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Background: Assessing dietary phenylalanine (Phe) tolerance is crucial for managing hyperphenylalaninemia (HPA) in children. However, traditionally, adjusting the diet requires significant time from clinicians and parents. This study aims to investigate the development of a machine-learning model that predicts a range of dietary Phe intake tolerance for children with HPA over 10 years following diagnosis. Methods: In this multicenter retrospective observational study, we collected the genotypes of phenylalanine hydroxylase (PAH), metabolic profiles at screening and diagnosis, and blood Phe concentrations corresponding to dietary Phe intake from over 10 years of follow-up data for 204 children with HPA. To incorporate genetic information, allelic phenotype value (APV) was input for 2965 missense variants in the PAH gene using a predicted APV (pAPV) model. This model was trained on known pheno-genotype relationships from the BioPKU database, utilizing 31 features. Subsequently, a multiclass classification model was constructed and trained on a dataset featuring metabolic data, genetic data, and follow-up data from 3177 events. The final model was fine-tuned using tenfold validation and validated against three independent datasets. Results: The pAPV model achieved a good predictive performance with root mean squared error (RMSE) of 1.53 and 2.38 on the training and test datasets, respectively. The variants that cause amino acid changes in the region of 200–300 of PAH tend to exhibit lower pAPV. The final model achieved a sensitivity range of 0.77 to 0.91 and a specificity range of 0.8 to 1 across all validation datasets. Additional assessment metrics including positive predictive value (0.68–1), negative predictive values (0.8–0.98), F1 score (0.71–0.92), and balanced accuracy (0.8–0.92) demonstrated the robust performance of our model. Conclusions: Our model integrates metabolic and genetic information to accurately predict age-specific Phe tolerance, aiding in the precision management of patients with HPA. This study provides a potential framework that could be applied to other inborn errors of metabolism. [ABSTRACT FROM AUTHOR]

Background: The GCH1 gene encodes the enzyme guanosine triphosphate cyclohydrolase I (GTPCH), which catalyzes the rate‐limiting step in the biosynthesis of tetrahydrobiopterin (BH4), a critical cofactor in the production of monoamine neurotransmitters. Autosomal dominant GTPCH (adGTPCH) deficiency is the most common cause of dopa‐responsive dystonia (DRD), whereas the recessive form (arGTPCH) is an ultrarare and poorly characterized disorder with earlier and more complex presentation that may disrupt neurodevelopmental processes. Here, we delineated the phenotypic spectrum of ARGTPCHD and investigated the predictive value of biochemical and genetic correlates for disease outcome. Objectives: The aim was to study 4 new cases of arGTPCH deficiency and systematically review patients reported in the literature. Methods: Clinical, biochemical, and genetic data and treatment response of 45 patients are presented. Results: Three phenotypes were outlined: (1) early‐infantile encephalopathic phenotype with profound disability (24 of 45 patients), (2) dystonia‐parkinsonism phenotype with infantile/early‐childhood onset of developmental stagnation/regression preceding the emergence of movement disorder (7 of 45), and (3) late‐onset DRD phenotype (14 of 45). All 3 phenotypes were responsive to pharmacological treatment, which for the first 2 must be initiated early to prevent disabling neurodevelopmental outcomes. A gradient of BH4 defect and genetic variant severity characterizes the 3 clinical subgroups. Hyperphenylalaninemia was not observed in the second and third groups and was associated with a higher likelihood of intellectual disability. Conclusions: The clinical spectrum of arGTPCH deficiency is a continuum from early‐onset encephalopathies to classical DRD. Genotype and biochemical alterations may allow early diagnosis and predict clinical severity. Early treatment remains critical, especially for the most severe patients. [ABSTRACT FROM AUTHOR]

Characterization of Kuvan tablets involved thorough analysis of physical, chemical, and dissolution properties. Formulation development encompassed pre-formulation studies, feasibility trials, and process optimization. Kuvan tablets demonstrated variability in sapropterin dihydrochloride concentration but rapid dissolution across different media. Consistent mechanical strength, low impurity levels, and appropriate pH values were observed, ensuring efficacy, safety, and reliability. [ABSTRACT FROM AUTHOR]

Abstract Background Assessing dietary phenylalanine (Phe) tolerance is crucial for managing hyperphenylalaninemia (HPA) in children. However, traditionally, adjusting the diet requires significant time from clinicians and parents. This study aims to investigate the development of a machine-learning model that predicts a range of dietary Phe intake tolerance for children with HPA over 10 years following diagnosis. Methods In this multicenter retrospective observational study, we collected the genotypes of phenylalanine hydroxylase (PAH), metabolic profiles at screening and diagnosis, and blood Phe concentrations corresponding to dietary Phe intake from over 10 years of follow-up data for 204 children with HPA. To incorporate genetic information, allelic phenotype value (APV) was input for 2965 missense variants in the PAH gene using a predicted APV (pAPV) model. This model was trained on known pheno-genotype relationships from the BioPKU database, utilizing 31 features. Subsequently, a multiclass classification model was constructed and trained on a dataset featuring metabolic data, genetic data, and follow-up data from 3177 events. The final model was fine-tuned using tenfold validation and validated against three independent datasets. Results The pAPV model achieved a good predictive performance with root mean squared error (RMSE) of 1.53 and 2.38 on the training and test datasets, respectively. The variants that cause amino acid changes in the region of 200–300 of PAH tend to exhibit lower pAPV. The final model achieved a sensitivity range of 0.77 to 0.91 and a specificity range of 0.8 to 1 across all validation datasets. Additional assessment metrics including positive predictive value (0.68–1), negative predictive values (0.8–0.98), F1 score (0.71–0.92), and balanced accuracy (0.8–0.92) demonstrated the robust performance of our model. Conclusions Our model integrates metabolic and genetic information to accurately predict age-specific Phe tolerance, aiding in the precision management of patients with HPA. This study provides a potential framework that could be applied to other inborn errors of metabolism.

Phenylalanine hydroxylase (PAH) is predominantly a hepatic enzyme that catalyzes phenylalanine (Phe) into tyrosine, which is the rate-limiting step in Phe catabolism. Biallelic variants in the PAH gene cause PAH enzyme deficiency. Phenylketonuria (PKU) is an autosomal recessive disorder that causes neurologic, behavioral, and dermatological findings. PKU could be divided clinically into three types based on the blood Phe levels: classic phenylketonuria (cPKU), mild-moderate phenylketonuria (mPKU), and mild hyperphenylalaninemia (MHP). This study aimed to determine the phenotypic and genotypic characteristics of Turkish PKU patients in the eastern region of Türkiye. Demographic characteristics, serum Phe levels, treatments, and PAH variants of 163 patients with PKU and hyperphenylalaninemia (HPA) were retrospectively evaluated. Blood Phe levels of the patients were analyzed with the high-performance liquid chromatography method. For PAH gene analysis, next-generation sequencing was performed. Of the 163 patients included in the study, 38 (23.3 %) had cPKU, 16 (9.8 %) had mPKU, and 109 (66.9 %) had MHP. Homozygous variants in the PAH gene were detected in 66 (40.5 %) of the patients, while compound heterozygous variants were detected in 97 (59.5 %) patients. Two novel and 35 recurrent variants in the PAH gene were detected. Of the two novel variants, one was missense (p.Phe351Leu) and the other was frameshift (p.Met276Cysfs*65). The most frequently detected variants were p.Thr380Met (18 %), p.Arg261Gln (16.8 %), and p.Ala300Ser (12.8 %). All patients with the homozygous c.1066-11G>A variant exhibited cPKU phenotype. The c.898G>T (p.Ala300Ser), c.1139C>T (p.Thr380Met), and c.1208C>T (p.Ala403Val) variants were statistically related to mild phenotype. On the other hand, c.592_613del (p.Tyr198Serfs*136), c.1028A>G (p.Tyr343Cys), and c.782G>A (p.Arg261Gln) variants were more frequently detected in the cPKU group. Our study, conducted with patients from the eastern region of Türkiye, demonstrates the genetic heterogeneity in the Turkish population. Simultaneously, our research contributes to genotype–phenotype correlation and expands the genotypic spectrum by identifying novel variants. [ABSTRACT FROM AUTHOR]

ObjectiveSapropterin dihydrochloride is the first drug for the therapy of phenylketonuria, which is a rare disease that occurs one of 10,000–15,000 newborns. As a result, detailed and comprehensive reports on the safety of sapropterin in large, real-world populations are required. The purpose of this study is to undertake a complete analysis of sapropterin’s adverse events (AEs) using the FDA Adverse Event Reporting System (FAERS) database.MethodsWe retrieved reports of adverse events with sapropterin as the principal suspect from FAERS between the first quarter of 2008 and the first quarter of 2024. The Reporting Odds Ratio (ROR), Proportional Reporting Ratio (PRR), and Bayesian Confidence Propagation Neural Network (BCPNN) were utilized to detect AE signals.ResultsThe study collected 4,953 suspected AE cases from the FAERS database, with sapropterin as the major suspect. A total of 130 positive signals were obtained utilizing the ROR, PRP, and BCPNN. The FAERS database revealed that common clinical AEs of sapropterin included vomiting, upper respiratory infection, rhinorrhea, and a reduction in amino acid concentrations. Furthermore, we detected probable unexpected adverse events (AEs) using disproportionality analysis, including gastroesophageal reflux disease, flatulence, influenza, ear infection, viral infection, pharyngitis streptococcal, spontaneous abortion, and nephrolithiasis.ConclusionBy analyzing huge amounts of real-world data from the FAERS database, we found potential novel AEs of sapropterin using disproportionate analysis. It is advantageous for healthcare professionals and pharmacists to focus on efficiently managing sapropterin’s high-risk adverse events, improving drug levels in clinical settings, and ensuring patient medication safety.

