Dietary interventions are essential for managing phenylketonuria (PKU) and may influence metabolic regulation beyond phenylalanine control. We characterized the urinary metabolomic fingerprint of pediatric participants (n=82) recruited into clinical phenotypes: PKU, PKU with response to tetrahydrobiopterin (BH4), and hyperphenylalaninemia (HPA), as well as sex- and age-matched healthy children controls. Untargeted metabolomics (HPLC-Q-TOF-MS/MS) revealed 59 discriminant metabolites across multiple biochemical pathways, including phenylalanine, tryptophan, and caffeine metabolisms, as well as metabolites related to dietary exposure or gut microbial metabolism. Distinct urinary signatures were described across phenotypes. Phenylalanine-related pathways predominated in PKU, accompanied by increased excretion of vitamin derivatives, consistent with protein substitute supplementation. In contrast, reduced levels of non-phenylalanine amino acid derivatives, methylhistidines, creatine, and branched-chain amino acid-related metabolites were observed in PKU, suggesting alterations in muscle metabolism or natural protein intake. Microbiota-derived metabolites were also less represented in PKU, indicating potential effects of dietary restrictions on gut-host metabolic interactions. HPA individuals showed a urinary fingerprint closer to controls, whereas the BH4 subgroup exhibited the greatest metabolic heterogeneity, reflecting variability in dietary and pharmacological treatment responses. These findings reveal metabolic diversity within the pediatric PKU spectrum driven by clinical phenotype and nutritional management. Urinary metabolomics may support more precise monitoring of metabolic health status and guide precision nutrition strategies in PKU and HPA from early life.

Ethnic Bridging of Sepiapterin in Chinese and Korean Populations Based on Predictions From Genetic Polymorphism of Breast Cancer Resistance Protein.

Mouse models of genetic diseases are important research tools. However, the genetic background of the mouse strain can significantly influence how a genetic mutation is expressed. Studies on preclinical models of phenylketonuria (PKU), an inherited metabolic disorder, have used two strains, BTBR and C57Bl/6, created via a chemically induced point mutation in the gene encoding the enzyme phenylalanine hydroxylase (BTBRenu2 and C57enu2, respectively). Despite having the same levels of hyperphenylalaninemia (HPA), published results indicate differences in neural and behavioral phenotypes between the two backgrounds. To explore this difference further, the current study examines the genome-wide transcriptome of the prefrontal cortex (pFC), the brain region which is the most vulnerable to the negative effects of HPA. Regardless of the strain, the enu2 mutation upregulated the expression of several aminoacyl-tRNA synthetases and eukaryotic translation initiation factors, suggesting an essential modification in the protein translation process and supporting the downregulation of gene programs related to myelination. Accordingly, we deepened the exploration of cognitive dysfunctions in C57enu2− mice, showing a previously unreported working memory impairment under increasing information load. These findings identify convergent pFC molecular and cognitive alterations induced by HPA across distinct genetic backgrounds, providing clinically relevant insights into mechanisms that may contribute to executive dysfunctions in PKU.

Hyperphenylalaninemia (HPA) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase, characterized by significantly elevated phenylalanine levels. Conventional mechanisms, such as neurotransmitter deficiency and dysmyelination, fail to fully explain the progressive neurological damages associated with HPA. Meanwhile, ferroptosis, an emerging form of iron-dependent regulated cell death, has proven to play an important role in neurodegenerative diseases. We hereby propose a hypothesis that tetrahydrobiopterin (BH4) depletion in HPA may lead to the collapse of intracellular antioxidant defenses. This process could induce ferroptosis, thereby serving as a pivotal mechanism underlying HPA-related neurological injury. This review has systematically summarized the pathological mechanisms of HPA, the biological features of ferroptosis, and the bridging role of BH4 between them, thereby establishing a novel "HPA-BH4-ferroptosis" theoretical framework and providing a rationale for developing new therapeutic strategies targeting ferroptosis.

