Congenic mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse

doi:10.1016/j.ymgme.2004.04.006

Molecular Genetics and Metabolism

Volume 82, Issue 3, July 2004, Pages 251-254

https://doi.org/10.1016/j.ymgme.2004.04.006 Get rights and content

Abstract

The hph-1 ENU-mutant mouse provides a model of tetrahydrobiopterin deficiency for studying hyperphenylalaninaemia, dopa–response dystonia, and vascular dysfunction. We have successively localized the hph-1 mutation to a congenic interval of 1.6–2.8 Mb, containing the GCH gene encoding GTP cyclohydrolase I (GTP-CH I). We used these data to establish a PCR method for genotyping wild type, hph-1 and heterozygote mice, and found that heterozygote animals have partial tetrahydrobiopterin deficiency. These new findings will extend the utility of the hph-1 mouse in studies of GTP-CH I deficiency.

Introduction

Tetrahydrobiopterin (BH4) is a required co-factor for the aromatic amino acid hydroxylases that are key enzymes in phenylalanine metabolism and neurotransmitter biosynthesis [1]. BH4 is also required by all nitric oxide synthase (NOS) isoforms for nitric oxide synthesis. BH4 biosynthesis proceeds from a de novo pathway from guanosine triphosphate (GTP), and the committing and rate-limiting enzyme is GTP cyclohydrolase I (GTP-CH I; EC 3.5.4.16) [1]. In humans, mutations in GCH lead to severe BH4 deficiency syndromes that include hyperphenylalaninaemia and dopa–responsive dystonia (DRD) [2]. Relative BH4 deficiency, which occurs in the endothelium in vascular disease states [3], [4], causes NOS uncoupling, characterized by reduced NO production and increased NOS-dependent superoxide production [5].

The hph-1 mouse, generated by ENU mutagenesis, has attracted considerable interest as a metabolic model of BH4 deficiency in relation to dopamine and serotonin biosynthesis, and more recently, vascular NOS dysfunction [6], [7], [8], [9], [10]. Following mutagenesis, (C57BL/6 × CBA/Ca) F₁ mice were selected for hyperphenylalaninaemia [11]. The biochemical defect in the hph-1 mouse is deficient GTP-CH I activity due to reduced GCH mRNA expression [12], [13], resulting in low tissue BH4 concentrations [9], [14]. Although the nature and site of the hph-1 allele is unknown, unpublished data cited by Gutlich et al. [13] suggested that it lies in CBA/Ca DNA. Linkage analysis indicated that the hph-1 allele and GCH are within 8 cM of each other on mouse chromosome 14 [15]. However, no mutation has been detected in the coding region or 746 bp upstream of GCH [13], [16].

The hph-1 strain has been crossed onto a C57BL/6 background, selecting offspring on the basis of hyperphenylalaninemia. Due to the lack of a validated genotyping assay, previous studies using hph-1 mice have used either separately maintained C57BL/6, (C57BL/6 × CBA), or CBA wild-type strains as controls [9], [16]. This is an important limitation, as it is recognized that strain differences may have major effects on biological responses and hence experimental results [17]. In addition to the benefit of using matched littermate controls, genotyping hph-1 mouse in breeding experiments would allow study of hph-1 heterozygotes and informative cross-breeding with other genetically modified mouse models.

Accordingly, we aimed to define the CBA congenic interval in the hph-1 mouse, and use these new data to develop a robust genotyping assay to facilitate hph-1 breeding programs. We further sought to successively localize the causative hph-1 mutation to a minimal congenic fragment in recombinant animals identified from hph-1/C57bl6 crosses, and to phenotype the degree of BH4 deficiency in heterozygote animals.

Section snippets

Materials and methods

C57BL/6, CBA/Ca, and hph-1 mice were used for mapping of the congenic interval. Hph-1 mice were mated with C57BL/6 mice to produce obligate heterozygotes, and these heterozygotes were then mated with each other to produce wild type (WT), hph-1 heterozygotes (+/−), and hph-1 littermates. All studies were conducted in accordance with UK Home Office Animals (Scientific Procedures) Act 1986.

DNA from tail tips was prepared by standard phenol–chloroform extraction. Using polymerase chain reaction

Results and discussion

Allelic lengths obtained for each marker with C57BL/6, CBA, and hph-1 strains are detailed in Table 1. The markers were able to differentiate between C57BL/6 and CBA strains. In the hph-1 mouse, a region on chromosome 14 containing the GCH locus, of maximum size 14.9 Mb and minimum size 11.9 Mb (position 38.4–50.3, and 38.1–53.0 Mb, respectively), was identified by markers D14MIT60, D14MIT121, D14MIT101, D14MIT61, D14MIT62, D14MIT142, D14MIT268, D14MIT83, and D14MIT234 to be of CBA origin (Table 1

Acknowledgements

Supported by the British Heart Foundation and The Wellcome Trust. J.P.K. is a Wellcome Trust Cardiovascular Research Initiative Clinical Training Fellow.

