[Analysis of large deletion of phenylalanine hydroxylase gene in Chinese patients with phenylketonuria].

OBJECTIVE: To analyze the types and distribution of large deletion of phenylalanine hydroxylase (PAH) gene in Chinese patients with phenylketonuria (PKU). METHODS: On the basis of 953 PKU patients from Peking Union Medical College and Gansu Province Medical Genetics Center, which were detected by directed sequencing of PAH gene between 2006 and 2014. Multiplex ligation-dependent probe amplification (MLPA) of PAH gene was performed in 43 patients with one or two unknown genotypes. And the deletion breakpoints were characterized by Gap PCR-sequencing. RESULTS: Twenty-four large deletion/duplication alleles were found in 22 patients, accounting for 51.1%(24/47)of the 47 unknown mutations of the 43 patients.There were 6 different large deletions, including Ex1del3758 (n=10), Ex4_5del (n=4), Ex4_7del (n=3), Ex1del5329ins56 (n=3), Ex3del6599ins8 (n=2), and Ex4del (n=1); and 1 duplication was found (Ex12dup, n=1). The most common large deletions in Chinese patients were Ex1del3758 (21.3%), Ex4_5del (8.5%), and Ex4_7del (6.4%). CONCLUSIONS: Large deletion mutations of PAH gene are present in Chinese PKU patients. It's important to detect the large del/dup mutation, and there are different hotspot mutation genotypes in Chinese patients.

Correlation between genotype and the tetrahydrobiopterin-responsive phenotype in Chinese patients with phenylketonuria.

BACKGROUND: A growing body of research has suggested that tetrahydrobiopterin (BH4) responsive phenotype can be predicted by the phenylalanine hydroxylase (PAH) genotype in patients with phenylketonuria (PKU), but data concerning the association between genotype and BH4 responsiveness are scarce in China. METHODS: A total of 165 PKU patients from China who had undergone a 24-h loading test with BH4 administration were recruited. Genotyping was performed by the next-generation sequencing (NGS) technique. Using the predicted residual PAH activity, we analyzed the association between genotype and BH4-responsiveness. RESULTS: Among the 165 patients, 40 patients (24.24%) responded to BH4. A total of 74 distinct mutations were observed, including 13 novel mutations. The mutation p.R241C was most frequently associated with response. Two known mutations (p.A322T and p.Q419R) and two novel mutations (p.L98V and IVS3-2A>T) were first reported as responsive to BH4. Residual PAH activity of at least 12.5% was needed for responsive genotypes. CONCLUSION: Genotype-based predictions of BH4-responsiveness are only for selecting potential responders. Accordingly, it is necessary to test potential responders with a long-term BH4 challenge.

Mutation analysis of PAH gene in patients with PKU in western Iran and its association with polymorphisms: identification of four novel mutations.

Phenylketonuria (PKU) is an autosomal recessive disorder characterized by a mutation in the phenylalanine hydroxylase (PAH) gene. Untreated PKU can lead to mental retardation, seizures, and other serious medical problems. This study was designed to investigate the status of molecular defects in the PAH gene and their association with polymorphisms in Kurdish patients with PKU in the Kermanshah province, western Iran. The study was conducted on 27 unrelated patients with PKU over a 2-year period (from 2010 to 2012). All 13 exons plus exon-intron boundaries of the PAH gene were analyzed and we identified 15 different mutations, including two novel mutations, in 51 of the 54 mutant alleles (diagnostic efficiency of 94.4 %). IVS4 + 1G > C (c.441 + 1G > C) and IVS7 - 5 T > C (c.843 - 5 T > C) are novel mutations that have not been reported in the academic literature or the PAH locus database ( http://www.pahdb.mcgill.ca ); therefore, they may be specific to the Kurdish population. IVS2 + 5G > C and IVS9 + 5G > A were the two most prevalent mutations in our sample, with frequencies of 26 % and 17 %, respectively. The second most common mutations were p.R261X, IVS10 - 11G > A, p.K363 > Nfs and IVS7 - 5 T > C, with each showing a relative frequency of 7.4 %. All other detected mutations, including p.F55 > Lfs, p.R176X, p.R243Q, p.V230I, p.R243X, p.R261Q, IVS8 - 7A > G and p.E390G had frequencies of less than 4 %. The present study showed that there is a distinct difference in the characteristics of PAH mutations between the Kermanshah province and other parts of Iran, suggesting that Kermanshah may have a unique population distribution of PAH gene mutations. Iran lies on the route of major ancient movements of the Caucasian people toward the Mediterranean basin, and Kermanshah has previously been called the gateway to Asia. Most of the mutations identified in this study are common in the Mediterranean region. Therefore, our findings are consistent with the historical and geographical links between the Iranian population and the populations of Mediterranean region.