BackgroundThe global incidence of Hyperphenylalaninemia (HPA) demonstrates significant geographical variations, exhibiting distinct regional and ethnic characteristics in both phenotypic manifestations and genotypic profiles. To date, there remains a paucity of data regarding the genotype-phenotype correlation in pediatric patients with phenylalanine hydroxylase deficiency (PAHD) from Southwest China. This study aims to conduct a retrospective analysis of neonatal HPA prevalence and characterize PAH gene mutations in Yunnan Province in Southwest China. These findings are expected to establish an evidence base for optimizing clinical follow-up protocols, facilitating genetic counseling, and enabling prenatal molecular diagnosis for affected children.MethodsFrom January 2013 to December 2023, neonatal screening data for HPA were retrospectively collected from the Yunnan Neonatal Screening Center. Neonates with presumptive positive screening results underwent confirmatory diagnosis through quantitative analysis of phenylalanine levels using tandem mass spectrometry. Subsequently, HPA-related genetic variants were identified by next-generation sequencing technology. Putative pathogenic mutations detected in probands were validated through Sanger sequencing of trios.ResultsA total of 1,261,043 newborn screening samples for phenylalanine were analyzed, with 125 cases confirmed as HPA. The overall incidence rates were 0.99 per 10,000 for HPA, 0.98 per 10,000 for PAHD, and 0.16 per 100,000 for tetrahydrobiopterin deficiency. Genetic analysis of hyperphenylalaninemia-related genes was performed in 84 children, revealing 49 PAH variants, 2 PTS variants, and 1 QDPR variant, with a total of 164 mutation sites identified. Missense mutations constituted the predominant variant type. The most frequent PAH mutations were c.728G > A/p.R243Q (26.88%), c.331C > T/p.R111* (9.38%), c.320A > G/p.H107R (8.13%), c.158G > A/p.A53H (7.50%), and c.441 + 2T > A/splicing (5.00%), with clustering observed in exons 7, 11, 6, and 3. A novel PAH mutation (c.60 + 4A > G/ p.?.) was identified.ConclusionsThe incidence and genetic mutation spectrum of HPA in Yunnan Province, Southwest China, exhibit distinctive characteristics when compared with other regions in China and international reports. These differences may be attributed to the unique geographical distribution of HPA patients and ethnic-specific genetic characteristics in this region. Furthermore, specific genetic mutations demonstrate potential associations with clinical phenotypes in PAHD. The novel PAH mutations identified in this study have expanded the current PAH gene database.

Background/Objectives: Skeletal impairment has been reported as a common finding in Hyperphenylalaninemia (HPA)/Phenylketonuria (PKU) patients regardless of age and method of diagnosis, both in children and adults. Quantitative Ultrasound (QUS) is a radiation-free and low-cost method for assessing bone quality, used in various chronic conditions. Methods: Bone quality was evaluated using a calcaneal QUS device. Auxological parameters, nutritional intakes, and plasma levels of key bone biomarkers were also registered. The population was divided into four groups: PKU patients under diet therapy and HPA patients on a free diet, both divided into receiving or not receiving single vitamin D supplementation. Results: All HPA/PKU patients had median bone quality index (BQI) Z- and T-score values lower than −1, with slightly better values in HPA children and PKU-supplemented adults. Dietary vitamin D intake in PKU patients was significantly higher than in HPA subjects (p < 0.001), due to protein substitute supplementation. However, plasma 25(OH) vitamin D levels, although increased compared to baseline, were still overlapping among groups (p = 0.845) after supplementation. Approximately a quarter of both pediatric and adult non-supplemented PKU patients had Z-score and T-score levels below −2, and this percentage decreased with vitamin D supplementation in all groups. In PKU-supplemented patients, the Broadband Ultrasound Attenuation (BUA) was significantly higher than in the other groups (p = 0.040). Conclusions: The improvement in BUA may represent preliminary evidence of the effect of vitamin D on bone architecture, which could encourage this supplementation to prevent the worsening of bone structure and reduce the risk of fractures.

