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Background: The GCH1 gene encodes the enzyme guanosine triphosphate cyclohydrolase I (GTPCH), which catalyzes the rate‐limiting step in the biosynthesis of tetrahydrobiopterin (BH4), a critical cofactor in the production of monoamine neurotransmitters. Autosomal dominant GTPCH (adGTPCH) deficiency is the most common cause of dopa‐responsive dystonia (DRD), whereas the recessive form (arGTPCH) is an ultrarare and poorly characterized disorder with earlier and more complex presentation that may disrupt neurodevelopmental processes. Here, we delineated the phenotypic spectrum of ARGTPCHD and investigated the predictive value of biochemical and genetic correlates for disease outcome. Objectives: The aim was to study 4 new cases of arGTPCH deficiency and systematically review patients reported in the literature. Methods: Clinical, biochemical, and genetic data and treatment response of 45 patients are presented. Results: Three phenotypes were outlined: (1) early‐infantile encephalopathic phenotype with profound disability (24 of 45 patients), (2) dystonia‐parkinsonism phenotype with infantile/early‐childhood onset of developmental stagnation/regression preceding the emergence of movement disorder (7 of 45), and (3) late‐onset DRD phenotype (14 of 45). All 3 phenotypes were responsive to pharmacological treatment, which for the first 2 must be initiated early to prevent disabling neurodevelopmental outcomes. A gradient of BH4 defect and genetic variant severity characterizes the 3 clinical subgroups. Hyperphenylalaninemia was not observed in the second and third groups and was associated with a higher likelihood of intellectual disability. Conclusions: The clinical spectrum of arGTPCH deficiency is a continuum from early‐onset encephalopathies to classical DRD. Genotype and biochemical alterations may allow early diagnosis and predict clinical severity. Early treatment remains critical, especially for the most severe patients. [ABSTRACT FROM AUTHOR]

This article discusses a case study of a 57-year-old Han Chinese male with dopa-responsive dystonia (DRD) caused by a large inversion affecting the GCH1 gene. The patient experienced abnormal head posturing and gait disturbance following a head injury at age 10. Despite not having an identifiable disease-causing variant through conventional gene sequencing, the patient responded well to low-dose levodopa treatment. The study highlights the importance of using whole genome sequencing, nanopore long-read sequencing, and optical genome mapping to detect and confirm complex structural variants in patients with DRD. [Extracted from the article]

Background: Pathogenic variants in several genes have been linked to genetic forms of isolated or combined dystonia. The phenotypic and genetic spectrum and the frequency of pathogenic variants in these genes have not yet been fully elucidated, neither in patients with dystonia nor with other, sometimes co‐occurring movement disorders such as Parkinson's disease (PD). Objectives: To screen >2000 patients with dystonia or PD for rare variants in known dystonia‐causing genes. Methods: We screened 1207 dystonia patients from Germany (DysTract consortium), Spain, and South Korea, and 1036 PD patients from Germany for pathogenic variants using a next‐generation sequencing gene panel. The impact on DNA methylation of KMT2B variants was evaluated by analyzing the gene's characteristic episignature. Results: We identified 171 carriers (109 with dystonia [9.0%]; 62 with PD [6.0%]) of 131 rare variants (minor allele frequency <0.005). A total of 52 patients (48 dystonia [4.0%]; four PD [0.4%, all with GCH1 variants]) carried 33 different (likely) pathogenic variants, of which 17 were not previously reported. Pathogenic biallelic variants in PRKRA were not found. Episignature analysis of 48 KMT2B variants revealed that only two of these should be considered (likely) pathogenic. Conclusion: This study confirms pathogenic variants in GCH1, GNAL, KMT2B, SGCE, THAP1, and TOR1A as relevant causes in dystonia and expands the mutational spectrum. Of note, likely pathogenic variants only in GCH1 were also found among PD patients. For DYT‐KMT2B, the recently described episignature served as a reliable readout to determine the functional effect of newly identified variants. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Objective: GTP cyclohydrolase 1(GCH1) was reported to protect against ferroptosis. However, it is not clear whether GCH1 reduced lipopolysaccharide (LPS)-induced macrophage polarization and inflammation by inhibition of ferroptosis. Methods: Bioinformatics analysis was used to screen differential expression genes (DEGs) and obtain the different pathways and biological features. Lasso cox regression analysis with ferroptosis related DEGs was established to screen the most relevant genes for disease risk. LPS induced Raw264.7 macrophage polarization model and GCH1-specific siRNA oligos transfection were performed to confirm the function of GCH1. Immunofluorescence staining, western blot and quantitative real-time PCR were performed to detect the expression of iNOS, CD206, GCH1, IL6, SLC2A6, F4/80, IL1β, TNFα, IL10, GPX4, ACSL4, AMPK and p-AMPK in macrophages. The levels of ROS, SOD, MDA and GSH were detected according to the instructions of the reagent kit, respectively. Results: 542 DEGs were screened from GSE40885 microarray. GO and KEGG pathway enrichment analysis showed that the upregulated DEGs induced by LPS in alveolar macrophage were closely associated with inflammatory and immune responses, the downregulated DEGs were related to lipid metabolism, insulin resistance and AMPK signal pathway. Lasso cox regression analysis screened GCH1, IL6, and SLC2A6. Our experimental results showed that the expression of GCH1 and IL6 in the LPS group was higher than that in the control group, but there was no difference in the expression of SLC2A6. Bioinformatics analysis with GSE112720 observed that ferroptosis was enriched in GCHfl/fl + LPS group compared with GCHfl/flTie2cre + LPS group and GCHfl/fl + control group. Silence of GCH1 increased the levels of IL6, TNF-α and IL-1β and decreased IL10 level. Silence of GCH1 increased iNOS level and decreased CD206 level. Moreover, silence of GCH1 raised ferroptosis induced by LPS in macrophages and suppressed the activity of AMPK pathway. Conclusions: GCH1 inhibited ferroptosis in LPS-stimulated macrophages, reduced macrophage toward to M1 polarization and inflammatory response. [ABSTRACT FROM AUTHOR]

Abstract Background Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM. Methods The expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay. Results We found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients’ poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4). Conclusions Our study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.

Abstract Background Methyltransferase 3 (METTL3)‐mediated N6‐methyladenosine (m6A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3‐mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer. Methods To investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6A‐modified RNA immunoprecipitation (Me‐RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre‐B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3‐PBX1‐GCH1 axis. ELISA and liquid chromatography‐mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4). Results Knockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3‐PBX1‐GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression. Conclusions This study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3‐PBX1‐GCH1 axis increasing BH4 levels in GC.

