Phenylketonuria Disease Research Articles

PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Selective Recognition of Aromatic Amino Acids by a Molecular Cleft in Water

AbstractThe development of water‐soluble hosts for the selective recognition of aromatic amino acids is highly desirable and may serve as a tool to facilitate drug discovery and enable fabrication of sensors for point‐of‐care monitoring in the context of phenylketonuria disease. This paper presents the synthesis and characterization of a water‐soluble molecular cleft which is demonstrated to selectively bind aromatic amino acid guests over other amino acids in aqueous medium, favoring ʟ‐Trp over ʟ‐Phe and ʟ‐Tyr by a factor of approximately five. Host/guest‐interaction forces were studied by 1H‐NMR titrations complemented by fluorescence titrations and isothermal titration calorimetry. The here presented results provide a starting point for future optimizations in our efforts to selectively identify and quantify individual aromatic amino acids in aqueous medium.

Evaluation of Decayed, Missing due to Caries, and Filled Teeth Index in Children with Phenylketonuria in Comparison to Normal Population.

Management of patients with phenylketonuria mainly includes limiting the content of phenylalanine in the diet. Therefore, the purpose of this study was to investigate oral problems in children with phenylketonuria compared to the healthy population as a case-control study. The subjects of the case and control groups were selected according to the inclusion criteria. First, the oral cavity and tooth were examined by a specialist dentist to indicate the decayed, missing due to caries, and filled teeth index in both groups. To inves- tigate the level of phenylalanine and evaluate other laboratory examinations, 2 mL of blood and saliva samples was taken from the subjects. Blood and saliva phenylalanine levels were measured by high-performance liquid chromatography. Phosphorus, calcium, and pH levels were investigated through calorimetric measurement. The data were analyzed using Statistical Package for Social Sciences software. There was no significant difference between the case and control groups in terms of age and sex. The average level of calcium and phosphorus in the case group was higher than in the control group. Also, the average decayed, missing due to caries, and filled teeth index in the case group was not significantly different compared to the control group. None of the above-investigated indicators had a significant relationship with each other. On the other hand, it was found that there was a positive and significant relationship between phenylalanine in blood, saliva, and pH as well as between saliva phenylalanine with decayed, missing due to caries, and filled teeth. The results of this study indicate a significant effect of phenylketonuria disease on calcium, phosphorus, and oral pH levels in children.

Detection of IVS4+1G>A mutation in phenylalanine hydroxylase gene in North of Iran using PCR-sequencing

Background and aims: Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main reason for the incidence of PKU. To date, more than 1180 variants have been detected in the PAH gene. Given that the distribution pattern of mutations in the PAH gene is specific to each population, the present study was conducted to detect exon 4 mutations and adjacent flanking regions of the PAH gene in northern Iran. Methods: This is a descriptive cross-sectional study, in which 24 unrelated PKU patients in Taleghani Hospital in Gorgan were enrolled for a one-year period. After extraction of genomic DNA from leukocytes, identification of exon 4 mutations and adjacent flanking regions was performed using polymerase chain reaction (PCR) and sequencing techniques. Results: In this study, IVS4+1G>A mutation was detected in one allele (2.08%) among 48 alleles. Moreover, IVS4+47C>T and IVS3-22C>T polymorphisms were observed in 12 alleles (25%) and eight alleles (16.7%), respectively. Conclusion: In the present study, IVS4+1G>A mutation was only found in 2% of chromosomes. Hence, different mutations are responsible for PKU disease in the north of Iran, and further studies are recommended to identify all mutations in the PAH gene in the region.

Design and Modelling a Graduated Dispenser for Metabolic Diseases—Phenylketonuria

In metabolic diseases such as phenylketonuria (a rare disease), a very important way to keep the patient healthy is the administration of amino acid substitutes. This dispenser was designed because in other places (except home) for patients, it is very difficult to take the substitute powder due to the custom weight, which depends on the body weight. We designed and made on a 3D printer a graduated dosing device (12 g) which can be used very easily and has all the elements to be transported. In this way, the necessary dose of amino acid substitutes can be administered to patients with phenylketonuria, including infants aged 6 months–1 year, at the kindergarten or any other places with the absence of a food scale. This dispenser is very easy to carry and very useful to patients.