This study, conducted in the Republic of North Ossetia-Alania (RNOA), aimed to explore the genetic landscape of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) in the Ossetian population using data from newborn screening (NBS). Through comprehensive molecular genetic analysis of 29 patients with HPA from diverse ethnic backgrounds, two major genetic variants in the PAH gene, P281L and P211T, were identified, constituting 50% of all detected pathogenic alleles in Ossetian patients. Remarkably, these variants exhibited an exceptionally high frequency in the Ossetian population, surpassing global prevalence rates. This study unveiled a notable prevalence of mild forms of HPA (78%), underscoring the importance of genetic counseling for carriers of pathogenic variants in the PAH gene. Moreover, the findings emphasized the necessity for ongoing monitoring of patients with mild forms, as they may lack significant symptoms for diagnosis, potentially impacting offspring. Overall, this research offers valuable insights into the genetic landscape of HPA and PKU in the Ossetian population. [ABSTRACT FROM AUTHOR]

Women with phenylketonuria (PKU) should maintain blood phenylalanine (phe) concentration within the recommended range before and during pregnancy to prevent maternal PKU syndrome (MPKUS) in their offspring. Women who gave birth to children with MPKUS symptoms were more likely to report elevated phe concentration before pregnancy, and barriers to accessing components of their dietary management during pregnancy, including blood phe testing, medical food, modified low-protein foods, and healthcare visits with PKU specialists.

Abstract Background Tetrahydrobiopterin (BH4) deficiency caused by 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is a rare disorder that is one of the major causes of hyperphenylalaninemia in Taiwan. Methods In this study, we reviewed the clinical courses of 12 adolescent and adult patients (7 females and 5 males) with PTPS deficiency. Results The patients were treated shortly after diagnosis through newborn screening with a combination of BH4, levodopa/carbidopa, and 5-OH-tryptophan. Their plasma phenylalanine and tyrosine levels were well controlled, and their prolactin levels were also decreased after treatment. However, their prolactin levels gradually rose as they grew into puberty, and at a current age of 27.5 [interquartile range (IQR 7.9)] years, five of the 12 patients had either highly elevated prolactin levels (> 100 ng/mL in one male patient, normal reference values, male

Abstract Background The risk of neuropsychological disorders appears to be high in hyperphenylalaninemia (HPA). The hypothesis of executive function impairment is prominent in accounting for the neuropsychological phenotype in phenylketonuria (PKU) and is suspected in moderate hyperphenylalaninemia (MHP). However, the issue of early onset of executive disorders remains. The aim of this study was to explore the hypothesis of early executive dysfunction in HPA patients and the possible links with certain metabolic variables according to the new international classifications for patients with PKU and MHP. A group of 23 HPA children (12 PKU, 11 MHP) aged 3 to 5 years was included and compared to 50 control children. The two groups were comparable in terms of socio-demographics (age, sex, parental education level). Executive functions were assessed using performance-based tests and daily life questionnaires (parents and teachers). Results Preschool HPA patients have comparable executive scores to control subjects. In contrast, PKU patients score significantly worse than MHP patients on 3 executive tests (verbal working memory, visual working memory and cognitive inhibition. There is no executive complaints in daily life (parents and teachers) for the 2 groups of patients. In addition, 3 correlations were identified between executive scores and Phe levels at inclusion, mean Phe level and variability of Phe levels throughout life. Conclusions Thus, there appears to be evidence of early executive dysfunction in PKU preschool-children, but not in MHP children. Occasionally, certain metabolic indicators can predict executive difficulties in young children with PKU.

Background: DNAJC12 co-chaperone protein deficiency has been recently described as a stand-alone metabolic disorder explaining many cases of mild hyperphenylalaninemia (HPA) that are not caused by variants in the PAH gene, which encodes for the hepatic enzyme phenylalanine hydroxylase (PAH), or inGCH1, PTS, QDPR, PCBD1 and DHPR, involved in tetrahydrobiopterin (BH4) biosynthesis and activity. Results: We describe two sisters born to consanguineous parents. The youngest sister (Patient 1), initially asymptomatic, tested positive at NewBorn Screening (NBS) for mild HPA. After variants in the PAH and BH4 related-genes were excluded, we performed DNAJC12 genetic analysis and found a previously described homozygous deletion [NM_021800.3: c.58_59del p.(Gly20Metfs*2)]. The older sister (Patient 2), homozygous for the same variant and exhibiting mild HPA, was diagnosed subsequently and presented with ataxia and repeated falls, upper limb dyskinesia, intentional tremor, and mild intellectual disability. Patient 1 was started on treatment with low Phenylalanine (Phe) diet, BH4, l-3,4-dihydroxyphenylalanine/carbidopa (L-DOPA) and 5-OH-Tryptophan, soon after diagnosis, and despite poor adherence to the dietary regimen, only manifested language impairment at last follow-up (age 5 years and 4 months). Patient 2, who started the same treatment at school age, experienced a minimal progression of neurological symptoms, with some improvement in her motor skills. Conclusions: These two new patients with DNAJC12-associated HPA, in addition to previous reports, point to DNAJC12 deficiency as a new metabolic syndrome that must be considered in patients with unexplained HPA.

Hyperphenylalaninemia is a biomarker for several monogenic neurotransmitter disorders where the body cannot metabolise phenylalanine to tyrosine. Biallelic pathogenic variants in DNAJC12, co-chaperone of phenylalanine, tyrosine, and tryptophan hydroxylases, leads to hyperphenylalaninemia and biogenic amines deficiency. A male firstborn to non-consanguineous Sudanese parents had hyperphenylalaninemia 247 µmol/L [reference interval (RI) < 200 µmol/L] at newborn screening. Dried blood spot dihydropteridine reductase (DHPR) assay and urine pterins were normal. He had severe developmental delay and autism spectrum disorder without a notable movement disorder. A low phenylalanine diet was introduced at two years without any clinical improvements. Cerebrospinal fluid (CSF) neurotransmitters at five years demonstrated low homovanillic acid (HVA) 0.259 µmol/L (reference interval (RI) 0.345–0.716) and 5-hydroxyindoleaetic acid (5HIAA) levels 0.024 µmol/L (reference interval (RI) 0.100–0.245). Targeted neurotransmitter gene panel analysis identified a homozygous c.78 + 1del variant in DNAJC12. At six years, he was commenced on 5-hydroxytryptophan 20 mg daily, and his protein-restricted diet was liberalised, with continued good control of phenylalanine levels. Sapropterin dihydrochloride 7.2 mg/kg/day was added the following year with no observable clinical benefits. He remains globally delayed with severe autistic traits. Urine, CSF neurotransmitter studies, and genetic testing will differentiate between phenylketonuria, tetrahydrobiopterin or DNAJC12 deficiency, with the latter characterised by a clinical spectrum ranging from mild autistic features or hyperactivity to severe intellectual disability, dystonia, and movement disorder, normal DHPR, reduced CSF HIAA and HVA. DNAJC12 deficiency should be considered early in the differential workup of hyperphenylalaninemia identified from newborn screening, with its genotyping performed once deficiencies of phenylalanine hydroxylase (PAH) and tetrahydrobiopterin (BH4) have been biochemically or genetically excluded. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study, conducted in the Republic of North Ossetia-Alania (RNOA), aimed to explore the genetic landscape of hyperphenylalaninemia (HPA) and phenylketonuria (PKU) in the Ossetian population using data from newborn screening (NBS). Through comprehensive molecular genetic analysis of 29 patients with HPA from diverse ethnic backgrounds, two major genetic variants in the PAH gene, P281L and P211T, were identified, constituting 50% of all detected pathogenic alleles in Ossetian patients. Remarkably, these variants exhibited an exceptionally high frequency in the Ossetian population, surpassing global prevalence rates. This study unveiled a notable prevalence of mild forms of HPA (78%), underscoring the importance of genetic counseling for carriers of pathogenic variants in the PAH gene. Moreover, the findings emphasized the necessity for ongoing monitoring of patients with mild forms, as they may lack significant symptoms for diagnosis, potentially impacting offspring. Overall, this research offers valuable insights into the genetic landscape of HPA and PKU in the Ossetian population.

