Tetrahydrobiopterin Deficiency Research Articles

Genetic analysis of eighteen patients from Gansu Province with Tetrahydrobiopterin deficiency

To explore the genetic basis of eighteen patients with Tetrahydrobiopterin deficiency (BH4D) from Gansu Province. Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing. All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c.259C>T (34.38%) and c.286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.259C>T was classified as a pathogenic variant, whilst the c.286G>A, c.166G>A, c.200C>T, c.272A>G, c.402A>C, c.421G>T, c.84_291A>G and c.317C>T were classified as likely pathogenic variants. A novel c.289_290insCTT variant was classified as likely pathogenic (PM1+PM2_Supporting+PM3+PP3+PP4). The two variants (c.478C>T and c.665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+PM2_Supporting+PP3+PP4). Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

The value of simultaneous determination of blood large neutral amino acids and tetrahydrobiopterin metabolites in the diagnosis of atypical hyperphenylalaninemia

Tetrahydrobiopterin deficiency in newborns with atypical hyperphenylalaninemia requires rapid and accurate diagnosis and the ability to distinguish it from the classical type to prevent early irreversible neurological damage. The study aimed to evaluate neopterin and biopterin (products of tetrahydrobiopterin recycling pathway) and amino acid profiles (used in supplementation therapy) in patients with hyperphenylalaninemia after optimizing ultra-performance liquid chromatography coupled with tandem mass spectrometry to simultaneously measure neopterin, biopterin, and amino acids in dried blood spots. The study enrolled preselected infants with classic (n = 46), atypical (n = 14) hyperphenylalaninemia, and a control group (n = 50).Result Tandem mass spectrometry detected neo/biopterin in the blood with a sensitivity and specificity of 100%. The mean neo/biopterin levels were significantly lower in the atypical cases (4 ± 1 and 3 ± 1 nmol/L) than the classic (49 ± 13 and 50 ± 12 nmol/L) and control (15.2 and 15.3 nmol/L) groups and correlated with phenylalanine and phenylalanine to tyrosine ratio (all P < 0.05). The study compared classic and atypical hyperphenylalaninemia cases with the control group. Both classic and atypical cases exhibited decreased levels of arginine, valine, and leucine compared to controls. Classic cases showed increased levels of citrulline, ornithine, and methionine, while atypical cases showed increased citrulline levels only. Comparing atypical versus classic cases, atypical cases exhibited decreased levels of citrulline, ornithine, methionine, arginine, leucine, and valine (all P < 0.05). Correlation analysis revealed negative associations between ornithine and biopterin and between arginine and neopterin in classic PKU cases. These findings highlight distinct metabolic differences between classic and atypical PKU.Conclusion The optimized method detected neo/biopterin in the blood with accuracy and precision. The characteristic pattern of neo/biopterin in the blood makes it possible to differentiate between classic and atypical hyperphenylalaninemia with a sensitivity and specificity of 100%. The amino acid profile could add value when treatment with large neutral amino acids is considered.

Inherited Pediatric Neurotransmitter Disorders: Case Studies and Long-Term Outcomes

AbstractPrimary pediatric neurotransmitter disorders include genetic defects of neurotransmitter metabolism that may mimic common neurological conditions in children. Our objective was to evaluate the clinical experience and outcomes of affected patients. Five patients with primary neurotransmitter defects were identified in the neurometabolic database between 2004 and 2022. Two patients with 6-pyruvoyltetrahydropterin synthase deficiency and one with pyridoxine-dependent epilepsy (PDE) presented in the neonatal period. One patient with succinic semialdehyde dehydrogenase (SSADH) deficiency and one with aromatic l-amino acid decarboxylase (AADC) deficiency presented in later life. A diagnosis of cerebral palsy was revised following biochemical confirmation of SSADH deficiency. AADC deficiency was confirmed via exome sequencing and reduced activity on enzyme assay. Late diagnosis in the latter two cases was likely due to a low index of suspicion and lack of access to diagnostic tests in the country of origin. In two children with tetrahydrobiopterin deficiency, newborn screening results and atypical clinical features prompted investigations. An early diagnosis of PDE was established based on presenting features, a high index of suspicion, the presence of an identifiable biochemical marker and molecular genetic testing. Pediatric neurotransmitter disorders can be diagnosed based on a high clinical index of suspicion, availability of biochemical markers, and molecular genetic testing. These disorders, though rare, need to be included in the differential diagnosis of common neurological presentations in children as they may be potentially treatable. Outcomes and influencing factors in the present series are discussed in comparison to published data.