Deubiquitinase USP19 enhances phenylalanine hydroxylase protein stability and its enzymatic activity.

Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism, deficiency of which is associated with the most common metabolic phenotype of phenylketonuria (PKU) and hyperphenylalaninemia (HPA). A bulk of PKU disease-associated missense mutations in the PAH gene have been studied, and the consequence of each PAH variant vary immensely. Prior research established that PKU-associated variants possess defects in protein folding with reduced cellular stability leading to rapid degradation. However, recent evidence revealed that PAH tetramers exist as a mixture of resting state and activated state whose transition depends upon the phenylalanine concentration and certain PAH variants that fail to modulate the structural equilibrium are associated with PKU disease. Collectively, these findings framed our understanding of the complex genotype-phenotype correlation in PKU. In the current study, we substantiate a link between PAH protein stability and its degradation by the ubiquitin-mediated proteasomal degradation system. Here, we provide an evidence that PAH protein undergoes ubiquitination and proteasomal degradation, which can be reversed by deubiquitinating enzymes (DUBs). We identified USP19 as a novel DUB that regulates PAH protein stability. We found that ectopic expression of USP19 increased PAH protein level, whereas depletion of USP19 promoted PAH protein degradation. Our study indicates that USP19 interacts with PAH and prevents polyubiquitination of PAH subsequently extending the half-life of PAH protein. Finally, the increase in the level of PAH protein by the deubiquitinating activity of USP19 resulted in enhanced metabolic function of PAH. In summary, our study identifies the role of USP19 in regulating PAH protein stability and promotes its metabolic activity. Graphical highlights 1. E3 ligase Cdh1 promotes PAH protein degradation leading to insufficient cellular amount of PAH causing PKU. 2. A balance between E3 ligase and DUB is important to regulate the proteostasis of PAH. 3. USP19 deubiquitinates and stabilizes PAH further protecting it from rapid degradation. 4. USP19 increases the enzymatic activity of PAH, thus maintaining normal Phe levels.

Present and future of lipid nanoparticle-mRNA technology in phenylketonuria disease treatment.

Phenylketonuria (PKU) is a metabolic rare disease characterized by a failure of the body to clear out the high levels of Phenylalanine (Phe), leading to devastating neurological defects and growth retardation. PKU was discovered in 1934 by AsbjrØrn FØlling, and even though there have been continuous efforts from the scientific community to find therapeutic approaches to modulate the high levels of phenylalanine found in the body of the PKU patients, an efficient therapy still needs to be developed. Current standard of care includes low phenylalanine diets, but the strict restrictions for patients and families makes it very difficult to adequately being implemented. FDA has approved two drugs to help reduce Phe levels in PKU patients: an enzyme substitution therapy, Palynziq® (PEGylated recombinant phenylalanine ammonia lyase), and Kuvan®, a supplemental tetrahydrobiopterin (BH4) cofactor that enhances residual enzyme activity. Both treatments are restricted to certain PKU patients' population, and, therefore, there are still high unmet needs for most of the patients. The present review will focus on current advancements in lipid nanoparticles (LNP)-mRNA technologies and their potential in treating the root cause of PKU, a therapeutic approach that will be analyzed in the context of other promising therapeutic approaches that are been developed for PKU.

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.

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.