DNAJC12 deficiency (OMIM# 617384) is a new cause of hyperphenylalaninemia (HPA). The deficiency of the co-chaperone protein DNAJC12 was identified in 2017. To date, only 43 patients have been reported. Here, we report four patients from a single family with DNAJC12 deficiency while being followed up with a diagnosis of HPA. Two of the patients, who were cousins, were diagnosed with HPA by newborn screening. And the other two patients were siblings of these patients. Neurological examinations were normal except for one patient with mild learning disability. A c.158-2A>T p.(?) biallelic pathogenic variant was detected in intron 2 of the DNAJC12 gene. In the 24 h tetrahydrobiopterin (BH4) challenge test, there was a significant decrease in phenylalanine levels, especially at the 16th hour. Three patients had decreased homovalinic acid (HVA) and 5-hydroxyindoleacetic acid (5HIAA) in cerebrospinal fluid (CSF), while only one had decreased 5HIAA. In treatment, sapropterin, levodopa/carbidopa and 5-OH tryptophan were started. We propose that it will be beneficial to evaluate the patients who have unexplained hyperphenylalaninemia for DNAJC12 deficiency. Patients with early diagnosis of neurotransmitter deficiency may be given a chance to be treated before clinical symptoms begin. [ABSTRACT FROM AUTHOR]

Background: The risk of neuropsychological disorders appears to be high in hyperphenylalaninemia (HPA). The hypothesis of executive function impairment is prominent in accounting for the neuropsychological phenotype in phenylketonuria (PKU) and is suspected in moderate hyperphenylalaninemia (MHP). However, the issue of early onset of executive disorders remains. The aim of this study was to explore the hypothesis of early executive dysfunction in HPA patients and the possible links with certain metabolic variables according to the new international classifications for patients with PKU and MHP. A group of 23 HPA children (12 PKU, 11 MHP) aged 3 to 5 years was included and compared to 50 control children. The two groups were comparable in terms of socio-demographics (age, sex, parental education level). Executive functions were assessed using performance-based tests and daily life questionnaires (parents and teachers). Results: Preschool HPA patients have comparable executive scores to control subjects. In contrast, PKU patients score significantly worse than MHP patients on 3 executive tests (verbal working memory, visual working memory and cognitive inhibition. There is no executive complaints in daily life (parents and teachers) for the 2 groups of patients. In addition, 3 correlations were identified between executive scores and Phe levels at inclusion, mean Phe level and variability of Phe levels throughout life. Conclusions: Thus, there appears to be evidence of early executive dysfunction in PKU preschool-children, but not in MHP children. Occasionally, certain metabolic indicators can predict executive difficulties in young children with PKU. [ABSTRACT FROM AUTHOR]

Background: Restless legs syndrome (RLS) is characterized by an unpleasant or painful sensation generally localized to lower limbs and relieved by movement. Its pathogenesis is hypothesized to involve the dopaminergic system, also in the light of the response of RLS to ex adiuvantibus treatment with dopamine agonists. DNAJC12 deficiency is a recently identified inherited metabolic disease coupling hyperphenylalaninemia to deficient dopaminergic and serotoninergic neurotransmission, due to the combined impairment of the three aromatic amino acids' (i.e., phenylalanine, tyrosine, and tryptophan) hydroxylases. DNAJC12 deficiency was reported in 43 patients so far, presenting with wide spectrum of clinical symptoms. Case presentation results: Here, we report RLS as a novel clinical manifestation of DNAJC12 deficiency, occurring in two adults while on treatment with l-dopa at longitudinal follow-up. The adjunct of low-dose pramipexole was effective in both patients to treat RLS. Besides, this treatment also allowed an improvement of dopaminergic homeostasis, as evidenced by clinical amelioration and stabilization of a peripheral short prolactin profile (a tool to indirectly evaluate dopaminergic homeostasis). Discussion: Besides including RLS as a new treatable clinical manifestation of DNAJC12, these observations may suggest the opportunity of a selective screening for DNAJC12 deficiency in patients with idiopathic RLS. [ABSTRACT FROM AUTHOR]

Phenylketonuria is the most common inborn error of amino acid metabolism. It is caused by the impossibility of converting the essential amino acid phenylalanine into tyrosine, most often due to insufficient production or a complete lack of the enzyme phenylalanine hydroxylase due to a mutation in its gene. In most developed countries, including Croatia, it is detected through mandatory newborn screening. Treatment is carried out with strictly regulated lifelong low-protein diet therapy. The case of a one-year-old girl with phenylketonuria, which is well-regulated, is presented.

Abstract Background Orthostatic tremor (OT) is a type of postural tremor of the lower extremities that has not been described in either phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Because little is known about the clinical features and therapeutic responses of OT in mild HPA, we describe a mild HPA patient who presented with OT as an initial symptom. Case presentation A 22-year-old male was admitted for bilateral leg tremor while standing, with symptom onset eight months prior. One month before admission, the tremor disappeared in the left leg but persisted in the right leg. Electromyography recorded from the right gastrocnemius revealed a 6–8 Hz tremor, which appeared when the patient was standing and disappeared when he was resting or walking. Blood screening showed a phenylalanine/tyrosine ratio of 2.06 and a phenylalanine level of 140 μmol/L. Urine metabolic screening was negative. Whole-exome sequencing confirmed the presence of a compound heterozygous mutation, c.158G > A and c.728G > A, in phenylalanine hydroxylase (PAH) gene. After three months of levodopa/benserazide tablets (250 mg, tid) and a low-phenylalanine diet treatment, the tremor disappeared. Conclusions Young-onset mild HPA is a relatively rare autosomal recessive metabolic disease, and slow OT is a rare clinical feature. Metabolic screening and genetic testing are the keys to early diagnosis and treatment. For adolescents and young adults, appropriate medication and long-term dietary therapy remain important treatments. This case expanded the disease spectrum of slow OT.

Background: Hyperphenylalaninemias (HPA) are due to several gene mutations, of which the PAH gene is the most frequently involved. Prevalence and incidence of disease vary between populations, with genotype/phenotype correlations not always capable to correctly predict disease severity. The aim of this study was to give an overview of PAH mutations among one of the largest cohort of patients among Europe, born in Lombardy (Italy) starting from late 1970 s and including over a 60 years of activity; furthermore, to evaluate and discuss identified genotype/phenotype correlations and related reliability. Patients/Methods: Eight hundred and twenty‐six HPA patients in current follow‐up at the San Paolo Hospital in Milan (Italy) were retrospectively reviewed, including molecular results and allelic phenotype and genotype values (attributed on the basis of the APV/GPV system) to verify genotype–phenotype correlations. Results: A total of 166 different PAH variants were reviewed; of those, seven variants were identified as not previously described in literature. Most frequently reported variant was p.Ala403Val, followed by p.Arg261Gln, p.Val245Ala, IVS10‐11 g>a, p.Tyr414Cys and p.Leu48Ser. Phenotype prediction, based on APV/GPV, matched the actual phenotype in most cases, but not always. Conclusion/Discussion: The cohort of patients included in this study constitute a representative sample of the HPA population worldwide. Studies on this sample may allow to improve clinical and genetic evaluation performances for affected patients, consequently to develop personalized medicine interventions and provide more precise indications on the correct treatment approach based on the accumulated evidence, also in light of a prognostically reliable but not always conclusive APV/GPV system. [ABSTRACT FROM AUTHOR]

DNAJC12-deficient hyperphenylalaninemia is a recently described inborn error of metabolism associated with hyperphenylalaninemia, neurotransmitter deficiency, and developmental delay caused by biallelic pathogenic variants of the DNAJC12 gene. The loss of the DNAJC12-encoded chaperone results in the destabilization of the biopterin-dependent aromatic amino acid hydroxylases, resulting in deficiencies in dopamine, norepinephrine, and serotonin. We present the case of a patient who screened positive for hyperphenylalaninemia on newborn screening and was discovered to be homozygous for a likely pathogenic variant of DNAJC12. Here, we review the management of DNAJC12-related hyperphenylalaninemia and compare our patient to other reported cases in the literature to investigate how early detection and management may impact clinical outcomes.