[Mutation analysis of the phenylalanine hydroxylase gene of phenylketonuria patients of Kemerovskaya Oblast' and Saha Republic].

Phenylketonuria (PKU) associated mutations in phenylalanine hydroxylase (PAH) gene were identified by direct DNA sequencing in 46 PKU patients and members of their families from Kemerovskaya Region and Saha Republic. Mutations found included both widespread known mutations (R158Q, R252W, R261Q, P281L, IVS10-11G>A, R408W, IVS12+1G>A) and several rare mutations (IVS2+5G>A, R155H, Y168H, W187R, E221_D222>Efs, A342T, Y386C, IVS11+1G>C). We observed the increase in diversity of PKU-associated alleles in the populations studied, probably due to their complex mixed ethnic structure.

Mutational spectrum of phenylketonuria in the Chinese Han population: a novel insight into the geographic distribution of the common mutations.

The absence of a comprehensive analysis for phenylketonuria (PKU) mutations in the Chinese Han population has resulted in continued studies during the past 18 y to elucidate the mutational spectrum in patients from virtually all Chinese regions. Our study systematically investigated 13 exons and their surrounding introns of the phenylalanine hydroxylase (PAH) gene in 212 unrelated patients using PCR and direct sequencing. A total of 79 different mutations were identified in 405 of 424 mutant PAH alleles including 15 novel ones. Eight mutations, R243Q, Ex6-96A>G, IVS4 - 1G>A, R413P, Y356X, R111X, R241C, and V399V, with a relative frequency of 3% or more, accounted for two thirds of the identified ones. The data presented in this study indicates that the total pool of mutant PAH alleles in China consisted of a small number of common mutations and a very high number of rare mutations. Moreover, by merging the findings of previous studies to generate a more composite data set for the Chinese mainland, it is shown that there are no significant differences of the common mutations between southern and northern except for R413P statistically, raising questions about the previous hypothesis that great variations on mutation frequencies exist between above regions.

Cognitive Outcomes and Relationships with Phenylalanine in Phenylketonuria: A Comparison between Italian and English Adult Samples.

We aimed to assess if the same cognitive batteries can be used cross-nationally to monitor the effect of Phenylketonuria (PKU). We assessed whether a battery, previously used with English adults with PKU (AwPKU), was also sensitive to impairments in Italian AwPKU. From our original battery, we selected a number of tasks that comprehensively assessed visual attention, visuo-motor coordination, executive functions (particularly, reasoning, planning, and monitoring), sustained attention, and verbal and visual memory and learning. When verbal stimuli/or responses were involved, stimuli were closely matched between the two languages for psycholinguistic variables. We administered the tasks to 19 Italian AwPKU and 19 Italian matched controls and compared results from with 19 English AwPKU and 19 English matched controls selected from a previously tested cohort. Participant election was blind to cognitive performance and metabolic control, but participants were closely matched for age and education. The Italian AwPKU group had slightly worse metabolic control but showed levels of performance and patterns of impairment similar to the English AwPKU group. The Italian results also showed extensive correlations between adult cognitive measures and metabolic measures across the life span, both in terms of Phenylalanine (Phe) levels and Phe fluctuations, replicating previous results in English. These results suggest that batteries with the same and/or matched tasks can be used to assess cognitive outcomes across countries allowing results to be compared and accrued. Future studies should explore potential differences in metabolic control across countries to understand what variables make metabolic control easier to achieve.

Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria.

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.

Dietary patterns, cost and compliance with low-protein diet of phenylketonuria and other inherited metabolic diseases.

BACKGROUND/OBJECTIVES: Phenylketonuria (PKU) and several other inherited metabolic diseases (IMD) require a lifelong low-protein diet (LPD), otherwise they lead to many health complications. LPDs, however, carry a significant economic burden for patients and their families. The objective of this study was to explore the costs of low-protein foods (LPFs) necessary for LPD as well as dietary patterns and compliance towards an LPD. SUBJECTS/METHODS: A detailed questionnaire was created in cooperation with National Association of PKU and other IMD (NSPKU), and consequently sent to all NSPKU members treated with an LPD (n=303). A total of 184 respondents from the Czech Republic were included in the study (174 had PKU, 10 had other IMD). RESULTS: The average daily consumption of LPF was equal to 411.7 g (PKU) and 345.6 g (other IMD), which corresponds to energy value of 5558 kJ and 4438 kJ, respectively, per patient per day. Patients mostly consumed low-protein flour (≈30% of energy intake), pasta (≈18%), basic pastry (≈15%) and sweets (≈10%). The average monthly costs of LPDs were equal to [euro ]130 (PKU) and [euro ]129 (other IMD) per patient per month. The compliance with LPD was decreasing with increasing age (P<0.0001). CONCLUSIONS: This is the largest study examining costs and dietary patterns of LPDs in patients with PKU and the first study of this kind in other IMD patients requiring an LPD. The study clearly showed that an LPD carries a very high economic burden for families, which may lead to less LPD compliance and potential severe health consequences.