An oral formulation of sepiapterin (Sephience™) is being developed by PTC Therapeutics for the treatment of phenylketonuria (PKU). Sepiapterin is a natural precursor of enzyme cofactor tetrahydrobiopterin (BH4), which is required for phenylalanine hydroxylase (PAH)-mediated phenylalanine metabolism. Sepiapterin enhances PAH activity, which is deficient in patients with PKU. Sepiapterin received its first approval on 19 June 2025 in the European Union, for use in hyperphenylalaninaemia (HPA) in adult and paediatric patients with PKU. This was followed by approval in the USA on 28 July 2025, for the treatment of HPA in adult and paediatric patients ≥ 1 month of age with sepiapterin-responsive PKU. This article summarizes the milestones in the development of sepiapterin leading to its first approval for HPA in adult and paediatric patients with PKU.

Portable collection of breath phenylpyruvic acid for noninvasive assessment of pediatric hyperphenylalaninemia by mass spectrometry.

Background: Phenylketonuria (PKU) is a rare autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, leading to hyperphenylalaninemia (HPA). Untreated, elevated phenylalanine (Phe) levels cause severe neurocognitive, developmental, and psychiatric complications. Management relies on a Phe-restricted diet, which is challenging to maintain, particularly in adolescents and adults. Sapropterin dihydrochloride, a synthetic form of tetrahydrobiopterin (BH4), can enhance residual PAH activity, lowering blood Phe levels and increasing dietary tolerance in responsive patients. However, real-world alignment with best practices remains underexplored. This study aims to report a tertiary referral center's experience with sapropterin treatment in PKU and assess adherence to international guidelines. Methods: We retrospectively analyzed 23 PKU patients treated with sapropterin from 2007 to 2025. Patients with baseline Phe levels of 360-2000 µmol/L underwent a 10 mg/kg/day loading test over two weeks. Responsiveness was defined as a ≥30% reduction in blood Phe levels. Phe levels were measured pre- and post-test, and dietary tolerance was evaluated. Adherence to best practices was critically reviewed. Results: All patients showed significant Phe reductions (mean 71.43%, p < 0.0001), exceeding responsiveness thresholds. Most achieved substantial increases in dietary Phe tolerance, with three patients partially responsive (800-1200 mg/day). Responsiveness was unrespectful of the patient's genotype, for those individuals for whom this was known (8/23 patients). Although effective, the test dose and duration differed from guideline recommendations (20 mg/kg/day). Neuropsychological and QoL assessments were not systematically performed, representing a key limitation. Conclusions: Sapropterin dihydrochloride effectively identified responders and improved dietary flexibility even with lower dosing protocols. Greater adherence to international standards, particularly regarding long-term neuropsychological monitoring, is needed to optimize patient care.