Abstract Background Hyperphenylalaninemias (HPA) are due to several gene mutations, of which the PAH gene is the most frequently involved. Prevalence and incidence of disease vary between populations, with genotype/phenotype correlations not always capable to correctly predict disease severity. The aim of this study was to give an overview of PAH mutations among one of the largest cohort of patients among Europe, born in Lombardy (Italy) starting from late 1970 s and including over a 60 years of activity; furthermore, to evaluate and discuss identified genotype/phenotype correlations and related reliability. Patients/Methods Eight hundred and twenty‐six HPA patients in current follow‐up at the San Paolo Hospital in Milan (Italy) were retrospectively reviewed, including molecular results and allelic phenotype and genotype values (attributed on the basis of the APV/GPV system) to verify genotype–phenotype correlations. Results A total of 166 different PAH variants were reviewed; of those, seven variants were identified as not previously described in literature. Most frequently reported variant was p.Ala403Val, followed by p.Arg261Gln, p.Val245Ala, IVS10‐11 g>a, p.Tyr414Cys and p.Leu48Ser. Phenotype prediction, based on APV/GPV, matched the actual phenotype in most cases, but not always. Conclusion/Discussion The cohort of patients included in this study constitute a representative sample of the HPA population worldwide. Studies on this sample may allow to improve clinical and genetic evaluation performances for affected patients, consequently to develop personalized medicine interventions and provide more precise indications on the correct treatment approach based on the accumulated evidence, also in light of a prognostically reliable but not always conclusive APV/GPV system.

Background: Tetrahydrobiopterin is a coenzyme of phenylalanine hydroxylase and other enzymes essential for the synthesis of tyrosine and other neurotransmitters. Despite proper nutritional regulation of blood phenylalanine levels, tetrahydrobiopterin deficiency results in progressive neurologic illness. Objective: The study aims to optimize a mass spec-dependent assay for quantitative measurement of biopterin and neopterin as distinctive markers of the atypical phenylketonuria due to tetrahydrobiopterin deficiency. Patients and methods: The study enrolled 46 patients with typical hyperphenylalaninaemia, 14 atypical cases, and 50 healthy children as a control group. Quantitative measurements of biopterin, neopterin, phenylalanine, and tyrosine were performed by ultra-performance liquid chromatography-mass spectrometry in the dried blood spots of patients and control samples. Results: Validation of the analytical protocol was performed at a biopterin and neopterin concentration range from 0 to 100 nmol/l. The regression coefficients for the linearity of the calibration curves exceeded 0.98. The lower limit of detection of biopterin and neopterin ranged from 1.5 to 2.5 nmol/l. The intra-day and inter-day precision and accuracy ranged from 96% - 105%, and from 97% - 110%. The mean recoveries were 105 ± 7% for biopterin and 106 ± 9% for neopterin. The short and long-term stability of the stored samples was seven days at room temperature and 12 weeks at -20 °C. Biopterin and neopterin were significantly higher in the classic group compared to the atypical and control group (p < 0.05), with their level being in the order classic > control > atypical group. Phenylalanine levels had a significant positive correlation with biopterin and neopterin levels in classic phenylketonuria (p < 0.05). Conclusions: The performance of the developed assay for biopterin and neopterin in the dried blood spot by ultraperformance liquid chromatography-mass spectrometry was accurate and precise thus, provide a legitimate diagnostic tool for cases of atypical phenylketonuria. [ABSTRACT FROM AUTHOR]

Background: Hyperphenylalaninemia (HPA) is an autosomal recessive disorder that results from a deficiency in the phenylalanine hydroxylase enzyme (PAH) or from a flaw in the genes that are responsible for the biosynthesis or regeneration of the cofactor tetrahydrobiopterin (BH4), including GCH1, SR, QDPR, PTS, and PCD. Identification of disease-causing variants in these genes can help physicians and clinical geneticists in differential diagnosis, appropriate prescription drugs, and saving time and cost. This study attempted to identify these genes' most prevalent disease-causing variants in Iranian HPA patients. Summary: This study was performed under the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Before it started, the flow work and inclusion/exclusion criteria were published as a protocol in PROSPERO (CRD42021273705). We conducted a comprehensive search on December 10, 2022, on international online databases, including Web of Science, Scopus, EMBASE, Science Direct, PubMed/Medline, Google Scholar, SID, ISC, and Magiran search engine, to find pertinent publications. Some studies were chosen based on inclusion and exclusion criteria. Altogether, 1,243 Iranian patients from 13 articles were considered. In total, we identified 129 distinct disease-causing variants in PAH (20 novel variants), 29 in QDPR (17 novel variants), 15 in PTS (seven novel variants), and one novel variant in PCD. Twenty disease-causing variants for PAH, 18 for QDPR, and 8 for PTS are included in the genes' proposed genetic diagnostic panels. These panels include more than 75% of the documented disease-causing variants in the Iranian population. Key Messages: The findings of this research illustrated the spectrum of disease-causing variants in the PAH, QDPR, PTS, and PCD genes identified in Iranian HPA patients. Common disease-causing variants of these genes may be chosen as a preliminary diagnostic panel for early diagnosis and lowering therapy costs. [ABSTRACT FROM AUTHOR]

INTRODUCTION: To evaluate the 2-year follow-up of hyperphenylalaninemia (HPA) patients born in 2019. METHODS: Growth, neuromotor development, and vitamin levels of 61 two-year-old babies followed up with the diagnosis of HPA in 2019 were evaluated. RESULTS: Thirty-six (59.02%) of the cases were female. The mean birth weight of the babies was 3198.7+-504 g and 23.1% (n=14) of them were preterm. The mean of initial National Newborn Screening Program phenylalanine (phe) levels was 3.85+-1.86 mg/dL. The mean day to diagnosis was 13.29 days (range 4-18). Forty-eight (78.7%) of the patients declared sufficient knowledge about HPA follow-up. Follow-up compliance of the families was 67%. Among the infants who participated in our study, 4 (6.6%) patients were diagnosed with sapropterin dihydrochloride (BH4) responsive mild phenylketonuria (MPKU). No patient was started on with phe-restricted diet therapy. The remaining 32 (52.4%) infants were followed up with the diagnosis of female HPA. The follow-up of 25 (41%) male infants was terminated. Anemia was found in 4 (6.6%) babies. Iron deficiency in 4 (6.6%) and B12 deficiency in 9 (14.8%) babies were detected. There was no significant difference between MPKU and HPA groups regarding growth parameters and vitamin levels. DISCUSSION AND CONCLUSION: No growth or neuromotor retardation was found in HPA patients. Vitamin D and, less frequently, vitamin B12 deficiencies are prominent. No difference was observed between the patients who passed from HPA to MPKU and HPA cases, in terms of growth, development, and nutritional status.

Background: Orthostatic tremor (OT) is a type of postural tremor of the lower extremities that has not been described in either phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Because little is known about the clinical features and therapeutic responses of OT in mild HPA, we describe a mild HPA patient who presented with OT as an initial symptom.Case Presentation: A 22-year-old male was admitted for bilateral leg tremor while standing, with symptom onset eight months prior. One month before admission, the tremor disappeared in the left leg but persisted in the right leg. Electromyography recorded from the right gastrocnemius revealed a 6-8 Hz tremor, which appeared when the patient was standing and disappeared when he was resting or walking. Blood screening showed a phenylalanine/tyrosine ratio of 2.06 and a phenylalanine level of 140 μmol/L. Urine metabolic screening was negative. Whole-exome sequencing confirmed the presence of a compound heterozygous mutation, c.158G > A and c.728G > A, in phenylalanine hydroxylase (PAH) gene. After three months of levodopa/benserazide tablets (250 mg, tid) and a low-phenylalanine diet treatment, the tremor disappeared.Conclusions: Young-onset mild HPA is a relatively rare autosomal recessive metabolic disease, and slow OT is a rare clinical feature. Metabolic screening and genetic testing are the keys to early diagnosis and treatment. For adolescents and young adults, appropriate medication and long-term dietary therapy remain important treatments. This case expanded the disease spectrum of slow OT. [ABSTRACT FROM AUTHOR]