Trace elements in dried blood spots as potential discriminating features for metabolic disorder diagnosis in newborns

Trace elements in dried blood spots (DBSs) from newborns were determined by laser ablation coupled with inductively coupled plasma mass spectrometry, and data were subjected to chemometric evaluation in an attempt to classify healthy newborns and newborns suffering from metabolic disorders. Unsupervised [principal component analysis (PCA) and cluster analysis (CA)] and supervised [linear discriminant analysis (LDA) and soft independent modeling by class analogy (SIMCA)] pattern recognition techniques were used as classification techniques. PCA and CA have shown a clear tendency to form two groups (healthy newborns and newborns suffering from metabolic disorders). LDA and SIMCA have predicted that 90.5% and 83.9% of originally grouped healthy newborn cases were correctly classified by LDA and SIMCA, respectively. In addition, these percentages were 97.6% (LDA) and 80.6% (SIMCA) for DBSs from newborns suffering from metabolic disorders. However, SIMCA has only detected one misclassified DBS from the healthy group, and the lower percentage is attributed to four DBSs from the healthy newborn group and five DBSs from newborns with disorders that were found as belonging to both categories (healthy newborns and newborns with disorders) in the training set. LDA also gave a percentage of grouped maple syrup urine disease (MSUD) cases correctly classified of 100%, although the percentage fells to 66.7% when classifying phenylketonuria (PKU) cases. Finally, essential elements such as Fe, K, Rb, and Zn were found to be matched (correlated) with the concentration of amino acids such as phenylalanine, valine, and leucine, biomarkers linked with MSUD and PKU diseases.

Imagining Life with a Genetic Disorder: The Challenge of Evaluating Health States that Exist from Birth

This article explores difficulties encountered by those with no personal experience of a chronic disease or disability in accurately evaluating the quality of life with a condition present from birth. In most countries, cost-effectiveness analysis relies on ratings of health states by members of the general population, who must try to imagine what life for those affected is like based on (usually brief) descriptions of the conditions. That task is challenging for reasons that have been well-discussed in the literature. This article employs a detailed case-study of dietary treatment for the genetic disease phenylketonuria to identify additional, unrecognized challenges that arise in the case of conditions experienced from birth (or early childhood) and considers some implications of these difficulties for practices in health-state valuation.

Pfizer invests in vector firm Homology

Pfizer will pay $60 million for a stake in Homology Medicines, a Bedford, Massachusetts–based firm developing HMI-102, a gene therapy for adults with the metabolic disease phenylketonuria. Homology delivers genes with a suite of adeno-associated virus vectors derived from human hematopoietic stem cells that it says are taken up by the body more readily than traditional vectors.

HEALTH HALO EFFECT OF FOOD LABELS

Sosyal psikolojide ilk etki olarak bilinen halo etkisi/ hatası bir nesne veya kişi ilk görüldüğünde edinilen anlık izlenim olup, kararların temelini oluşturur. Günümüzde birçok gıda şirketi, ürün etiketlerinde yer alan FOP (front on package) bilgisini kullanarak bir çeşit sağlıklı algılanma etkisi yaratmayı satış stratejisi olarak kullanmaktadır. Örneğin gıda etiketleri üzerinde yer alan, düşük yağlı, tuzsuz, düşük kalorili, glutensiz, yüksek proteinli, yüksek lifli, GDO’suz, gibi gıdanın içerik bilgilerinin yanında; ürünün üretim sürecindeki tercihleri ifade eden organik, doğal, katkısız, "süper gıda" ya da "fair trade" gibi etik ifadeler kullanılmaktadır. Sağlıklı algılanma etkisi ile tüketici tercihleri manipüle edilmektedir. Çölyak hastalığı, fenilketonürü hastalığı gibi özel hastalıklar için hazırlanan ürünler ve laktozsuz, tuzsuz vb durumlar hariç; sağlıklı algılanma etkisi yaratan zihinsel kısa yolları devre dışı bırakmak için "düz paketleme" önerilmektedir.

From in vitro to in silico: Modeling and recombinant production of DT-Diaphorase enzyme

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni–NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done.

