Phenylalanine hydroxylase genotype-phenotype associations in the United States: A single center study.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the phenylalanine hydroxylase gene (PAH). The correlation between genotype and phenotype can be complex and sometimes variable but often very useful for categorizing and predicting dietary tolerance and potential outcome. We reviewed medical records for 367 patients diagnosed with PKU or persistent mild hyperphenylalaninemia (MHP) between 1950 and 2015 who had PAH genotyping. In 351 we had the full PAH genotype as well as phenotypic characteristics such as phenylalanine (Phe) concentrations (at newborn screening, confirmation, and highest known), and dietary Phe tolerance. On 716 mutant chromosomes, including 14 in genotypes with only one identified variant, we identified 114 different pathogenic variants. The most frequent, p.R408W, was present in 15.4% of the alleles; other frequent variants were c.1315 + 1G > A (6.1%), p.I65T (5.7%), and p.R261Q (5.7%). Three variants, c.142 T > G (p.L48 V), c.615G > C (p.E205D), and c.1342_1345delCTCC, were novel. We used the phenotypic parameters of variants paired with null alleles (functional hemizygotes) to assign the variants as classic PKU, moderate PKU, mild PKU, MHP-gray zone, or MHP. We also included the phenotype association(s) for all of the full genotypes. In 103 patients, we also could assign sapropterin dihydrochloride responsiveness, which is a synthetic form of the tetrahydrobiopterin (BH4) PAH cofactor. This compilation from a single metabolic center provides further information on PAH variants in the United States and the correlations between genotype and phenotype.

Clinical characterization of tremor in patients with phenylketonuria.

BACKGROUND: Phenylketonuria (PKU) is due to the deficit of the enzyme phenylalanine hydroxylase, the first step of dopamine synthesis. If not early treated the disease results in severe neurological impairment. Minor neurological signs have been reported in early treated PKU (ETPKU) subjects. Prolactin level is affected by (and reflects) brain dopamine availability. Object of the study was to assess the occurrence, age at onset, distribution, associated neurological signs, and possible pathogenetic biomarkers of tremor in ETPKU. METHODS: Fifty-nine ETPKU and 43 control subjects (age range 7-54) underwent individual and familiar tremor history, clinical assessment of tremor by means of the Fahn-Tolosa-Marin Tremor Rating Scale, and IQ evaluation. Historical and concomitant biochemical data (blood levels of Phe) and serum prolactin were included in the analysis. RESULTS: Thirty-two percent of ETPKU patients were affected by postural and kinetic tremor. We found a significant correlation between severity of tremor and: prolactin level at the day of examination (part A: rs = 0.320; p = .014; part C: rs = 0.319; p = .014), Phe fluctuations from 12 years onwards (part B: rs = 0.300; p = .036). We also found a significant correlation between prolactin (18.2 ±â€¯9.6 ng/ml) and Phe levels (852 ±â€¯472 µmol/l) on the day of assessment (rs = 0.470; p < .001). CONCLUSIONS: The main clinical features of tremor in ETPKU evoke those of essential tremor, although with a higher prevalence and an earlier onset than in general population. The severity of tremor was related to concomitant prolactin rather than Phe levels. This pattern suggests that metabolic alterations associated with PKU may result in an anticipation of the tremor onset in subjects who are possibly prone to this disorder.

Toward mechanistic models for genotype-phenotype correlations in phenylketonuria using protein stability calculations.

Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild-type PAH of 20 selected PKU-linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild-type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4 ), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single-site missense variants using the full-length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease-linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.

Haplotype-based Noninvasive Prenatal Diagnosis of Hyperphenylalaninemia through Targeted Sequencing of Maternal Plasma.

Here we developed a haplotype-based noninvasive prenatal diagnosis method for hyperphenylalaninemia (HPA) and demonstrated its accuracy and feasibility during early pregnancy. Capture sequencing was performed on genomic DNA from parents and probands using customized hybridization probes targeting highly heterozygous single-nucleotide polymorphisms located within the 1 M region flanking phenylalanine hydroxylase (PAH) and 6-pyruvoyltetrahydropterin (PTS) and its coding region to determine the parental haplotypes and linkage to pathogenic mutations. Maternal plasma DNA obtained at 12-20 weeks of gestation was also subjected to targeted sequencing to deduce the fetal haplotypes based on the parental haplotypes. The fetal genotypes were further validated by invasive prenatal diagnosis. Haplotype-based noninvasive prenatal testing was successfully performed in 13 families. Five fetuses were identified to harbor bi-allelic pathogenic variants of PAH, four fetuses were carriers of one heterozygous PAH variant, three fetuses were normal, and the fetus of the 6-pyruvoyl tetrahydrobiopterin synthase family was identified as normal. The fetal genotypes at two gestational weeks from the same PAH family were identical. All results were consistent with the prenatal diagnosis based on amniotic fluid. Haplotype-based noninvasive prenatal testing for HPA through targeted sequencing is accurate and feasible during early gestation.

