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.
OBJECTIVE: 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.

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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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.

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 (BH4), 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.

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.

OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.

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.

OBJECTIVE: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022.
METHODS: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 μIU/mL was considered positive for CH, while Phe>120 μmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests.
RESULTS: The overall neonatal screening rate during 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21 176. Ninety-seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation.
CONCLUSIONS: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.
目的: 了解浙江省新生儿先天性甲状腺功能减退症（CH）及高苯丙氨酸血症（HPA）的筛查及发病情况。方法: 收集1999年9月至2022年12月浙江省新生儿疾病筛查中心11 922 318名新生儿血促甲状腺素（TSH）及苯丙氨酸（Phe）检测的资料。其中，血TSH浓度采用荧光法检测，血Phe浓度采用荧光法或串联质谱法检测。TSH在9 μIU/mL及以上的新生儿为CH筛查阳性，血Phe浓度超过120 μmol/L和（或）血Phe与酪氨酸（Tyr）比值大于2.0为HPA筛查阳性，召回复查。采用高通量测序和MassARRAY技术检测基因变异。结果: 1999—2022年共筛查新生儿11 922 318名，总筛查率为89.41%，筛查率由1999年6.46%提升至2022年的100.00%。检出CH患儿8924例，患病率为1/1336，检出HPA患儿563例，患病率为1/21 176，其中508例为经典型苯丙酮尿症（cPKU），55例为四氢生物蝶呤缺乏症（BH4D）。97例CH患儿完善基因检测，检出9种CH相关基因，双氧化酶2（DUOX2）基因检出频率最高（69.0%），其中c.3329G>A（p.R1110Q）（18.2%）和c.1588A>T（p.K530X）（17.3%）为热点突变。250例cPKU患者中检出81种苯丙氨酸羟化酶（PAH）基因变异，其中c728G>A（p.R243Q）（24.4%）和c.721C>T（p.R241C）（15.0%）为热点突变，7种为新发变异：c.107C>A（p.S36*）、c.137G>T（p.G46V）、c.148A>G（p.K50E）、c.285C>T（p.I95I）、c.843-10delTTCC、exon4-7del和c.1066-2A>G。36例BH4D患者中检出12种6-丙酮酰四氢蝶呤合成酶（PTS）基因变异，热点突变为c.259C>T（p.P87S）（31.9%）。结论: 浙江省新生儿筛查率逐年升高，CH发病率明显增高，HPA发病率与全国平均水平接近。DUOX2基因变异为CH患儿最常见基因变异，c728G>A（p.R243Q）为cPKU患者的热点突变，c.259C>T（p.P87S）为BH4D患者的热点突变。.