Phenylalanine Hydroxylase Research Articles

Development of an mRNA replacement therapy for phenylketonuria

Phenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy. A full-length mRNA encoding human PAH (hPAH) is encapsulated in our proprietary lipid nanoparticle LUNAR and delivered to a Pahenu2 mouse model that carries a missense mutation in the mouse PAH gene. Animals carrying this missense mutation develop hyperphenylalanemia and hypotyrosinemia in plasma, two clinical features commonly observed in the clinical presentation of PKU. We show that intravenous infusion of LUNAR-hPAH mRNA can generate high levels of hPAH protein in hepatocytes and restore the Phe metabolism in the Pahenu2 mouse model. Together, these data establish a proof of principle of a novel mRNA replacement therapy to treat PKU.

Characterization and expression of phenylalanine hydroxylase in rabbits under different feeding conditions of faba bean (Vicia faba L.)

Characterization and expression of phenylalanine hydroxylase in rabbits under different feeding conditions of faba bean (Vicia faba L.)

A culture model for the assessment of phenylalanine neurotoxicity in phenylketonuria

ObjectivePhenylketonuria (PKU) is caused by a specific mutation of the phenylalanine hydroxylase (PAH) gene. The deficiency of PAH results in high phenylalanine levels (Phe), low tyrosine levels (Tyr), and reduced catecholamine neurotransmitters. The majority of PKU patients, if untreated, develop severe mental retardation. The specific contribution of high Phe and low Tyr levels in mental retardation is largely unknown. In this study, we used organic hippocampal slice cultures in an optimized medium as an adequate culture model to decipher the precise role of high Phe and low Tyr levels on synaptic and glial integrity in PKU. The hippocampus is closely related to learning and memory and reduced catecholamine neurotransmitter levels can be neglected since these neurotransmitters do not derive from the hippocampus. Cultures exposed to physiological concentrations of Phe were compared with cultures exposed to doses of Phe/Tyr, as in the cerebral fluid of PKU patients.MethodsUsing capillary western blot analysis and immunohistochemistry, followed by quantitative image analysis, we tested the expression of various pre- and postsynaptic proteins (PSD95, synaptopodin, SNAP25, synaptophysin), glial cell markers (GFAP, Iba1, P2Y12, CD68, C3b), and the morphology of glial cells.ResultsWe found a downregulation of the postsynaptic protein PSD95 and the presynaptic protein SNAP25 in the presence of high/low Phe/Tyr levels after 3 weeks, which, then however, recovered after 6 weeks in culture. Furthermore, no change in the expression pattern of glial proteins was observed.ConclusionOur results show that high Phe levels/low Tyr levels alone are unlikely to substantially contribute to mental retardation in PKU. The direct neurotoxic potency of high Phe/low Tyr concentrations is almost negligible since the effects are transient. The transient character in the presence of unchanged levels of high Phe/low Tyr points to a role of reduced catecholamine derivate neurotransmitters, rather than of high Phe/low Tyr levels in PKU.

Mutation analysis of exon 5 of PAH gene in phenylketonuria patients from Golestan Province, Iran

Background and aims: In the phenylalanine hydroxylase (PAH) gene, various mutations are mainly responsible for phenylketonuria (PKU). After thalassemia, PKU is considered as the most common autosomal recessive disease among Iranian population. Therefore, identifying the mutations that cause the disease in this population is of great importance. This study aimed to identify exon 5 mutations of PAH gene in PKU patients from Golestan, the northern province of Iran, and compare these mutations with those onesa found by studies carried out in other parts of the country. Methods: During a one-year period, 25 unrelated PKU patients aged 1-23 years and from different parts of Golestan province were included in the study. Then, their genomic DNAs were extracted from their blood samples and PCR-sequencing method was used to identify the mutations. The sequencing results were analyzed using Chromas and CLC Main Workbench v3.5 software. Results: In this study, R158Q mutation was detected with a frequency of 6%. This mutation was homozygous in one PKU patient, but it was heterozygous in another one. These patients had cPKU phenotype. Conclusion: Evaluation of mutations proved to be a useful technique for molecular diagnosis of PKU and identification of disease carriers in the given population. Taking into account the fact that only one exon of PAH gene was explored in this study, however, it is recommended that further studies be conducted to investigate other exons in order for obtaining the complete mutation spectrum of this gene in PKU patients in Golestan province.

