrate unambiguously that the mutation occurred after racial divergence of Orientals and Caucasians and suggest that the allele has spread throughout the Orient by a founder effect. Previous protein polymorphism studies in eastern Asia have led to the hypothesis that "northern Mongoloids" represented a founding population in Asia. Our results are compatible with this hypothesis in that the PKU mutation might have occurred in northern Mongoloids and subsequently spread to the Chinese and Japanese populations.
The classic form of phenylketonuria (PKU) is a typical inborn error of metabolism in humans. The biochemical defect in this disease is a severe diminution or complete absence of hepatic phenylalanine hydroxylase [PAH; phenylalanine 4-monooxygenase; L-phenylalanine,tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1], which catalyzes the conversion of phenylalanine to tyrosine. As a direct consequence of this PAH deficiency, individuals with PKU accumulate high concentrations of phenylalanine in blood and other tissues. Clinically, untreated phenylketonurics exhibit profound, irreversible mental retardation (1-3). Many clinical studies have demonstrated that this mental retardation can be ameliorated by long-term restriction of protein intake and a diet supplemented with tyrosine (4). These observations led to the implementation of mass neonatal screening programs for PKU throughout the Western world. As a result, collective PKU frequencies of -1 in 10,000 births among Caucasians and -1 in 100,000 births in Japan have been documented (5-7). More recently, PKU
MATERIALS AND METHODS Patients. The proband selected for gene analysis is a classic PKU child from northern China. The RFLP haplotypes at the PAH gene locus in this family are as follows: father 4/4, mother 1/4, proband 1/4. The 52 Chinese (29 from the North and 23 from the South) and 11 Japanese PKU kindreds analyzed in this study were evaluated biochemically and clinically in their home institutes as exhibiting a typical PKU phenotype according to standard criteria (5, 13, 26). Twentyfive Caucasian PKU families from different European countries were also used for population studies (13).
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Abbreviations: PKU, phenylketonuria; RFLP, restriction fragment length polymorphism; GAWTS, gene amplification with transcript sequencing.
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Genetics: Wang et al. Genomic Amplification with Transcript Sequencing (GAWTS) of Individual Exons of the PAH Gene. Primers were designed for the PCR-mediated amplification of individual exons and flanking intronic sequences of the human PAH gene. A T7 promoter sequence [29 base pairs (bp) long] was added to the 5' end of one of the PCR primers (Fig. 1). A standard 34 cycles of amplification were performed in a 100-jA vol using a thermocycler (Cetus) (24). The amplified products were purified by using the Centricon-30 system (Amicon) and one-fifth of the reaction mixture was transcribed into RNA in a 20-.ul vol with an SP6-T7 transcription kit (Boehringer Mannheim). Transcript sequencing was performed with avian myeloblastosis virus reverse transcriptase according to a modification of the method developed by Stoflet (27). Mutation Analysis in PKU Families Using Allele-Specific Oligonucleotide Probes. Genomic DNA from core PKU families was PCR amplified and dotted onto a Zeta-Probe membrane (Bio-Rad). Oligonucleotide probes (17-mers) specific for the normal or mutant sequence were end-labeled with [-32P]ATP. Hybridization and washing procedures have been described (28). Site-Directed Mutagenesis and Expression Analysis of Mutant Aileles. A full-length human PAH cDNA (PAH 247) was cloned into the EcoRP site of M13mpl8. Site-directed mutagenesis was carried out on the sense-strand vector with a PAH cDNA insert using an antisense mutant oligonucleotide specific for this mutation (29). Successful introduction of the mutant was confirmed by sequence analysis. The mutant PAH cDNA was then subcloned into the EcoRI site of the eukaryotic expression vector pcDNA I. One hundred micrograms of either pcDNA I, pcDNA I containing the wild-type PAH cDNA insert, or pcDNA I containing the mutant PAH cDNA insert was then transfected into COS cells by the manufacturer's standard electroporation protocol (Promega). The transfected cells were harvested after 72 hr of transient expression. PAH enzyme assays were performed on crude extracts of cells transfected with pcDNA I containing either normal or mutant PAH cDNA insert (30). Transcribed PAH RNA was quantified by Northern blot analysis usingthe PAH cDNA probe (15). PAH protein was analyzed by Western blot using an anti-rat PAH antibody (31).
