Lysosomal storage diseases presenting as transient or persistent hydrops fetalis.

Two cases of beta-glucuronidase deficiency (mucopolysaccharidosis VII), presented with fetal hydrops at 20 and 26 weeks of gestation. The enzyme deficiency was observed in cultured amniotic fluid cells and in fetal plasma from cord-blood and was confirmed after termination of pregnancy. A third case presented with transient ascites at 6.5 months of gestation. Mild dysmorphic features at birth and gradual neurological deterioration were observed. Deficiency of beta-galactosidase was documented confirming a GM1 gangliosidosis. Evidence has accumulated that fetuses affected by lysosomal diseases, may present with transient or persistent hydrops fetalis. The exact frequency is however not known. Further diagnostic studies in persistent or transient hydrops fetalis, looking for lysosomal and other metabolic diseases, whenever major causes of hydrops fetalis have been excluded, are therefore indicated. Amniocentesis and cordocentesis should always be performed.     

Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.     

Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations

We have performed molecular genetic analyses on 160 Brazilian patients diagnosed with cystic fibrosis (CF). Screening of mutations in 320 CF chromosomes was performed through single strand conformation polymorphism (SSCP) and heteroduplex analyses assay followed by DNA sequencing of the 27 exons and exon/intron boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of CFTR variants of T-tract length of intron 8 (IVS8 Tn) was also investigated. This analysis enabled the detection of 232/320 CF mutations (72.2%) and complete genotyping of 61% of the patients. The deltaF508 mutation was found in 48.4% of the alleles. Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 621+1G-->T, V232D, 1717-1G-->A, 2347 delG, R851L, 2789+5G-->A, and W1089X. Five novel mutations were identified, V201M (exon 6a), Y275X (exon 6b), 2686 insT (exon 14a), 3171 delC (exon 17a), and 3617 delGA (exon 19). These results contribute to the molecular characterization of CF in the Brazilian population. In addition, the identification of the novel mutation Y275X allowed prenatal diagnosis in a high-risk fetus.     

Molecular genetic assay of mucopolysaccharidosis IVA in South China

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Molecular mutational analysis was performed by PCR product sequencing for fourteen exons and exon–intron boundaries of GALNS gene in 21 patients from 19 unrelated families with severe MPS IVA in South China. We identified fifteen different mutations, including 10 reported mutations (p.P125L, p.G290S, p.M318R, p.G340D, p.L366P, p.R386C, p.A392V, c.1243-1G>C, p.L440RfsX54 and p.X523E) and five novel mutations (p.N177S, p.G290R, p.F306S, p.W403_T404delinsCS, p.W520X). All five novel mutations were inherited from parents of the patients and not found in 100 normal control alleles. Three mutations, p.M318R, p.L366P and p.R386C were common, accounting for 36.8% of mutant alleles investigated. One patient homozygous of p.A392V and the other two unrelated patients homozygous of p.L366P presented classical disease course. The results show that the GALNS gene has a different mutational spectrum in South China as compared to other regions. The p.A392V and p.L366P mutations were associated with severe phenotype of MPS IVA.     

Low β-glucuronidase enzyme activity and mutations in the human β-glucuronidase gene in mild mucopolysaccharidosis type VII, pseudodeficiency and a heterozygote

Deficiency of β-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced β-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low β-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the β-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and ³⁵SO₄-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low β-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low β-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low β-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801ΔT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold. Received: 31 May 1997 / Accepted: 26 Augsut 1997     

Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity

We identified two different exonic point mutations causing beta-glucuronidase (beta G1) deficiency in two Japanese patients with mucopolysaccharidosis type VII (MPSVII). Enzyme assay of lysates of the lymphocytes and cultured fibroblasts showed little residual activity. The beta G1-specific mRNA levels were normal, as determined by northern blot analysis. Mutated cDNA clones, including the entire coding sequence, were isolated using the polymerase chain reaction (PCR) products derived from beta G1-deficient fibroblasts. Sequence analysis of the full-length mutated cDNAs showed C----T transitions, which resulted in a single Ala619----Val change (case 1, a 24-year-old male) and a Arg382----Cys change (case 2, a 7-year-old female). The former change was revealed by a loss of the cleavage site for the Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient (case 1) was a homozygote with this mutation. The mutational change in patient 2 was confirmed by direct sequencing and by demonstrating heterozygosity for the mutation in her parents. The Ala619----Val and Arg382----Cys mutations each disrupt a different domain which is highly conserved among human, rat, and Escherichia coli beta G1s. Each of these two amino acid changes reduced the beta G1 activity of the corresponding mutant beta G1 expressed following transfection of COS cells with expression vectors harboring the mutated cDNAs.     

Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.     

PCR-based restriction fragment length polymorphism and haplotype of the most common mutation L176F in the beta-glucuronidase gene

Mucopolysaccharidosis type VII or Sly syndrome is an autosomal recessive disorder of glycosaminoglycan storage leading to variable clinical symptoms, such as hepatosplenomegaly, bone deformities, hearing loss, corneal opacities, mental retardation, and hydrops fetalis in affected individuals. The disease is caused by approximately 40 different mutations in the beta-glucuronidase gene. Detection of the most common mutation L176F by single-strand conformation polymorphism (SSCP) was not always successful. Although DNA sequencing followed by PCR amplification can easily detect this mutation, accessibility to a DNA sequencer or useful reagents in the sequencing procedure is not readily available in many countries. A PCR-based restriction fragment length polymorphism (RFLP) developed in this report would allow rapid and easier detection of this mutation for screening new patients or neonates of heterozygous parents. Analysis of intragenic polymorphic sites in the L176F patients identified two distinct alleles; the predominant one probably originated in Spain.     

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.     

Characterization of the defective beta-glucuronidase activity in canine mucopolysaccharidosis type VII

Canine mucopolysaccharidosis type VII results from deficient activity of lysosomal beta-glucuronidase. Residual enzymatic activity (0.2-1.7% of normal) was detected in tissue homogenates from affected dogs. In contrast, serum and urine from affected animals had up to 15% residual activity. To further characterize the nature of the defective enzyme, hepatic beta-glucuronidase was partially purified from normal and MPS VII dogs for determination of their physical and kinetic properties. About 65% of the total beta-glucuronidase in normal canine liver required detergent for solubilization (i.e., membrane-associated), whereas only 22% of the residual activity in canine MPS VII liver was membrane-associated. Compared to the normal hepatic enzyme, the Km towards 4-methylumbelliferyl-beta-glucuronide was markedly increased in MPS VII dogs (i.e., 0.48 versus greater than 2.5 mmol/l). In contrast, the thermo-, cryo-, and pH stability properties, as well as the pH optimum (approximately 4.6), were essentially unaffected. In addition, the canine MPS VII hepatic residual activity was unresponsive to sulfhydryl reducing reagents and divalent cations, despite the fact that incubation of normal canine beta-glucuronidase with dithiothreitol and magnesium and/or calcium enhanced the activity more than 15-fold.     

Mutational analysis in longest known survivor of mucopolysaccharidosis type VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive disorder caused by the deficiency of beta-glucuronidase leading to the intralysosomal storage of heparan, dermatan, and chondroitin sulfate. Here, we report the identification of two novel missense mutations K350N and R577L in a 37-year-old patient with beta-glucuronidase deficiency and a relatively mild MPS VII phenotype. Expression of the K350N mutation in baby hamster kidney cells has revealed residual enzymatic activity and normal transport of the enzyme to the lysosome. However, expression of the R577L or the double mutant K350N/R577L results in rapid degradation of the enzyme in early biosynthetic compartments and a total loss of enzymatic activity. We attribute the mild phenotype to the residual catalytic activity provided by the K350N mutant. At the time of her death at the age of 37 years, this patient was the longest known survivor with MPS VII.     

alpha-L-iduronidase premature stop codons and potential read-through in mucopolysaccharidosis type I patients

alpha-L-Iduronidase is a glycosyl hydrolase involved in the sequential degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. A deficiency in alpha-L-iduronidase results in the lysosomal accumulation and urinary secretion of partially degraded glycosaminoglycans and is the cause of the lysosomal storage disorder mucopolysaccharidosis type I (MPS I; Hurler and Scheie syndromes; McKusick 25280). The premature stop codons Q70X and W402X are two of the most common alpha-l-iduronidase gene (IDUA) mutations accounting for up to 70% of MPS I disease alleles in some populations. Here, we have reported a new mutation, making a total of 15 different mutations that can cause premature IDUA stop codons and have investigated the biochemistry of these mutations. Natural stop codon read-through was dependent on the fidelity of the codon when evaluated at Q70X and W402X in CHO-K1 cells, but the three possible stop codons TAA, TAG and TGA, had different effects on mRNA stability and this effect was context dependent. In CHO-K1 cells expressing the Q70X and W402X mutations, the level of gentamicin-enhanced stop codon read-through was slightly less than the increment in activity caused by a lower fidelity stop codon. In this system, gentamicin had more effect on read-through for the TAA and TGA stop codons when compared to the TAG stop codon. In an MPS I patient study, premature TGA stop codons were associated with a slightly attenuated clinical phenotype, when compared to classical Hurler syndrome (e.g. W402X/W402X and Q70X/Q70X genotypes with TAG stop codons). Natural read-through of premature stop codons is a potential explanation for variable clinical phenotype in MPS I patients. Enhanced stop codon read-through is a potential treatment strategy for a large sub-group of MPS I patients.     

