In vitro and in vivo correlations for I65T and M1V mutations at the phenylalanine hydroxylase locus.

Mutations at the phenylalanine hydroxylase (PAH) locus are the major cause of hyperphenylalaninemia. We have previously described four mutations (M1V, IVS12nt1, R408W, and S349P) at the PAH locus in French Canadians with ancestry in eastern Quebec. Here we report (1) identification of another mutation, on a haplotype 9 chromosome, which converts codon 65 from isoleucine (ATT) to threonine (ACT), (2) expression analysis of the I65T mutation in COS cells demonstrating 75% loss of both immunoreactive protein and enzyme activity, and (3) expression analysis of the most prevalent PKU allele (M1V) in eastern Quebec, showing nondetectable levels of PAH protein and activity, a finding compatible with a mutation in the translation initiation codon. Homozygosity for M1V and codominant inheritance of I65T/R408W were both associated with classical phenylketonuria.     

Mutation spectrum and phenylalanine hydroxylase RFLP/VNTR background in 44 Romanian phenylketonuric alleles

The mutation spectrum and polymorphic haplotype background in 22 Romanian families have been analysed in this study using the restriction digestion of phenylalanine hydroxylase (PAH) regions specifically amplified or the DGGE/direct sequencing methods. Eleven PAH mutations specifically associated with six mutant haplotypes were detected. In spite of the relative heterogeneity of the molecular defects in the PAH gene, three mutations covered almost 70% of all alleles: R408W, 47.72%, 21/44; K363fsdelG 13.63%, 6/44; and P225T 6.81%, 3/44. Among these, R408W, the most frequent mutation in our population, represented 50% of all the phenylketonuric (PKU) chromosomes. Splice mutation IVS12nt1g→a affected two PAH alleles (4.54%); the remaining seven mutations were rare, each having an effect on just one chromosome (1/44), resulting in a relative frequency of 2.27%. A high frequency was observed in our PKU samples for the relatively uncommon mutations, K363fsdelG and P225T mutation, suggesting a possible founder effect at origin. Within the investigated panel, these mutations, both very rare among other Caucasians were exclusively linked to haplotype 5.8 and 1.7, respectively. These results provide a basis for the development of a routine molecular analysis of Romanian PKU families. Hum Mutat 12:314–319, 1998.© 1998 Wiley-Liss, Inc.     

Mutation analysis of the phenylalanine hydroxylase gene and its clinical implications in two Japanese patients with non-phenylketonuria hyperphenylalaninemia

We describe a mutation analysis for the phenylalanine hydroxylase gene and the clinical outcome of two Japanese patients with non-phenylketonuria (PKU) hyperphenylalaninemia (serum phenylalanine level below 600 μmol/l under a free diet) detected by a mass-screening program. Single strand conformation polynorphism analysis and direct sequencing of their genomic DNAs revealed that non-PKU hyperphenylalaninemia resulted from compound heterozygosity for a mutation causing classical PKU and a mutation with a milder effect on phenylalanine hydroxylase activity. The mutations were R241C and R243Q in exon 7, and R413P in exon 12. The mutation genotypes of the two patients were R241C/R243Q and R241C/R413P. It has been demonstrated that homozygosity for the R243Q or R413P mutation is associated with a severe phenotype of PKU and low in vitro expression activity. In contrast, the R241C mutation has much less effect on phenylalanine hydroxylase activity. The metabolic consequence of each variant allele was confirmed by a phenylalanine loading test in the patients and their parents. The patients achieved normal results in all intellectual and neurologic tests. Brain magnetic resonance imaging revealed no abnormalities. The dietary restriction was continued until 10 years of age in order to maintain the serum phenylalanine level below 400 μmol/l. The genetic analysis to distinguish non-PKU hyperphenylalaninemia from classical PKU helps to determine the principles of dietary management in the early infantile period. Received: June 2, 1998 / Accepted: July 17, 1998     