Background: Pterin profiles or molecular analyses of hyperphenylalaninemia (HPA) caused by phenylalanine hydroxylase (PAH) deficiency or tetrahydrobiopterin deficiency (BH4D) are not always available in low- or middle-income countries, including Mexico, limiting information regarding the phenotypic and genotypic characteristics of patients exhibiting BH4D. Objective: To report the genotypes underlying BH4D and the clinical presentation in unrelated Mexican HPA pediatric patients with normal PAH genotypes who attended a single metabolic reference center in Mexico. Methods: Automated Sanger sequencing of the PTS, QDPR, and PCBD1 genes of 14 HPA patients was performed. Predicted effects on protein structure caused by missense variants were assessed by in silico protein modeling. Results and discussion: A high prevalence of BH4D was noted in our HPA cohort (9.8%, N = 14/142). Clinically relevant biallelic genotypes were identified in the PTS (N = 7/14 patients), QDPR (N = 6/14 patients), and PCBD1 (N = 1/ 14 patients) genes. Four novel QDPR variants [c.714dup or p.(Leu239Thrfs*44), c.106-1G>T or p.(?), c.214G>T or p.(Gly72*), and c.187_189dup or p.(Gln63dup)] were identified. In silico protein modeling of six missense variants of PTS [p.(Thr67Met), p.(Glu81Ala), and p.(Tyr113Cys)], QDPR [p.(Cys161Phe) and p.(Pro172Leu)], and PCBD1 [p.(Glu97Lys)] supports their pathogenicity. Progressive neurological symptoms (mainly intellectual and motor impairment and even death in three patients) were noted in all patients with biallelic QDPR genotypes and in 5/7 patients bearing biallelic PTS genotypes. The single homozygous PCBD1 p.(Glu97Lys) patient remains asymptomatic. Conclusion: A higher proportion of BH4D (9.8 vs. 1%--2% worldwide), attributable to a heterogeneous mutational spectrum and wide clinical presentation, was noted in our Mexican HPA cohort, with the PTS-related HPA disorder being the most frequent. Sequencing-based assays could be a reliable approach for diagnosing BH4D in our population. [ABSTRACT FROM AUTHOR]

• The incidence of HPA in Quanzhou, China was estimated to be 1 in 10,365 newborns. • The incidences of PAHD and BH 4 D in Quanzhou were 1:12,350 and 1:64,496, respectively. • The incidence of BH 4 D was as high as 15.1% in the southern Chinese population. • The most common PAH variants were c.158G > A (22.3 %) and c.721C > T (14.9 %). • The most common PTS variants were c.155A > G (50.0 %) and c.259C > T (33.3 %). Hyperphenylalaninemia (HPA) is the most common congenital amino acid metabolism-related defect, but its incidence differs substantially between northern and southern China. We aimed to elucidate the incidence, proportion, and genetic features of HPA in a southern Chinese population. We analyzed the HPA screening results for 580,460 newborns from 2014 to 2021. Of the 296 newborns who tested HPA positive, 56 were diagnosed with HPA, including 47 with phenylalanine hydroxylase deficiency and nine with tetrahydrobiopterin deficiency (BH 4 D). HPA incidence was estimated to be 1:10,365 newborns. All patients had elevated Phe and Phe/Tyr levels. Thirty-three PAH variants and five PTS variants were detected in HPA patients; 80.6 % PAH variants and 100 % PTS variants were classified as pathogenic or likely pathogenic. In silico tools predicted the remaining variants to be damaging. PAH variants clustered in exons 3, 5, 7, 11, and 12 and PTS variants clustered in exons 2 and 5. The most common PAH variants were c.158G > A (p.R53H, 22.3 %) and c.721C > T (p.R241C, 14.9 %). The most common PTS variants were c.155A > G (p.N52S, 50.0 %) and c.259C > T (p.P87S, 33.3 %). Newborn screening is an effective method for early detection of HPA, but differential diagnosis of BH 4 D is necessary. [ABSTRACT FROM AUTHOR]

Background: This systematic review study aims to evaluate the cognitive function of patients with mild hyperphenylalaninemia (mHPA) without treatment.Methods: A systematic literature search was done from the 1st of May to the 30th of July in 2021 on published studies. The search strategy was ((hyperphenylalaninemia) OR (phenylketonuria) AND (cognition)). We use PubMed, Scopus, and the Web of Science databases. Studies which reported their findings regarding the cognitive function of patients with mHPA (screening serum phenylalanine > 120 and < 600 μmol/L) were included and reviewed.Results: From initially retrieved 2805 studies, finally, 15 studies (10 on untreated patients with Phe levels below 360 μmol/L, 7 on untreated patients with Phe levels between 360 and 600 μmol/L, four 120-600 μmol/L) were selected. Most of the studies used the Wechsler Intelligence Scale for IQ evaluation, two (2/15) of them used the Stanford-Binet test and two used both tests. Four studies have reported a worse cognitive outcome compared to the control group, and in one study, relative defects in attention and working memory were reported. Other studies have reported normal IQ levels and no significant cognitive defects.Conclusion: It is suggested that Phe levels between 120 and 360 μmol/L are generally safe. Some studies showed that untreated patients with higher levels might show some degrees of cognitive impairment. In conclusion, current knowledge is insufficient to state that treatment is not required for HPA patients to preserve their cognitive status, especially in patients with Phe levels of 360-600 μmol/L. Further studies with a larger sample size and standardized cognitive function evaluation tools are needed. [ABSTRACT FROM AUTHOR]

Abstract Tetrahydrobiopterin (BH4) is a cofactor that participates in the biogenesis reactions of a variety of biomolecules, including l‐tyrosine, l‐3,4‐dihydroxyphenylalanine, 5‐hydroxytryptophan, nitric oxide, and glycerol. Dihydropteridine reductase (DHPR, EC 1.5.1.34) is an enzyme involved in the BH4 regeneration. DHPR deficiency (DHPRD) is an autosomal recessive disorder, leading to severe and progressive neurological manifestations, which cannot be exclusively controlled by l‐phenylalanine (l‐Phe) restricted diet. In fact, the supplementation of neurotransmitter precursors is more decisive in the disease management, and the administration of sapropterin dihydrochloride may also provide positive effects. From the best of our knowledge, there is limited information regarding DHPRD in the past 5 years in the literature. Here, we describe the medical journey of the first patient to have DHPRD confirmed by molecular diagnostic methods in Brazil. The patient presented with two pathogenic variants of the quinoid dihydropteridine reductase (QDPR) gene—which codes for the DHPR protein, one containing the in trans missense mutation c.515C>T (pPro172Leu) in exon 5 and the other containing the same type of mutation in the exon 7 (c.635T>C [p.Phe212Ser]). The authors discuss their experience with sapropterin dihydrochloride for the treatment of DHPRD in this case report.

Background: Pterin profiles or molecular analyses of hyperphenylalaninemia (HPA) caused by phenylalanine hydroxylase (PAH) deficiency or tetrahydrobiopterin deficiency (BH4D) are not always available in low- or middle-income countries, including Mexico, limiting information regarding the phenotypic and genotypic characteristics of patients exhibiting BH4D.Objective: To report the genotypes underlying BH4D and the clinical presentation in unrelated Mexican HPA pediatric patients with normal PAH genotypes who attended a single metabolic reference center in Mexico.Methods: Automated Sanger sequencing of the PTS, QDPR, and PCBD1 genes of 14 HPA patients was performed. Predicted effects on protein structure caused by missense variants were assessed by in silico protein modeling.Results and discussion: A high prevalence of BH4D was noted in our HPA cohort (9.8%, N = 14/142). Clinically relevant biallelic genotypes were identified in the PTS (N = 7/14 patients), QDPR (N = 6/14 patients), and PCBD1 (N = 1/14 patients) genes. Four novel QDPR variants [c.714dup or p.(Leu239Thrfs*44), c.106-1G>T or p.(?), c.214G>T or p.(Gly72*), and c.187_189dup or p.(Gln63dup)] were identified. In silico protein modeling of six missense variants of PTS [p.(Thr67Met), p.(Glu81Ala), and p.(Tyr113Cys)], QDPR [p.(Cys161Phe) and p.(Pro172Leu)], and PCBD1 [p.(Glu97Lys)] supports their pathogenicity. Progressive neurological symptoms (mainly intellectual and motor impairment and even death in three patients) were noted in all patients with biallelic QDPR genotypes and in 5/7 patients bearing biallelic PTS genotypes. The single homozygous PCBD1 p.(Glu97Lys) patient remains asymptomatic.Conclusion: A higher proportion of BH4D (9.8 vs. 1%–2% worldwide), attributable to a heterogeneous mutational spectrum and wide clinical presentation, was noted in our Mexican HPA cohort, with the PTS-related HPA disorder being the most frequent. Sequencing-based assays could be a reliable approach for diagnosing BH4D in our population.