Early Screening for Tetrahydrobiopterin Responsiveness in Phenylketonuria.

Since 2007, synthetic tetrahydrobiopterin (BH4) has been approved as a therapeutic option in BH4-responsive phenylketonuria (PKU) and since 2015 extended to infants younger than 4 years in Europe. The current definition of BH4 responsiveness relies on the observation of a 20% to 30% blood phenylalanine (Phe) decrease after BH4 administration, under nonstandardized conditions. By this definition, however, patients with the same genotype or even the same patients were alternatively reported as responsive or nonresponsive to the cofactor. These inconsistencies are troubling, as frustrating patient expectations and impairing cost-effectiveness of BH4-therapy. Here we tried a quantitative procedure through the comparison of the outcome of a simple Phe and a combined Phe plus BH4 loading in a series of infants with PKU, most of them harboring genotypes already reported as BH4 responsive. Under these ideal conditions, blood Phe clearance did not significantly differ after the 2 types of loading, and a 20% to 30% decrease of blood Phe occurred irrespective of BH4 administration in milder forms of PKU. Such early screening for BH4 responsiveness, based on a quantitative assay, is essential for warranting an evidence-based and cost-effective therapy in those patients with PKU eventually but definitely diagnosed as responsive to the cofactor.

DNA methylated alleles of the phenylalanine hydroxylase promoter remodeled at elevated phenylalanine levels in newborns with hyperphenylalaninemia.

OBJECTIVES: Although high phenylalanine (phe) exposure has been shown to influence the DNA methylation status of leukocytes in hyperphenylalaninemia (HPA), the potential of DNA methylation changes as a biomarker of pretreatment high phe exposure in diet free newborns with HPA has not been explored. We therefore investigated the DNA methylation pattern of the phenylalanine hydroxylase (PAH) gene promoter at different phe levels, and the possibility of DNA methylation pattern changes being a biomarker of high phe exposure in diet free newborns with HPA. DESIGN AND METHODS: With a combination of methylated PCR, high resolution melting, and sequencing, the cytosine phosphodiester bond guanine (CpG) dinucleotides in the 5' untranslated region of the PAH gene were analysed 2-15days after birth using leukocyte DNA from diet free 16 newborns with HPA and 16 healthy controls. RESULTS: In 2-3days blood cards, GTGTG and GTGC/TG alleles were both detected at similar low mean phe levels in healthy controls (59.39±14.62 and 55.33±13.43µmol/L) and non-phenylketonuria (PKU) HPA (265.00 and 244.25±73.73µmol/L). In HPA with PKU, the GTGTG and GTGC/TG alleles were both detected at dissimilar elevated mean phe levels (380.80±64.62 and 589.00±191.96µmol/L). In ≥7day blood cards, GTGTG and GTGC/TG alleles were both detected at similar excess mean phe levels in HPA with PKU (2297±374.38 and 1562.66±718.23µmol/L). CONCLUSION: The demethylated GTGTG and partial methylated GTGC/TG alleles are not pathogenic alleles. Our results suggest a specific remodeling of the DNA methylated alleles of the PAH promoter at elevated, but not excess phe levels in diet free newborns with PKU.

Prenatal diagnosis of Chinese families with phenylketonuria.

The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.

[Mutations of the phenylalanine hydroxylase gene in phenylketonuria patients from Shaanxi].