Disease spectrum and pathogenic genes of inherited metabolic disorder in Gansu Province of China

To investigate the disease spectrum and pathogenic genes of inherited metabolic disorder (IMD) among neonates in Gansu Province of China. A retrospective analysis was conducted on the tandem mass spectrometry data of 286 682 neonates who received IMD screening in Gansu Provincial Maternal and Child Health Hospital from January 2018 to December 2021. A genetic analysis was conducted on the neonates with positive results in tandem mass spectrometry during primary screening and reexamination. A total of 23 types of IMD caused by 28 pathogenic genes were found in the 286 682 neonates, and the overall prevalence rate of IMD was 0.63 (1/1 593), among which phenylketonuria showed the highest prevalence rate of 0.32 (1/3 083), followed by methylmalonic acidemia (0.11, 1/8 959) and tetrahydrobiopterin deficiency (0.06, 1/15 927). In this study, 166 variants were identified in the 28 pathogenic genes, with 13 novel variants found in 9 genes. According to American College of Medical Genetics and Genomics guidelines, 5 novel variants were classified as pathogenic variants, 7 were classified as likely pathogenic variants, and 1 was classified as the variant of uncertain significance. This study enriches the database of pathogenic gene variants for IMD and provides basic data for establishing an accurate screening and diagnosis system for IMD in this region.

Results of neonatal screening for congenital hypothyroidism and hyperphenylalaninemia in Zhejiang province from 1999 to 2022.

To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022. 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. 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. 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.

Dihydropteridine Reductase Deficiency - A Rare and Potentially Treatable Cause Mimicking Cerebral Palsy.

Dihydropteridine reductase deficiency (DHPRD) is a rare genetic disorder of tetrahydrobiopterin (BH4) regeneration, a cofactor for several enzymes, including phenylalanine hydroxylase. Due to hyperphenylalaninemia (HPA), patients can be detected by the newborn metabolic screening, when available. The most common symptoms of DHPRD may mimic cerebral palsy: developmental/cognitive impairment, hypotonia, peripheral hypertonia, dystonia, feeding difficulties, epilepsy, and microcephaly. The long-term neurodevelopmental outcome is strongly influenced by the early initiation of effective treatment. A 2-year-old boy, born in Guinea, was evaluated in our center with the diagnosis of "cerebral palsy". He was born after a prolonged labor, and had feeding difficulties and severe developmental delay. Examination revealed microcephaly, axial hypotonia, and dyskinetic movements without hypertension. No seizures or oculogyric crisis were reported. Brain MRI showed slight brain atrophy and hyperintensity T2/FLAIR in basal ganglia. The diagnosis of cerebral palsy was questioned, and further investigation was carried out. HPA raised the possibility of BH4 deficiency, supported by increased biopterin in urine, decreased neurotransmitters in CSF, and low DHPR enzyme activity. A variant (128_130del (p.(Val43del)) in apparent homozygosity was later detected in the QPDR gene. At 4 years old, he started L-dopa/carbidopa, oxitriptan, and a phenylalanine-restrictive diet with moderate clinical improvement. When the diagnosis of "cerebral palsy" is questionable, other etiologies should be investigated, particularly disorders that have specific disease-modifying treatment. In our patient, the atypical constellation of neurological signs, brain MRI findings, and the nonexistence of newborn metabolic screening in the country of origin supported additional investigation. The presence of HPA-associated dystonia was crucial to the investigation and was later confirmed as DHPRD. Unfortunately, at this stage, the reversibility of the neurological damage in response to treatment is doubtful.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

Biochemical and molecular features of tetrahydrobiopterin deficiency in Fujian Province, southeastern China.