• The antioxidative activities of CTRP13 alleviate ox-LDL-induced endothelial cell ferroptosis. • CTRP13 reduced the progression of atherosclerosis and increases the stability of atherosclerotic plaques. • CTRP13 treatment reduced ox-LDL-induced mitochondrial oxidative stress. • CTRP13 exerted its anti-atherosclerosis effects via GCH1-BH4 pathway activation. C1q/TNF-related protein 13 (CTRP13) is a secreted adipokine that has been shown to play an important role in a variety of cardiovascular diseases. However, the effect of CTRP13 on ferroptosis of endothelial cells and its underlying mechanism remain unclear. In the present study, we analyzed the effects of CTRP13 on endothelial dysfunction in high-lipid-induced ApoE−/− mice and ox-LDL-induced mouse aortic endothelial cells (MAECs). In vivo experiment: Male ApoE−/− mice fed high fat were given C1ql3 gene overexpression adeno-associated virus. The atherosclerotic plaque size, lipid content, collagen fiber proportion and iron deposition level were measured. In vitro, CTRP13 combined with ox-LDL was used to pretreat MAECs to detect cell survival rate, lipid peroxidation, iron ion deposition and mitochondrial level. In this study, CTRP13 was found to inhibit ferroptosis of endothelial cells, demonstrated by up-regulated the expression of ferroptosis protective protein glutathione Peroxidase 4 (GPX4), and decreased the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) protein. Mechanistically, gtp cyclohydrolase 1(GCH1) silencing or tetrahydrobiopterin (BH4) inhibiting may counteract the protective effect of CTRP13 on ox-LDL-induced ferroptosis of endothelial cells, which is characterized by decreased cell activity, mitochondrial damage, increased iron ion deposition and lipid peroxidation, decreased GPX4 expression, and increased ACSL4 expression. This study demonstrated for the first time that CTRP13 can improve mitochondrial oxidative stress, inhibit ferroptosis of endothelial cells and improve endothelial cell dysfunction by activating the GCH1/BH4 signaling pathway, thereby inhibiting the progression of atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM. Methods: The expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay. Results: We found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients' poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4). Conclusions: Our study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM. [ABSTRACT FROM AUTHOR]

What is known and objective: Significant individual differences have been observed in pain sensitivity and analgesic effect of opioids. Previous studies have shown that genetic factors contributed to analgesics requirement obviously. Therefore, we investigated the role of genetic polymorphisms in the sensitivity to the analgesic effect of remifentanil in this study. Methods: One hundred thirty‐seven patients undergoing gynaecological surgery were observed. Before procedures, we measured the basal pain threshold of each patient, including the pressure pain threshold and pressure pain tolerance threshold. Subsequently, patients received a continuous remifentanil infusion for 15 min at a constant rate of 0.2 μg/(kg min). The pain thresholds were measured again after the remifentanil infusion. Moreover, respiratory depression was estimated using oxygen saturation during infusion. DNA was extracted from peripheral venous blood and genotyped using SNaPshot technology. Results and discussion: Polymorphisms were found in genes associated with the individual variation in analgesia. Participants carrying OPRM1 rs9397685 AA, ADRB1 rs1801253 CC, and GCH1 rs8007267 CC polymorphisms showed higher sensitivity to analgesic effect induced by remifentanil, and the participants carrying the OPRD1 rs2234918 TT showed lower sensitivity to remifentanil‐related respiratory depression. Moreover, individual susceptibility to remifentanil increases with age. What is new and conclusion: Gene variation in OPRM1 rs9397685 AA, ADRB1 rs1801253 CC, GCH1 rs8007267 CC, and OPRD1 rs2234918 TT were related to the conspicuous interindividual differences in the analgesia and respiratory depression of remifentanil, mainly by affecting the target protein receptors and relative metabolic enzymes. [ABSTRACT FROM AUTHOR]

Dopa-responsive dystonia (DRD) comprises a group of rare but treatable dystonias that exhibit diurnal fluctuation. The GCH1 gene encodes GTP cyclohydrolase-1 (GTPCH-I), a protein that catalyzes the first rate-limiting step of tetrahydrobiopterin biosynthesis. Pathogenic variants in GCH1 are the most common causes of DRD. However, the autosomal recessive form of DRD caused by biallelic GCH1 variants is very rare. Homozygous GCH1 variants have been associated with two clinically distinct human diseases: hyperphenylalaninemia, and DRD with or without hyperphenylalaninemia. Here, we describe one patient who presented during infancy with severe truncal hypotonia and motor developmental regression but without diurnal fluctuation and hyperphenylalaninemia. Treatment with levodopa/carbidopa resulted in the complete and persistent remission of clinical symptoms without any side effects. This was accompanied by age-appropriate neurological development during follow-up. A homozygous GCH1 variant (c.604G>A/p.V202I) was identified in the patient. To our knowledge, this is the first Chinese case of DRD caused by a homozygous GCH1 variant, thus expanding the spectrum of DRD phenotypes. Autosomal recessive DRD that is associated with homozygous GCH1 variants should be considered in patients with severe truncal hypotonia, with or without diurnal fluctuation, even if there is an absence of limb dystonia and hyperphenylalaninemia. [ABSTRACT FROM AUTHOR]

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes. [ABSTRACT FROM AUTHOR]

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes.

Ferroptosis has recently been discovered as an iron-dependent and non-apoptotic regulated mechanism of cell death. The induction of ferroptosis in tumor cells improves tumor treatment, making it a current research hotspot. Mechanistically, it starts by lipid peroxidation, iron accumulation, reactive oxygen species (ROS) production, and glutathione deprivation, highlighting novel treatment opportunities for many tumors and neurodegenerative disorders. Several tumor cell lines are resistant to ferroptosis inducers, even when the ferroptosis key enzyme glutathione peroxidase 4 (GPX4) is blocked, indicating that other important elements are also involved in this process. Ferroptosis-suppressor-protein 1 (FSP1) was discovered to be one of these elements in addition to a few others such as ferroptotic gatekeepers like GTP cyclohydrolase 1 (GCH1) and dihydroorotate dehydrogenase (DHODH). Osteosarcoma is the most common primary malignant bone tumor observed most frequently in children and adolescents. Several studies demonstrated that ferroptosis plays a critical role in the treatment of osteosarcoma, in particular drug-resistant osteosarcoma cells. We outlined four primary regulators involved in ferroptosis in this article, reviewed previously published studies of ferroptosis in osteosarcoma to provide covert insights about osteosarcoma treatment, and highlighted several critical issues to point out future research possibilities. [ABSTRACT FROM AUTHOR]