OBJECTIVE: To investigate the feature of phenylalanine hydroxylase (PAH) gene mutations and provide guidance for genetic and prenatal diagnosis of patients with phenylketonuria from Shaanxi. METHODS: For 55 patients whose blood Phe concentration was over 2.0 mg/dL, potential mutations in 13 exons and flanking sequences of the PAH gene were detected by PCR and DNA sequencing. RESULTS: A total of 98 mutations were detected in 110 PAH alleles, with the detection rate being 89.10%. Nine mutations have been identified in exon 7, which accounted for 33.67% of all. Exon 12 (14.29%) and exon 3 (12.24%) have followed. Thirty eight mutations, locating in exon2-exon12 and the flanking sequence, were detected in the 55 PKU patients. p.R243Q (24.49%) was the commonest mutation, whilstp.A47E, p.I65S and p.A259T were first discovered in China. After querying international databases including PAHdb and HGMD, the p.C334X was verified as the novel PAH gene mutation. CONCLUSION: The mutation spectrum of the PAH gene in Shaanxi has been identified. And a novel mutation has been identified. This may facilitate the diagnosis of PKU in the future.

Variations in genotype-phenotype correlations in phenylalanine hydroxylase deficiency in Chinese Han population.

BACKGROUND: The value of genotyping to predict variant phenotypes in patients with phenylalanine hydroxylase (Pah) deficiency is a matter of debate. However, there exists no comprehensive population relationship study focused on the Han Chinese. METHODS: We analyzed genotype-phenotype correlation for 186 different genotypes in 338 unrelated Chinese patients harboring 109 different Pah mutations. Two systems were used in this process. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation. The second was a pair-wise correlation analysis. The observed phenotype for AV analysis was the corresponding metabolic phenotype stratified according to the pretreatment phenylalanine (Phe) value. RESULTS: We found that the observed phenotype matched the predicted phenotype in 54.41% of 272 patients for whom AV information was available; the highest degree of concordance (61.83%) was found in patients with null/null genotypes, whereas the lowest "concordance rate" (32.69%) was observed for patients with expected mild-PKU phenotype. There are repeated inconsistencies for such mutations as R241C, R243Q, R261Q, V388M, V399V, R408Q, A434D and EX6-96A>G which are associated with variable phenotypes in patients with identical genotype. Significant correlations were disclosed between pretreatment Phe values and predicted residual activity (r=-0.45643, P<0.0001) or AV sum (r=-0.59523, P<0.0001). CONCLUSION: Our study supports the notion that the Pah mutation genotype is the main determinant of metabolic phenotype in most patients in a particular population, and provided novel insights into the values that underpin the subsequent treatment and the prognosis of PKU in Chinese.

Processing speed and executive abilities in children with phenylketonuria.

OBJECTIVE: Phenylketonuria (PKU) is a hereditary metabolic disorder that often results in neuropsychological impairment, even in individuals treated early and continuously. This study was conducted to examine processing speed, variability in processing speed, and the relationship between processing speed variables and executive abilities in children with early and continuously treated PKU. METHOD: Participants were 42 children with PKU and 81 typically developing children from 7 to 18 years of age. Children completed 3 computerized reaction time (RT) tasks (simple RT, go/no-go, stimulus-response compatibility) and 7 tasks assessing executive abilities (working memory, inhibitory control, strategic processing). RESULTS: Performance of children with PKU was significantly slower and more variable than that of controls across the 3 tasks administered. When age was considered, it was shown that processing speed improved with age to a comparable degree for both groups. Variability in processing speed, however, decreased more with age for the PKU than control group, reflecting the fact that variability in younger, but not older, children with PKU was greater than that of controls. With regard to executive abilities, processing speed and variability contributed to performance on most, but not all, executive tasks; and after controlling for processing speed and variability, executive impairments were still identified in working memory and inhibitory control (not strategic processing). CONCLUSIONS: These findings indicate that information processing is slower and less efficient in children with PKU. In addition, processing speed and variability contribute to some, but not all, of the impairments in executive abilities observed in children with PKU.

START, a double blind, placebo-controlled pharmacogenetic test of responsiveness to sapropterin dihydrochloride in phenylketonuria patients.

