Newborn screening and genetic features of patients with hyperphenylalaninemia in a southern Chinese population.

BACKGROUND AND AIMS: 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. MATERIALS AND METHODS: We analyzed the HPA screening results for 580,460 newborns from 2014 to 2021. RESULTS: Of the 296 newborns who tested HPA positive, 56 were diagnosed with HPA, including 47 with phenylalanine hydroxylase deficiency and nine with tetrahydrobiopterin deficiency (BH4D). 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 %). CONCLUSION: Newborn screening is an effective method for early detection of HPA, but differential diagnosis of BH4D is necessary.

[Analysis of GCH1 gene variant in a consanguineous Chinese pedigree affected with tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis for a child featuring tetrahydrobiopterin deficiency and global developmental delay. METHODS: Clinical and laboratory examinations were carried out for the child. Genomic DNA of the patient was subjected to high-throughput sequencing to identify genetic variants associated with hyperphenylalaninemia. Candidate variants were verified by Sanger sequencing. RESULTS: The result of blood tandem mass spectrometry showed that the Phenylalanine in the blood was 642.7 µmol/l, and the ratio of Phenylalanine/Tyrosine was 5.42. Analysis of urinary pterin: neopterin 0.09 mmol/mol Cr, biopterin 0.04 mmol/mol Cr, biopterin% 77%, which suggested tetrahydrobiopterin deficiency. The parents of the proband were first cousins. DNA sequencing revealed that the proband has harbored homozygous c.353A>T variants in exon 2 of the GCH1 gene, for which his great grandmother, grandfather, mother, uncle, father and elder brother were heterozygous carriers with normal phenotype and no clinical symptoms associated with dopa responsive dystonia. CONCLUSION: The homozygous c.353A>T variant of the GCH1 gene probably underlay the tetrahydrobiopterin deficiency in this pedigree of consanguineous marriage.

Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth.

[Figure: see text].

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2-•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

The Genetic Landscape and Epidemiology of Phenylketonuria.

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066-11G>A (IVS10-11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G>A];[1066-11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.

Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli.

Tetrahydrobiopterin-dependent hydroxylation of aromatic amino acids is the first step in the biosynthesis of many neuroactive compounds in humans. A fundamental challenge in building these pathways in Escherichia coli is the provision of the non-native hydroxylase cofactor, tetrahydrobiopterin. To solve this, we designed a genetic selection that relies on the tyrosine synthesis activity of phenylalanine hydroxylase. Using adaptive laboratory evolution, we demonstrate the use of this selection to discover: (1) a minimum set of heterologous enzymes and a host folE (T198I) mutation for achieving this type of hydroxylation chemistry in whole cells, (2) functional complementation of tetrahydrobiopterin by indigenous cofactors, and (3) a tryptophan hydroxylase mutation for improving protein abundance. Thus, the goal of having functional aromatic-amino-acid hydroxylation in E. coli was achieved through directed metabolic pathway evolution.

Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase.

The molecular genetics of well-characterized inherited diseases, such as phenylketonuria (PKU) and hyperphenylalaninemia (HPA) predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene, is often complicated by the identification of many novel variants, often with no obvious impact on the associated disorder. To date, more than 1100 PAH variants have been identified of which a substantial portion have unknown clinical significance. In this work, we study the functionality of seven yet uncharacterized PAH missense variants p.Asn167Tyr, p.Thr200Asn, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, p.Ala342Pro, and p.Ile406Met first identified in the Czech PKU/HPA patients. From all tested variants, three of them, namely p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met, exerted residual enzymatic activity in vitro similar to wild type (WT) PAH, however, when expressed in HepG2 cells, their protein level reached a maximum of 72.1% ± 4.9%, 11.2% ± 4.2%, and 36.6% ± 7.3% compared to WT PAH, respectively. Remaining variants were null with no enzyme activity and decreased protein levels in HepG2 cells. The chaperone-like effect of applied BH4 precursor increased protein level significantly for p.Asn167Tyr, p.Asp229Gly, p.Ala342Pro, and p.Ile406Met. Taken together, our results of functional characterization in combination with in silico prediction suggest that while p.Asn167Tyr, p.Thr200Asn, and p.Ile406Met PAH variants have a mild impact on the protein, p.Asp229Gly, p.Gly239Ala, p.Phe263Ser, and p.Ala342Pro severely affect protein structure and function.