Background: Pathogenic variants in 5 genes (GCH1, TH, PTS, SPR, and QDPR), involved in dopamine/tetrahydrobiopterin biosynthesis or recycling, have been linked to Dopa‐responsive dystonia (DRD). Diagnosis and treatment are often delayed due to high between‐ and within‐group variability. Objectives: Comprehensively analyzed individual genotype, phenotype, treatment response, and biochemistry information. Methods: 734 DRD patients and 151 asymptomatic GCH1 mutation carriers were included using an MDSGene systematic literature review and an automated classification approach to distinguish between different forms of monogenic DRDs. Results: Whereas dystonia, L‐Dopa responsiveness, early age at onset, and diurnal fluctuations were identified as red flags, parkinsonism without dystonia was rarely reported (11%) and combined with dystonia in only 18% of patients. While sex was equally distributed in autosomal recessive DRD, there was female predominance in autosomal dominant DYT/PARK‐GCH1 patients accompanied by a lower median age at onset and more dystonia in females compared to males. Accordingly, the majority of asymptomatic heterozygous GCH1 mutation carriers (>8 years of age) were males. Multiple other subgroup‐specific characteristics were identified, showing high accuracy in the automated classification approach: Seizures and microcephaly were mostly seen in DYT/PARK‐PTS, autonomic symptoms appeared commonly in DYT/PARK‐TH and DYT/PARK‐PTS, and sleep disorders and oculogyric crises in DYT/PARK‐SPR. Biochemically, homovanillic acid and 5‐hydroxyindoleacetic acid in CSF were reduced in most DRDs, but neopterin and biopterin were increased only in DYT/PARK‐PTS and DYT/PARK‐SPR. Hyperphenylalaninemia was seen in DYT/PARK‐PTS, DYT/PARK‐QDPR, and rarely reported in autosomal recessive DYT/PARK‐GCH1. Conclusions: Our indicators will help to specify diagnosis and accelerate start of treatment. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Introduction: GCH1 mutations have been linked to decreased striatal dopamine and development of dopa-responsive dystonia (DRD) and Parkinsonism. Sensory and sensorimotor integration impairments have been documented in various forms of dystonia. DRD patients with confirmed GCH1 mutations have demonstrated normal short-latency afferent inhibition (SAI), a measure of sensorimotor inhibition, under chronic dopaminergic replacement therapy (DRT), but reduced inhibition after a single l-dopa dose following 24 h withdrawal. Studies have revealed normal SAI in other forms of dystonia but reductions with DRT in Parkinson's disease. Longitudinal changes in sensorimotor inhibition are unknown.Methods: We analyzed sensorimotor inhibition using two different measures: SAI and somatosensory-motor inhibition using dual-site transcranial magnetic stimulation (ds-TMS). SAI was measured using digit stimulation 25 ms prior to contralateral primary motor cortex (M1) TMS. DS-TMS was measured using TMS over the somatosensory cortex 1 or 2.5 ms prior to ipsilateral M1 stimulation. A total of 20 GCH1 mutation carriers and 20 age-matched controls were included in the study. SAI and ds-TMS were evaluated in GCH1 mutation carriers both OFF and ON DRT compared to controls. Furthermore, longitudinal changes of SAI were examined in a subset of the same individuals that were measured ∼five years earlier.Results: Neither SAI nor ds-TMS were significantly different in GCH1 mutation carriers relative to controls. No effects of DRT on SAI or ds-TMS were seen but SAI decreased over time in mutation carriers OFF DRT.Conclusion: Our longitudinal results suggest changes in SAI that could be associated with plasticity changes in sensorimotor networks. [ABSTRACT FROM AUTHOR]

Introduction: In 2009, we described a possible founder effect of autosomal dominant Segawa disease in Córdoba (Spain) due to mutation c.265C>T (p. Q89*) in the GCH1 gene. We present a retrospective multicentre study aimed at improving our knowledge of Segawa disease in Spain and providing a detailed phenotypic-genotypic description of patients.Methods: Clinical-genetic information were obtained from standardized questionnaires that were completed by the neurologists attending children and/or adults from 16 Spanish hospitals.Results: Eighty subjects belonging to 24 pedigrees had heterozygous mutations in GCH1. Seven genetic variants have been described only in our cohort of patients, 5 of which are novel mutations. Five families not previously described with p. Q89* were detected in Andalusia due to a possible founder effect. The median latency to diagnosis was 5 years (IQR 0-16). The most frequent signs and/or symptoms were lower limb dystonia (38/56, 67.8%, p = 0.008) and diurnal fluctuations (38/56, 67.8%, p = 0.008). Diurnal fluctuations were not present in the phenotypes other than dystonia. Fifty-three of 56 symptomatic patients were treated with a levodopa/decarboxylase inhibitor for (mean ± SD) 12.4 ± 8.12 years, with 81% at doses lower than 350 mg/day (≤5 mg/kg/d in children). Eleven of 53 (20%) patients had nonresponsive symptoms that affected daily life activities. Dyskinesias (4 subjects) were the most prominent adverse effects.Conclusion: This study identifies 5 novel mutations and supports the hypothesis of a founder effect of p. Q89* in Andalusia. New insights are provided for the phenotypes and long-term treatment responses, which may improve early recognition and therapeutic management. [ABSTRACT FROM AUTHOR]

Abstract Background Common and rare variants of guanosine triphosphate cyclohydrolase 1 (GCH1) gene may play important roles in Parkinson’s disease (PD). However, there is a lack of comprehensive analysis of GCH1 genotypes, especially in non-coding regions. The aim of this study was to explore the genetic characteristics of GCH1, including rare and common variants in coding and non-coding regions, in a large population of PD patients in Chinese mainland, as well as the phenotypic characteristics of GCH1 variant carriers. Methods In the first cohort of this case-control study, we performed whole-exome sequencing in 1555 patients with early-onset or familial PD and 2234 healthy controls; then in the second cohort, whole-genome sequencing was performed in sporadic late-onset PD samples (1962 patients), as well as 1279 controls. Variants at target GCH1 regions were extracted, and then genetic and detailed phenotypic data were analyzed using regression models and the sequence kernel association test. We also performed a meta-analysis to correlate deleterious GCH1 variants with age at onset (AAO) in PD patients. Results For coding variants, we identified a significant burden of GCH1 deleterious variants in early-onset or familial PD cases compared to controls (1.2% vs 0.1%, P

Ferroptosis has recently been discovered as an iron-dependent and non-apoptotic regulated mechanism of cell death. The induction of ferroptosis in tumor cells improves tumor treatment, making it a current research hotspot. Mechanistically, it starts by lipid peroxidation, iron accumulation, reactive oxygen species (ROS) production, and glutathione deprivation, highlighting novel treatment opportunities for many tumors and neurodegenerative disorders. Several tumor cell lines are resistant to ferroptosis inducers, even when the ferroptosis key enzyme glutathione peroxidase 4 (GPX4) is blocked, indicating that other important elements are also involved in this process. Ferroptosis-suppressor-protein 1 (FSP1) was discovered to be one of these elements in addition to a few others such as ferroptotic gatekeepers like GTP cyclohydrolase 1 (GCH1) and dihydroorotate dehydrogenase (DHODH). Osteosarcoma is the most common primary malignant bone tumor observed most frequently in children and adolescents. Several studies demonstrated that ferroptosis plays a critical role in the treatment of osteosarcoma, in particular drug-resistant osteosarcoma cells. We outlined four primary regulators involved in ferroptosis in this article, reviewed previously published studies of ferroptosis in osteosarcoma to provide covert insights about osteosarcoma treatment, and highlighted several critical issues to point out future research possibilities.