Characterization of phenylketonuria alleles in the Italian population.

In order to identify the molecular basis of phenylketonuria (PKU) in Italy, we screened the entire coding sequence of the phenylalanine hydroxylase gene in 20 Italian PKU patients, whose origins are scattered throughout Italy. The frequency of each identified mutation and of 5 other European mutations was determined within a panel of 92 Italian PKU patients. This approach allowed us to identify 20 different PKU mutations and characterize 64% of the Italian PKU chromosomes. Eleven mutations (IVS10nt546, L48S, R158Q, R261Q, P281L, R261X, R252W, delta T55, IVS7nt1, IVS12nt1, Y414C) represent 55.4% of the Italian PKU alleles, the most common mutations being IVS10nt546 (12.4%) and L48S (9%). All the other mutations are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations.

Phenylalanine hydroxylase gene mutation R408W is present on 84% of Estonian phenylketonuria chromosomes.

Phenylalanine hydroxylase (PAH) is the enzyme which converts phenylalanine into tyrosine. In case of its deficiency, hyperphenylalaninemia is observed, which leads to phenylketonuria (PKU), a disease causing mental retardation, unless treated with a low-phenylalanine diet since early childhood. In Estonia, PKU is among the most common inherited metabolic diseases. The data from retrospective study and newborn screening show an approximate incidence of 1 in 6,000 newborns. Molecular analysis of 34 Estonian patients has revealed high genotypic homogeneity in this group, as 84% of the mutant alleles carry the R408W mutation. The high rate of this mutation in the Estonian population rises the speculation of Finno-Ugric contribution to the East European pool of mutant PAH alleles. Five more mutations-IVS12nt1, R261Q, R252W, R158Q, S349P-have been detected. The mutation detection rate was 92% among the studied patients.

'Celtic' phenylketonuria chromosomes found? Evidence in two regions of Quebec Province.

We analyzed mutations, RFLP haplotypes (H), and a VNTR polymorphism at the phenylalanine hydroxylase locus (PAH) in 12 French-Canadian patients with phenylketonuria (PKU) from the eastern region of Quebec province and 13 non-French-Canadian PKU patients from the Montreal region. There were 10 different mutation/H/VNTR haplotype combinations on the 50 PKU chromosomes: one set of 5 and another of 8 accounted for 88 and 77% of these chromosomes in the French-Canadian and non-French-Canadian patients, respectively. The differences in PKU haplotypes between the two groups of probands reflect the different histories of the two populations. Three PKU haplotype combinations were shared by the two groups: IVS12nt1:H-3:VNTR-8, I65T:H-9:VNTR-8, and R408W:H-1:VNTR-8. The IVS12nt1 mutation (18% of the total sample) is prevalent in northern Europeans. The I65T-H-9 and R408W:H-1 haplotypes have seldom been reported in Europeans but when encountered tend to be found in northwestern regions. The R408W mutation is usually on H-2 in Europeans. In Quebec the R408W:H-1 and I65T:H-9 haplotypes accounted for 20% of PKU chromosomes, clustered in two geographic regions, and in every family where they occurred an Irish or Scottish ('Celtic') ancestor could be inferred. We propose that I65T:H-9:VNTR-8 and R408W:H-1:VNTR-8 chromosomes are markers for a diaspora of 'Celtic' descendants. Our findings predict the distributions of these unusual PKU haplotypes in contemporary Europeans.

Gypsy phenylketonuria: a point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia.

A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. We analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 "classical PKU" patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group we detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served us effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects.

Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan.

Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.

[Mutation in the structure of exon 7 of the phenylalanine hydroxylase in phenylketonuria patients from the Novosibirsk area]. / Mutatsii v strukture ékzona 7 gena fenilalaningidroksilazy u bol'nykh fenilketonuriei Novosibirskoi oblasti.

