Targeted metabolomics unravels altered phenylalanine levels in piglets receiving total parenteral nutrition.

Parenteral nutrition, received by many patients with intestinal failure, can induce hepatobiliary complications, which is termed as parenteral nutrition-associated liver disease (PNALD). The spectrum of PNALD ranges from cholestasis and steatosis to fibrosis and cirrhosis. Although many factors contribute to the pathogenesis of PNALD, the underlying mechanisms remain unclear. In this study, we performed targeted metabolomics to characterize the metabolomic profile in neonatal piglets receiving total parenteral nutrition (TPN) or enteral nutrition (EN) for 1 or 2 weeks. Overall, the metabolomic signature of TPN groups differed from EN groups at both time points. Among the 20 acylcarnitines identified, a majority of them were significantly reduced in TPN groups. KEGG pathway analysis showed that phenylalanine metabolism-associated pathways were dysregulated accompanied by more progressive liver steatosis associated with TPN. Next, we evaluated phenylalanine catabolism and its association with fatty acid oxidation in piglets and rats with PNALD. We showed that the hepatic expression of phenylalanine-degrading enzyme phenylalanine hydroxylase (PAH) was reduced and systemic phenylalanine levels were increased in both animal models of PNALD. Moreover, carnitine palmitoyltransferase 1A, a central regulator of fatty acid oxidation, was downregulated and its expression was negatively correlated with phenylalanine levels in TPN-fed animals. To explore the effects of phenylalanine accumulation on lipid metabolism, we treated HepG2 cells with phenylalanine co-cultured with sodium palmitate or soybean oil emulsion to induce lipid accumulation. We found that phenylalanine treatment exacerbated lipid accumulation by inhibiting fatty acid oxidation without affecting fatty acid synthesis. In summary, our findings establish a pathogenic role of increased phenylalanine levels in driving liver steatosis, linking dysregulation of phenylalanine catabolism with lipid accumulation in the context of PNALD.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Long-term safety of sapropterin in paediatric and adult individuals with phenylalanine hydroxylase deficiency: Final results of the Kuvan® Adult Maternal Paediatric European Registry multinational observational study.

Phenylketonuria is a rare inherited disorder that disrupts the metabolism of phenylalanine (Phe) to tyrosine by phenylalanine hydroxylase (PAH). Sapropterin dihydrochloride (Kuvan®) is approved for use in Europe to reduce blood Phe levels and improve Phe tolerance in sapropterin-responsive individuals. KAMPER (NCT01016392) is an observational, multinational registry assessing long-term safety and efficacy of sapropterin. Five hundred and seventy-six participants with PAH deficiency were enrolled from nine European countries (69 sites; December 2009-May 2016). Participants were aged <4 years (n = 11), 4 to <12 years (n = 329), 12 to <18 years (n = 141), and ≥18 years (n = 95) at enrolment. Overall, 401 (69.6%) participants experienced a total of 1960 adverse events; 61 events in 42 participants were serious, and two were considered sapropterin-related by the investigator. Mean (standard deviation) actual dietary Phe intake increased from baseline across all age groups: 957 (799) mg/day to a maximum of 1959 (1121) mg/day over a total study period of 11 years. Most participants exhibited an increase in Phe tolerance while blood Phe levels remained in the target range for their age (120-360 µmol/L for <12 years; 120-600 µmol/L for ≥12 years). Most participants exhibited normal growth for height, weight, and body mass index. No additional safety concerns were identified. As an observational study, limitations include variability in routine care practices and inconsistent availability of data. Long-term sapropterin use demonstrates a favourable safety profile in real-world settings and increases Phe tolerance in participants with PAH deficiency while maintaining blood Phe levels in the target ranges.

[In vitro expression and functional analyses of the mutants p.R243Q, p.R241C and p.Y356X of the human phenylalanine hydroxylase]. / äººè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶çªå˜ä½“p.R243Q、p.R241C和p.Y356Xçš„ä½“å¤–è¡¨è¾¾åŠåŠŸèƒ½ç ”ç©¶.

OBJECTIVES: To study the in vitro expression of three phenylalanine hydroxylase (PAH) mutants (p.R243Q, p.R241C, and p.Y356X) and determine their pathogenicity. METHODS: Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein. Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells. Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants, and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay. RESULTS: Bioinformatics analysis predicted that all three mutants were pathogenic. The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control (P>0.05), while the mRNA expression level of the p.Y356X mutant significantly decreased (P<0.05). The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control (P<0.05). The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control (P<0.05), while there was no significant difference between the p.R243Q mutant and the wild type control (P>0.05). CONCLUSIONS: p.R243Q, p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells, with p.R241C and p.Y356X mutants also affecting the function of PAH protein. These three PAH mutants are to be pathogenic.