A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

ABSTRACT The majority of long noncoding RNAs (lncRNAs) contain transposable elements (TEs). PAHAL, a nuclear-retained lncRNA that is inserted by a Gypsy retrotransposon, has been shown to be a vital regulator of phenylalanine hydroxylase (PAH) gene expression that controls dopamine biosynthesis and behavioural aggregation in the migratory locust. However, the role of the Gypsy retrotransposon in the transcriptional regulation of PAHAL remains unknown. Here, we identified a Gypsy retrotransposon (named Gypsy element) as an inverted long terminal repeat located in the 3′ end of PAHAL, representing a feature shared by many other lncRNAs in the locust genome. The embedded Gypsy element contains a RNA nuclear localization signal motif, which promotes the stable accumulation of PAHAL in the nucleus. The Gypsy element also provides high-affinity SRSF2 binding sites for PAHAL that induce the recruitment of SRSF2, resulting in the PAHAL-mediated transcriptional activation of PAH. Thus, our data demonstrate that TEs provide discrete functional domains for lncRNA organization and highlight the contribution of TEs to the regulatory significance of lncRNAs.

Allelic dropout in PAH affecting the results of genetic diagnosis in phenylketonuria.

Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism. It is mainly caused by a deficiency in phenylalanine hydroxylase (PAH) and frequently diagnosed with Sanger sequencing. To some extent, allelic dropout can explain the inconsistency in genotype and phenotype. Three families were evaluated through DNA sequence analysis, multiplex ligation-dependent probe amplification (MLPA) and prenatal diagnosis technologies. The possibility of inconsistency in phenotype and genotype with c.331C>T variant was analysed. Through pedigree analysis, three mothers carried a homozygous c.331C>T variant, which was a false-positive result. New primers were used, and this error was caused by allelic dropout. In this case, c.158G>A was likely a benign variant. Sequence variants in primer-binding regions could cause allelic dropout, creating unpredictable errors in genotyping. Our results emphasised the need for careful measures to treat genotype-phenotype inconsistencies.

Engineering Organoids for in vitro Modeling of Phenylketonuria.

Phenylketonuria is a recessive genetic disorder of amino-acid metabolism, where impaired phenylalanine hydroxylase function leads to the accumulation of neurotoxic phenylalanine levels in the brain. Severe cognitive and neuronal impairment are observed in untreated/late-diagnosed patients, and even early treated ones are not safe from life-long sequelae. Despite the wealth of knowledge acquired from available disease models, the chronic effect of Phenylketonuria in the brain is still poorly understood and the consequences to the aging brain remain an open question. Thus, there is the need for better predictive models, able to recapitulate specific mechanisms of this disease. Human induced pluripotent stem cells (hiPSCs), with their ability to differentiate and self-organize in multiple tissues, might provide a new exciting in vitro platform to model specific PKU-derived neuronal impairment. In this review, we gather what is known about the impact of phenylalanine in the brain of patients and highlight where hiPSC-derived organoids could contribute to the understanding of this disease.

Frequency of PAH Mutations Among Classic Phenylketon Urea Patients in Mazandaran and Golestan Provinces, North of Iran

Phenylketonuria (PKU) is the most common aminoacidopathy with an autosomal recessive inheritance pattern. A global PKU prevalence is estimated about 6.002 in 100,000 newborns. In Iran, the prevalence of PKU is estimated at about 1 in 4,698, and it shows an increasing trend from north (0.0015%) to south (0.02%) of the country. Untreated PKU causes mental retardation, microcephaly, and seizure. PAH gene mutations located at chromosome 12q23 are responsible for the classical type of this disease. The spectrum of PAH mutations is varied in different ethnicities and different parts of the world. The aim of this study was to investigate the frequency of PAH mutation in the Mazandaran province, which could be useful for genetic counseling and prenatal diagnosis. A total of 66 individuals from 33 families from two provinces (9 families from Golestan and 24 families from Mazandaran) from north of Iran participated in this study. After genomic DNA extraction, PAH gene analysis was carried out using DNA sequencing of both coding and non-coding regions by ABI 3130XL genetic analyzer. Twenty-six different mutations were identified in the PAH gene in this study. Four mutations including IVS10-11 (c.1066-11G>A), c.727C>T (p.Arg243X), c.898G>T (p.Ala300Ser), and c.601C>T (p.His201Tyr) were the most common mutations with 37.48% frequency in Mazandaran province. Most frequent mutations in Golestan province were IVSI0-11 (c.1066-11G>A), c.722delG (p.Arg241fs), c.842C>T (p.Pro281Leu), and IVSII+5 (G>A) with frequency 58.57%. The results from the present study verify heterogeneity of the PAH gene and may help to diagnose tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.