Phenylketonuria (PKU) is a common genetic metabolic disorder that causes phenylalanine accumulation in the blood. The most serious symptoms are related to the brain, as intellectual disability, seizure, and microcephaly are commonly found in poorly treated PKU patients and the babies of maternal PKU. However, the mechanism of hyperphenylalaninemia on human neurodevelopment is still unclear. Here we utilized human induced pluripotent stem cell (iPSC)-derived cerebral organoids to investigate the neurotoxicity of hyperphenylalaninemia. Cerebral organoids at days 40 or 100 were treated with different concentrations of phenylalanine for 5 days. After phenylalanine treatments, the cerebral organoids displayed alterations in organoid size, induction of apoptosis, and depletion of neural progenitor cells. However, phenylalanine did not have an impact on neurons and glia, including astrocytes, immature oligodendrocytes, and mature oligodendrocytes. Remarkably, a reduction in the thickness of the cortical rosettes and a decrease in myelination at the intermediate zone were inspected with the elevated phenylalanine concentrations. RNA-seq of phenylalanine-treated organoids revealed that gene sets related to apoptosis, p53 signaling pathway, and TNF signaling pathway via NF-kB were enriched in upregulated genes, while those related to cell cycle and amino acid metabolism were enriched in downregulated genes. In addition, there were several microcephaly disease genes, such as ASPM , LMNB1 , and CENPE , ranked at the top of the downregulated genes. These findings indicate that phenylalanine exposure may contribute to microcephaly, abnormal cortical expansion, and myelination lesions in the developing human brain. [ABSTRACT FROM AUTHOR]

Abstract Background Phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disorder that results in elevated concentrations of phenylalanine (Phe) in the blood. If left untreated, the accumulation of Phe can result in profound neurocognitive disability. The objective of this systematic literature review and meta-analysis was to estimate the global birth prevalence of PAH deficiency from newborn screening studies and to estimate regional differences, overall and for various clinically relevant Phe cutoff values used in confirmatory testing. Methods The protocol for this literature review was registered with PROSPERO (International prospective register of systematic reviews). Pubmed and Embase database searches were used to identify studies that reported the birth prevalence of PAH deficiency. Only studies including numeric birth prevalence reports of confirmed PAH deficiency were included. Results From the 85 publications included in the review, 238 birth prevalence estimates were extracted. After excluding prevalence estimates that did not meet quality assessment criteria or because of temporal and regional overlap, estimates from 45 publications were included in the meta-analysis. The global birth prevalence of PAH deficiency, estimated by weighting regional birth prevalences relative to their share of the population of all regions included in the study, was 0.64 (95% confidence interval [CI] 0.53–0.75) per 10,000 births and ranged from 0.03 (95% CI 0.02–0.05) per 10,000 births in Southeast Asia to 1.18 (95% CI 0.64–1.87) per 10,000 births in the Middle East/North Africa. Regionally weighted global birth prevalences per 10,000 births by confirmatory test Phe cutoff values were 0.96 (95% CI 0.50–1.42) for the Phe cutoff value of 360 ± 100 µmol/L; 0.50 (95% CI 0.37–0.64) for the Phe cutoff value of 600 ± 100 µmol/L; and 0.30 (95% CI 0.20–0.40) for the Phe cutoff value of 1200 ± 200 µmol/L. Conclusions Substantial regional variation in the birth prevalence of PAH deficiency was observed in this systematic literature review and meta-analysis of published evidence from newborn screening. The precision of the prevalence estimates is limited by relatively small sample sizes, despite widespread and longstanding newborn screening in much of the world.

Abstract Objectives The present study describes clinical, biochemical, molecular genetic data, current treatment strategies and follow‐up in nine patients with tetrahydrobiopterin (BH4) deficiency due to various inherited genetic defects. Methods We analyzed clinical, biochemical, and molecular data of nine patients with suspected BH4 deficiency. All patients were diagnosed at Ege University Faculty of Medicine in Izmir, Turkey and comprised data collected from 2006 to 2019. The diagnostic laboratory examinations included blood phenylalanine and urinary or plasma pterins, dihydropteridine reductase (DHPR) enzyme activity measurement in dried blood spots, folic acid and monoamine neurotransmitter metabolites in cerebrospinal fluid, as well as DNA sequencing. Results Among the nine patients, we identified one with autosomal recessive GTP cyclohydrolase I (ar GTPCH) deficiency, two with 6‐pyruvoyl‐tetrahydropterin synthase (PTPS) deficiency, three with sepiapterin reductase (SR) deficiency, and three with DHPR deficiency. Similar to previous observations, the most common clinical symptoms are developmental delay, intellectual disability, and movement disorders. All patients received treatment with l‐dopa and 5‐hydroxytryptophan, while only the ar GTPCH, the PTPS, and one DHPR deficient patients were supplemented in addition with BH4. The recommended dose range varies among patients and depends on the type of disease. The consequences of BH4 deficiencies are quite variable; however, early diagnosis and treatment will improve outcomes. Conclusions As BH4 deficiencies are rare group of treatable neurometabolic disorders, it is essential to diagnose the underlying (genetic) defect in newborns with hyperphenylalaninemia. Irreversible brain damage and progressive neurological deterioration may occur in untreated or late diagnosed patients. Prognosis could be satisfying in the cases with early diagnose and treatment.

Objective To explore the gene mutation frequency and characteristics of phenylalanine hydroxylase (PAH) in PAH deficiency children in some regions of Chongqing in order to provide scientific reference for the diagnosis and treatment of the disease. Methods A retrospective analysis was conducted on 45 children with PAH deficiency who were diagnosed in our hospital from January 1, 2014 to October 25, 2020. They was assigned into classic phenylketonuria (PKU), mild PKU and mild hyperphenylalaninemia (HPA). We analyzed the mutations of PAH gene by second-generation high-throughput sequencing and multiplex ligase probe dependent amplification (MLPA) technique, and the results were verified by the detection of corresponding mutation sites of their parents by Sanger sequencing. Results ① There were 2 mutation sites in 43 cases, including 40 cases of compound heterozygous mutation and 3 cases of homozygous mutation. All mutations were detected in the corresponding mutation sites of their parents. The left 2 cases had heterozygous mutations, and had only 1 mutation site. ② There were 34 types of mutations detected in 45 patients with PAH deficiency. Missense mutation was the main mutation type (52.9%). The mutation frequency of c.728G>A was the highest (15.9%, 14/88), followed by c.158G>A (11.4%, 10/ 88), c.1197A>T (9.1%, 8/88), and c.721C > T (9.1%, 8/88). The region of high frequency mutation was in exon 7, which contained 4 mutations and 26 mutations sites (29.5%). ③ There were 11 PAH gene mutations in 13 patients with classic PKU. The mutation frequency of c.728G> A (8/25, 32%) was the highest. There were 9 PAH gene mutations in 8 patients with mild PKU. The mutation frequency of c.728G>A (3/15, 20%) was the highest. There were 24 PAH gene mutations in 24 patients with mild HPA. The mutation frequency of c.158G>A (10/48, 20.8%). Conclusion The mutations in children with PAH deficiency in some regions of Chongqing are mainly compound heterozygosity. Missense mutation is the main type, with obvious hot spot mutations (c.728G>A, c.158G>A, c.721C > T and c.1197A>T).

Abstract Background Hyperphenylalaninemia (HPA) is the most common amino acid metabolic disease involving phenylalanine hydroxylase (PAH, OMIM*612,349) deficiency or coenzyme tetrahydrobiopterin (BH4) deficiency. Patients with severe HPA often have a difficult life. Early diagnosis of HPA before the development of symptoms is possible via neonatal screening, facilitating appropriate treatment and reducing mortality and disability rates. This study revealed the prevalence, mutational and phenotypic spectrum, and prognosis of HPA by neonatal screening from January 2001 to September 2020 in Nanjing, Jiangsu Province, China. Methods Through a retrospective analysis of the information available in the neonatal screening database, the clinical presentations, laboratory data, molecular characteristics and treatment follow-up data of HPA patients detected by neonatal screening were evaluated. Results We diagnosed 181 patients with HPA from 1 to 957 newborns, giving an incidence of 1:6873. Among these patients, 177 were identified as PAH deficient and four patients were BH4 deficient. The average current age of the patients was 6.38 years old. The most common mutations of PAH were c.728 C > A/ p.Arg243Gln (13.83 %), c.158G > A/ p.Arg53His (9.57 %), c.611 A > G/ p.Tyr204Cys (7.44 %), and c.721 C > T/ p.Arg241Cys (6.38 %). Conclusions This study revealed the prevalence, phenotype-genotype, and prognosis of HPA in China and contributes to the updating of PAHD data for China and worldwide. Our study not only expanded the spectrum of phenotypes and genotype but also provided a valuable tool for improved genetic counseling and management of future cases.