Incidence of the mucopolysaccharidoses in Northern Ireland

An epidemiological study of the mucopolysaccharidoses (MPS) in Northern Ireland using multiple ascertainment sources was carried out and the incidence rate for the period 1958–1985 was estimated. An incidence of approximately 1 in 76 000 live births was obtained for MPS 1H (Hurler phenotype); 1 in 280 000 for MPS 1 H/S (Hurler/Scheie phenotype); 1 in 140 000 live births (1 in 72 000 male live births) for MPS II (Hunter syndrome); 1 in 280 000 for MPS III (Sanfilippo syndrome) and 1 in 76 000 for MPS IV A (Morquio syndrome type A). No cases of MPS IS (Scheie phenotype), MPS IV B (Morquio syndrome type B) or MPS VI (Maroteaux–Lamy syndrome) were ascertained during the study period. Three cases of non-immune hydrops fetalis born to consanguineous parents were thought to be due to β-glucuronidase deficiency (MPS VII) on the basis of placental histology and enzyme studies on both parents but no living cases of MPS VII were ascertained. The overall incidence for all types of mucopolysaccharidosis was approximately 1 in 25 000 live births. A comparison is made with incidence estimates obtained from other published studies. Received: 25 May 1997 / Accepted: 22 August 1997     

Three novel α-L-iduronidase mutations in 10 unrelated Chinese mucopolysaccharidosis type I families

Mucopolysaccharidosis type I (MPS I) arises from a deficiency in the α-L-iduronidase (IDUA) enzyme. Although the clinical spectrum in MPS I patients is continuous, it was possible to recognize 3 phenotypes reflecting the severity of symptoms, viz., the Hurler, Scheie and Hurler/Scheie syndromes. In this study, 10 unrelated Chinese MPS I families (nine Hurler and one Hurler/Scheie) were investigated, and 16 mutant alleles were identified. Three novel mutations in IDUA genes, one missense p.R363H (c.1088G > A) and two splice-site mutations (c.1190-1G > A and c.792+1G > T), were found. Notably, 45% (nine out of 20) and 30% (six out of 20) of the mutant alleles in the 10 families studied were c.1190-1G > A and c.792+1G > T, respectively. The novel missense mutation p.R363H was transiently expressed in CHO cells, and showed retention of 2.3% IDUA activity. Neither p.W402X nor p.Q70X associated with the Hurler phenotype, or even p.R89Q associated with the Scheie phenotype, was found in this group. Finally, it was noted that the Chinese MPS I patients proved to be characterized with a unique set of IDUA gene mutations, not only entirely different from those encountered among Europeans and Americans, but also apparently not even the same as those found in other Asian countries.     

Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.     

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

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.     

Rapid mutation screening of phenylketonuria by polymerase chain reaction-linked restriction enzyme assay and direct sequence of the phenylalanine hydroxylase gene: clinical application in northern Japan and northern China

We describe a simple and technically feasible method for mutation screening of the phenylalanine hydroxylase (PAH) gene and its application to Japanese and Chinese patients with hyperphenylalaninemia. The strategy is based on the identification of a nucleotide substitution by restriction enzyme analysis, coupled with PCR and direct sequencing of exon 7 of the PAH gene. Because the detection of various mutations can proceed simultaneously using the same technique, it is quite rapid and reproducible, making it possible to perform effective molecular diagnosis and carrier screening in most laboratories. Using this procedure, we found that the most common molecular defects were R413P in Hokkaido, Japan (35 %) and R243Q in Heilongjiang, China (50%). R111X, IVS4nt-1, and five mutations in exon 7 (R241C, R243Q, R252W, A259T, and S273P) accounted for 55% of phenylketonuria (PKU) alleles in Hokkaido. In Heilongjiang, the R111X, Y356X, and R408W mutations accounted for 35% of PKU alleles. Clinically, homozygotes or compound heterozygotes of null alleles, which express nonfunctional enzyme activity, were all associated with classic PKU. On the other hand, patients heterozygous for the R241C allele had a benign phenotype of mild hyperphenylalaninemia. The DNA diagnosis in early infancy can predict various PKU phenotypes, and can prove useful in decision-making concerning dietary therapy.