Mutation Analysis of The Phenylalanine Hydroxylase Gene in Phenylketonuria Patients from Different Regions of Azerbaijan

The present study aimed to determine frequencies of mutations in the phenylalanine hydroxylase gene (PAH) in 30 patients diagnosed with phenylketonuria, from 633 person who presented to Scientific Research Pediatric Institute of the Ministry of Health of Azerbaijan and hospitals of different regions of Azerbaijan over the period from 2016 to 2020. More than 600 mutations in the PAH gene in exons 6, 7, 8, 11 and 12 have been detected in various countries around the world. Among them, 18 different mutations were found in Azerbaijani patients (V245V, R261Q, Q232Q, V245V, P281L, R241C, L385L, V399V, E280K, R261X, A434D, R176X, Ex6-96A> G, R241C, R243Q, R256Q). The R261Q mutation in exon 7 was observed in seven patients from 30 patients. In this study, we identified mutations in the PAH gene and to evaluate the genetic heterogeneity of PKU disease in 30 Azerbaijanian patients who had been referred to Lankaran, Masalli (Southern region of Azerbaijan), Qabala, Oguz, Zagatala, Balakan (North-western region of Azerbaijan), Guba, Gusar, Khachmaz and Khudat (Northern region of Azerbaijan) regions. From this experiment, 7 of 30 PKU patients had the same mutation. Our analysis of the R261Q mutations was nearly similar to that observed in Southern region of Azerbaijan. The R261Q mutation in exon 7 with a relative frequency of 23.3 % is G → A substitution leads to conservation of arginine amino acid to glutamine amino acid at codon 782 of PAH. The another major mutation in our study was R241C (10%), which is similar to the data obtained in population of Northern region of Azerbaijan, R243Q (6.6%) and R252Q (6.6%). Studies have shown that this mutation is most common in the Azerbaijani population. Genetic drift and founder effect may have also played a role in the mutation spectrum we observed. The data obtained in our study can also be used in the development of genetic tests for phenylketonuria. Phenylketonuria (PKU) is an autosomal recessive genetic disease, caused by the phenylalanine hydroxylase (PAH) deficiency in the metabolic pathway, which prevents phenylalanine from being converted into tyrosine, leading to a large amount of phenylalanine discharged from the urine. Therefore, it is necessary to establish a simple, fast, accurate and reliable PKU molecular diagnostic method for clinical diagnosis.

Deep learning program to predict protein functions based on sequence information

Deep learning technologies have been adopted to predict the functions of newly identified proteins in silico. However, most current models are not suitable for poorly characterized proteins because they require diverse information on target proteins. We designed a binary classification deep learning program requiring only sequence information. This program was named ‘FUTUSA’ (function teller using sequence alone). It applied sequence segmentation during the sequence feature extraction process, by a convolution neural network, to train the regional sequence patterns and their relationship. This segmentation process improved the predictive performance by 49% than the full-length process. Compared with a baseline method, our approach achieved higher performance in predicting oxidoreductase activity. In addition, FUTUSA also showed dramatic performance in predicting acetyltransferase and demethylase activities. Next, we tested the possibility that FUTUSA can predict the functional consequence of point mutation. After trained for monooxygenase activity, FUTUSA successfully predicted the impact of point mutations on phenylalanine hydroxylase, which is responsible for an inherited metabolic disease PKU. This deep-learning program can be used as the first-step tool for characterizing newly identified or poorly studied proteins.•We proposed new deep learning program to predict protein functions in silico that requires nothing more than the protein sequence information.•Due to application of sequence segmentation, the efficiency of prediction is improved.•This method makes prediction of the clinical impact of mutations or polymorphisms possible.

Food Regime for Phenylketonuria: Presenting Complications and Possible Solutions.