The estimated prevalence of tetrahydrobiopterin deficiency (BH4D) and the mutational spectrum of the causal 6-pyruvoyl-tetrahydropterin synthase (PTS) gene vary widely according to race and region. This study assessed the prevalence and genetic characteristics of BH4D in Fujian Province, southeastern China. A total of 3,204,067 newborns were screened between 2012 and 2022 based on the phenylalanine level and the phenylalanine/tyrosine ratio in dried blood spots. Differential diagnosis was determined by the urine purine spectrum, dihydropteridine reductase activity in red blood cells, and genetic testing. The PTS mutation spectrum and genotypes were determined by next-generation sequencing. A total of 189 newborns were diagnosed with hyperphenylalaninemia (HPA) over the study period, including 159 with phenylalanine hydroxylase deficiency and 30 with BH4D. Therefore, the prevalence of BH4D in Fujian was 9.36 per 1,000,000 live births (30/3,204,067) and the proportion of BH4D among patients with HPA was 15.87% (30/189). A total of 58 PTS alleles were identified in the 29 patients with PTS deficiency (PTPSD), and those alleles were composed of 10 different variants, including eight missense variants and two splice-site variants. The most prevalent variants were c.155A>G, p.Asn52Ser (44.83%); c.259C>T, p.Pro87Ser (39.66%); and c.84-291A>G, p.Tyr27Argfs*8 (3.45%). The predominant genotype was c [155A>G]; [259C>T] (11/29, 37.93%). The prevalence of BH4D and the spectrum of associated PTS mutations were successfully determined for the first time in Fujian Province, southeastern China. Since the mutation spectrum of PTS is region-specific, such data will facilitate molecular diagnosis and genetic counseling in PTPSD cases.

Levodopa-refractory hyperprolactinemia and pituitary findings in inherited disorders of biogenic amine metabolism.

Elevated serum prolactin concentrations occur in inherited disorders of biogenic amine metabolism because dopamine deficiency leads to insufficient inhibition of prolactin secretion. This work from the International Working Group on Neurotransmitter Related Disorders (iNTD) presents the results of the first standardized study on levodopa-refractory hyperprolactinemia (LRHP; >1000 mU/L) and pituitary magnetic resonance imaging (MRI) abnormalities in patients with inherited disorders of biogenic amine metabolism. Twenty-six individuals had LRHP or abnormal pituitary findings on MRI. Tetrahydrobiopterin deficiencies were the most common diagnoses (n = 22). The median age at diagnosis of LRHP was 16 years (range: 2.5-30, 1st-3rd quartiles: 12.25-17 years). Twelve individuals (nine females) had symptoms attributed to hyperprolactinemia: menstruation-related abnormalities (n = 7), pubertal delay or arrest (n = 5), galactorrhea (n = 3), and decreased sexual functions (n = 2). MRI of the pituitary gland was obtained in 21 individuals; six had heterogeneity/hyperplasia of the gland, five had adenoma, and 10 had normal findings. Eleven individuals were treated with the dopamine agonist cabergoline, ameliorating the hyperprolactinemia-related symptoms in all those assessed. Routine monitoring of these symptoms together with prolactin concentrations, especially after the first decade of life, should be taken into consideration during follow-up evaluations. The potential of slow-release levodopa formulations and low-dose dopamine agonists as part of first-line therapy in the prevention and treatment of hyperprolactinemia should be investigated further in animal studies and human trials. This work adds hyperprolactinemia-related findings to the current knowledge of the phenotypic spectrum of inherited disorders of biogenic amine metabolism.

Cardiomyocyte tetrahydrobiopterin synthesis regulates fatty acid metabolism and susceptibility to ischaemia-reperfusion injury.