Ferroptosis, a type of cell death triggered by excessive accumulation of iron-dependent lipid peroxidation, possesses an excellent potential in cancer treatment. However, many colorectal cancer (CRC) cell lines are resistant to ferroptosis induced by erastin and RSL3, the classical ferroptotic inducers. Moreover, the underlying mechanism of resistance remains poorly elucidated. This study sought to discover the major factor contributing to ferroptosis resistance in CRC. The study findings will help design strategies for triggering ferroptosis for application in individualized tumor therapy. Here, we show that tetrahydrobiopterin (BH4) determines the sensitivity of CRC cells to ferroptosis induced by erastin. GTP cyclohydrolase-1 (GCH1) is the first rate-limiting enzyme of BH4. Genetic or pharmacological inhibition of GCH1 decreased BH4 and assisted erastin in cell death induction, lipid peroxidation enhancement, and ferrous iron accumulation. BH4 supplementation completely inhibited ferroptotic features resulting from GCH1 knockdown. Unexpectedly, GCH1 knockdown failed to enhance RSL3-induced cell death in CRC. Mechanistically, GCH1 knockdown drastically activated ferritinophagy during erastin treatment rather than RSL3 treatment. Administration of an autophagy inhibitor reversed erastin resistance in GCH1-knockdown cells. GCH1 inhibitor and erastin co-treatment in vivo synergistically inhibited tumor growth in CRC. Overall, our results identified GCH1/BH4 metabolism as a burgeoning ferroptosis defense mechanism in CRC. Inhibiting GCH1/BH4 metabolism promoted erastin-induced ferroptosis by activating ferritinophagy, suggesting that combining GCH1 inhibitors with erastin in the treatment of CRC is a novel therapeutic strategy.

Dopa-responsive dystonia (DRD), also known as Segawa syndrome, is a phenotypically and genetically heterogeneous group of neurological disorders that typically presents as early-onset lower limb dystonia with diurnal fluctuation, and exhibits a marked, persistent response to levodopa. Heterozygous loss-of-function mutations in the guanosine triphosphate cyclohydrolase 1 (GCH1) are the most common cause of DRD. In addition to the classic form of the disease, there have been a number of studies addressing atypical clinical features of GCH1 related DRD with variable age of onset. This report describes a 37-year-old Japanese male patient with a 10-year history of focal upper limb dystonia that initially emerged as task-specific, guitarist’s cramp. The dystonic symptoms responded very well to levodopa treatment, and genetic analysis identified a novel heterozygous mutation in the C-terminal catalytic domain of GCH1. Insufficient recognition of this treatable condition often leads to misdiagnosis, which causes delays in the patient receiving adequate dopamine replenishing therapy. A diagnostic trial with levodopa should be considered in all patients with relatively young-onset dystonia, whether they have classic features of DRD or not.

GCH1 mutations have been associated with dopa‐responsive dystonia (DRD), Parkinson's disease (PD) and tetrahydrobiopterin (BH4)‐deficient hyperphenylalaninemia B. Recently, GCH1 mutations have been reported in five patients with hereditary spastic paraplegia (HSP). Here, we analyzed a total of 400 HSP patients (291 families) from different centers across Canada by whole exome sequencing (WES). Three patients with heterozygous GCH1 variants were identified: monozygotic twins with a p.(Ser77_Leu82del) variant, and a patient with a p.(Val205Glu) variant. The former variant is predicted to be likely pathogenic and the latter is pathogenic. The three patients presented with childhood‐onset lower limb spasticity, hyperreflexia and abnormal plantar responses. One of the patients had diurnal fluctuations, and none had parkinsonism or dystonia. Phenotypic differences between the monozygotic twins were observed, who responded well to levodopa treatment. Pathway enrichment analysis suggested that GCH1 shares processes and pathways with other HSP‐associated genes, and structural analysis of the variants indicated a disruptive effect. In conclusion, GCH1 mutations may cause HSP; therefore, we suggest a levodopa trial in HSP patients and including GCH1 in the screening panels of HSP genes. Clinical differences between monozygotic twins suggest that environmental factors, epigenetics, and stochasticity could play a role in the clinical presentation. [ABSTRACT FROM AUTHOR]

A large number of patients are affected by classical heart failure (HF) symptomatology with preserved ejection fraction (HFpEF) and multiorgan syndrome. Due to high morbidity and mortality rate, hospitalization and mortality remain serious socioeconomic problems, while the lack of effective pharmacological or device treatment means that HFpEF presents a major unmet medical need. Evidence from clinical and basic studies demonstrates that systemic inflammation, increased oxidative stress, and impaired mitochondrial function are the common pathological mechanisms in HFpEF. Tetrahydrobiopterin (BH4), beyond being an endogenous co-factor for catalyzing the conversion of some essential biomolecules, has the capacity to prevent systemic inflammation, enhance antioxidant resistance, and modulate mitochondrial energy production. Therefore, BH4 has emerged in the last decade as a promising agent to prevent or reverse the progression of disorders such as cardiovascular disease. In this review, we cover the clinical progress and limitations of using downstream targets of nitric oxide (NO) through NO donors, soluble guanylate cyclase activators, phosphodiesterase inhibitors, and sodium-glucose co-transporter 2 inhibitors in treating cardiovascular diseases, including HFpEF. We discuss the use of BH4 in association with HFpEF, providing new evidence for its potential use as a pharmacological option for treating HFpEF. [Display omitted] [ABSTRACT FROM AUTHOR]

Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo , while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC. [Display omitted] • Epigenetic silencing of GCH1 is predictive of poor prognosis of HCC patients in two independent cohorts. • BH4 inhibits HCC growth and decreases intracellular superoxide anion levels in a dose-dependent manner. • GCH1 silencing promotes HCC growth via activating superoxide anion-mediated ASK1/P38 signaling. [ABSTRACT FROM AUTHOR]

The genetic combined dystonias are a clinically and genetically heterogeneous group of neurologic disorders defined by the overlap of dystonia and other movement disorders such as parkinsonism or myoclonus. The number of genes associated with combined dystonia syndromes has been increasing due to the wider recognition of clinical features and broader use of genetic testing. Nevertheless, these diseases are still rare and represent only a small subgroup among all dystonias. Dopa-responsive dystonia (DYT/PARK-GCH1), rapid-onset dystonia-parkinsonism (DYT/PARK-ATP1A3), X-linked dystonia-parkinsonism (XDP, DYT/PARK-TAF1), and young-onset dystonia-parkinsonism (DYT/PARK-PRKRA) are monogenic combined dystonias accompanied by parkinsonian features. Meanwhile, MYC/DYT-SGCE and MYC/DYT-KCTD17 are characterized by dystonia in combination with myoclonus. In the past, common molecular pathways between these syndromes were the center of interest. Although the encoded proteins rather affect diverse cellular functions, recent neurophysiological evidence suggests similarities in the underlying mechanism in a subset. This review summarizes recent developments in the combined dystonias, focusing on clinico-genetic features and neurophysiologic findings. Disease-modifying therapies remain unavailable to date; an overview of symptomatic therapies for these disorders is also presented. [ABSTRACT FROM AUTHOR]