To explore the spectrum of genetic variants and phenotypes of Phenylalanine hydroxylase deficiency (PAHD) in Lianyungang area and the correlation between genotype and phenotypes among the patients. Eighty children with Hyperphenylalaninemia (HPA) diagnosed at the Lianyungang Branch of Jiangsu Provincial Newborn Screening Center between January 2015 and December 2022 were enrolled. Peripheral blood samples were collected for genetic analysis using next generation sequencing (NGS), Sanger sequencing, and multiplex ligation-dependent probe amplification (MLPA) to identify the variants of PAH gene. Clinical and phenotypic data were concurrently analyzed to investigate the correlation between the types of PAH gene variant and phenotypes. This study was approved by the Medical Ethics Committee of Lianyungang Maternal and Child Health Care Hospital (Ethics No.: XM2022041). PAH gene variants were identified in 93.75% (75/80) of the children, classified as PAHD cases, while 6.25% (5/80) harbored PTS gene variants. Of the 150 PAH alleles from 75 PAHD children, a total of 152 variants (55 distinct types) were detected, with a detection rate of 100%. 80.26% (122/152) of the variants were located in exons, with the main types being missense variants (67.11%, 102/152). 53.29% (81/152) of coding sequence variants have occurred in the PAH gene's catalytic center region, while 19.74% (30/152) of the variants involved non-coding sequences. The phenotypes of the 75 PAHD children were evenly distributed. The re-screened Phe concentrations and Phe/Tyr ratios of classic-phenylketonuria (CPKU) and mild-phenylketonuria (MPKU) patients were markedly higher than initial screening values (P < 0.001, P < 0.001; P = 0.004, P = 0.016). The genotypes of the PAHD patients mostly occurred as compound heterozygotes, and different mutation positions and variant types have significantly affected the phenotypes (P = 0.042, P = 0.045). APV/GPV genotype-phenotype analysis of 61 patients showed high consistency between predicted and actual phenotypes (κ = 0.755, P < 0.001). PAH gene variants were detected in most HPA children from Lianyungang area. The location and type of PAH gene variants has correlated with the severity of the phenotype, and the non-coding sequence variants and non-missense variants may aggravate the phenotype, and the APV/GPV model has predicted the phenotype with high consistency with the actual phenotype.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder characterized as deficiencies in phenylalanine hydroxylase, leading to neurotoxic effects and neurodevelopmental challenges. Sleep, crucial for cognitive and behavioral development, remains underexplored in PKU populations. This study evaluates sleep characteristics and influencing factors in school-aged children with PKU compared to those with hyperphenylalaninemia (HPA) and healthy controls. A total of 101 children aged 5–10 years participated: 37 with PKU, 31 with HPA, and 33 healthy controls. Sleep quality and disturbances were assessed using the Children’s Sleep Habits Questionnaire (CSHQ) and parent-reported data. Additional factors, including phenylalanine levels and sleep hygiene practices, were analyzed. Despite no significant differences in total CSHQ scores across groups, children with PKU exhibited unique patterns, such as being less likely to awaken by himself or herself. Phenylalanine levels showed no significant correlations with overall sleep characteristics, except for an association with reduced sleep anxiety. Distinct sleep hygiene influences emerged in the HPA and control groups, while no such relationships were observed in PKU. This study underscores the complexity of sleep disturbances in PKU, highlighting the need for future research integrating biological, behavioral, and environmental factors. Identifying determinants of sleep problems will aid in developing tailored interventions to enhance the quality of life for patients with PKU.What is known:• Children with PKU, in addition to the risks associated with chronic illness, may be more susceptible to sleep disturbances due to metabolic and neurochemical imbalances.• Prior studies on PKU and sleep have mainly focused on adult populations, with limited data available for school-aged children.What is new:• The sleep characteristics of PKU, HPA, and healthy controls were generally similar, except for awakening by themselves.• Distinct sleep hygiene influences were identified in the HPA and control groups, while no such associations were observed in the PKU group, highlighting the need for a broader perspective to explore alternative biological, behavioral, and environmental factors affecting sleep regulation in PKU.

ObjectivesThe aim of this study is to evaluate neurodevelopmental and cognitive outcomes in patients diagnosed with different types of hyperphenylalaninemia (HPA), identify the factors influencing these outcomes, and contribute to the debate regarding the thresold for initiating dietary treatment based on plasma phenylalanine (Phe) levels.MethodsPatients with hyperphenylalaninemia (HPA) who were followed up and had developmental and/or cognitive evaluations at the Division of Pediatric Metabolism and Nutrition, Department of Pediatrics, between 1984 and 2018, were retrospectively assessed. The study included patients with mild (Phe:360-600 μmol/L), moderate (Phe:600-1200 μmol/L), or classic Phenylketonuria (PKU) (Phe ≥1200 μmol/L) treated with diet and/or tetrahydrobiopterin (BH4), along with untreated HPA patients (Phe:240-360 μmol/L). This classification was based on plasma Phe levels measured at the time of diagnosis. Denver Developmental Screening Test (DDST), Stanford-Binet test, and Wechsler Intelligence Scale for Children (WISC-R) adapted for Turkish children were applied for developmental and cognitive evaluation. Intellectual disability or developmental delay (ID/DD) was defined as a full-scale intelligence quotient (IQ) <70 on the Stanford-Binet or WISC-R, or as delay in two or more developmental domains on the DDST, with children meeting any of these criteria classified as having ID/DD. The relationships between ID/DD, age at diagnosis, diagnostic methods, plasma Phe levels, and brain MRI findings were analyzed.ResultsA total of 342 patients were included in the study, comprising 182 (53.2%) females and 160 (46.8%) males. Of these, 53 (15.5%) had mild PKU, 97 (28.4%) had moderate PKU, 102 (29.8%) had classic PKU, and 90 (26.3%) were diagnosed with HPA. A significant association was found between ID/DD and both the age at diagnosis and diagnostic method in patients treated with diet and/or BH4 (p < 0.001 and p < 0.01, respectively). In patients with ID/DD, the median plasma Phe levels at the first, third, and last years of follow-up were significantly higher compared to patients without ID/DD (p < 0.024). White matter abnormalities observed on brain MRI were significantly associated with PKU severity, the presence of ID/DD, and the median plasma Phe levels in the last year of follow-up (p = 0.01, p < 0.001, and p < 0.001, respectively). Notably, 9 (10%) of untreated HPA patients exhibited ID/DD, despite regular follow-up and the absence of known risk factors.ConclusionIn addition to early diagnosis and treatment, lifelong adherence and regular follow-up are essential for achieving normal neurodevelopmental and cognitive outcomes in individuals with PKU. However, clinical management remains heterogeneous across centers. The presence of developmental delay in 10% of untreated HPA patients underscores the need to urgently re-evaluate current plasma Phe thresholds for treatment initiation and follow-up.