Mutational landscape of phenylketonuria in Iran.

To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: "phenylketonuria", "hyperphenylalaninemia", and "PKU" in combination with "Iran", "Iranian population", "mutation analysis", and "Molecular genetics". Among the literature-related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066-11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.

Phenylalanine hydroxylase deficiency treatment and management: A systematic evidence review of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: Elevated serum phenylalanine (Phe) levels due to biallelic pathogenic variants in phenylalanine hydroxylase (PAH) may cause neurodevelopmental disorders or birth defects from maternal phenylketonuria. New Phe reduction treatments have been approved in the last decade, but uncertainty on the optimal lifespan goal Phe levels for patients with PAH deficiency remains. METHODS: We searched Medline and Embase for evidence of treatment concerning PAH deficiency up to September 28, 2021. Risk of bias was evaluated based on study design. Random-effects meta-analyses were performed to compare IQ, gestational outcomes, and offspring outcomes based on Phe ≤ 360 µmol/L vs > 360 µmol/L and reported as odds ratio and 95% CI. Remaining results were narratively synthesized. RESULTS: A total of 350 studies were included. Risk of bias was moderate. Lower Phe was consistently associated with better outcomes. Achieving Phe ≤ 360 µmol/L before conception substantially lowered the risk of negative effect to offspring in pregnant individuals (odds ratio = 0.07, 95% CI = 0.04-0.14; P < .0001). Adverse events due to pharmacologic treatment were common, but medication reduced Phe levels, enabling dietary liberalization. CONCLUSIONS: Reduction of Phe levels to ≤360 µmol/L through diet or medication represents effective interventions to treat PAH deficiency.

Predictors of eventual requirement of phenylalanine-restricted diet in young infants with phenylalanine hydroxylase deficiency initially managed with sapropterin monotherapy.

BACKGROUND: Phenylalanine (Phe)-restricted diet is associated with lower quality of life for patients with phenylketonuria (PKU), and a concern for caregivers of recently-diagnosed infants. Sapropterin is an oral drug used as an alternative or adjunct to dietary treatment. We have observed that some of the young infants initially managed successfully with sapropterin monotherapy have required dietary treatment in long-term follow-up. We aimed to determine the baseline factors associated with future initiation of dietary treatment in these patients. METHODS: Data were obtained retrospectively from the medical records of 80 PKU patients started on sapropterin monotherapy before 3 months of age between 2011 and 2021. RESULTS: The patients were followed for a median of 3.9 years (Q1-Q3: 2.5-5.75 years). Sapropterin was tapered down and discontinued in 5 patients (6.3%) as their Phe levels remained below 360 µmol/L without treatment. Sapropterin monotherapy was sufficient in 62 patients (77.5%), while 13 (16.2%) required dietary treatment. Phe and tyrosine (Tyr) levels, and Phe:Tyr ratios differed significantly among the patients maintained on sapropterin monotherapy and those started on dietary treatment, but the Phe:Tyr ratio at diagnosis was the most important independent baseline variable (OR: 1.61, 95% CI: 1.15-2.27, p = 0.006), with Phe:Tyr ratio at diagnosis >5.25 associated with dietary treatment (sensitivity: 90.0%, specificity: 81.8%). Genotypic phenotype value (GPV), unavailable at baseline, was also associated with dietary treatment (median GPV 9.2 vs. 3.8, p = 0.006), but some genotypes were not specific to the final treatment modality. DISCUSSION: We propose that the Phe:Tyr ratio at diagnosis is an important indicator to predict dietary requirement in young infants initially managed with sapropterin monotherapy.

Genetic etiology and clinical challenges of phenylketonuria.