Isolated dystonia is a common movement disorder often caused by genetic mutations, although it is predominantly sporadic in nature. Common variants of dystonia-related genes were reported to be risk factors for idiopathic isolated dystonia. In this study, we aimed to analyse the roles of previously reported GTP cyclohydrolase (GCH1) and Torsin family 1 member A (TOR1A) polymorphisms in an Indian isolated dystonia case-control group. A total of 292 sporadic isolated dystonia patients and 316 control individuals were genotyped for single-nucleotide polymorphisms (SNPs) of GCH1 (rs3759664:G > A, rs12147422:A > G and rs10483639:C > G) and TOR1A (rs13300897:G > A, rs1801968:G > C, rs1182:G > T and rs3842225:G > Δ) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by direct Sanger sequencing. The statistical significance of allelic, genotypic and haplotypic associations of all of the SNPs were evaluated using the two-tailed Fisher exact test. The minor allele (A) of rs3759664 is significantly associated with isolated limb dystonia as a risk factor (p = 0.005). The minor allele (C) of rs1801968 is strongly associated with isolated dystonia (p < 0.0001) and most of its subtypes. The major allele of rs3842225 (G) may act as a significant risk factor for Writer's cramp (p = 0.03). Four different haplogroups comprising of either rs1182 or rs3842225 or in combination with rs1801968 and rs13300897 were found to be significantly associated with isolated dystonia. No other allelic, genotypic or haplotypic association was found to be significant with isolated dystonia cohort or its endophenotype stratified groups. Our study suggests that TOR1A common variants have a significant role in isolated dystonia pathogenesis in the Indian population, whereas SNPs in the GCH1 gene may have a limited role. [ABSTRACT FROM AUTHOR]

Background: Common and rare variants of guanosine triphosphate cyclohydrolase 1 (GCH1) gene may play important roles in Parkinson's disease (PD). However, there is a lack of comprehensive analysis of GCH1 genotypes, especially in non-coding regions. The aim of this study was to explore the genetic characteristics of GCH1, including rare and common variants in coding and non-coding regions, in a large population of PD patients in Chinese mainland, as well as the phenotypic characteristics of GCH1 variant carriers. Methods: In the first cohort of this case-control study, we performed whole-exome sequencing in 1555 patients with early-onset or familial PD and 2234 healthy controls; then in the second cohort, whole-genome sequencing was performed in sporadic late-onset PD samples (1962 patients), as well as 1279 controls. Variants at target GCH1 regions were extracted, and then genetic and detailed phenotypic data were analyzed using regression models and the sequence kernel association test. We also performed a meta-analysis to correlate deleterious GCH1 variants with age at onset (AAO) in PD patients. Results: For coding variants, we identified a significant burden of GCH1 deleterious variants in early-onset or familial PD cases compared to controls (1.2% vs 0.1%, P < 0.0001). In the analysis of possible regulatory variants in GCH1 non-coding regions, rs12323905 (P = 0.001, odds ratio = 1.19, 95%CI 1.07–1.32) was significantly associated with PD, and variant sets in untranslated regions and intron regions, GCH1 brain-specific expression quantitative trait loci, and two possible promoter/enhancer (GH14J054857 and GH14J054880) were suggestively associated with PD. Genotype-phenotype correlation analysis revealed that the carriers of GCH1 deleterious variants manifested younger AAO (P < 0.0001), and had milder motor symptoms, milder fatigue symptoms and more autonomic nervous dysfunctions. Meta-analysis of six studies demonstrated 6.4-year earlier onset in GCH1 deleterious variant carriers (P = 0.0009). Conclusions: The results highlight the importance of deleterious variants and non-coding variants of GCH1 in PD in Chinese mainland and suggest that GCH1 mutation can influence the PD phenotype, which may help design experimental studies to elucidate the mechanisms of GCH1 in the pathogenesis of PD. [ABSTRACT FROM AUTHOR]

Dopa and tetrahydrobiopterin (BH4) supplementation are recommended therapies for the dopa-responsive dystonia caused by GTP cyclohydrolase 1 (GCH1 , also known as GTPCH) deficits. However, the efficacy and mechanisms of these therapies have not been intensively studied yet. In this study, we tested the efficacy of dopa and BH4 therapies by using a novel GTPCH deficiency mouse model, Gch1 KI/KI , which manifested infancy-onset motor deficits and growth retardation similar to the patients. First, dopa supplementation supported Gch1 KI/KI mouse survival to adulthood, but residual motor deficits and dwarfism remained. Interestingly, RNAseq analysis indicated that while the genes participating in BH4 biosynthesis and regeneration were significantly increased in the liver, no significant changes were observed in the brain. Second, BH4 supplementation alone restored the growth of Gch1 KI/KI pups only in early postnatal developmental stage. High doses of BH4 supplementation indeed restored the total brain BH4 levels, but brain dopamine deficiency remained. While total brain TH levels were relatively increased in the BH4 treated Gch1 KI/KI mice, the TH in the striatum were still almost undetectable, suggesting differential BH4 requirements among brain regions. Last, the growth of Gch1 KI/KI mice under combined therapy outperformed dopa or BH4 therapy alone. Notably, dopamine was abnormally high in more than half, but not all, of the treated Gch1 KI/KI mice, suggesting the existence of variable synergetic effects of dopa and BH4 supplementation. Our results provide not only experimental evidence but also novel mechanistic insights into the efficacy and limitations of dopa and BH4 therapies for GTPCH deficiency. [ABSTRACT FROM AUTHOR]

Atherosclerosis and ensuing cardiovascular disease are major causes of death with insufficient treatment options. In search for pathomechanisms of atherosclerosis, we investigated the impact of the B2 bradykinin receptor, Bdkrb2, on atherosclerotic lesion formation, because to date it is not clear whether the B2 bradykinin receptor is atheroprotective or atherogenic. As a model of atherosclerosis, we used hypercholesterolemic ApoE-deficient (apolipoprotein E-deficient) mice, which develop atherosclerotic lesions in the aorta with increasing age. The role of Bdkrb2 in atherosclerosis was studied in ApoE-deficient mice, which were either Bdkrb2-deficient, or had moderately increased aortic B2 bradykinin receptor protein levels induced by transgenic BDKRB2 expression under control of the ubiquitous CMV promoter. We found that Bdkrb2 deficiency led to a significantly decreased atherosclerotic plaque area whereas transgenic BDKRB2 expression enhanced atherosclerotic lesion formation in the aorta of ApoE-deficient mice at an age of 8 months. Concomitantly, the aortic content of reactive oxygen species (ROS) was higher in BDKRB2-expressing mice whereas Bdkrb2 deficiency decreased aortic ROS levels of ApoE-deficient mice. In addition, aortic nitrate as a marker of nitric oxide activity and the endothelial nitric oxide synthase (eNOS) co-factor, tetrahydrobiopterin (BH4) were reduced in BDKRB2-expressing ApoE-deficient mice. The decreased aortic BH4 content could be a consequence of increased ROS generation and down-regulated aortic expression of the BH4-synthesizing enzyme, Gch1 (GTP cyclohydrolase 1). In agreement with a causal involvement of decreased BH4 levels in the atherogenic function of BDKRB2, we found that treatment with the BH4 analog, sapropterin, significantly retarded atherosclerotic plaque formation in BDKRB2-expressing ApoE-deficient mice. Together our data show that the B2 bradykinin receptor is atherogenic, and the atherosclerosis-promoting function of BDKRB2 is partially caused by decreased aortic BH4 levels, which could account for eNOS uncoupling and further enhancement of ROS generation.