BackgroundAssessing 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.MethodsIn 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.ResultsThe 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.ConclusionsOur 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.

Background: In 2011, a European phenylketonuria (PKU) survey reported that the blood phenylalanine (Phe) levels were well controlled in early life but deteriorated with age. Other studies have shown similar results across the globe. Different target blood Phe levels have been used throughout the years, and, in 2017, the European PKU guidelines defined new targets for blood Phe levels. This study aimed to evaluate blood Phe control in patients with PKU across Europe. Methods: nine centres managing PKU in Europe and Turkey participated. Data were collected retrospectively from medical and dietetic records between 2012 and 2018 on blood Phe levels, PKU severity, and medications. Results: A total of 1323 patients (age range:1–57, 51% male) participated. Patient numbers ranged from 59 to 320 in each centre. The most common phenotype was classical PKU (n = 625, 48%), followed by mild PKU (n = 357, 27%) and hyperphenylalaninemia (HPA) (n = 325, 25%). The mean percentage of blood Phe levels within the target range ranged from 65 ± 54% to 88 ± 49% for all centres. The percentage of Phe levels within the target range declined with increasing age (<2 years: 89%; 2–5 years: 84%; 6–12 years: 73%; 13–18 years: 85%; 19–30 years: 64%; 31–40 years: 59%; and ≥41 years: 40%). The mean blood Phe levels were significantly lower and the percentage within the target range was significantly higher (p < 0.001) in patients with HPA (290 ± 325 μmol/L; 96 ± 24%) and mild PKU (365 ± 224 μmol/L; 77 ± 36%) compared to classical PKU (458 ± 350 μmol/L, 54 ± 46%). There was no difference between males and females in the mean blood Phe levels (p = 0.939), but the percentage of Phe levels within the target range was higher in females among school-age children (6–12 years; 83% in females vs. 78% in males; p = 0.005), adolescents (13–18 years; 62% in females vs. 59% in males; p = 0.034) and adults (31–40 years; 65% in females vs. 41% in males; p < 0.001 and >41 years; 43% in females vs. 28% in males; p < 0.001). Patients treated with sapropterin (n = 222) had statistically significantly lower Phe levels compared to diet-only-treated patients (mean 391 ± 334 μmol/L; percentage within target 84 ± 39% vs. 406 ± 334 μmol/L; 73 ± 41%; p < 0.001), although a blood Phe mean difference of 15 µmol/L may not be clinically relevant. An increased frequency of blood Phe monitoring was associated with better metabolic control (p < 0.05). The mean blood Phe (% Phe levels within target) from blood Phe samples collected weekly was 271 ± 204 μmol/L, (81 ± 33%); for once every 2 weeks, it was 376 ± 262 μmol/L, (78 ± 42%); for once every 4 weeks, it was 426 ± 282 μmol/L, (71 ± 50%); and less than monthly samples, it was 534 ± 468 μmol/L, (70 ± 58%). Conclusions: Overall, blood Phe control deteriorated with age. A higher frequency of blood sampling was associated with better blood Phe control with less variability. The severity of PKU and the available treatments and resources may impact the blood Phe control achieved by each treatment centre.