This review discusses the epidemiology, pathophysiology, genetic etiology, and management of phenylketonuria (PKU). PKU, an autosomal recessive disease, is an inborn error of phenylalanine (Phe) metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. The prevalence of PKU varies widely among ethnicities and geographic regions, affecting approximately 1 in 24,000 individuals worldwide. Deficiency in the PAH enzyme or, in rare cases, the cofactor tetrahydrobiopterin results in high blood Phe concentrations, causing brain dysfunction. Untreated PKU, also known as PAH deficiency, results in severe and irreversible intellectual disability, epilepsy, behavioral disorders, and clinical features such as acquired microcephaly, seizures, psychological signs, and generalized hypopigmentation of skin (including hair and eyes). Severe phenotypes are classic PKU, and less severe forms of PAH deficiency are moderate PKU, mild PKU, mild hyperphenylalaninaemia (HPA), or benign HPA. Early diagnosis and intervention must start shortly after birth to prevent major cognitive and neurological effects. Dietary treatment, including natural protein restriction and Phe-free supplements, must be used to maintain blood Phe concentrations of 120-360 µmol/L throughout the life span. Additional treatments include the casein glycomacropeptide (GMP), which contains very limited aromatic amino acids and may improve immunological function, and large neutral amino acid (LNAA) supplementation to prevent plasma Phe transport into the brain. The synthetic BH4 analog, sapropterin hydrochloride (i.e., Kuvan®, BioMarin), is another potential treatment that activates residual PAH, thus decreasing Phe concentrations in the blood of PKU patients. Moreover, daily subcutaneous injection of pegylated Phe ammonia-lyase (i.e., pegvaliase; PALYNZIQ®, BioMarin) has promised gene therapy in recent clinical trials, and mRNA approaches are also being studied.

Serotonin modulates insect gut bacterial community homeostasis.

BACKGROUND: Metazoan guts are in permanent contact with microbial communities. However, the host mechanisms that have developed to manage the dynamic changes of these microorganisms and maintain homeostasis remain largely unknown. RESULTS: Serotonin (5-hydroxytryptamine [5-HT]) was found to modulate gut microbiome homeostasis via regulation of a dual oxidase (Duox) gene expression in both Bactrocera dorsalis and Aedes aegypti. The knockdown of the peripheral 5-HT biosynthetic gene phenylalanine hydroxylase (TPH) increased the expression of Duox and the activity of reactive oxygen species, leading to a decrease in the gut microbiome load. Moreover, the TPH knockdown reduced the relative abundance of the bacterial genera Serratia and Providencia, including the opportunistic pathogens, S. marcescens and P. alcalifaciens in B. dorsalis. Treatment with 5-hydroxytryptophan, a precursor of 5-HT synthesis, fully rescued the TPH knockdown-induced phenotype. CONCLUSIONS: The findings reveal the important contribution of 5-HT in regulating gut homeostasis, providing new insights into gut-microbe interactions in metazoans.

Analysis of gene variation and long-term follow-up in children with phenylalanine hydroxylase deficiency diagnosed by newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥ç¡®è¯Šè‹¯ä¸™æ°¨é ¸ç¾ŸåŒ–é ¶ç¼ºä¹ç—‡æ‚£è€ åŸºå› å˜å¼‚åŠé•¿æœŸéšè®¿åˆ†æž.

OBJECTIVES: To retrospectively analyze the variation and characteristics of phenylalanine hydroxylase (PAH) gene, and to observe the long-term treatment effect and follow-up of newborns with PAH deficiency. METHODS: Clinical data, treatment and follow-up results of 198 patients with PAH deficiency diagnosed by newborn screening in Jinan from 1996 to 2021 were collected. The genetic analysis of 55 patients with PAH deficiency diagnosed by newborn screening in Jinan and 213 patients referred from the surrounding areas of Jinan were summarized. Gene variations were checked by a customized Panel gene detection method. Blood phenylalanine-concentration and physical development indicators including height and weight were regularly monitored. Intellectual development was assessed using a neuropsychological development scale for patients aged 0-6 years and academic performance, and brain injury in patients was assessed using brain magnetic resonance imaging. RESULTS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. The genotype of c.158G>A variation is compound heterozygous variation, with mainly a mild hyperpheny-lalaninemia. 168 patients with PAH deficiency who were followed-up regularly had normal physical development without dwarfism or malnutrition. Among the 33 preschool patients who underwent mental development assessment, 2 were mentally retarded and the initial treatment age was older than 6 months. Nine patients with an average age of (17.13±2.42) years completed brain magnetic resonance imaging, one case was normal, and 8 cases were abnormal. There were patchy or patchy hyperintense foci near the bilateral lateral ventricles on T2WI, and the intellectual development was normal. Compared with the other eight patients, the blood phenylalanine concentration of the normal child was better and stably controlled within the ideal range. CONCLUSIONS: c.728G>A, c.158G>A, c.721C>T, c.1068C>A, c.611A>G variations were common in PAH gene. After standardized treatment, most patients with PAH deficiency diagnosed by screening can obtain normal growth and intellectual development in adolescence, but there are different degrees of organic lesions in the cerebral white matter.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

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-Pahenu2/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 Pahenu2/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.

Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

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

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

Characterization of phenylalanine hydroxylase gene variants and analysis of genotype-phenotype correlation in patients with phenylalanine hydroxylase deficiency from Fujian Province, Southeastern China.

BACKGROUND: Phenylalanine hydroxylase deficiency (PAHD) is the most prevalent inherited disorder of amino acid metabolism in China. Its complex phenotype includes many variants and genotypes among different populations. METHODS AND RESULTS: In this study, we analyzed the phenylalanine hydroxylase gene (PAH) variants in a cohort of 93 PAHD patients from Fujian Province. We also assessed genotype and phenotype correlation in patients with PAHD. A total of 44 different pathogenic variants were identified, including five novel variants. The three most prevalent variants among all patents were c.158G > A, p.(Arg53His) (18.03%), c.721C > T, p.(Arg241Cys) (14.75%), and c.728G > A, p.(Arg243Gln) (7.65%). The frequency of the c.158G > A, p.(Arg53His) variant was highest in patients with mild hyperphenylalaninemia, whereas the frequency of the c.1197A > T, p.(Val399 =) and c.331C > T, p.(Arg111Ter) variants was highest in patients with classic phenylketonuria. The most abundant genotypes observed in PAHD patients were c.[158G > A];[728G > A], c.[158G > A];[442-1G > A], and c.[158G > A];[721C > T]. Comparing allelic phenotype to genotypic phenotype values yielded fairly accurate predictions of phenotype, with an overall consistency rate was 85.71% for PAHD patients. CONCLUSIONS: Our study identified a PAH variant spectrum in PAHD patients from Fujian Province, Southeastern China. Quantitative correlation analysis between genotype and phenotype severity is helpful for genetic counseling and management.

Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin.

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4 BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4 Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina.

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.

Repetitive transcranial magnetic stimulation in the treatment of resistant depression: changes of specific neurotransmitter precursor amino acids.

Repetitive transcranial magnetic stimulation (rTMS) for treatment-resistant major depression offers an alternative therapy, since more than every third patient is not responding to adequate antidepressive treatment. In this interventional study safety, symptom development and changes of serum concentrations of neurotransmitter precursor amino acids, of immune activation and inflammation markers, of brain-derived neurotrophic factor (BDNF), nitrite as well as of salivary amylase were measured before and after a frontal polar cortex stimulation using rTMS as add-on treatment in 38 patients with treatment-resistant depression. Out of these, 17 patients received sham stimulation as a control. Treatment was well tolerated: with the exception of one patient of the verum group, who described discomfort during the second treatment, no serious adverse effects were observed. Improvement of depression with a significant decrease in the HAMD-7 scale (p = 0.001) was found in patients treated with rTMS, but not in sham-treated patients. Furthermore, serum phenylalanine and tyrosine dropped significantly (p = 0.03 and p = 0.027, respectively) in rTMS-treated patients. The kynurenine to tryptophan ratio (Kyn/Trp) tended to decrease under rTMS (p = 0.07). In addition, associations between concentrations of BDNF and neopterin as well as serum nitrite levels were found in patients after rTMS treatment, which indicates an influence of immune regulatory circuits on BDNF levels. In the sham-treated patients, no changes of biomarker concentrations were observed. Results show that rTMS is effective in the treatment of resistant depression. rTMS appears to influence the enzyme phenylalanine hydroxylase, which plays a central role in the biosynthesis of neurotransmitter precursors tyrosine and dihydroxyphenylalanine (DOPA).

Molecular characterization of Thai patients with phenylalanine hydroxylase deficiency and in vitro functional study of two novel PAH variants.

Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This study aimed to assess the specific heterogeneity of PAH variants found in Thai population as well as evaluate enzyme activity and expression of novel variants. PAH gene from 13 patients was analyzed by PCR amplification and direct Sanger-sequencing of 13 exons of the coding region. The novel variants were transiently transfected in COS-7 cells for functional verification. Eleven different PAH variants were identified: all pathogenic variants were missense variants, of which the most frequent variant was p.R169L, accounting for 24% (6/25) of all identified alleles. Two novel variants p.R169L and p.Y317N and previously reported variants with mutated residues at the same positions (p.R169H and p.Y317H) were expressed in COS-7 cells. These showed mildly impaired residual activity levels (42.3-63.1% of wild type), while the protein levels were well expressed (82.8-110%), except for p.R169L, which showed decreased protein expression of 55.7% compared to the wild type enzyme. All subjects with p.R169L identified in at least one of pathogenic alleles (one case is homozygous) had a metabolic phenotype of mild hyperphenylalaninemia (HPA). Our data has expanded the information on the genetic heterogeneity of Thai patients with PAH deficiency. This finding emphasizes the importance of genotyping in patients with HPA, and in vitro studies can provide additional information for prediction of phenotype.

Spectrum of PAH gene mutations in 1547 phenylketonuria patients from Iran: a comprehensive systematic review.

As one of the highest prevalence rates in the world, the prevalence of Phenylketonuria (PKU) in Iran has been estimated at 16.5 per 100,000 neonates. The objective of this study was to evaluate the spectrum and frequency of mutations of the phenylalanine hydroxylase (PAH) gene in Iranian PKU patients. A systematic review was carried out on previous studies on PAH gene mutations in Iranian PKU patients. A complete search was carried out on the on-line databases of Scopus, Web of Science, PubMed/Medline, ProQuest, Science Direct, Magiran, SID and the search engine Google Scholar. The keywords of Phenylketonuria, PKU, Phenylalanine Hydroxylase, PAH, and Iran, as well as their Persian equivalents, in all possible combinations were used. Finally, a total of 21 eligible articles with a sample size of 1547 Iranian PKU patients, published between 2003 and 2020, were included in our systematic review. A total of 129 different PAH gene mutations including, IVS10-11G > A (c.1066-11G > A) (19.23%), p.R261Q (c.782G > A) (7.63%), p.P281L (c.842C > T) (6.24%), IVS2 + 5G > C (c.168 + 5G > C) (5.75%), p.R243* (c.727C > T) (3.59%), IVS9 + 5G > A (c.969 + 5G > A) (2.84%), p.R176* (c.526C > T) (2.42%), p.Lys363Nfs*37 (c.1089delG) (2.13%), IVS11 + 1G > C (c.1199 + 1G > C) (2.07%) and p.L48S (c.143 T > C) (2.04%) were identified. The spectrum and frequency of mutations observed in Iran were closer to those observed in the Mediterranean countries. Our results are valuable in planning panel-based studies in provinces with incomplete data on PAH gene mutations. This study is a good reference for genetic counselors and physicians who advise couples in making decisions to maintain or terminate a pregnancy.

DNACJ12 deficiency in patients with unexplained hyperphenylalaninemia: two new patients and a novel variant.

In addition to tetrahydrobiopterin deficiencies and phenylalanine hydroxylase deficiency (phenylketonuria) due to PAH variants, the deficiency of the co-chaperone protein DNAJC12 was identified in 2017 as a novel cause of inherited hyperphenylalaninemia, revealing the genetic etiology in previously unresolved cases. In this study, we aimed to investigate DNAJC12 deficiency in non-tetrahydrobiopterin-deficient persistent hyperphenylalaninemia cases without biallelic PAH variants in a single pediatric metabolic center. It was determined retrospectively that 471 patients with non-tetrahydrobiopterin deficiency-hyperphenylalaninemia had undergone PAH gene sequencing and 451 patients had biallelic variants in PAH. DNAJC12 sequencing was performed in the remaining 20 patients, identifying a previously reported homozygous splice-site variant (c.158-2A > T) in one patient with axial hypotonia and developmental delay, and a novel, homozygous c.404del (p.Arg135Lysfs*21) frameshift variant in an asymptomatic patient. In segregation analysis, the asymptomatic patient's both parents were also found to be homozygous for this variant and hyperphenylalaninemic. The parents may have had academic difficulties but intellectual disability could not be confirmed due to lack of cooperation. The symptomatic patient significantly benefited from treatment with sapropterin dihydrochloride and neurotransmitter precursors. DNAJC12 deficiency might be responsible for approximately 10% or more of cases with unexplained hyperphenylalaninemia. The phenotypic spectrum is broad, ranging from early infantile hypotonia to incidental diagnosis in adulthood. Similar to tetrahydrobiopterin deficiencies, early diagnosis and treatment with sapropterin dihydrochloride and neurotransmitter precursors can be beneficial, supporting the analysis of DNACJ12 gene in patients with unexplained hyperphenylalaninemia.