Mutation screening of phenylketonuria in the Far East of Russia

We analyzed mutant genotypes at the human phenylalanine hydroxylase (PAH) locus among phenylketonuria (PKU) patients in the Far East of Russia. A total of 60 variant alleles from 30 PKU families were analyzed for prevalent Caucasian mutations and restriction fragment length polymorphism/variable number of tandem repeats (RFLP/VNTR) haplotypes. Seventy-eight percent of all variant alleles carried six mutations. The most prevalent mutation was R408W (63%), with a haplotype background of 2.3. It also showed a very high degree of homozygosity (43%). The other five mutations (R158Q, R261Q, R252W, R261X, and IVS12nt-1) accounted for 1.7%–6.7% of all PKU alleles, and a single haplotype was associated with each genotype, except for R261Q. The genetic structure of PKU patients in the Far East of Russia seems to be relatively homogeneous, compared with that in the other Slavic and Oriental populations of surrounding countries. Prediction of a clinical phenotype and carrier detection will be feasible using DNA tests. Received: June 30, 1999 / Accepted: August 10, 1999     

The molecular basis of phenylalanine hydroxylase deficiency in Croatia

We present the results of a comprehensive analysis of mutations, polymorphisms and haplotypes in the phenylalanine hydroxylase (PAH) gene in 39 Croatian families with phenylketonuria (PKU). A total of 21 disease-causing mutations was identified on 78 out of 79 independent chromosomes. The commonest mutation, R408W on haplotype 2 was found with a relative frequency of 37 %. P281L accounted for 11 %, R261Q and E390G each for 9 % of mutant chromosomes. There were three novel mutations: L249P (c.746T>C) in exon 7, IVS8+2T>C (c.912T>C) in intron 8, and F402L (c.1206T>G) in exon 12 of the PAH gene. Two known PKU mutations were found in cis on the same chromosome in one family, highlighting the need to perform full mutation scanning in recessive disease genes for molecular diagnosis even if two known mutations have been identified in a patient. This is the first comprehensive report on PKU mutations in southeastern Europe, adding to the growing bulk of molecular data for population genetic investigations.     

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype-phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction-restriction fragment length polymorphism and 'broad-range' denaturing-gradient gel electrophoresis/DNA sequencing analysis, 19 disease-causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype-phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.     

Molecular heterogeneity at the phenylalanine hydroxylase locus in the population of the south-west of England.

The phenylalanine hydroxylase gene locus has been studied in 35 independent phenylketonuric families in the south-west of England using RFLP haplotype patterns and allele specific oligonucleotide probes. Haplotype 3 was the most common pattern on mutant chromosomes and there was strict linkage disequilibrium between this haplotype and the splice mutation in exon 12. The R408W mutation in exon 12 occurred on both haplotypes 1 and 2. The R126Q mutation in exon 7 was found only on a rare haplotype 28 pattern. No gene carried the R158Q mutation. More than 60% of mutant genes did not carry these four mutations which were originally described in other European populations. We suggest that the splice mutation arose as a single event and spread throughout northern Europe by population migration and admixture. In addition, we believe the haplotype/mutation associations seen in our population are a reflection of the mixed ancestry of the inhabitants of the British Isles.     

Spectrum of CBS mutations in 16 homocystinuric patients from the Iberian Peninsula: high prevalence of T191M and absence of I278T or G307S

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency has been extensively studied, but to date, no spectrum of CBS mutations of Spanish homocystinuric patients has been reported. Here we present a mutation analysis of thirteen Spanish and three Portuguese unrelated homocystinuric patients. Ten mutations were found to account for the thirty-two mutant alleles and five of these (C275Y, L338P, S349N, R379Q, and L456P) are reported here for the first time. All five novel mutations were found to affect evolutionarily conserved residues suggesting that they may impair enzyme function. Interestingly, neither of the two common CBS mutations, I278T and G307S, was detected in this series, and no patient was found to respond to pyrodoxine. Enzyme activities in cultured fibroblasts from 10 of the patients were assayed, and they ranged from 0 to 13 % of controls analyzed in parallel. The T191M mutation (which has only ever been reported once before in a Spanish patient) accounted for 50% of the mutant alleles. Comparison of the clinical data of seven patients homozygous for T191M indicated that this genotype is a poor predictor of the phenotype. A common haplotype was identified in all the T191M chromosomes of Spanish origin, while a different one was present in the four T191M chromosomes from Portuguese patients.     