What is the central question of this study? What are the physiological roles of cardiomyocyte-derived tetrahydrobiopterin (BH4) in cardiac metabolism and stress response? What is the main finding and its importance? Cardiomyocyte BH4 has a physiological role in cardiac metabolism. There was a shift of substrate preference from fatty acid to glucose in hearts with targeted deletion of BH4 synthesis. The changes in fatty-acid metabolic profile were associated with a protective effect in response to ischaemia-reperfusion (IR) injury, and reduced infarct size. Manipulating fatty acid metabolism via BH4 availability could play a therapeutic role in limiting IR injury. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) synthases in which its production of NO is crucial for cardiac function. However, non-canonical roles of BH4 have been discovered recently and the cell-specific role of cardiomyocyte BH4 in cardiac function and metabolism remains to be elucidated. Therefore, we developed a novel mouse model of cardiomyocyte BH4 deficiency, by cardiomyocyte-specific deletion of Gch1, which encodes guanosine triphosphate cyclohydrolase I, a required enzyme for de novo BH4 synthesis. Cardiomyocyte (cm)Gch1 mRNA expression and BH4 levels from cmGch1 KO mice were significantly reduced compared to Gch1flox/flox (WT) littermates. Transcriptomic analyses and protein assays revealed downregulation of genes involved in fatty acid oxidation in cmGch1 KO hearts compared with WT, accompanied by increased triacylglycerol concentration within the myocardium. Deletion of cardiomyocyte BH4 did not alter basal cardiac function. However, the recovery of left ventricle function was improved in cmGch1 KO hearts when subjected to ex vivo ischaemia-reperfusion (IR) injury, with reduced infarct size compared to WT hearts. Metabolomic analyses of cardiac tissue after IR revealed that long-chain fatty acids were increased in cmGch1 KO hearts compared to WT, whereas at 5min reperfusion (post-35min ischaemia) fatty acid metabolite levels were higher in WT compared to cmGch1 KO hearts. These results indicate a new role for BH4 in cardiomyocyte fatty acid metabolism, such that reduction of cardiomyocyte BH4 confers a protective effect in response to cardiac IR injury. Manipulating cardiac metabolism via BH4 could play a therapeutic role in limiting IR injury.

Two Cases of 6-Pyruvoyl Tetrahydropterin Synthase Deficiency: Case Report and Literature Review

6-pyruvoyl tetrahydropterin synthase deficiency (PTPSD) is a rare neurometabolic disease that can be diagnosed in newborn screening (NBS) and is part of the family of tetrahydrobiopterin deficiency disorders (BH4Ds). It is essential to diagnose and treat this disease early to prevent permanent neurological damage secondary to this neurotransmitter disorder. We present the first two cases of PTPSD in Romania that were genetically confirmed and treated late. Improving the diagnosis and monitoring procedures in Romania with correct metabolic management will prevent severe neurological impairment from PTPSD or other BH4Ds.

Endothelial cell vasodilator dysfunction mediates progressive pregnancy-induced hypertension in endothelial cell tetrahydrobiopterin deficient mice

Background and purposePregnancy-associated vascular remodelling is essential for both maternal and fetal health. We have previously shown that maternal endothelial cell tetrahydrobiopterin (BH4) deficiency causes poor pregnancy outcomes. Here, we investigated the role and mechanisms of endothelial cell-mediated vasorelaxation function in these outcomes. Experimental approachThe vascular reactivity of mouse aortas and uterine arteries from non-pregnant and pregnant endothelial cell-specific BH4 deficient mice (Gch1fl/flTie2cre mice) was assessed by wire myography. Systolic blood pressure was assessed by tail cuff plethysmography. Key resultsIn late pregnancy, systolic blood pressure was significantly higher (∼24 mmHg) in Gch1fl/flTie2cre mice compared with wild-type littermates. This was accompanied by enhanced vasoconstriction and reduced endothelial-dependent vasodilation in both aorta and uterine arteries from pregnant Gch1fl/flTie2cre mice. In uterine arteries loss of eNOS-derived vasodilators was partially compensated by upregulation of intermediate and large-conductance Ca2+-activated K+ channels. In rescue experiments, oral BH4 supplementation alone did not rescue vascular dysfunction and pregnancy-induced hypertension in Gch1fl/flTie2cre mice. However, combination with the fully reduced folate, 5-methyltetrahydrofolate (5-MTHF), restored endothelial cell vasodilator function and blood pressure. Conclusions and implicationsWe identify a critical requirement for maternal endothelial cell Gch1/BH4 biosynthesis in endothelial cell vasodilator function in pregnancy. Targeting vascular Gch1 and BH4 biosynthesis with reduced folates may provide a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension.

Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin, and zinc, in mouse oocytes

To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age, resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. Experimental laboratory study. Laboratory. B6D2F1 mice. Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Mutation spectrum of PTS gene in patients with tetrahydrobiopterin deficiency from jiangxi province.

Background: Hyperphenylalaninemia (HPA) is the most common inborn error in amino acid metabolism. It can be primarily classified into phenylalanine hydroxylase (PAH) deficiency and tetrahydrobiopterin (BH4) deficiency. BH4 deficiency (BH4D) is caused by genetic defects in enzymes involved in the biosynthesis and regeneration of BH4. 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), which is encoded by the PTS gene, participates in the biosynthesis of BH4. PTPS deficiency (PTPSD) is the major cause of BH4D. In this study, we investigated that the prevalence of BH4D in Jiangxi province was approximately 12.5 per 1,000,000 live births (69/5,541,627). Furthermore, the frequency of BH4D was estimated to be 28.8% (69/240) in the HPA population of Jiangxi. In this study, we aimed to characterize the mutational spectrum of the PTS gene in patients with PTPSD from Jiangxi province. Method: Newborn screening data of Jiangxi province from 1997 to 2021 were analyzed and 53 families with PTPSD were enrolled for the analysis of the PTS gene variants by Sanger sequencing. Results: 106 variants were identified in 106 alleles of 53 patients with PTPSD, including 13 types of variants reported previously, and two novel variants (c.164-36A>G and c.146_147insTG). The predominant variant was c.259C>T (47.2%), followed by c.84-291A>G (19.8%), c.155A>G (8.5%), c.286G>A (6.6%) and c.379C>T (4.7%). Conclusion: The results of this study can not only provide guidance for the molecular diagnosis and genetic counseling in cases of PTPS deficiency but also enrich the PTS mutation database.

Expression of BmDHFR is up-regulated to trigger an increase in the BH4/BH2 ratio when the de novo synthesis of BH4 is blocked in silkworm, Bombyx mori

Tetrahydrobiopterin (BH4) is a vital coenzyme for several enzymes involved in diverse enzymatic reactions in animals. BH4 deficiency can lead to metabolic and neurological disorders due to dysfunction in its metabolism. Sepiapterin reductase (SPR) and dihydrofolate reductase (DHFR) are crucial enzymes in the BH4 de novo synthesis pathway and salvage pathway, respectively. Dihydrobiopterin (BH2) is an oxidized product of BH4 metabolism. The ratio of BH4/BH2 is a key indicator of the stability of BH4 levels. The de novo pathway of BH4 synthesis is well-defined; however, little is known about the mechanisms of the salvage pathway in insects. Herein, we used the natural BmSPR mutant silkworm (lem) as a resource material. Our results reveal that the BmDHFR expression and the BH4/BH2 ratio were remarkably higher in lem as compared to the wild-type silkworm. In BmN cells, knockdown of BmSpr showed increased BmDHFR expression, while the BH4/BH2 ratio decreased after BmDhfr knockdown by RNAi. Furthermore, simultaneous RNAi of BmSpr and BmDhfr showed a further decrease in the BH4/BH2 ratio. These manifest that the expression of BmDHFR is up-regulated to trigger an increase in the BH4/BH2 ratio when the de novo synthesis of BH4 is blocked in silkworm. Additionally, the knockdown of BmSpr in wild-type silkworms also showed an increased BmDHFR level and BH4/BH2 ratio. Taken together, when the silkworm BH4 de novo synthesis pathway is blocked, the salvage pathway is activated, and BmDHFR plays an important role in maintaining the metabolic balance of silkworm BH4. This study enriches our understanding of the molecular mechanism of the BH4 salvage pathway and lays a good foundation for further studies on BH4 using the silkworm as a model insect.