In the category of rare inherited genetic disorders, phenylketonuria is a prominent example. Here, the defective phenylalanine hydroxylase enzyme fails to catalyze conversion of phenylalanine to tyrosine. This leads to not only excess deposition of phenylalanine leading to phenylalanine toxicity but also precludes the production of important glutamatergic and cholinergic neurotransmitters, leading to epileptic disorders, microcephaly, low intelligence quotient etc. For long, specialized food products are considered as preferred solution to prevent disease outcome. Different medical diets are developed for managing phenylketonuria includes amino acid mixtures, protein hydrolysates, cofactor-based therapy, large neutral amino acids and glycomacropeptides. However, despite the advent of alternate forms of diet products, the central form of treatment has still been free amino acid mixture. The formulated diet is by and large expensive and in-depth evaluation of several factors which contribute to the expense of medicated diet is requisite to create effective yet affordable avenues for management of disease. For this, we have discussed the role of various factors involved in increasing price of medicated diet and presented possible solutions to it. We have also extensively reviewed prevalence of disease, commercial diet for PKU patients, and their associated limitations. Overall, this is the first attempt to present a holistic view of balance between the overall impact of diet associated therapy and weighing it against the associated finances incurred.

PTC923 (sepiapterin) lowers elevated blood phenylalanine in subjects with phenylketonuria: a phase 2 randomized, multi-center, three-period crossover, open-label, active controlled, all-comers study

Background & aimPTC923 (formerly CNSA-001), an oral formulation of sepiapterin, a natural precursor of intracellular tetrahydrobiopterin (BH4), has been shown in humans to induce larger increases in circulating BH4 vs. sapropterin dihydrochloride. Sapropterin reduces blood phenylalanine (Phe) by ≥20–30% in a minority of subjects with PKU. This was a Phase 2 randomized, multicenter, three-period crossover, open-label, active controlled, all-comers [regardless of phenylalanine hydroxylase (PAH) variants] comparison of PTC923 60 mg/kg, PTC923 20 mg/kg and sapropterin 20 mg/kg in 24 adults with phenylketonuria (PKU) and hyperphenylalaninemia. MethodsEligible subjects were adult men or women (18–60 y) with PKU. Subjects enrolled received 7 days of once-daily oral treatment with PTC923 20 mg/kg/day, PTC923 60 mg/kg/day and sapropterin dihydrochloride 20 mg/kg/day each in a random order. Treatments were separated by a 7-day washout. Subjects maintained their usual pre-study diet, including consumption of amino acid mixtures. Blood Phe was measured on Day 1 (predose baseline), Day 3, Day 5, and Day 7 of each treatment period. ResultsLeast squares mean changes (SE) from baseline in blood Phe were: −206.4 (41.8) μmol/L for PTC923 60 mg/kg (p < 0.0001); −146.9 (41.8) μmol/L for PTC923 20 mg/kg (p = 0.0010); and − 91.5 (41.7) μmol/L for sapropterin (p = 0.0339). Effects of PTC923 60 mg/kg on blood Phe vs. sapropterin were significantly larger (p = 0.0098) and faster in onset with a significantly larger mean reduction in blood Phe at day 3 of treatment, p = 0.0135 (20 mg/kg) and p = 0.0007 (60 mg/kg). Only PTC923 60 mg/kg reduced blood Phe in classical PKU subjects (n = 11, p = 0.0287). The mean blood Phe reduction (PTC923 60 mg/kg) in a cofactor responder analysis (n = 8; baseline Phe ≥300 μmol/L and blood Phe reduction ≥30%) was −463.3 μmol/L (SE 51.5) from baseline. Adverse events were mostly mild to moderate, transient, and similar across treatment groups with no serious adverse events or discontinuations. ConclusionsThe substantially significantly better effect of PTC923 60 mg/kg on blood Phe reduction vs. sapropterin supports further clinical development of PTC923 for PKU; ANZCTR number, ACTRN12618001031257.

Preliminary Investigation of Microbiome and Dietary Differences in Patients with Phenylketonuria on Enzyme Substitution Therapy Compared to Traditional Therapies

