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Introduction In 2009, we described a possible founder effect of autosomal dominant Segawa disease in Cordoba (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 non-responsive 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.

Introducción. En 2009, neuropediatras y neurólogos del Hospital Universitario Reina Sofia describieron un posible efecto fundador de la enfermedad de Segawa autosomica dominante en Córdoba (España) debido a la mutación c.265C> T (p. Q89*) en el gen GCH1. Se ha coordinado un estudio multicéntrico con el objetivo de mejorar el conocimiento de la situación de la enfermedad de Segawa en España y proporcionar una descripción fenotípica y genotípica detallada de los pacientes así como la respuesta al tratamiento y valorar posibles biomarcadores. Métodos. La información clínico-genética se obtuvo a partir de cuestionarios estandarizados que fueron completados por los neurólogos que atendieron a niños y/o adultos de 16 hospitales españoles. Se realizo el estudio in silico de las variantes genéticas noveles. El efecto fundador de la variante p.Q89* se analizó mediante el estudio de haplotipos satélites. Se analizaron prolactina sérica y pterinas en orina y en sangre seca como posibles biomarcadores para el diagnóstico y seguimiento de la enfermedad. Resultados. Ochenta sujetos pertenecientes a 24 familias mostraron mutaciones/deleciones en el gen GCH1 compatibles con la enfermedad de Segawa. Se describieron siete variantes genéticas exclusivas en la cohorte de pacientes, 5 de las cuales son mutaciones noveles. Se detectaron cinco familias con p.Q89*, no descritas previamente, en Andalucía debido a un posible efecto fundador. La mediana de latencia diagnostica fue de 5 anos (RIC 0- 16). Nueve de las 24 familias tenían miembros diagnosticados de la enfermedad de Parkinson en sus antecedentes familiares. Los signos y/o síntomas más frecuentes fueron distonía de miembros inferiores (38/56, 67,8%, p = 0,008) y fluctuaciones diurnas de la sintomatología motora (38/56, 67,8%, p = 0,008). Las fluctuaciones diurnas no estuvieron presentes en los fenotipos distintos de la distonía. Cincuenta y tres de 56 pacientes sintomáticos fueron tratados con un L-Dopa/inhibidor de decarboxilasa periférica durante (media +- DE) 12,4 +-} 8,12 años. En un 81% de casos se emplearon dosis inferiores a 350 mg/día (o ≤5 mg/kg/día en niños). Once de 53 (20%) pacientes presentaron síntomas que no respondían a L-Dopa/inhibidor de decarboxilasa periférica y que les afectaban en las actividades de la vida diaria. Las discinesias (en 4 sujetos) fueron los efectos adversos más destacados. No se observó deterioro de la respuesta al tratamiento con LDopa/ inhibidor de decarboxilasa periférica a largo plazo. Se analizo la prolactina sérica como posible biomarcador de seguimiento en 14 individuos, resultando normales en todos los casos. Se analizaron pterinas en orina y en sangre seca en 15 individuos afectos de la enfermedad y se comparó con un grupo control, observándose que presentaron valores significativamente más bajos tanto para la excreción de neopterina como de biopterina en orina (prueba t de Student; p T (p.Q89*) mutation in the GCH1 gene. A multicenter study has been coordinated to improve the knowledge of the situation of Segawa disease in Spain and to provide a detailed phenotypic and genotypic description of the patients as well as the response to treatment and to assess possible biomarkers. Methods. The clinical-genetic information was obtained from standardized questionnaires that were completed by neurologists who cared for children and/or adults from 16 Spanish hospitals. The in silico study of the novel genetic variants was carried out. The founder effect of p.Q89* was analyzed by studying satellite haplotypes. Serum prolactin and pterins in urine and dried blood were analyzed as possible biomarkers for the diagnosis and follow-up of the disease. Results. Eighty subjects belonging to 24 families showed mutations in GCH1 compatible with Segawa disease. Seven exclusive genetic variants have been described in the cohort of patients, 5 of which are novel mutations. Five previously undescribed families with p.Q89* were detected in Andalusia due to a possible founder effect. The median latency to diagnosis was 5 years (IQR 0-16). Nine of the 24 families had members diagnosed with Parkinson's disease in their family history. The most frequent signs and/or symptoms were lower limb dystonia (38/56, 67.8%, p = 0.008) and diurnal fluctuations in motor symptoms (38/56, 67.8%, p = 0.008). Diurnal fluctuations were not present in phenotypes other than dystonia. Fifty-three of 56 symptomatic patients were treated with an L Dopa/periferic decarboxylase inhibitor for (mean +-SD) 12.4 +-8.12 years. In 81% of cases, doses lower than 350 mg/day (or ≤5 mg / kg / day in children) were used. Eleven of 53 (20%) patients had non-responsive symptoms and that affected daily life activities. Dyskinesias (in 4 subjects) were the most prominent adverse effects. No deterioration in response to long-term L-Dopa/periferic decarboxylase inhibitor treatment was observed. Serum prolactin was analyzed as a possible follow-up biomarker in 14 individuals, all results were normal. Pterins were analyzed in urine and in dried blood in 15 individuals affected by the disease and compared with a control group, observing that they presented significantly lower values for both the excretion of neopterin and biopterin in urine (Student's t test; p < 0.0001). Conclusión. This study identified 5 novel disease-causing mutations in the GCH1 gene and 7 uniquely described in this cohort. Five new families carrying the p.Q89* mutation were detected, which supports the hypothesis of a founder effect of p.Q89* in Andalusia. New insights are provided about phenotypes and long-term response to treatment, which can improve early recognition and therapeutic management. It is noteworthy that the diurnal fluctuations of motor symptoms were found exclusively in early ages and phenotypes with dystonia. Despite being a disease with good response to treatment, 20% of patients described non-responsive symptoms to L-Dopa that affected their daily life. It was found a high prevalence of Parkinson's disease in families affected by Segawa disease. Serum prolactin does not seem to be useful in monitoring the disease, however, urine pterins could be of interest as a peripheral biomarker for biochemical diagnosis.