Genetic evaluation of hyperphenylalaninemia patients with tetrahydrobiopterin deficiency in Iranian population: Identification of four novel disease-causing variants.

Hyperphenylalaninemia (HPA) is the most common inborn error of amino acid metabolism worldwide. At least 2% of HPA cases are caused by a deficiency in tetrahydrobiopterin (BH4) metabolism. Genes such as QDPR and PTS are essential in the BH4 metabolism. This study aims to identify disease-causing variants in HPA patients, which may be helpful in genetic counseling and prenatal diagnosis. A total of 10 HPA patients were enrolled in this study. The coding and adjacent intronic regions of PTS and QDPR genes were examined using Sanger sequencing. Protein modeling was also performed for novel identified variants. Ten patients and a total of 20 alleles were studied, which led to the identification of 10 different variants. All variants identified in PTS and QDPR were missense, except for the c.383_407del variant in the QDPR. Also, three novel variants were identified in the QDPR, including c.79G>T, c.383_407del and c.488G>A, and a novel variant, c.65C>G, in the PTS. Despite the genetic similarities in the disease-causing variants, differences were observed in the Asian and European populations with our populations; As a result, similar but more extensive studies are needed to investigate the distribution of disease-causing variants in genes involved in non-PKU hyperphenylalaninemia.

Genotypic spectrum underlying tetrahydrobiopterin metabolism defects: Experience in a single Mexican reference center.

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

Exploring the Mystery of the Tetrahydrobiopterin Synthetic Defect Lethal Mutant leml from Birth to Death in the Silkworm Bombyx mori.

Tetrahydrobiopterin (BH4) is a vital coenzyme for several enzymes involved in diverse enzymatic reactions in animals, and BH4 deficiency can lead to metabolic and neurological disorders due to dysfunction in its metabolism. In the silkworm natural homozygous mutant leml, the key enzyme sepiapterin reductase (BmSPR) in the de novo synthesis pathway of BH4 is inactivated, resulting in severe deficiency of BH4 synthesis. However, it is not known why the leml larvae can survive to the second-instar stage and which pathways lead to their death when BH4 is deficient. Here, we quantified BH4 and found that the fertilized eggs contained large amounts of BH4 transferred from the mother to the offspring, maintaining its normal development in the embryo and the first instar. Subsequently, we investigated the multiple pathways in which BH4 is involved as a cofactor. The results showed that BH4 deficiency in silkworms blocked the melanin synthesis pathway, caused an insufficient degree of epidermal sclerosis, disordered tyrosine metabolism, and damaged mitochondria. On the other hand, BH4 deficiency led to the uncoupling of nitric oxide synthase (BmNOS), a reduced NO production, and a significantly reduced fat in fat body catalyzation by phospholipase A2, resulting in an impaired immune system. Meanwhile, the uncoupling of BmNOS increased the O2− content, damaged the DNA, and caused the apoptosis of the body cells. Taken together, BH4 is critical for the life and death of leml mutants. This study lays a foundation for the further exploration of lepidopteran insects and provides an important basis for the treatment of human BH4 deficiency-related diseases.

Role of BH4 deficiency as a mediator of oxidative stress-related endothelial dysfunction in menopausal women.