Post‐herpetic neuralgia (PHN) is a well‐established clinical problem with potential severe personal and socioeconomic implications. GTP cyclohydrolase 1 (GCH1) gene, which encodes the rate‐limiting enzyme in tetrahydrobiopterin synthesis, has been strongly implicated to be associated with neuropathic pain in previous animal and human studies. The rs3783641 (T > A) single‐nucleotide polymorphism (SNP) in the GCH1 gene is functional. Here we examine the association between rs3783641 and PHN. A total of 292 subjects including 103 PHN patients, 87 herpes zoster (HZ) patients and 102 healthy controls were enrolled in this study. The rs3783641 polymorphisms were detected via the high‐resolution melting curve (HRM) method. There were statistical differences between PHN group and the other two groups in genotype distribution (P = 0.029 and 0.017, respectively) and allele frequency (P = 0.032 and 0.005, respectively) of rs3783641. The proportion of subjects with AA genotype in the PHN group was significantly lower compared to HZ group and control group (P = 0.026 and 0.016, respectively). The frequency of A allele was lower in the PHN group than in control group (P = 0.005), and the frequency of T allele in the PHN group was higher than in HZ group and control group (P = 0.001 and 0.003, respectively). The results of this study suggest that the rs3783641 SNP in the GCH1 gene is associated with PHN, and the AA genotype showed a protective effect in PHN. [ABSTRACT FROM AUTHOR]

Although xanthophores are found widely among poikilothermic species, the developmental and biochemical pathways underlying differentiation of these pteridine‐ and carotenoid‐containing cells remain murky. I have identified a recessive zebrafish mutant, camembert (cmm), which displays defective xanthophore pigmentation during embryonic and larval stages with cells appearing grayish rather than yellow, but as an adult appears to have normally pigmented xanthophores and wild‐type stripe pattern. Examination of molecular markers reveals that xanthophores are present in typical numbers and position in cmm embryos; however, the localization of transcripts for the gene GTP cyclohydrolase 2 (gch2), encoding a critical protein in the pteridine biosynthetic pathway, is strikingly altered. RT‐PCR analysis indicates that gch2 transcripts in mutant embryos skip an exon or retain the intron upstream and that no correctly spliced mRNA is made. Sequencing of genomic DNA reveals that the skipped exon is intact, but the retained intron contains a deletion of approximately 180 base pairs, just upstream of the splice acceptor. Microinjection of a gch2 BAC clone rescues yellow pigmentation in camembert larvae, confirming that the pigmentation defect is due to mutation of gch2. [ABSTRACT FROM AUTHOR]

Introduction: Dopa-responsive dystonia (DRD) related to GCH1 mutation is a biochemical disorder. DRD is majorly characterized by dystonia and/or parkinsonism. Although clinical disorders show a dramatic positive response to levodopa, there are controversies over the residual signs following treatment. This study was designed to investigate the residual signs following levodopa treatment in Korean DRD patients with GCH1 mutation.Methods: A structured questionnaire was prepared to obtain information about demographic factors, clinical characteristics, genetic data, neuroimaging data and residual signs following levodopa treatment of the patients, and was sent to movement specialists at tertiary hospitals. The data collected from the returned forms were analyzed using appropriate statistical methods such as Student's t-test, Mann-Whitney U test, Chi-square test or Fisher's exact test.Results: Thirty-nine DRD Korean patients with GCH1 mutation were recruited. One patient was presented with only parkinsonism. Dystonia was completely resolved in 32 out of 38 patients following treatment, while parkinsonism improved without residual signs in 8 out of 9 patients. The frequency of the residual signs in Korean patients (15.8% for dystonia and 11.1% for parkinsonism) is similar to that observed in Chinese patients, but lower in Western patients. Furthermore, these signs were more frequent in those patients with a delay in their diagnosis, and those who were relatively older at the time of diagnosis.Conclusions: Ethnic differences, age at diagnosis, and a temporal gap between the onset and diagnosis in Korean patients may influence the remaining neurologic abnormalities of DRD. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To explore the effect of micro ribonucleic acid (miR)-124 on the spinal neuronal apoptosis and to explore its related mechanism. MATERIALS AND METHODS: The rat model of spinal cord injury (SCI) was established, agomir-124 was injected intrathecally and the effect of agomir-124 on motor function recovery of rats was evaluated using the Basso-Beattie-Bresnahan (BBB) score. The gene expression levels of miR-124 and GTP-cyclohydrolase 1 (GCH1) in spinal cord tissues were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), and the correlation between them was detected using the Pearson correlation coefficient. Then, the direct interaction between miR-124 and GCH1 mRNA was detected using the TargetScan software and luciferase reporter assay. The changes in apoptosis in each group were examined via flow cytometry and Western blotting. Moreover, the changes in the tetrahydrobiopterin (BH4) content in each group were detected via high-performance liquid chromatography, and the changes in the nitrite level in the supernatant in each group were detected using the Griess reagent. Finally, the changes in the activity of the inducible nitric oxide synthase (iNOS) protein were detected using the iNOS kit. RESULTS: Compared with that in the model group, the BBB score was significantly increased in agomir-124 group at 21, 28, 35 and 42 d. In the agomir-124 group, the relative expression level of miR 124 in spinal cord tissues was significantly increased at 7-28 d and reached the peak at 21 d, while the mRNA level of GCH1 in spinal cord tissues declined and touched the bottom at 21 d. According to the Pearson correlation coefficient, there was a significant negative correlation between the expression of miR-124 and mRNA expression of GCH1 (r =- 0.87, p = 1.5e-6). It was found in the prediction using TargetScan software that GCH1 might be a potential target for miR-124, which was further verified by the luciferase reporter assay. The results of flow cytometry and Western blotting showed that miR-124 significantly reduced the LPS-induced primary spinal neuronal apoptosis, while the miR-124 inhibitor remarkably increased the primary spinal neuronal apoptosis. Moreover, it was also found that the knockout of GCH1 reduced the LPS-induced spinal neuronal apoptosis. In addition, the GCH1 overexpression assay revealed that miR-124 inhibited spinal neuronal apoptosis by suppressing the GCH1 expression. LPS + miR-124 remarkably decreased the BH4 content, nitrite level, and iNOS activity while LPS + miR-124 + GCH1 remarkably increased the BH4 content, nitrite level, and iNOS activity. CONCLUSIONS: MiR-124 inhibits neuronal apoptosis in SCI by binding to GCH1. The results in the present study may provide a new mechanism for the therapeutic effect of miR-124, and miR-124 may have a potential therapeutic value in the treatment of SCI in the future. [ABSTRACT FROM AUTHOR]