UNLABELLED: Sapropterin dihydrochloride, a synthetic tetrahydrobiopterin (BH4), works as a chaperone of phenylalanine hydroxylase (PAH) in phenylketonuria (PKU) to facilitate and stabilize folding of PAH into its most active conformation. No standard pharmacogenetic tests exist to identify responsive genotypes. Previous studies have failed to identify genotypes that consistently predict response; they are weakened by varied: 1) doses; 2) response definitions; 3) duration; 4) phenylalanine (PHE) test times during different protein catabolic states; 5) control of dietary PHE. START (sapropterin therapy actual response test) protocol is a double blind, placebo-controlled, 4-week clinical test that obviates the confounders aforementioned. START results were evaluated for response-genotype correlates and trends in molecular characteristics. RESULTS: Seventy-four patients completed START. Thirty-six patients (48.6%) responded, 55 patients' genotypes are known, 38 unique genotypes are present. Alleles consistently associated with response include Y414C (8/8 patients, 6 genotypes) and I65T (9/9 patients, 6 genotypes). The p.R408W mutation, in which substitution of straight chain arginine with bulky aromatic amine, tryptophan, at the crux of a strategic hinge site activating folding of PAH, amino acid sequence 408, was strongly associated with non-response (21/29 patients non-responsive, 12/17 genotypes non-responsive). Genotypes containing at least one allele with ≥25% residual activity compared to wild type, were strongly associated with response. CONCLUSIONS: The START protocol provides a rigorous pharmacogenetic test to identify sapropterin responsiveness and genotypes associated with responsiveness and non-responsiveness. Some genotypes were found to be predictive of responsiveness or non-responsiveness, and responsiveness was associated with specific alleles. The START protocol provides a reliable test for sapropterin responsiveness and will continue to improve understanding of how PKU mutations impact PAH protein-folding dynamics and enhance understanding of PKU disease and its management.

[Phenylketonuria]. / Fenyyliketonuria.

Owing to our special genetic heritage, phenylketonuria is very rare in Finland, but the situation will change as the number of immigrants from populations with a larger incidence increases. In persons with this disorder, the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine to tyrosine, is not functioning normally, leading to the accumulation phenylalanine within the body. High levels of phenylalanine are toxic to the central nervous system. A newborn affected with phenylketonuria is asymptomatic but will rapidly become disabled without therapy. The leading principle of the therapy is careful limitation of phenylalanine intake.

Genotype and natural history in unrelated individuals with phenylketonuria and autistic behavior.

We describe three unrelated individuals, two males (ages 35 and 9) and a female (age 8) presenting with late diagnosed phenylketonuria (PKU) and autistic behavior, all showing poor adhesion to the dietary treatment resulting in high plasmatic phenylalanine levels, particularly in the oldest subject. Clinical findings included hair hypopigmentation, microcephaly, severe mental retardation with absent development of verbal language and autistic symptoms in all three patients, whereas variable neurological signs such as seizures, spasticity, ataxia, aggressivity, and hyperactivity were individually found. Homozygosity for the IVS10nt11g/a (IVS10nt546) was found in all. This is the first report of molecular findings in subjects with PKU also presenting with autistic features. The authors discuss if this mutation is particularly involved in the association of autistic symptoms in untreated PKU individuals.

Genotype and natural history in unrelated individual with phenylketonuria and autistic behavior

We describe three unrelated individuals, two males (ages 35 and 9) and a female (age 8) presenting with late diagnosed phenylketonuria (PKU) and autistic behavior, all showing poor adhesion to the dietary treatment resulting in high plasmatic phenylalanine levels, particularly in the oldest subject. Clinical findings included hair hypopigmentation, microcephaly, severe mental retardation with absent development of verbal language and autistic symptoms in all three patients, whereas variable neurological signs such as seizures, spasticity, ataxia, aggressivity, and hyperactivity were individually found. Homozygosity for the IVS10nt11g/a (IVS10nt546) was found in all. This is the first report of molecular findings in subjects with PKU also presenting with autistic features. The authors discuss if this mutation is particularly involved in the association of autistic symptoms in untreated PKU individuals.

Descrevemos três indivíduos não aparentados, dois do sexo masculino (com idades de 35 e nove anos) e um do sexo feminino (com idade de oito anos) apresentando fenilcetonúria diagnosticada tardiamente e comportamento autístico, todos com adesão limitada ao tratamento dietético resultando em altos níveis plasmáticos de fenilalanina, especialmente no indivíduo mais velho. Os achados clínicos incluem hipopigmentação de cabelos, retardo mental grave com ausência de desenvolvimento da linguagem verbal e sintomas autísticos nos três pacientes, enquanto outros achados neurológicos como convulsões, espasticidade, ataxia, agressividade e hiperatividade são descritos em um indivíduo, cada. Homozigose para a mutação IVS10nt11g/a (IVS10nt546) foi encontrada em todos. Este é o primeiro relato de achados moleculares em indivíduos com fenilcetonúria que desenvolveram características autísticas. Discute-se se essa mutação estaria particularmente envolvida no desenvolvimento de sintomas autísticos em indivíduos com fenilcetonúria não tratada.

Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism.

[Lipids composition diet in phenylketonuric children with early diagnosis]. / Composición lipidica de la dieta de niños fenilquetonúricos diagnosticados precozmente.

Phenylketonuria (PKU) is a genetic disorder caused by a partial or complete mutation of the enzyme phenylalanine hydroxylase (PHA), fact that produces high levels of phenylalanine in blood resulting in mental retardation if not diagnosed during the neonatal period. Treatment consists of a phenylalanine (Phe) restricted diet. Several studies have shown that due to restriction of animal protein, this diet is deficient in fatty acids such as alfalinolenic acid (ALA) and provides high levels of linoleic acid (LA). The objective of this study was to determine the lipid composition of the diet consumed by children with early-diagnosed PKU. Lipid composition of the Phenylalanine restricted diet consumed by 29 children with PKU and in follow-up at INTA, University of Chile, were analyzed. Children were paired by sex and age with a control group. A twenty-four hour dietary recall was performed for 3 consecutive days and total fatty acid intake, including saturated, monounsaturated, polyunsaturated, LA and ALA, were calculated. In the restricted diet of children with PKU, 31.8% of total calories are from fat, 13% of which are LA and 0.2% ALA, showing significant differences as compared to the control group. The ratio of saturated:monounsaturated:polyunsaturated fatty acids was 1:1.7:3.9 and the ratio of LA:ALA was ten-fold higher than the recommended ratio of 115:1. It is concluded that the Phenyalanine restricted diet of Chilean children with PKU is high in LA and low in ALA.

Optimal management of phenylketonuria: a centralized expert team is more successful than a decentralized model of care.

OBJECTIVE: To compare phenylketonuria (PKU) management by a centralized, expert team in the Province of Nova Scotia (NS) with the decentralized approach in New Brunswick (NB). STUDY DESIGN: Retrospective chart review documented frequency of outpatient visits, phenylalanine (Phe) concentration, and medical formula use. Structured telephone interviews with the 8 regional NB dietitians (NB-D) documented their knowledge and support in PKU management. Patients with PKU (n=108; age, birth to 42 years) reside in NB (n=69) and NS (n=39). More were lost to contact in NB than in NS (9/69 vs 1/39) and more were completely off diet in NB than in NS (24/60 vs 1/38, P=.05). All 15 children <2 years old followed by a PKU team in either NS or Saint John, NB had optimal Phe levels. Children 2 to 12 years of age in NS had better Phe control and more medical visits than in NB (P <.01). Older patients had more episodes of elevated Phe levels (P=.01). Formula was dispensed in appropriate yearly amounts to 52% in NB and >95% in NS. Mental handicap or borderline intelligence was common in both NB (44%) and NS (42%). All NB-D wished additional specialized medical, nursing, or social work assistance. CONCLUSIONS: PKU management appears to be more effective with an expert, coordinated team approach.

[Evaluation of the program for prevention and health promotion in phenylketonuria patients in Brazil]. / Avaliação do programa de prevenção e promoção da saúde de fenilcetonúricos.

OBJECTIVE: To evaluate the results of systematizing preventive and health promotion actions among phenylketonuria (PKU) patients. METHODS: Results of phenylketonuria patients attended in the Prenatal PKU Screening Program in the State of Paraná, Brazil, from 1996 to 2001, were evaluated. Socioeconomic data were investigated and the gross motor function measure was applied to determine the motor score among 32 PKU infants with early diagnosis and treatment. Pearson's correlation coefficient was adopted to investigate the relationship between the target variable (motor score) and other quantitative variables (mean post-treatment serum phenylalanine level, parents' educational level, infant's age at the start of treatment, and family income). RESULTS: Among all the children evaluated, 93.7% showed development within normal limits as reported in the literature. Treatment was initiated in the first month of life in 71.9% of the PKU cases. Socioeconomic data showed 39.5% of parents having completed the fourth grade of primary school or less. There was a significant correlation between infant's motor score and parents' educational level (N=32), as well as between motor score and early treatment (N=27). CONCLUSIONS: The results highlighted the program's effectiveness. The correlation between parents' low educational level and lower motor score emphasizes the importance of support for parents in their use of diet therapy. The association between motor score and early initiation of treatment confirms the need for immediate admission into the program. The paucity of other evaluation studies in the literature makes generalization of the results difficult.