Endothelial function (brachial artery flow-mediated dilation [FMD]) is reduced in estrogen-deficient postmenopausal women, mediated, in part, by reduced nitric oxide (NO) bioavailability, secondary to tetrahydrobiopterin (BH4) deficiency and oxidative stress. FMD is increased, but not fully restored, in postmenopausal women after acute intravenous vitamin C (VITC; superoxide scavenger) or oral BH4 supplementation. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite. To investigate mechanisms of endothelial dysfunction in women, we assessed the separate and combined effects of VITC and BH4 to determine whether coadministration of VITC + BH4 improves FMD in healthy postmenopausal women (n = 19, 58 ± 5 yr) to premenopausal (n = 14, 36 ± 9 yr) levels, with exploratory testing in perimenopausal women (n = 8, 51 ± 3 yr). FMD was measured during acute intravenous infusions of saline (control) and VITC (∼2-3 g) ∼3 h after a single dose of oral BH4 (KUVAN, 10 mg/kg body wt) or placebo (randomized crossover, separated by ∼1 mo). Under the placebo condition, FMD was reduced in postmenopausal compared with premenopausal women during the saline infusion (5.6 ± 0.7 vs. 11.6 ± 0.9%, P < 0.001) and increased in postmenopausal women during VITC (+3.5 [1.4, 5.6]%, P = 0.001) and acute BH4 (+1.8 [0.37, 3.2]%, P = 0.01) alone. Coadministration of VITC + BH4 increased FMD in postmenopausal women (+3.0 [1.7, 4.3]%, P < 0.001), but FMD remained reduced compared with premenopausal women (P = 0.02). Exploratory analyses revealed that VITC + BH4 did not restore FMD in perimenopausal women to premenopausal levels (P = 0.045). Coadministration of VITC + BH4 does not restore FMD in menopausal women, suggesting that additional mechanisms may be involved.NEW & NOTEWORTHY Endothelial function is reduced across the menopausal stages related to increased oxidative stress associated with estrogen deficiency. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite; however, this remains untested in humans. We demonstrate that the coadministration of BH4 + VITC does not restore endothelial function in perimenopausal and postmenopausal women to the level of premenopausal women, suggesting that other mechanisms contribute.

Identification and molecular analysis of 11 cases of the PTS gene variants associated with tetrahydrobiopterin deficiency.

Background: Tetrahydrobiopterin deficiency (BH4D) is a rare autosomal recessive amino acid metabolic disease that belongs to a kind of hyperphenylalaninemia (HPA), and 6-pyruvyltetrahydrotrexate synthase (PTPS) deficiency is the most common type of BH4D. This study investigates the clinical and genetic characteristics of 11 PTPS deficiency cases in the Beijing area, identifies the genetic pathogenic factors, and evaluates the value of high-throughput sequencing in the precise diagnosis of PTPS deficiency. Methods: The Beijing Neonatal Disease Screening Center diagnosed patients with HPA. The study used phenylalanine (Phe) in blood, the ratio of Phe to Thr, urotrexate spectrum analysis, erythrocyte dihydrotrexate reductase (DHPR) activity determination, and high-throughput sequencing as methods. Bioinformatics software analyzed the variants’ pathogenicity and used RT-PCR to identify deep intron variants’ pathogenicity. Result: Among 635 cases with HPA, 38 cases were diagnosed with BH4D, of which the incidence in HPA was 5.98%. Nine kinds of PTS gene variants were detected, including seven missense variants, one splicing variant, and one deletion variant. The splicing variant c.84–291A>G had three splicing results in vivo: normal length, 79bp pseudoexon insertion, and exon 3 skipping. Bioinformatics and Sanger sequencing were performed to verify the identified variants. Conclusion: High-throughput sequencing is a helpful tool for clinical diagnosis and differential diagnosis of BH4D. This study confirms that c.84–291A>G is the hot spot variant of PTPS deficiency, and it is the first reported variant with a new splicing pattern in vivo. A novel deletion variant c.84_163del (p.Lys29Cysfs∗9) was found to enrich the genetic variant spectrum of the disease.