Dopa-responsive dystonia (DRD), a movement disorder, is characterized by young onset dystonia and dramatic response to levodopa treatment. However, the wide range of phenotypic spectrum of the disease often leads to misdiagnosis. DRD is usually caused by mutation in GCH1 gene coding for GTP cyclohydrolase 1 (GTPCH1) enzyme, which is involved in biosynthesis of tetrahydrobiopterin (BH4) and dopamine. In this study, the entire GCH1 gene was screened in 14 Indian DRD patients and their family members. A family was identified where the proband was found to be a compound heterozygote for GCH1 (p.R184H and p.V204I) variants; the former variant being inherited from the father and the latter from the mother. All other family members harboring one of these GCH1 variants were asymptomatic except for one (heterozygous for p.R184H) who was diagnosed with DRD. In silico analyses predicted these two variants to be pathogenic and disruptive to GCH1enzymatic activity. This proband was misdiagnosed as cerebral palsy and remained untreated for 25 years. He developed retrograde movements and gait problems in lower limbs, deformity in upper limbs, and difficulty in swallowing, and became mute. However, most of his symptoms were alleviated upon levodopa administration. Our study confirms the variability of DRD phenotype and the reduced penetrance of GCH1 mutations. It also emphasizes the need of molecular diagnostic test and L-dopa trial especially for those with atypical DRD phenotype. [ABSTRACT FROM AUTHOR]

Abstract Background GTP cyclohydrolase I (GTPCH) deficiency could impair the synthesis of tetrahydrobiopterin and causes metabolic diseases involving phenylalanine catabolism, neurotransmitter synthesis, nitric oxide production and so on. Though improvements could be achieved by tetrahydrobiopterin and neurotransmitter precursor levodopa supplementation, residual motor and mental deficits remain in some patients. An appropriate GTPCH deficiency animal model with clinical symptoms, especially the motor impairments, is still not available for mechanism and therapy studies yet. Objectives and methods To investigate whether the heterozygous GTPCH missense mutation p.Leu117Arg identified from a patient with severe infancy-onset dopa-responsive motor impairments is causative and establish a clinical relevant GTPCH deficiency mouse model, we generated a mouse mutant mimicking this missense mutation using the CRISPR/Cas9 technology. Series of characterization experiments on the heterozygous and homozygous mutants were conducted. Results The expressions of GTPCH were not significantly changed in the mutants, but the enzyme activities were impaired in the homozygous mutants. BH4 reduction and phenylalanine accumulation were observed both in the liver and brain of the homozygous mutants. Severer metabolic disturbance occurred in the brain than in the liver. Significant reduction of neurotransmitter dopamine, norepinephrine and serotonin was observed in the brains of homozygous mutants. Live-born homozygous mutants exhibited infancy-onset motor and vocalization deficits similar to the disease symptoms observed in the patient, while no obvious symptoms were observed in the young heterozygous mutant mice. With benserazide-levodopa treatment, survival of the homozygous mutants was improved but not completely rescued. Conclusions The GTPCH p.Leu117Arg missense mutation is deleterious and could cause tetrahydrobiopterin, phenylalanine and neurotransmitter metabolic disturbances and infancy-onset motor dysfunctions recessively. This is the first GTPCH deficiency mouse model which could be live-born and exhibits significant motor impairments. The different extents of BH4 reduction and phenylalanine accumulation observed between liver and brain in response to GTPCH deficiency gives potential new insights into the vulnerability of brain to GTPCH deficiency. Highlights • The novel GCH1 missense mutation p.Leu117Arg is a deleterious recessive mutation. • Established the first GTPCH deficiency animal model with motor impairments • Severer BH4 reduction and phenylalanine accumulation in the brain than the liver [ABSTRACT FROM AUTHOR]

DYT-GCH1 is the most common form of Dopa-responsive dystonia. We analysed a three-generation Portuguese family with the same variant of DYT-GCH1. Dystonia and parkinsonism were found, along with non-motor symptoms. There was a wide array of phenotypic manifestations. Further studies are needed to explain the wide clinical expression of this entity. [ABSTRACT FROM AUTHOR]

Guanosine triphosphate cyclohydrolase I (GCH1) mutations are associated with increased risk for dopa-responsive dystonia (DRD) and Parkinson’s disease (PD). Herein, we investigated the frequency of GCH1 mutations and clinical symptoms in patients with clinically diagnosed PD and DRD. We used the Sanger method to screen entire exons in 268 patients with PD and 26 patients with DRD, with the examinations of brain magnetic resonance imaging scans, striatal dopamine transporter scans, and [123I] metaiodobenzylguanidine (MIBG) myocardiac scintigraphy scans. We identified 15 patients with heterozygous GCH1 mutations from seven probands and five sporadic cases. The prevalence of GCH1 mutations in probands was different between PD [1.9% (5/268)] and DRD [26.9% (7/26)] (p value < 0.0001). The onset age tends to be different between PD and DRD patients: 35.4 ± 25.3 and 16.5 ± 13.6, respectively (average ± SD; p = 0.08). Most of the patients were women (14/15). Dystonia was common symptom, and dysautonomia and cognitive decline were uncommon in our PD and DRD. All patients presented mild parkinsonism or dystonia with excellent response to levodopa. Seven of seven DRD and three of five PD presented normal heart-to-mediastinum ratio on MIBG myocardial scintigraphy. Five of six DRD and three of four PD demonstrated normal densities of dopamine transporter. Our findings elucidated the clinical characteristics of PD and DRD patients due to GCH1 mutations. PD patients with GCH1 mutations also had different symptoms from those seen in typical PD. The patients with GCH1 mutations had heterogeneous clinical symptoms. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is an important molecule for vasomotor tone, and elevated NO signaling was previously hypothesized as a unique and adaptive physiological change in highland Tibetans. However, there has been lack of NO data from Tibetans living at low altitude and lowlander immigrants living at high altitude, which is crucial to test this hypothesis. Here, through cross-altitude (1990–5018 m) and cross-population (Tibetans and Han Chinese) analyses of serum NO metabolites (NOx) of 2086 individuals, we demonstrate that although Tibetans have a higher serum NOx level compared to lowlanders, Han Chinese immigrants living at high altitude show an even higher level than Tibetans. Consequently, our data contradict the previous proposal of increased NO signaling as the unique adaptive strategy in Tibetans. Instead, Tibetans have a relatively lower circulating NOx level at high altitude. This observation is further supported by data from the hypoxic experiments using human umbilical vein endothelial cells and gene knockout mice. No difference is detected between Tibetans and Han Chinese for endothelial nitric oxide synthase (eNOS), the key enzyme for circulating NO synthesis, suggesting that eNOS itself is unlikely to be the cause. We show that other NO synthesis-related genes (e.g. GCH1) carry Tibetan-enriched mutations significantly associated with the level of circulating NOx in Tibetans. Furthermore, gene network analysis revealed that the downregulation and upregulation of NOx is possibly achieved through distinct pathways. Collectively, our findings provide novel insights into the physiological and genetic mechanisms of the evolutionary adaptation of Tibetans to high-altitude hypoxia. [ABSTRACT FROM AUTHOR]