Abstract Background Reliable measurement of phenylalanine (Phe) is a prerequisite for adequate follow‐up of phenylketonuria (PKU) patients. However, previous studies have raised concerns on the intercomparability of plasma and dried blood spot (DBS) Phe results. In this study, we made an inventory of differences in (pre‐)analytical methodology used for Phe determination across Dutch laboratories, and compared DBS and plasma results. Methods Through an online questionnaire, we assessed (pre‐)analytical Phe measurement procedures of seven Dutch metabolic laboratories. To investigate the difference between plasma and DBS Phe, participating laboratories received simultaneously collected plasma‐DBS sets from 23 PKU patients. In parallel, 40 sample sets of DBS spotted from either venous blood or capillary fingerprick were analyzed. Results Our data show that there is no consistency on standard operating procedures for Phe measurement. The association of DBS to plasma Phe concentration exhibits substantial inter‐laboratory variation, ranging from a mean difference of −15.5% to +30.6% between plasma and DBS Phe concentrations. In addition, we found a mean difference of +5.8% in Phe concentration between capillary DBS and DBS prepared from venous blood. Conclusions The results of our study point to substantial (pre‐)analytical variation in Phe measurements, implicating that bloodspot Phe results should be interpreted with caution, especially when no correction factor is applied. To minimize variation, we advocate pre‐analytical standardization and analytical harmonization of Phe measurements, including consensus on application of a correction factor to adjust DBS Phe to plasma concentrations.

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants. [ABSTRACT FROM AUTHOR]

The present study aimed to conduct a comprehensive meta-analysis to assess the diagnostic value of fluorometric assays and tandem mass spectrometry (MS/MS) for hyperphenylalaninemia (HPA) and its subtypes. The PubMed, Embase and Cochrane Library databases were searched from inception to October 2023. The present study included studies that reported the newborn screening and genetic features of patients with HPA and excluded duplicate publications, studies without full text, studies with incomplete information, studies from which it was not possible to extract data, animal experiments, reviews and systematic reviews. STATA 15.1 was used to analyze the data. The pooled results revealed that 0.04% [95% confidence interval (CI): 0.019-0.069] of neonatal HPA fluorometric assays and MS/MS. The positive predictive value (PPV) of neonatal HPA screening using fluorometric assays and tandem mass spectrometry was 31.7% (95% CI: 19.6-45.2). Notably, the PPV of neonatal HPA screening using fluorometric assays was 8.3% (95% CI: 7.1-9.6), while the PPV of neonatal HPA screening using tandem mass spectrometry was 31.8% (95% CI: 16.4-49.4). Additionally, the pooled results showed that the incidence of tetrahydrobiopterin deficiency (BH4D) in HPA patients was 12.43% (95% CI: 3.28-25.75) and the incidence of phenylalanine hydroxylase deficiency (PAHD) in HPA patients was 88.65% (95% CI: 78.84-95.86). Newborn screening is an effective method for the early detection of HPA and MS/MS has a greater PPA than fluorometric assays for diagnosing HPA. In addition, in the screening of HPA, the proportion of HPA patients with PAHD was significantly higher than that of patients with BH4D. [ABSTRACT FROM AUTHOR]

• In this review, we have discussed about an autosomal recessive metabolic disorder, Phenylketonuria (PKU), resulting from deficient phenylalanine hydroxylase (PAH) enzyme activity, leading to impaired phenylalanine (Phe) metabolism. • This review provides insights into pathophysiology, current treatment and diagnostic approaches. • We have further explored the wide array of sensors used till date for diagnosis of PKU. Phenylketonuria (PKU) is an autosomal recessive metabolic disorder resulting from deficient phenylalanine hydroxylase (PAH) enzyme activity, leading to impaired phenylalanine (Phe) metabolism. This condition can lead to intellectual disability, epilepsy, and behavioural issues. Treatment typically involves strict dietary restrictions on natural protein intake, supplemented with chemically manufactured protein substitutes containing amino acids other than Phe. Various approaches, including casein glycomacropeptide (GMP), tetrahydrobiopterin (BH 4), phenylalanine ammonia-lyase (PAL) therapy, large neutral amino acid (LNAA) supplementation, enzyme therapy, gene therapy, and medical therapies, aim to prevent Phe transport in the brain to potentially treat PKU. Although newborn screening programs and early dietary interventions have enhanced outcomes of the potential treatment strategies, limitations still persist in this direction. These involve potent accuracy concerns in diagnosis due to the existence of antibiotics in blood of PKU patients, affecting growth of the bacteria in the bacterial inhibition assay. Monitoring involves complex methods for instance, mass spectrometry and high-pressure liquid chromatography, which involve shortcomings such as lengthy protocols and the need for specialized equipment. To address these limitations, adaptable testing formats like bio/nano sensors are emerging with their cost-effectiveness, biodegradability, and rapid, accurate, and sensitive detection capabilities, offering promising alternatives for PKU diagnosis. This review provides insights into current treatment and diagnostic approaches, emphasizing on the potential applications of the diverse sensors intended for PKU diagnosis. [ABSTRACT FROM AUTHOR]

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6- Pah enu2/enu2 mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults. 2) Cognitive deficits reappear in mice treated with weekly pegvaliase from birth but in which pegvaliase is discontinued at 3 months age. 3) Twice weekly pegvaliase injection also prevented cognitive deficits but again cognitive deficits emerged in early-treated animals following discontinuation of pegvaliase treatment during adulthood, particularly in females. In all studies, pegvaliase treatment was associated with complete correction of brain monoamine neurotransmitter content and with improved overall growth of the mice as measured by body weight. Mean total brain weight however remained low in all PAH deficient mice regardless of treatment. Application of enzyme substitution therapy with pegvaliase, initiated near birth and continued into adulthood, to PAH-deficient Pah enu2/enu2 mice models contemporary early-treated human PKU. This model will be useful for exploring the differential pathophysiologic effects of HPA at different developmental stages of the murine brain. [ABSTRACT FROM AUTHOR]

Background. Phenylketonuria (PKU), is an autosomal recessive disease leading to the conversion defect of phenylalanine (Phe) into tyrosine. Severe neurocognitive and behavioral outcomes are observed in untreated cases. The present paper aims to review clinical experiences and expert recommendations in diagnosis, treatment, and follow-up of pediatric PKU patients in Turkey. Methods. Two advisory board meetings were held in the year 2016 and 2017 with contributions of four leading experts in this field, and an online update meeting was held for final decisions about statements, and conclusions in January 2021. Considering management gaps in diagnosis, treatment, and follow-up of PKU, discussion points are defined. The Committee members then reviewed the Turkish and general literature and the final statements were formulated. Results. The diagnostic cut-off for dried blood spots should remain at 2 mg/dl. Treatment cut-off value is acceptable at 6 mg/dl. Compliance with an ideal follow-up list is strongly recommended. Total protein intake should not be limited. Age-related safe levels of protein intake should be encouraged with an additional 40% from L-amino acids supplements, a 20% compensatory factor to account for the digestibility and utilization of amino acids from the supplement, and a further 20% compensation to optimize Phe control. Cognitive impairment and intelligence quotient evaluations should be performed at least twice before 3 years of age. In pregnant women, the target Phe level should be < 5 mg/dl, and they should be followed-up weekly in the first trimester, then every 2 weeks after organogenesis. Novel pharmacological treatments are promising, but some of them have limitations for our country. Conclusions. Early diagnosis and treatment initiation; determination and standardization of diagnostic and treatment thresholds; treatment modalities and follow-up parameters are significant steps in treating PKU in the long term. PKU follow-up is a dynamic process with uncertainties and differences in clinical practice. [ABSTRACT FROM AUTHOR]