Phenylketonuria mutations in Europe

Phenylketonuria (PKU) is heterogeneous. More than 400 different mutations in the phenylalanine hydroxylase (PAH) gene have been identified. In a systematic review of the molecular genetics of PKU in Europe we identified 29 mutations that may be regarded as prevalent in European populations. Comprehensive regional data for these mutations were collated from all available studies. The spectrum of mutations found in individual regions results from a combination of factors including founder effect, range expansion and migration, genetic drift, and probably heterozygote advantage. Common mutations include R408W on a haplotype 2 background in Eastern Europe, IVS10-11G>A in the Mediterranean, IVS12+1G>A in Denmark and England, Y414C in Scandinavia, I65T in Western Europe, and R408W on haplotype 1 in the British Isles. Molecular data from mild hyperphenylalaninemia (MHP) patients are available from a number of countries, but it is currently not possible to calculate relative allele frequencies. The available data on PAH mutations are useful for the understanding of both the clinical features and the population genetics of PAH deficiency in Europe.     

Novel mutations and structural implications in R-type pyruvate kinase-deficient patients from southern Italy

Deficiency of the R-type pyruvate kinase (R-PK) causes an autosomal recessive, hereditary, nonspherocytic hemolytic anemia (HNSHA). We screened seven unrelated patients from the south of Italy for the known mutations and found one patient homozygous for the 1529A (R510Q) mutation, two others bearing the 1456T (R486W) mutation, one homozygous and another heterozygous, and two heterozygotes for the 994A mutation (G332S). We also found three novel mutations at the heterozygote status: a G to C transversion in position 1010 (1010C; R337P) and a C to T transition in position 1492 (1492T; R498C), which are missense, and a T to G transversion in position 1523 (1523G; L508Z), which produces a stop codon with a subsequent loss of the C-terminal protein domain. The structural features of R-PK in the mutation-bearing regions were examined. In all cases the mutations altered the local conformation of the enzyme. Both G332S and R337P are in highly conserved sequence regions. In particular, the R337P mutation significantly affects the intersubunit interactions, because it is located in a region subjected to a large conformational change that occurs during the R→T allosteric transition, which is essential for the enzyme activity. The R486W mutation affects an external pocketlike region, producing only a local conformational change; the R498C mutation changes the interactions among neighbouring residues; the R510Q mutation involves the loss of interdomain interactions that may reduce enzyme stability and activity. Our data also indicate that in patients from Southern Italy, pyruvate kinase deficiency is heterogeneous, the 1529A mutation, which is the most frequent mutation in the U.S. Caucasian population, having a lower frequency. Hum Mutat 11:127–134, 1998. © 1998 Wiley-Liss, Inc.     

Phenylketonuria mutations in Northern China

Mutation spectrum of phenylalanine hydroxylase (PAH) gene in patients with phenylketonuria (PKU) in Northern China is described with a discussion on genotype-phenotype correlation. By using PCR/SSCP and DNA sequencing, all exons of PAH gene in the 185 unrelated patients with PKU from Northern China were studied. A total of 70 different mutations, including 42 missense, 12 splice, 7 nonsense, 5 deletion, 3 insertion, and 1 silence/splice mutations, were detected in 349/370 mutant alleles (94.3%). Deletion, insertion, and frameshift mutations were found for the first time in China PKU patients. The mutations R243Q, EX6-96A>G, R111X, Y356X, and R413P were the prevalent mutations with relative frequencies of 22.2, 11.1, 8.7, 6.5, and 6.5%, respectively. Fifteen novel mutations were identified in this study: I38fsX19, IVS4+3G>C, Y154H, R157K, R157I, T200fsX6, Q267H, Q267E, F302fsX39, G346R, S349A, L367L, R400K, IVS12+4A>G, and IVS12+6T>A. Each of them occurs at very low frequency (0.3-1.1%). The mutation spectrum of PKU in Chinese is similar to other Asian populations but significantly different from European populations. Altogether, 70 different mutations are found in 109 genotypes distributed among 185 PKU patients. As shown by the analysis, the predicted residual activity found in the majority of PKU individuals match their in vivo phenotypes, though evidence is also found for both phenotypic inconsistencies among subjects with similar genotypes and discordance between the in vitro and in vivo effects of some mutant alleles. The study enables us to construct a national database in China serving as a valuable tool for genetic counseling and prognostic evaluation of future cases of PKU.     

Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V

Kharrat M, Trabelsi S, Chaabouni M, Maazoul F, Kraoua L, Ben Jemaa L, Gandoura N, Barsaoui S, Morel Y, M’rad R, Chaabouni H. Only two mutations detected in 15 Tunisian patients with 11β‐hydroxylase deficiency: the p.Q356X and the novel p.G379V. Steroid 11β‐hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11β‐hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11β‐hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population.     