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.

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 twonovel 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.

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.

To explore the pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase (PAH) gene among patients with PAH deficiency. Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed. Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c.158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c.158G>A variants and a heterozygous c.842+2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c.[158G>A,842+2T>A]/c.728G>A and c.[158G>A,842+2T>A]/c.611A>G, respectively, and one with c.[158G>A, c.722G>A]/c.728G>A. The c.158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. When the c.158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

Background: Hyperphenylalaninemia (HPA) is defined as blood phenylalanine (Phe) levels exceeding the normal values (>120 μmol/L or >2 mg/dL) and is caused by a deficiency in the enzyme phenylalanine hydroxylase (PAH). The widespread screening of Phe levels in newborn screening programs has led to a very high number of patients with HPA. Methods: The samples were collected at various ages, not at the point of diagnosis. Nine pterin derivatives, including isoxanthopterin, sepiapterin, xanthopterin, primapterin, biopterin, neopterin, 7,8-dihydrobiopterin, 7,8-dihydroneopterin, and tetrahydrobiopterin (BH4), were analyzed in different HPA classes in serum, dried blood spots (DBS), and urine samples. A total of 18 patients, including six classical phenylketonuria (PKU), eight BH4-responsive PKU, and four mild HPA patients, were included in the study. Results: Among the nine pterin derivatives measured, a significant increase was observed in the levels of isoxanthopterin, biopterin, and 7,8-dihydrobiopterin in serum, dried blood spots (DBS), and urine samples of patients with HPA compared to the control group. However, elevations in isoxanthopterin, biopterin, and 7,8-dihydrobiopterin were observed in all HPA groups, although the extent of elevation varied among the different disease groups. There were also significant differences between HPA subgroups among these high values. Conclusion: In this study, it might be suggested that pterin profiling shows promising potential for its effective utilization in the differential diagnosis of HPA. Pterin profiling demonstrated its efficacy in accurately categorizing patients into distinct subtypes. This approach offers several notable advantages, including the ability to simultaneously screen multiple HPA subtypes through a single test, establish disease decision limits for pterins, shorten the time required for HPA differential diagnosis, reduce the risk of misdiagnosis, and increase overall diagnostic accuracy. This study is the most comprehensive study examining the association between HPA pterin in the literature. In our study, samples obtained from BH4-responsive PKU patients were on treatment. This may have affected the results. Preliminary findings on pterin profiles may need to be replicated in a prospective cohort of samples collected at the time of diagnosis to confirm the results.

We aimed to evaluate the diagnostic capabilities of Chinese laboratories for inherited metabolic disorders (IMDs) using gas chromatography-mass spectrometry (GC-MS) on urine samples. Meanwhile, based on the result of the pilot external quality assessment (EQA) scheme, we hope to establish a standardized and reliable procedure for future EQA practice. We recruited laboratories that participated in the EQA of quantitative analysis of urinary organic acids with GC-MS before joining the surveys. In each survey, a set of five real urine samples was distributed to each participant. The participants should analyze the sample by GC-MS and report the "analytical result", "the most likely diagnosis", and "recommendation for further tests" to the NCCL before the deadline. A total of 21 laboratories participated in the scheme. The pass rates were 94.4% in 2020 and 89.5% in 2021. For all eight IMDs tested, the analytical proficiency rates ranged from 84.7% - 100%, and the interpretational performance rate ranged from 88.2% - 97.0%. The performance on hyperphenylalaninemia (HPA), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), and ethylmalonic encephalopathy (EE) samples were not satisfactory. In general, the participants of this pilot EQA scheme are equipped with the basic capability for qualitative organic acid analysis and interpretation of the results. Limited by the small size of laboratories and samples involved, this activity could not fully reflect the state of clinical practice of Chinese laboratories. NCCL will improve the EQA scheme and implement more EQA activities in the future.