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.

Gene mutation and pedigree analysis of tetrahydrobiopterin deficiency in a Uygur family of China.

BACKGROUND: Tetrahydrobiopterin (BH4 ) deficiency is an autosomal recessive disorder, which is caused by an enzyme deficiency involved in its synthetic or metabolic pathways. Clinical symptoms may include microcephaly, hypoevolutism, severe ataxia, and seizures. The purposes of this study are to analyze the genotype-phenotype and the pedigree of the first case of BH4 deficiency in the Uygur of China. METHODS: (a) This patient received tandem mass spectrometry, urinary neopterin and biopterin analysis, and determination of dihydropteridine reductase (DHPR) activity in dried blood spots. (b) Blood DNA samples of this patient and her three family members were collected for gene sequencing and mutation analysis. RESULTS: (a) The basic urinary neopterin and biopterin were 1.07 mmol/mol Cr and 3.12 mmol/mol Cr, respectively, and biopterin percentage was 74.42%. The DHPR activity of this patient was 31.11% of normal control. (b) Sanger sequencing of PAH gene in this patient was negative but positive of her sister, which carries 2 heterozygous mutation c.781C>T and c.1238G>C. Next-generation sequencing on the patient identified a homozygous mutation in the quinoid dihydropteridine reductase (QDPR) gene at c.508G>A, which was confirmed by Sanger sequencing. CONCLUSION: (a) The patient was the first case of clinical diagnosis of BH4 deficiency in the Uighur. And there are two types of hyperphenylalaninemia (HPA) in the same family. (b) The mild HPA patient with severe nervous system damage should pay more attention to the BH4 deficiency. (c) Using next-generation sequencing technology can increase the mutation detection rate when the hereditary diseases are highly suspected in clinic.

Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

[Research progress on phenotype and genotype of hyperphenylalaninemia].

Hyperphenylalaninemia(HPA), an autosomal recessive disease, is the most common inborn error of amino acid metabolism, caused by the deficiency of phenylalanine hydroxylase(PAH) or tetrahydrobiopterin(BH4) which induced by mutations of genes. The accumulation of the clinical database and genetic information will enhance the development of novel personalized medicine and to provide more accurate and timely diagnostic and therapeutic approaches for HPA. This paper summarizes the correlations between HPA metabolism and PAH, BH4, pathogenic genes and their distributions in HPA, as well as the phenotypes and genotypes of HPA, so as to provide reference for personalized medicine for HPA.

An Overview of Traditional and Novel Therapeutic Options for the Management of Phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by the deficiency of phenylalanine hydroxylase enzyme that catalyzes the conversion of L-phenylalanine to L-tyrosine using tetrahydrobiopterin (BH4) as a cofactor. Among aminoacidopathies, PKU is one of the most prevalent disorders in different populations. It may be caused by deficiency of BH4 or mutations in PAH. About 98% of PKU patients have mutations in the PAH, while the remaining have BH4 deficiency. If PKU is diagnosed earlier in life using advance analytical techniques (e.g., high performance liquid chromatography, mass spectrometry, and polymerase chain reaction), then it is potentially treatable by special diets (L-phenylalanine-free medical formula). However, some complications such as vitamin B12 deficiency, cardiovascular problems, and neurodevelopmental problems have been reported in PKU patients when they ate special diets for a long period. Hence, special diet alone is not a good option for proper treatment. Next generation therapies require structure-function based development. For therapies which target PAH gene (e.g., gene therapy, RNAi, gene editing), a lot of research has yet to be done. Treatment with BH4 therapy is safe and effective but only in BH4-responsive PKU patients. Therefore, research efforts should be focused on the development of more targeted pharmacological and genetic therapies especially PAH gene therapy, which can reduce the burden or deleterious effects of this disease in affected patients.

Metabolic pathway engineering for high-level production of 5-hydroxytryptophan in Escherichia coli.

Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of Griffonia Smplicifolia. This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coli. Firstly, human tryptophan hydroxylase I (TPH1) gene was functionally expressed. For endogenous supply of the cofactor tetrahydrobiopterin (BH4), human BH4 biosynthesis and regeneration pathway was reconstituted. Whole-cell bioconversion resulted in high-level production of 5-HTP (~1.2 g/L) from 2 g/L L-tryptophan in shake flasks. Further metabolic engineering efforts were employed to achieve 5-HTP biosynthesis from simple carbon sources. The whole biosynthetic pathway was divided into three functional modules, L-tryptophan module, the hydroxylation module, and the BH4 module. By reducing the copy number of L-tryptophan module, replacing TPH1 with a more stable mutant form, and promoter regulation of the BH4 module, 5-HTP was produced at a final titer of 1.3 g/L in the shake flask and 5.1 g/L in a fed-batch fermenter with glycerol as the carbon source, both of which were the highest ever reported for microbial production of 5-HTP.

Secondary BH4 deficiency links protein homeostasis to regulation of phenylalanine metabolism.

Metabolic control of phenylalanine concentrations in body fluids is essential for cognitive development and executive function. The hepatic phenylalanine hydroxylating system is regulated by the ratio of l-phenylalanine, which is substrate of phenylalanine hydroxylase (PAH), to the PAH cofactor tetrahydrobiopterin (BH4). Physiologically, phenylalanine availability is governed by nutrient intake, whereas liver BH4 is kept at constant level. In phenylketonuria, PAH deficiency leads to elevated blood phenylalanine and is often caused by PAH protein misfolding with loss of function. Here, we report secondary hepatic BH4 deficiency in Pah-deficient mice. Alterations in de novo synthesis and turnover of BH4 were ruled out as molecular causes. We demonstrate that kinetically instable and aggregation-prone variant Pah proteins trap BH4, shifting the pool of free BH4 towards bound BH4. Interference of PAH protein misfolding with metabolite-based control of l-phenylalanine turnover suggests a mechanistic link between perturbation of protein homeostasis and disturbed regulation of metabolic pathways.

Tetrahydrobiopterin in antenatal brain hypoxia-ischemia-induced motor impairments and cerebral palsy.

Antenatal brain hypoxia-ischemia, which occurs in cerebral palsy, is considered a significant cause of motor impairments in children. The mechanisms by which antenatal hypoxia-ischemia causes brain injury and motor deficits still need to be elucidated. Tetrahydrobiopterin is an important enzyme cofactor that is necessary to produce neurotransmitters and to maintain the redox status of the brain. A genetic deficiency of this cofactor from mutations of biosynthetic or recycling enzymes is a well-recognized factor in the development of childhood neurological disorders characterized by motor impairments, developmental delay, and encephalopathy. Experimental hypoxia-ischemia causes a decline in the availability of tetrahydrobiopterin in the immature brain. This decline coincides with the loss of brain function, suggesting this occurrence contributes to neuronal dysfunction and motor impairments. One possible mechanism linking tetrahydrobiopterin deficiency, hypoxia-ischemia, and neuronal injury is oxidative injury. Evidence of the central role of the developmental biology of tetrahydrobiopterin in response to hypoxic ischemic brain injury, especially the development of motor deficits, is discussed.

Organic anion transporters, OAT1 and OAT3, are crucial biopterin transporters involved in bodily distribution of tetrahydrobiopterin and exclusion of its excess.

Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

A fat-derived metabolite regulates a peptidergic feeding circuit in Drosophila.

Here, we show that the enzymatic cofactor tetrahydrobiopterin (BH4) inhibits feeding in Drosophila. BH4 biosynthesis requires the sequential action of the conserved enzymes Punch, Purple, and Sepiapterin Reductase (Sptr). Although we observe increased feeding upon loss of Punch and Purple in the adult fat body, loss of Sptr must occur in the brain. We found Sptr expression is required in four adult neurons that express neuropeptide F (NPF), the fly homologue of the vertebrate appetite regulator neuropeptide Y (NPY). As expected, feeding flies BH4 rescues the loss of Punch and Purple in the fat body and the loss of Sptr in NPF neurons. Mechanistically, we found BH4 deficiency reduces NPF staining, likely by promoting its release, while excess BH4 increases NPF accumulation without altering its expression. We thus show that, because of its physically distributed biosynthesis, BH4 acts as a fat-derived signal that induces satiety by inhibiting the activity of the NPF neurons.

Tetrahydrobiopterin deficiency in the pathogenesis of Fabry disease.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.