Periaxin mutation in Japanese patients with Charcot-Marie-Tooth disease

Periaxin (PRX) plays an important role in the myelination of the peripheral nerve and consequently in the pathogenesis of Charcot-Marie-Tooth disease (CMT). To date, nine nonsense or frameshift PRX mutations have been reported in eight families with CMT. The patients with PRX mutations appeared to show characteristic clinical features with early onset but slow or no progression, a common result of mutations that lead to missing a C-terminal acidic domain. Here, we report a Japanese CMT patient with these characteristic clinical features, who was a compound heterozygote for PRX R1070X and L132FsX153 mutations. We previously reported that three Japanese isolated families also had the homozygous R1070X mutation. To examine the potential founder effect of the R1070X mutation in the Japanese population, we performed haplotype analysis and found that each R1070X allele lay on a different haplotype background in these four families. Therefore, the high frequency of the R1070X mutation among the Japanese population is not likely the consequence of a founder effect, but probably a result of a mutation hot spot.     

In vitro expression analysis of mutations in phenylalanine hydroxylase: Linking genotype to phenotype and structure to function

Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell-free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site-directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site ( http://www.mcgill.ca/pahdb ). Hum Mutat 11:4–17, 1998. © 1998 Wiley-Liss, Inc.     

Three prevalent mutations in a patient with phenylalanine hydroxylase deficiency: implications for diagnosis and genetic counselling.

Mutation analysis in a patient with mild hyperphenylalaninaemia showed three distinct base substitutions in exon 12 of the phenylalanine hydroxylase (PAH) gene. All three mutations, R413P, Y414C, and D415N, have previously been described as being independently associated with PAH deficiency. Family studies and independent analysis of the PAH alleles of the patient showed cosegregation of the R413P and Y414C mutations. Data on the ethnic background of the family provide evidence that the R413P mutation has occurred on a PAH allele carrying the Y414C mutation. Using current methods for mutation identification, the presence of two known mutations on a single PAH allele implies the risk of misdiagnosis of PAH deficiency and complicates genetic counselling. Our results stress the need for comprehensive mutation scanning of the PAH gene in diagnostic settings.     

Population genetics of hyperphenylalaninaemia resulting from phenylalanine hydroxylase deficiency in Portugal.

In order to elucidate the molecular basis of phenylketonuria (PKU) in Portugal, a detailed study of the Portuguese mutant phenylalanine hydroxylase (PAH) genes was performed. A total of 222 mutant alleles from 111 PKU families were analysed for 26 mutations and restriction fragment length polymorphismlvariable number tandem repeat (RFLP/VNTR) haplotypes. It was possible to characterise 55% of the mutant alleles, in which 14 different mutations (R261Q, V388M, IVS10nt-11, I65T, P281L, R252W, R158Q, L348V, Y414C, L311P, Y198fsdel22bp, R408W, R270K, and R261X) and three polymorphisms (Q232Q, V245V, and L385L) were identified. A total of 14 different haplotypes were observed, with a high prevalence of haplotype 1 among mutant and normal alleles. The results reported in this study show considerable genetic heterogeneity in the Portuguese PKU population, as has also been described for other southern European populations.     

Molecular and Biochemical Basis of Galactosemia

Galactosemia is a clinically heterogeneous autosomal recessive inborn error of metabolism caused by deficiency of galactose-1-phosphate uridylyltransferase (GALT). Despite the numerous point mutations identified in the GALT gene, the prevalence of these mutations in different ethnic groups has not been studied. Reports on genotype/phenotype correlation are not consistent due to the small sample sizes studied and the lack of a sensitive enzyme assay. We applied multiplex PCR/ASO dot blot analysis to screen 293 galactosemic patients for 17 known point mutations in exons 5, 6, and 10. Our data demonstrate that only 7 of these mutations were detected in our patients, accounting for 65% of the GALT mutant alleles. Although Q188R is the most common mutation in Caucasian and Hispanic patients, the S135L mutation is most common in African-Americans. Another mutation, F171S, was observed only among African-American patients. An improved, sensitive, and accurate method was used to measure GALT activity in patient's red blood cells. The results indicated that patients homozygous for Q188R have no enzyme activity while those homozygous for S135L had residual enzyme activity. Interestingly, both Q188R/S135L and S135L/F171S compound heterozygotes demonstrated zero enzyme activity. Overall, 85% of Q188R compound heterozygotes also did not have any enzyme activity, whereas the remaining Q188R and the majority of S135L compound heterozygotes expressed variable amounts of GALT activity. We speculate that heterodimeric subunit interaction plays an important role in determining the overall enzymatic activity. Various genotypes thus result in biochemical and clinical heterogeneity among the patients.     