RESULTS Identification of a Missense Mutation in Exon 12 by GAWTS. The amplification of exon 12 of the human PAH gene and its flanking introns has been reported by using PCR primers 12A and 12B (28). A 29-bp T7 promoter sequence was added to primer 12A during oligonucleotide synthesis (Fig. 1). The exon 12-containing region was successfully amplified by 34 cycles of PCR and transcribed into RNA with T7 RNA polymerase (data not shown). A nested sequencing primer of 17 bp was designed (5'-CCGAGTGGCCTCGTAAG-3'). Transcription products were then sequenced directly with avian myeloblastosis virus reverse transcriptase (27). The left portion of Fig. 1 (Upper) shows normal exon 12 sequence (32) and the right portion shows the corresponding region of the patient's DNA. An extra C band appeared in the patient's sequence with equal intensity to the corresponding G band in the normal individual, indicating that one of the two alleles in the patient has a G-to-C transition at this position (Fig. 1). This nucleotide substitution results in a change from arginine to proline at residue 413 of the PAH protein (R413P). Kindred Analysis Using Allele-Specific Oligonucleotide Probe. Genomic DNA samples from the proband and both parents of three PKU families were PCR-amplified for the exon 12-containing region. Amplified products were dotblotted onto Zeta-Probe membrane and hybridized separately with oligonucleotide probes corresponding to either
Proc. Natl. Acad. Sci. USA 88 (1991) normal
2147
mutant
GATC GATC G+ C
NORMAL
413 Arq 5'-CAGTTCGgTACGA-3'
MUTANT
5
-
CCC 413 Pro
31
FIG. 1. Identification of the missense mutation in exon 12 of the human PAH gene by GAWTS. Primers used for the PCR amplification are as follows: 5'-ggatcctaatacgactcactatagggagaATGCCACTGAGAACTCTCTT-3' (T12A) and 5'-AGTCTTCGATTACTGAGAAA-3' (12B). An internal primer was designed for GAWTS (5'-CCGAGTGGCCTCGTAAG-3'). The left portion of the figure is part of the normal exon 12 sequence (32). (Upper) The right portion of the figure is the corresponding region of exon 12 containing the mutation. The C and G bands appear at the same position in the mutant sequence with equal but reduced intensity, indicating that only one of the two alleles in this patient bears the mutation. The G-to-C transition results in the substitution of Pro413 for Arg413
(R413P).
the normal or the mutant sequences. The genomic DNA samples of all three families were successfully amplified as indicated by hybridization with the normal probe (Fig. 2). In family A from China (Fig. 2A), both the patient and paternal samples hybridized with the R413P probe, indicating that this mutation is indeed present in the genomic DNA of the family and was transmitted from the father to the patient. Since
A Fa m i l i e s
Normal Probe
C
B
TO
*.P.*
0
a
0
Mutant Probe
S..0
*
S
0
FIG. 2. Transmission of the R413P mutation in three PKU families. Genomic DNAs were isolated from leukocytes of different family members. PCR amplification of the exon 12-containing region was performed followed by dot-blot hybridization with allele-specific oligonucleotide probes. The probes used for detecting the mutation were as follows: 5'-GGTCGTAGCGAACTGAG-3' (normal) and 5'-GGTCGTAGGGAACTGAG-3' (mutant). Both probes are complementary to the sense strand. The pedigree patterns of these three families are shown at the top of the figure. Solid symbols, R413P mutant allele; hatched symbols, unknown PKU alleles; open symbols, normal alleles. The dot-blot hybridization signals using normal and mutant probes are shown at the bottom of the figure.
Genetics: Wang et al.