BackgroundPhenylketonuria (PKU) is an inborn error of metabolism that impairs the function of the enzyme phenylalanine hydroxylase. Historical treatment includes limiting dietary phenylalanine (Phe) consumption while supplementing with medical food; however, this treatment has been associated with complications, such as nutritional deficiencies and disruptions in the gut microbiota. ObjectiveThe study aim was to compare dietary and gut microbiome differences between adult patients on a traditional PKU diet with those receiving the enzyme substitution therapy Palynziq on a liberalized diet while controlling blood Phe levels to <600 μmol/L (to convert to mg/dL divide by 60.5). DesignA cross-sectional study was conducted comparing patients on a traditional Phe-restricted diet with patients receiving Palynziq eating a liberalized diet. Participants/settingSix patients eating a traditional Phe-restricted diet with medical food and 6 patients on Palynziq eating a liberalized diet without medical food intake for more than 3 years were selected from the University of Kentucky Metabolic Clinic from August to December 2019. Main outcome measuresNutrient intake from 3-day diet records and fecal microbiome taxonomic abundances were analyzed. Statistical analysisMann-Whitney U tests were used for dietary data analysis. Differential abundance analysis for microbiome taxa and pathway data was done using DESeq2 analysis. ResultsDietary data showed patients receiving Palynziq consumed a lower percent of kilocalories from total protein and lower amounts of most micronutrients, but consumed greater amounts of intact protein and cholesterol (P < .05). Microbiome data revealed a greater abundance of the phylum Verrucomicrobia and genus Lachnobacterium in the Traditional group and a greater abundance of the genus Prevotella in the Palynziq group (P < .05). Pathway analysis depicted greater enrichment in carotenoid and amino acid metabolism pathways in the Traditional group (P < .05). Protein (% kcal), dietary fiber (g), fat (% kcal), linolenic acid (% Dietary Reference Intakes), and age were correlated with the underlying microbial community structure for both groups combined. ConclusionsPatients with PKU treated with Palynziq on a liberalized diet manifest significant differences in diet composition compared with those treated with traditional Phe-restricted diets. Several of these dietary differences may affect the microbiome architecture.

Phenylalanine hydroxylase (PAH) plays a positive role during WSSV and Vibrio parahaemolyticus infection in Litopenaeus vannamei

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei, with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l-DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l-DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei.

Optical Coherence Tomography to Assess Neurodegeneration in Phenylalanine Hydroxylase Deficiency.

In phenylalanine hydroxylase (PAH) deficiency, an easily feasible method to access the progression of neurodegeneration is warranted to contribute to current discussions on treatment indications and targets. The objective of the present study was to investigate whether optical coherence tomography (OCT) measures as markers of neurodegeneration differ between patients with PAH deficiency and healthy controls (HCs) according to phenotype and metabolic control. In this single-center cross-sectional study, 92 patients with different phenotypes of PAH deficiency [PAH deficiency not requiring treatment, early treated phenylketonuria (ETPKU), and late-diagnosed phenylketonuria (PKU)] compared with 76 HCs were examined using spectral-domain OCT. Indices of phenylalanine elevation and variability were correlated with OCT parameters. Late-diagnosed PKU patients showed reduced peripapillary retinal nerve fiber layer (pRNFL) thickness and combined ganglion cell and inner plexiform layer (GCIPL) volume. Adult ETPKU patients were found to have lower GCIPL volume (p = 0.016), which correlated with the indices of phenylalanine control. In pediatric ETPKU patients with poor metabolic control, pRNFL was significantly reduced (p = 0.004). Patients with PAH deficiency not requiring treatment did not exhibit retinal degeneration. Inner nuclear layer (INL) was significantly increased in the pediatric ETPKU patients, driven by those with current poor metabolic control (p = 0.006). Our data provide evidence of retinal neuroaxonal degeneration and INL swelling, depending on the phenotype, current age, and metabolic control. These findings suggest that OCT is suitable to investigate neurodegeneration in PKU and we propose OCT as a sensitive, reliable, safe, low-burden, and low-cost examination for future multicenter studies.

Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

BackgroundPhenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment.MethodsHere we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion.ResultsThis technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid.ConclusionsThe results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Blood phenylalanine reduction reverses gene expression changes observed in a mouse model of phenylketonuria

Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH) in liver resulting in blood phenylalanine (Phe) elevation and neurotoxicity. A pegylated phenylalanine ammonia lyase (PEG-PAL) metabolizing Phe into cinnamic acid was recently approved as treatment for PKU patients. A potentially one-time rAAV-based delivery of PAH gene into liver to convert Phe into tyrosine (Tyr), a normal way of Phe metabolism, has now also entered the clinic. To understand differences between these two Phe lowering strategies, we evaluated PAH and PAL expression in livers of PAHenu2 mice on brain and liver functions. Both lowered brain Phe and increased neurotransmitter levels and corrected animal behavior. However, PAL delivery required dose optimization, did not elevate brain Tyr levels and resulted in an immune response. The effect of hyperphenylalanemia on liver functions in PKU mice was assessed by transcriptome and proteomic analyses. We observed an elevation in Cyp4a10/14 proteins involved in lipid metabolism and upregulation of genes involved in cholesterol biosynthesis. Majority of the gene expression changes were corrected by PAH and PAL delivery though the role of these changes in PKU pathology is currently unclear. Taken together, here we show that blood Phe lowering strategy using PAH or PAL corrects both brain pathology as well as previously unknown lipid metabolism associated pathway changes in liver.