Mutation analysis of the 6-pyruvoyl-tetrahydropterin synthase gene in Chinese hyperphenylalaninemia caused by tetrahydrobiopterin synthesis deficiency

Hyperphenylalaninemia (HPA) may be caused by deficiency of phenylalanine hydroxylase or tetrahydrobiopterin (BH₄), the essential cofactor for the aromatic amino acid hydroxylases. 6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is a major cause of BH₄ deficient HPA. In this study, seven single base mutations at nucleotides 73 (C>G), 155 (A>G), 166 (G>A), 209 (T>A), 259 (C>T), 286 (G>A), and 317 (C>T) on PTPS cDNA were detected in Chinese PTPS-deficient HPA by polymerase chain reaction and solid phase DNA sequencing. These nucleotide alterations result in R25G, N52S, V56M, V70D, P87S, D96N, and T106M amino acid substitutions, respectively. The R25G, V56M, V70D, and T106M were novel mutations found in PTPS gene. By analysis of 38 PTPS mutant alleles from 19 unrelated Chinese PTPS-deficient HPA families, the allele frequency of these mutations in Chinese PTPS-deficient HPA were determined to be ˜5.3% (R25G), 34.2% (N52S), 7.9% (V56M), 2.6% (V70D), 36.8% (P87S), 7.9% (D96N), and 2.6% (T106M), respectively. Two common mutations, N52S and P87S, were found to account for 71% of the Chinese PTPS mutant alleles. The N52S mutation accounts for 48% of the southern Chinese PTPS mutation, but only one (9%) of the northern Chinese PTPS mutant allele was found to be N52S, which suggested that the N52S mutation might be southern Chinese. Clinically, the V56M mutation was found to associate with the mild form of PTPS deficiency. However, the R25G, N52S, P87S, and D96N were found mainly in the patients with severe clinical symptom. Using polymerase chain reaction-based mutation analysis, a fetus at risk of PTPS deficiency was diagnosed prenatally to be a carrier of N52S mutation. Hum Mutat 11:76–83, 1998. © 1998 Wiley-Liss, Inc.     

Phenylketonuria: variable phenotypic outcomes of the R261Q mutation and maternal PKU in the offspring of a healthy homozygote.

Phenylketonuria (PKU) and benign hyperphenylalaninaemia (HPA) result from a variety of mutations in the gene for the hepatic enzyme phenylalanine hydroxylase. PKU has been found in the Israeli population in two variants, classical and atypical. The two are clinically indistinguishable and require treatment with low phenylalanine diet to prevent mental retardation, but show differences in serum phenylalanine levels and in tolerance to this amino acid. Maternal PKU is a syndrome of congenital anomalies and mental retardation that appears in offspring of PKU mothers as a result of fetal exposure to the high phenylalanine level in the maternal blood. We studied a family in which two children with severe, classical PKU and their unaffected brother showed mild signs of maternal PKU. Their mother had no clinical signs of PKU, but the phenylalanine concentration in her serum reached a level that usually characterises PKU patients. This woman represents a rare phenotype, benign atypical PKU. Such 'hidden' PKU in women may lead to maternal PKU in the offspring, similar to overt PKU. Special attention should therefore be paid to women having children with any of the clinical hallmarks of maternal PKU, and to children born to women known to have benign HPA. The mother was also found to be homozygous for a missense mutation at the phenylalanine hydroxylase locus, R261Q, which does not abolish enzymatic activity completely. In two other families, homozygosity for this mutation resulted in atypical PKU in four children. This observation suggests that mutations that do not completely destroy phenylalanine hydroxylase activity may exhibit variable phenotypic expression which is unpredictable. Compound heterozygosity for R261Q and other mutations led in other patients either to classical PKU or to mild benign HPA.