The spectrum and frequency of mutations of exon 7 of the gene for phenylalanine hydroxylase (PAH) were studied in 34 phenylketonuria (PKU) patients living in Novosibirsk oblast. The five most prevalent mutations constituted 17.64% of defective alleles: R243Q (1.47%), R252W (1.47%), R261Q (5.88%), E280K (1.47%), and P281L (7.35%). A neutral polymorphic locus V245V was found within exon 7.

[Genetic diagnosis of phenylketonuria. III. Mutations of phenylalanine hydroxylase gene in Orientals].

Phenylketonuria (PKU) is an autosomal recessive disorder caused by lesions in the phenylalanine hydroxylase (PAH) gene. The recent studies on PAH mutations show the genetic drift of PKU alleles among some Oriental populations. Therefore, we searched for PKU mutations among Japanese, Chinese and Taiwanese. Direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid-phase technology involving the biotin-streptavidin system. Two new mutations (R241C and G247V) and two of the known mutant alleles (Y204C and R243Q) were found in two Taiwanese and two Chinese PKU patients, and three known mutations (R111X, Y204C and R413P) were recognized in three Japanese; two new mutations were identified in exon 7 of the PAH gene at codon 241 and codon 247, where the single base changes from C to T and from G to T substituted cysteine for arginine and valine for glycine, respectively. Further all the PAH mutations detected are common in Oriental populations as they have been thus far unreported among Caucasians. From these data as well as the clinical phenotype of the patients, we suggest that the R241C and G247V substitutions may interfere with proper enzyme function, although we have not yet performed functional studies. More detailed studies would be needed to clarify the regional distribution of mutant chromosomes in Oriental populations and other unidentified mutations.

[Genetic diagnosis of phenylketonuria. IV. Mutations of phenylalanine hydroxylase gene in Caucasian and Gypsy populations in Czech and Slovakia Republics].

Direct sequencing was conducted on the regions of the exon 7 and 12 in the phenylalanine hydroxylase (PAH) gene amplified by the polymerase chain reaction, using solid-phase technology involving the biotin streptavidin system. A novel mutation and seven previously known mutations were identified in the PAH genes among 15 Caucasians and 10 Gypsies in the Czech and Slovakia republics, affected with classical phenylketonuria (PKU). Two of these substitutions (R243X and G272X) resulted in the generation of a premature stop codon, and a single base transition of G to A at codon 261 resulted in the substitution of Arg for Gln (R261Q). These three mutations together accounted for 16.7% of PKU alleles among 15 Caucasians. The R252W mutation was detected in these two groups: two Caucasians were compound heterozygous for the P281L or R408W mutations (6.7% of all mutant alleles). However, all 10 Gypsy PKU patients were homozygous for the R252W transition. The R408W mutation accounted for 43% of PKU alleles in 15 Caucasian PKU patients. A novel heterozygous C-to-G transversion at the third base of codon 263 in the exon 7, resulting in the substitution of Phe for Leu (F263L), was detected in a Caucasian PKU patient (3.3% of all mutant alleles). In this study, we revealed a novel PKU mutation of the F263L in Caucasian populations and a high-risk PKU mutation of the R252W in Gypsy populations.

Ethnic and gender patterns for the five congenital disorders in Texas from 1992 through 1998.

The Texas Department of Health's Newborn Screening Program screens for five inherited disorders: phenylketonuria (PKU), congenital adrenal hyperplasia (CAH), congenital hypothyroidism (CH), galactosemia (GAL), and sickle cell disease (SCD). The objective of this study was to determine the prevalence of these disorders and to describe ethnic and gender patterns in their distribution. Cases were identified from blood specimens collected at birth from live births in Texas from 1992 through 1998. During this time, the overall prevalence of these disorders per 10,000 live births was 0.70 for PKU, 0.21 for GAL, 4.18 for CH, 1.03 for CAH, and 3.92 for SCD. Ethnic and gender disparities were observed among PKU, CH, CAH, and SCD prevalence. Results suggest that unidentified mutations and environmental factors may exist that contribute to these patterns. This warrants further investigation to determine possible modifiable risk factors for populations with higher prevalence.

Preliminary mutation analysis in the phenylalanine hydroxylase gene in Greek PKU and HPA patients.

The presence of nine mutations in the phenylalanine hydroxylase (PAH) gene, previously described in phenylketonuria (PKU) patients of other Mediterranean and European populations, was assessed in 47 Greek PKU and 3 hyperphenylalaninaemia (HPA) patients. Of the nine mutations investigated, only five were detected, characterizing 31% of the PKU alleles in our patients.

Linkage disequilibrium between RFLP haplotype 2 and the affected PAH allele in PKU families from the Berlin area of the German Democratic Republic.

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.