CAMP/PKA-CREB-BDNF signaling pathway in hippocampus of rats subjected to chemically-induced phenylketonuria.

Phenylketonuria (PKU) is an inborn error disease in phenylalanine metabolism resulting from defects in the stages of converting phenylalanine to tyrosine. Although the pathophysiology of PKU is not elucidated yet, the toxic effect of phenylalanine on the brain causes severe mental retardation. In relation to learning and memory, the hippocampal PKA / CREB / BDNF pathway may play a role in learning deficits in PKU patients. This study aimed to investigate PKA/CREB/BDNF pathway in hippocampus of chemically induced PKU rats with regard to gender. Sprague-Dawley rat pups were randomized into two groups of both genders. To chemically induce PKU, animals received subcutaneous administration of phenylalanine (5.2mmol / g) plus p-chlorophenylalanine, phenylalanine hydroxylase inhibitor (0.9mmol / g); control animals received 0.9% NaCl. Injections started on the 6th day and continued until the 21st day after which locomotor activity, learning and memory were tested. In male PKU rats, locomotor activity was reduced. There were no differences in learning and memory performances of male and female PKU rats. In PKU rats, pCREB / CREB levels in males was unchanged while it decreased in females. Elevated PKA activity, BDNF levels and decreased pCREB/CREB ratio found in female PKU rats were not replicated in PKU males in which BDNF is decreased. Our results display that in this disease model a gender specific differential activation of cAMP/PKA-CREB-BDNF signaling pathway in hippocampus occurs investigation of which can help us to a better understanding of disease pathophysiology.

Untreated PKU patients without intellectual disability: SHANK gene family as a candidate modifier

Phenylketonuria (PKU) is an inborn error of metabolism caused by variants in the phenylalanine hydroxylase (PAH) gene and it is characterized by excessively high levels of phenylalanine in body fluids. PKU is a paradigm for a genetic disease that can be treated and majority of developed countries have a population-based newborn screening. Thus, the combination of early diagnosis and immediate initiation of treatment has resulted in normal intelligence for treated PKU patients. Although PKU is a monogenic disease, decades of research and clinical practice have shown that the correlation between the genotype and corresponding phenotype is not simple at all. Attempts have been made to discover modifier genes for PKU cognitive phenotype but without any success so far.We conducted whole genome sequencing of 4 subjects from unrelated non-consanguineous families who presented with pathogenic mutations in the PAH gene, high blood phenylalanine concentrations and near-normal cognitive development despite no treatment. We used cross sample analysis to select genes common for more than one patient. Thus, the SHANK gene family emerged as the only relevant gene family with variants detected in 3 of 4 analyzed patients. We detected two novel variants, p.Pro1591Ala in SHANK1 and p.Asp18Asn in SHANK2, as well as SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro and SHANK3:p.Pro1716Thr variants that were previously described. Computational analysis indicated that the identified variants do not abolish the function of SHANK proteins. However, changes in posttranslational modifications of SHANK proteins could influence functioning of the glutamatergic synapses, cytoskeleton regulation and contribute to maintaining optimal synaptic density and number of dendritic spines.Our findings are linking SHANK gene family and brain plasticity in PKU for the first time. We hypothesize that variant SHANK proteins maintain optimal synaptic density and number of dendritic spines under high concentrations of phenylalanine and could have protective modifying effect on cognitive development of PKU patients.