The aim of our study was to define the genotype–phenotype correlations of mutations in the PAH gene among the Turkey's Central Anatolian region. Demographic characteristics of 108 patients with hyperphenylalaninemia (HPA) and 94 patients whose diagnosis was confirmed by PAH gene analysis (Sanger DNA Sequence Analysis and Next-Generation Sequencing) were determined retrospectively. Blood phenylalanine levels were analyzed using the high-performance liquid chromatography method. Mild HPA-not-requiring-treatment (NT) was found in 50.9% of the patients, and a classical phenylketonuria (PKU) phenotype was found in 25.9%. Forty-seven types of variants were identified. The predominant variants were p.Ala403Val (9.9%), p.Ala300Ser (9.4%), and c.1066-11G>A (splicing) (9.4%). Missense mutations accounted for 68% of mutations and attenuated the clinical impact; splice variations were found in 14.8% of cases with severe features. The p.Thr380Met allele was specific to the mild HPA-NT group. The c.1066-11G>A (splicing) allele was associated with classical PKU, whereas the p.Arg408Trp allele was linked to severe symptoms. Three variations of unknown clinical significance were discovered: c.706+4A>T (splicing), c.843-5T>C (splicing), and p.Thr323=. Of these variants, the patient who was homozygous for the c.843-5T>C (splicing) allele related to the classical PKU phenotype. 70% of the patients who underwent tetrahydrobiopterin (BH4) test were responsive. Phenotypes that responded to BH4 treatment were mostly mild phenotypes. The PAH genotype is the main factor that determines the phenotype of PKU. Establishing the relationship between the identified genetic mutations and phenotypic characteristics will provide very important data for each patient in terms of the specific management style. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr−/− mice to elucidate the physiological significance of the regeneration of BH4. We found that the Qdpr−/− mice exhibited mild hyperphenylalaninemia and monoamine deficiency in the brain, despite the presence of substantial amounts of BH4 in the liver and brain. Hyperphenylalaninemia was ameliorated by exogenously administered BH4, and dietary phenylalanine restriction was effective for restoring the decreased monoamine contents in the brain of the Qdpr−/− mice, suggesting that monoamine deficiency was caused by the secondary effect of hyperphenylalaninemia. Immunohistochemical analysis showed that QDPR was primarily distributed in oligodendrocytes but hardly detectable in monoaminergic neurons in the brain. Finally, we performed a behavioral assessment using a test battery. The Qdpr−/− mice exhibited enhanced fear responses after electrical foot shock. Taken together, our data suggest that the perturbation of BH4 metabolism should affect brain monoamine levels through alterations in peripheral amino acid metabolism, and might contribute to the development of anxiety‐related psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15398 [ABSTRACT FROM AUTHOR]

The aim is to evaluate the clinical, demographic and laboratory data of the patients we followed up with phenylalanine metabolism disorder. In this study, patients with phenylalanine metabolism disorder who applied to Bursa Uludag University Faculty of Medicine, Department of Pediatrics, Pediatric Metabolism Department between 2011 and 2021 were retrospectively examined. The files of 397 patients who were followed up in our pediatric metabolism outpatient clinic and were found to have phenylalanine metabolism disorder by plasma phenylalanine level and molecular genetic analysis were evaluated. According to the highest plasma phenylalanine levels at admission, mild hyperphenylalaninemia phenotype constituted the largest group of 397 patients with 282 cases (71.1%), while the least common phenotype was malignant phenylketonuria (BH4 metabolism disorder) with four patients (1.0%). The number of patients with classical phenylketonuria was 90 (22.6%). 61 (62.8%) of 97 phenylalanine metabolism disorder cases who underwent BH4 loading test had a response. The mean phenylalanine level of the patients was 3.62 ± 1.31 mg/dL in mild hyperphenylalaninemia, 7.98 ± 3.99 mg/dL in mild phenylketonuria and 11.71 ± 4.39 mg/dL in classical phenylketonuria. While 241 (76%) of 317 patients younger than 8 years old were in the well-controlled group, 76 (24%) were in the poorly-controlled group. While 41 (53.9%) of 76 patients older than 8 years of age were in the well-controlled group, 35 (46.1%) were in the poorly-controlled group. In our study, the largest patient group consisted of patients with mild hyperphenylalaninemia, and the least common phenotype was mild phenylketonuria. [ABSTRACT FROM AUTHOR]

More than 1280 variants in the phenylalanine hydroxylase (PAH) gene are responsible for a broad spectrum of phenylketonuria (PKU) phenotypes. While the genotype-phenotype correlation is reaching 88%, for some inconsistent phenotypes with the same genotype additional factors like tetrahydrobiopterin (BH 4), the PAH co-chaperone DNAJC12, phosphorylation of the PAH residues or epigenetic factors may play an important role. Very recently an additional player, the long non-coding RNA (lncRNA) transcript HULC , was described to regulate PAH activity and enhance residual enzyme activity of some PAH variants (e.g. , the most common p.R408W) by using HULC mimics. In this review we present an overview of the lncRNA function and in particular the interplay of the HUCL transcript with the PAH and discuss potential applications for the future treatment of some PKU patients. [ABSTRACT FROM AUTHOR]

Blood phenylalanine-to-tyrosine (Phe/Tyr) ratio is an important indicator of metabolic control in phenylketonuria patients. We present the data that highlights the role of Phe/Tyr-ratio in the evaluation of tetrahydrobiopterin (BH4)-responsiveness in patients with hyperphenylalaninemia. Our data complements the results from the original research article by Tansek et al., 2012 [1]. We performed a BH4-loading test in 32 patients after four days of increased protein intake (2000 mg/kg body weight). Blood sampling was performed 96, 72, 48, 24, 16 h, and moments before oral administration of BH4 in a dose of 20 mg/kg body weight. Additional blood samples were collected 8 and 24 h after its administration. Phenylalanine (Phe) and Tyrosine (Tyr) levels were determined from dried blood spots by tandem mass spectrometry. Phe/Tyr-ratio reached a plateau after three days of increased dietary protein intake. Fifteen patients (47%) responded to BH4, defined as a decrease of Phe-of at least 30% after 24 h of BH4 administration. Phe/Tyr-ratios were significantly higher in non-responders compared to responders. In the responder group, Phe/Tyr-ratios decreased in average of 67% (p = 0.001) and 45% (p = 0.001) after 8 and 24 h of BH4 administration, respectively. Phe/Tyr-ratio decreased after 8 h of drug administration also in the non-responder group, but not 24 h after administration.

Objectives: To evaluate the 2-year follow-up of hyperphenylalaninemia (HPA) patients born in 2019. Methods: Growth, neuromotor development, and vitamin levels of 61 two-year-old babies followed up with the diagnosis of HPA in 2019 were evaluated. Results: Thirty-six (59.02%) of the cases were female. The mean birth weight of the babies was 3198.7±504 g and 23.1% (n=14) of them were preterm. The mean of initial National Newborn Screening Program phenylalanine (phe) levels was 3.85±1.86 mg/dL. The mean day to diagnosis was 13.29 days (range 4-18). Forty-eight (78.7%) of the patients declared sufficient knowledge about HPA follow-up. Follow-up compliance of the families was 67%. Among the infants who participated in our study, 4 (6.6%) patients were diagnosed with sapropterin dihydrochloride (BH4) responsive mild phenylketonuria (MPKU). No patient was started on with phe-restricted diet therapy. The remaining 32 (52.4%) infants were followed up with the diagnosis of female HPA. The follow-up of 25 (41%) male infants was terminated. Anemia was found in 4 (6.6%) babies. Iron deficiency in 4 (6.6%) and B12 deficiency in 9 (14.8%) babies were detected. There was no significant difference between MPKU and HPA groups regarding growth parameters and vitamin levels. Conclusion: No growth or neuromotor retardation was found in HPA patients. Vitamin D and, less frequently, vitamin B12 deficiencies are prominent. No difference was observed between the patients who passed from HPA to MPKU and HPA cases, in terms of growth, development, and nutritional status. [ABSTRACT FROM AUTHOR]

Phenylketonuria is an inherited metabolic disease that can be treated if detected early and can easily be screened with phenylalanine as a biomarker. Phenylketonuria is included in the National Neonatal Screening Program. We aimed to investigate the possible effects of the pandemic on phenylketonuria screening in our country during coronavirus disease 2019 (COVID-19) era. We retrospectively evaluated patients who were referred to our center due to elevated blood phenylalanine levels detected by national screening program retrospective- and prospectively within one-year as of 11 March 2020, the date when first COVID-19 case was detected in our country. Multivariate analysis showed birth region and phenylalanine levels to be independently associated with the time period until the first admission to hospital, but none of the parameters were significantly associated with the pandemic. There was no evidence that the COVID-19 pandemic affected the screening of newborn phenylketonuria, however since the data was limited to our center and to draw generalized conclusions larger scaled multicentric studies are needed. [ABSTRACT FROM AUTHOR]