Human studies have demonstrated a correlation between noncoding polymorphisms of "the pain protective" haplotype in the GCH1 gene that encodes for GTP cyclohydrolase I (GTPCH1)-which leads to reduced tetrahydrobiopterin (BH4) production in cell systems-and a diminished perception of experimental and clinical pain. Here, we investigate whether heterozygous mutations in the GCH1 gene which lead to a profound BH4 reduction in patients with dopa-responsive dystonia (DRD) have any effect on pain sensitivity. The study includes an investigation of GCH1-associated biomarkers and pain sensitivity in a cohort of 22 patients with DRD and 36 controls. The patients with DRD had, when compared with controls, significantly reduced levels of BH4, neopterin, biopterin, and GTPCH1 in their urine, blood, or cytokine-stimulated fibroblasts, but their pain response with respect to non-painful stimulation, (acute) stimulus-evoked pain, or pain response after capsaicin-induced sensitization was not significantly different. A family-specific cohort of 11 patients with DRD and 11 controls were included in this study. The patients with DRD were heterozygous for the pain protective haplotype in cis with the GCH1 disease-causing mutation, c.899T>C. No effect on pain perception was observed for this combined haplotype. In conclusion, a reduced concentration of BH4 is not sufficient to alter ongoing pain sensitivity or evoked pain responses. [ABSTRACT FROM AUTHOR]

Background/aims: Cardiac autonomic nerve remodeling (ANR) is an important mechanism of atrial fibrillation (AF). GTP cyclohydrolase I, encoded by GCH1, is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthesis. Previous studies reported that increased BH4 and NO content negatively regulated nerve regeneration. This study investigated the effects of GCH1 on ANR via BH4 pathway, regulated by microRNA-206 (miR-206). Methods and results: In canines, atrial tachypacing (A-TP), together with miR-206 overexpression, increased PGP9.5 level and inhibited GCH1 expression by quantitative real-time polymerase chain reaction and western blot analysis. GCH1 was validated to be a direct target of miR-206 by luciferase assays. Meanwhile, miR-206 overexpression by lentiviruses infection into right superior pulmonary vein fat pad decreased GCH1 expression to ~40% and further reduced BH4 and NO content compared with the control canines. After infection of GCH1 overexpression lentiviruses for two weeks, atrial effective refractory period was increased compared with the control group (105.8 ± 1.537 ms vs 99.17 ± 2.007 ms, P < 0.05). Moreover, GCH1 overexpression attenuated canines' atrial PGP9.5 level to ~56% of the controls. In myocardial cells, transfection of GCH1 overexpression lentiviruses also decreased PGP9.5 expression to 26% of the control group. In patients, plasma was collected and miR-206 expression was upregulated in AF patients (n = 18) than the controls (n = 12). Conclusions: Our findings suggested that GCH1 downregulation exacerbated ANR by decreasing atrial BH4 and NO content modulated by miR-206 in A-TP canines. This indicates that GCH1 may prevent the initiation of AF through inhibiting ANR. [ABSTRACT FROM AUTHOR]

Background: GTP cyclohydrolase 1-deficient DOPA-responsive dystonia, caused by autosomal dominant mutation in the gene coding for GTP cyclohydrolase 1, is a rare disorder with a reported prevalence of 0.5 per million. A correct diagnosis of DRD is crucial, given that this is an exquisitely treatable neurogenetic disorder. Although genetic testing is now widely available, we hypothesize that DRD is still underdiagnosed and its prevalence underestimated.Methods: Molecular genetic analysis of the GCH1 gene was performed in a representative cohort of 47 Serbian patients with clinical features of DRD and in their 16 available relatives. The DRD prevalence rate in Serbia was estimated based on population size, catchment area, and the centralized Serbian referral system for rare diseases.Results: We identified 9 different GCH1 mutations in 23 individuals from 11 families, 5 of which are novel. Patients displayed a broad range of clinical phenotypes. The estimated prevalence of GCH1-related DOPA-responsive dystonia in Serbia was 2.96 per million individuals and there was no evidence for a common founder.Conclusions: Our data expand the genotypic spectrum of GCH1 and confirm the broad phenotypic spectrum of DRD in the Serbian population. The number of detected mutation carriers in this sample implies that the frequency of DRD in the Serbian population is considerably higher than expected based on published prevalence rates, suggesting that the prevalence of this treatable disease should be revisited also in other populations. [ABSTRACT FROM AUTHOR]

A large meta-analysis recently identified six new loci associated with risk of PD, but subsequent studies have given discrepant results. Here we conducted a case-control study in a Han Chinese population in an attempt to clarify risk associations in Chinese. Among the four single-nucleotide polymorphisms (SNPs) that we examined – VPS13C-rs2414739, MIR4697-rs329648, GCH1-rs11158026, and SIPA1L2- rs10797576 we detected a significant association between rs329648 and risk of developing PD in a recessive model. This association remained significant after adjusting for gender and age (OR 1.87, 95%CI 1.295–2.694, p = 8.21 × 10 −4 ) or Bonferroni correction. The T allele of rs329648 occurred significantly more frequently among patients with PD than among healthy controls (OR 1.22, 95%CI 1.033–1.443, p = 0.02), while there was no statistic significant after Bonferroni correction. Subgroup analysis showed a significant association specifically among males in a recessive model (OR 1.943, 95%CI 1.200–3.147, p = 0.007). In contrast, genotye and allele frequencies at rs329648 did not differ significantly between female patients with PD and healthy female controls, or between patients with early-onset or late-onset PD. Our results suggest that rs329648 is associated with risk of developing PD in the Han Chinese population. Our findings should be verified in further studies, and they highlight the need for functional studies of MIR4697. [ABSTRACT FROM AUTHOR]

Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene, Gch1 , is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers. Gch1 is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced Gch1 expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell-derived sensory neurons. Graphical overview., Competing Interests: Competing interestsThe authors have no competing financial interests., (Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC.)

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Prevalence of PD increases steadily with age. A recent meta-analysis of genome-wide association studies has identified six new loci to be linked with PD. Here we investigated the association of four of these new loci, SIPA1L2 , MIR4697 , GCH1 and VPS13C with PD in an Iranian population. Through a case-control study a total of 1800 subjects comprising 600 PD patients and 1200 unrelated healthy controls were recruited. Rs10797576, rs329648, rs11158026 and rs2414739 related to SIPA1L2, MIR4697, GCH1 and VPS13C loci respectively, were genotyped in all subjects. The difference of genotype and allele frequencies between case and control groups were investigated using chi-square test and logistic regression models with R software. Genotype and allele frequencies were significantly different in PD patients and control group for rs329648, rs11158026 and rs2414739 ( p -value = 0.018, 0.025, and 0.009 respectively for allele frequency differences). There was no difference in genotype nor allele frequencies between the two groups for rs10797576. We replicated the association of three new loci which are proposed for PD. More studies in other populations and also functional analysis are required to clear the role of these variants in PD. [ABSTRACT FROM AUTHOR]