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Proc. Natl. Acad. Sci. USA 88 (1991)
1
2
_
0 go 40 ft
3
4
5
6
4
Normal PAH
40 4D 40 MO 4p 40 Mutant 413
FIG. 3. PAH
enzyme assay
for the R413P mutation. Protein
extract from COS cells (0, 10, 20, 50, 100, and 200 ,g) transfected
with either the normal (Upper, lanes 1-6, respectively) or R413P mutant (Lower, lanes 1-6, respectively) PAH cDNA constructs were assayed for PAH activity. Both the substrate [14C]phenylalanine (upper band of each portion) and the product [14C]tyrosine (lower band of each portion) are shown by autoradiography after their separation by thin-layer chromatography. mutant haplotype 4 is paternal in origin and mutant haplotype 1 is maternal in origin in this family, this mutation must occur on a haplotype 4 background. The maternal sample did not hybridize with the mutant probe, demonstrating the presence of an unidentified mutation in this mutant allele (Fig. 2A). A
second Chinese PKU family and a Japanese PKU family also analyzed (Fig. 2 B and C). In the family in Fig. 2B, the R413P mutant allele was transmitted from the mother and, in family C (Fig. 2C), both parents were carriers of this mutant allele and the patient is a homozygote. It is clear that the mutant alleles in these two families are also linked to RFLP haplotype 4. Site-Directed Mutagenesis and Expression Analysis of the R413P Mutant Protein. Successful site-directed mutagenesis on M13mpl8 with full-length PAH cDNA insert was confirmed by sequence analysis. The mutant PAH cDNA was then subcloned into pcDNA I for expression analysis. COS cells transfected with either the normal or mutant construct were harvested 72 hr after transfection and crude cell extract was analyzed for PAH activity (Fig. 3). Tyrosine production was directly proportional to the addition of cellular extract after transfection with the normal pcDNA I/human PAH cDNA construct (Fig. 3 Upper). The amount of tyrosine formed by increasing amounts of extracts from cells transfected with the R413P mutant construct (Fig. 3 Lower) was not significantly different from that of the negative control (lane 1), indicating a severe diminution or complete absence of PAH enzyme activity. This observation was further supported by Western blot analysis, which failed to reveal significant PAH immunoreactivity in extracts of cells transfected with the R413P construct, compared with the normal construct (data not shown). However, a comparable amount of PAH RNA transcript could be detected by Northern blot analysis in extracts from cells transfected with either the were
normal or R413P mutant constructs, but not in extracts from untransfected cells (data not shown). Correlation Between PAH Genotype and PKU Phenotype. The biochemical and clinical phenotypes of six PKU patients, including two homozygotes and four heterozygotes for the R413P mutation, are summarized in Table 1. The two homozygous patients are severe in clinical course with serum phenylalanine >20 mg/dl. The four compound heterozygous patients also manifest a classic form of PKU with serum phenylalanine ranging from 20 to 35 mg/dl. Thus, the severe diminution of PAH activity produced by the R413P mutation is compatible with the severe biochemical and clinical phenotypes of patients bearing these mutant alleles. Population Genetics of the R413P Mutation Among Orientals and Caucasians. A total of 52 Chinese, 11 Japanese, and 25 Caucasian families with a history of PKU were analyzed with oligonucleotide probes specific for this mutation. Eight R413P mutant alleles were detected among northern Chinese samples, one allele among southern Chinese samples, six among the Japanese samples, and none among the Caucasian samples (Table 2). The missense mutation was not present on any of the normal alleles, and all of the mutant alleles are associated with haplotype 4. This mutant allele accounts for an average of 9% of total PKU alleles among Chinese populations (13.8% of total PKU alleles in northern Chinese and 2.2% of that in southern Chinese) and 27% in the Japanese population.
DISCUSSION A new molecular lesion in the human PAH gene has been identified in a Chinese PKU patient who is a RFLP haplotype 1/4 heterozygote. This mutation, a C-to-G transition at the second base of codon 413, results in the substitution of proline for arginine in the enzyme (R413P). Expression analysis indicates that the mutant enzyme produces