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA–mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1β and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.

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.

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1(fl/fl)) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1(fl/fl)Tie2cre). Macrophages from Gch1(fl/fl)Tie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1(fl/fl)Tie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1(fl/fl)Tie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1(fl/fl)Tie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1(fl/fl)Tie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1(fl/fl)Tie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

Background GTP cyclohydrolase I (GCH1) mutations are the commonest cause of Dopa-responsive dystonia (DRD). Clinical phenotypes can be broad, even within a single family. Methods We present clinical, genetic and functional imaging data on a British kindred in which affected subjects display phenotypes ranging from DRD to Parkinson's disease (PD). Twelve family members were studied. Clinical examination, dopamine transporter (DAT) imaging, and molecular genetic analysis of GCH1 and the commonest known familial PD-related genes were performed. Results We have identified a novel missense variant, c.5A > G, p.(Glu2Gly), within the GCH1 gene in affected family members displaying a range of phenotypes. Two affected subjects carrying this variant had abnormal DAT imaging. These two with abnormal DAT imaging had a PD phenotype, while the remaining three subjects with the novel GCH1 variant had normal DAT imaging and a DRD phenotype. Conclusions We propose that this GCH1 variant is pathogenic in this family and these findings suggest that similar mechanisms involving abnormal GTP cyclohydolase I may underlie both PD and DRD. GCH1 genetic testing should be considered in patients with PD and a family history of DRD., Highlights • We have identified a novel missense variant, c.5A > G, p.(Glu2Gly), within the GCH1 gene in family with Dopa responsive dystonia (DRD) and parkinsonism. • Those with parkinsonism had abnormal DaTscans, indicating nigrostriatal neurodegeneration. • These findings suggest that similar mechanisms involving abnormal GTP cyclohydolase I may underlie both Parkinson's disease and Dopa responsive dystonia.

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation., Graphical abstract, Highlights • Gch1−/− macrophages lack GTPCH protein expression, rendering cells BH4 deficient. • Gch1−/− cells express normal iNOS protein levels, but lack nitric oxide production. • BH4-deficient cells exhibit higher cellular ROS production. • Alterations in NO and ROS production can be reversed by pharmacological BH4 rescue. • Stimulated BH4-deficient cells show defective NRF2-dependent gene expression.

Background: Provoked vestibulodynia (PVD) is a pain disorder localized in the vestibular mucosa. It is the most common cause of dyspareunia among young women and it is associated with general pain hypersensitivity and other chronic pain conditions. Polymorphism in the guanosine triphosphate cyclohydrolase ( GCH1) gene has been found to influence general pain sensitivity and the risk of developing a longstanding pain condition. The aim of this study was to investigate GCH1-polymorphism in women with PVD and healthy controls, in correlation to pain sensitivity. Results: We found no correlation between the previously defined pain-protective GCH1-SNP combination and the diagnosis of PVD. Nor any correlation with pain sensitivity measured as pressure pain thresholds on the arm, leg and in the vestibule, coital pain scored on a visual analog scale and prevalence of other bodily pain conditions among women with PVD (n = 98) and healthy controls (n = 102). However, among patients with current treatment (n = 36), there was a significant interaction effect of GCH1-gene polymorphism and hormonal contraceptive (HC) therapy on coital pain (p = 0.04) as well as on pressure pain thresholds on the arm (p = 0.04). PVD patients carrying the specified SNP combination and using HCs had higher pain sensitivity compared to non-carriers. In non-HC-users, carriers had lower pain sensitivity. Conclusions: The results of this study gave no support to the hypothesis that polymorphism in the GCH1-gene contributes to the etiology of PVD. However, among patients currently receiving treatment an interaction effect of the defined SNP combination and use of hormonal contraceptives on pain sensitivity was found. This finding offers a possible explanation to the clinically known fact that some PVD patients improve after cessation of hormonal contraceptives, indicating that PVD patients carrying the defined SNP combination of GCH1 would benefit from this intervention.