Establishing a Remedy for Phenylketonuria Disease from Indian Ayurvedic Herbs

The phenylketonuria (PKU) disease is an inherited disorder that increases the levels of a substance called phenylalanine in the blood and if not treated, phenylalanine can build up to harmful levels in the body. People with this disorder can't break down the amino acid phenylalanine. This phenylalanine, then builds up in the blood and brain causing intellectual disability and other serious health problems. It is rare but a serious inherited disorder. The main objective of this study is to establish a remedy for the phenylketonuria disease (novel drug leads for phenylketonuria disease’s receptors viz. ASCL1 gene (achaete-scute family bHLH transcription factor 1), GCH1 gene (GTP cyclohydrolase 1) and MAOB (Monoamine Oxidase B)) using phytocompounds from ayurvedic herbs. To achieve this objective we performed virtual screening with phytocompounds from ayurvedic herbs against the phenylketonuria disease’s receptors followed by ADME studies on the phytocompounds selected by virtual screening. Based on the analysis of the results of virtual screening and subsequent ADME studies on the phytocompounds it is seen that curcumin can be successfully considered as novel drug lead for treating phenylketonuria disease.

A comprehensive study of phenylalanine hydroxylase gene mutations in the Iranian phenylketonuria patients

Phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. After thalassemia, PKU is considered as the most common autosomal recessive diseases in the Iranian population. Therefore, an efficient diagnostic strategy is required to identify disease-causing mutations in this population. Following our first report in 2003, here we presented a comprehensive study on the mutation spectrum of the PAH gene in the Iranian population. This study was performed on 280 unrelated chromosomes from 140 Iranian patients with classic PKU. All 13 exons as well as exon-intron boundaries of the PAH gene were analyzed by direct DNA sequencing. Thirty four different mutations were identified by a mutation detection rate of 100%. IVS10-11G > A, p.P281L, R261Q, p.F39del and IVS11+1G > C were the most prevalent mutations with frequencies of 26.07%, 19.3%, 12.86%, 6.07 and 3.93%, respectively. All other mutations represented a relative frequency less than 3.5%. The data from this study provided a comprehensive spectrum of the PAH gene mutations which can facilitate carrier detection and prenatal diagnosis of PKU disease in the Iranian population.

Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Study on the Detection of the Content of the Phenylalanine by Rare Earth Ions Fluorescence Probe Method

Phenylketonuria(PKU) is a kind of genetic and metabolic disease which caused by mutations. High concent of phenylalanine and its metabolites in the blood which result in impairment of brain development, even mental retardation. In recent years, due to the impact factors of diet and environment, PKU disease was significantly increased. Studies have shown that early detection of abnormal levels of phenylalanine and treatment will greatly reduce the rate of mental retardation. Therefore, monitoring of blood levels of phenylalanine is particularly important for phenylketonuria patients. This study discussed a new method of terbium rare earth ions fluorescence probe to detect the content of phenylalanine. At the same time, the experimental conditions were optimized. The results showed that the optimal pH of the system was 8, the optimum surfactant was SDBS(1.0×10-3 mol/L) and the optimum concentration of Tb3+ was 3 × 10-2 mol/L. Under optimal test conditions, the fluorescence intensity had a good liner relationship with the concentration of phenylalanine in the range of 6×10-7∼3 ×10-5 mol/L. And the linear equation as follows: F = 1.0979CPhe +23.50268, r = 0.998. It provided a new theoretical approach for the clinical test of Phenylketonuria.

A 3-Step Process to Estimate Phenylalanine in Commercial Foods for PKU Management

Phenylalanine (Phe) is a key nutrient in the dietary management of the metabolic disease phenylketonuria (PKU). To give more freedom to PKU patients, we propose a numerical process to estimate the Phe content of a commercial food using the information printed on the label (Nutritional Fact Label and ordered ingredient list). The process is amenable to implementation as a smart phone/Web app and could be integrated into a personalized dietary management software. Our tests show that the results are very accurate (±13 mg per serving) in the vast majority of foods considered. Our approach can be modified to handle any other amino acid for the management of other inborn errors of metabolism. A similar approach can also be used for improving the precision (i.e., increasing the quantization resolution) of the nutrients already listed (e.g., protein).