Диференціальна діагностика фенілкетонурії і порушень обміну птеринів у хворих із гіперфенілаланінемією

Актуальність. Гіперфенілаланінемія (HPA), або підвищення концентрації фенілаланіну (Phe) в крові, є наслідком порушення перетворення фенілаланіну в тирозин. Яскравим прикладом цього клінічного стану є класична фенілкетонурія (PKU). У 1970-х роках стало зрозуміло, що в частини хворих із PKU, незважаючи на ефективну дієтотерапію, розвивалися тяжкі неврологічні порушення. Ці випадки верифікували як атипові, або злоякісні, варіанти PKU, що, як було досліджено, спричинялось мутаціями в генах, які регулюють синтез та регенерацію тетрагідробіоптерину (ВН4). Порушення метаболізму тетрагідробіоптерину, або ВН4-дефіцитні синдроми, — гетерогенна група спадкових захворювань ЦНС, спричинених дефіцитом моноамінів-нейротрансмітерів, які в низці випадків супроводжуються гіперфенілаланінемією. ВН4-дефіцитні синдроми, які є рідкісними вродженими порушеннями метаболізму, спричиненими недостатністю нейротрансмітерів, за відсутності своєчасного лікування призводять до прогресуючої, нерідко летальної, енцефалопатії. Упровадження в медичну практику ранньої диференціальної клінічної та лабораторної діагностики гіперфенілаланінемій дозволить запобігти розвитку летальних енцефалопатій та суттєво підвищити ефективність їх лікування. Матеріали та методи. Були обстежені 59 пацієнтів із діагнозом PKU віком від 2 до 16 років. Гіперфенілаланінемія була загальним симптомом і встановлена в усіх обстежених пацієнтів. Усім хворим було проведено молекулярно-генетичне обстеження на панель мутацій у гені РАН (R408W, Y414C (12-й екзон), R158Q (5-й екзон), Ivs10nt546 (5-й інтрон), Ivs12nt1 (12-й інтрон), R252W, R261W, R261Q, R261X, G272X, P281L, S273F (7-й екзон). Усі хворі були обстежені на спектр птеринів (біоптерин та неоптерин) в крові та сечі. У 4 пацієнтів із вираженими неврологічними симптомами додатково визначали активність дигідроптеринредуктази в сухих краплях крові. Двом пацієнтам проводилося вимірювання концентрації гомованілової кислоти та 5-гідроксііндолоцтової кислоти в спинномозковій рідині методом високоефективної рідинної хроматографії. Усі пацієнти лікувались за допомогою дієтотерапії з використанням продуктів спеціалізованого лікувального харчування. Результати. 59 пацієнтів із гіперфенілаланінемією були обстежені на птериновий профіль. У 55 пацієнтів був установлений діагноз PKU, у 4 пацієнтів із прогресуючим ураженням нервової системи та порушенням птеринового профілю було встановлено діагноз ВН4-дефіцитних синдромів: 3 пацієнти — із діагнозом «PTPS-синдром», 1 пацієнт — «DHPR-синдром». Основні клінічні прояви ВН4-дефіцитних синдромів у виявлених пацієнтів були такими: затримка розумового розвитку, судоми, прогресування екстрапірамідних симптомів, порушення тонусу м’язів і пози, ковтання, гіперсалівація. Перші симптоми виникали частіше протягом перших місяців життя дитини. Головним біохімічним маркером на початку захворювання була гіперфенілаланінемія. При застосуванні дієтотерапії рівень Phe швидко досягав рекомендованих безпечних для нервової системи значень, але хвороба прогресувала. Висновки. Гіперфенілаланінемія — основний прояв гетерогенної групи рідкісних нейрометаболічних захворювань, які вимагають подальшої діагностичної верифікації. У новонароджених, у яких під час проведення масового неонатального скринінгу на фенілкетонурію виявлений підвищений рівень фенілаланіну, необхідно застосовувати додатковий діагностичний протокол (визначення птеринового профілю в крові та сечі, обов’язкове дослідження активності дигідроптеринредуктази у сухій краплі крові). Дослідження птеринів необхідне в пацієнтів із раніше встановленим діагнозом «фенілкетонурія» за неефективності дієтотерапії, прогресування когнітивних і неврологічних порушень, зниження тонусу м’язів, виникнення судомного синдрому, наявності екстрапірамідних симптомів. Рання діагностика ВН4-дефіцитних синдромів та своєчасно розпочате лікування дозволяють суттєво покращити стан хворих. Для деяких ВН4-дефіцитних синдромів гіперфенілаланінемія не характерна, що унеможливлює раннє виявлення таких пацієнтів під час неонатального скринінгу. Тому виявлення прогресуючих неврологічних симптомів у пацієнтів віком до 1 року й у ранньому дитинстві, які супроводжуються втратою когнітивних функцій на тлі м’язової гіпотонії, дистонічних порушень і судом, зумовлює необхідність дослідження птеринового профілю в крові й сечі.