References (19)

J.E Barker et al.
Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse
Brain Res.
(1998)
M Gutlich et al.
Molecular characterization of HPH-1: a mouse mutant deficient in GTP cyclohydrolase I activity
Biochem. Biophys. Res. Commun.
(1994)
C.S Montanez et al.
Linkage analysis of the hph-1 mutation and the GTP cyclohydrolase I structural gene
Mol. Genet. Metab.
(1999)
T Maeda et al.
Studies on the genotype-phenotype relation in the hph-1 mouse mutant deficient in guanosine triphosphate (GTP) cyclohydrolase I activity
Brain Dev.
(2000)
B Thony et al.
Tetrahydrobiopterin biosynthesis, regeneration and functions
Biochem. J.
(2000)
A Niederwieser et al.
GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia
Eur. J. Pediatr.
(1984)
F Cosentino et al.
Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries
Circulation
(1995)
N.J Alp et al.
Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I over-expression
J. Clin. Invest.
(2003)
J Vasquez-Vivar et al.
Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors
Proc. Natl. Acad. Sci. USA
(1998)

There are more references available in the full text version of this article.

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GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age
2021, Microvascular Research
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The mutant hyperphenylalaninemic mouse (hph-1) created by Simon and his team in 1987 (Bode et al., 1988) was selected for the evaluation of BH4 deficiency on vascular function. The hph-1 mouse displayed 90% deficiency of GTP-CH-1 expression in the whole body (McDonald et al., 1988; Khoo et al., 2004). The GTP-CH activity is decreased at least by 50% in aorta and mesenteric artery leading to similar decrease in BH4 level (Cosentino et al., 2001; D'Uscio et al., 2011).
The present study assessed the impact of impaired tetrahydrobiopterin (BH₄) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH₄ biosynthesis. BH₄ is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity.
In GTP-CH deficient mice, the aortic BH₄ levels were decreased, by −77% in 12 week-middle-aged mice (young) and by −83% in 35–45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (−46%), in flow-mediated vasodilation of mesenteric artery (−31%), in coronary hyperemia response measured in isolated heart following transient ischemia (−27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (−69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age.
Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH₄ was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH₄-deletion impact on the microcirculation.
A requirement for Gch1 and tetrahydrobiopterin in embryonic development
2015, Developmental Biology
Citation Excerpt :
This is a critical question to devise new treatment strategies. Previous studies of the importance of BH4 biosynthesis have used systemic pharmacologic inhibitors of GTPCH (Cotton, 1986; Mitchell et al., 2003), or the hph-1 hyperphenylalaninemic mouse, generated by ENU mutagenesis, that has moderate systemic BH4 deficiency due to reduced gene expression from the GCH1 locus (Cosentino et al., 2001; Khoo et al., 2004). However, in the hph-1 mouse, variable BH4 deficiency in different cell and tissue types, and the moderate reduction in GTPCH activity, results in no gross abnormality in foetal development or survival, and limit the interpretation of the role in GTPCH in development (McDonald and Bode, 1988).
GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo.
Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2–3 of the Gch1 gene. Gch1^fl/fl mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1^−/− embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1^−/− embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA.
These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4.
Anxiety- and depression-like phenotype of hph-1 mice deficient in tetrahydrobiopterin
2014, Neuroscience Research
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Moreover, previous studies report that hph-1 mice exhibit lower concentrations of BH4 and monoamines in the brain (Hyland et al., 1996), as well as decreased NO formation in brain astrocytes and blood (Barker et al., 1998; Lam et al., 2007). The hph-1 mutation has been localised to an interval of 1.6–2.8 Mb on chromosome 14, containing the Gch1 gene (Khoo et al., 2004), although the exact location is still undefined. To elucidate if the reported affective disturbances in DRD patients translates back to the hph-1 mouse model of DRD, this study investigated if hph-1 mice display altered phenotype in behavioural tests related to anxiety and depression.
Decreased tetrahydrobiopterin (BH4) biosynthesis has been implicated in the pathophysiology of anxiety and depression. The aim of this study was therefore to characterise the phenotype of homozygous hph-1 (hph) mice, a model of BH4 deficiency, in behavioural tests of anxiety and depression as well as determine hippocampal monoamine and plasma nitric oxide levels. In the elevated zero maze test, hph mice displayed increased anxiety-like responses compared to wild-type mice, while the marble burying test revealed decreased anxiety-like behaviour. This was particularly observed in male mice. In the tail suspension test, hph mice of both sexes displayed increased depression-like behaviours compared to wild-type counterparts, whereas the forced swim test showed a trend towards increased depression-like behaviours in male hph mice, but significant decrease in depression-like behaviours in female mice. This study provides the first evidence that congenital BH4 deficiency regulates anxiety- and depression-like behaviours. The altered responses observed possibly reflect decreased hippocampal serotonin and dopamine found in hph mice compared to wild-type mice, but also reduced nitric oxide formation. We propose that the hph-1 mouse may be a novel tool to investigate the role of BH4 deficiency in anxiety and depression.
Endothelial nitric oxide synthase in the vascular wall: Mechanisms regulating its expression and enzymatic function
2011, Artery Research
Endothelial nitric oxide synthase (eNOS) is the main source of nitric oxide (NO) in the vascular wall, a molecule with anti-inflammatory, antithrombotic, vasorelaxant, antioxidant and finally antiatherogenic properties. eNOS is expressed in vascular endothelium, and it uses l-arginine as a substrate, while it also requires the presence of multiple co-factors such as tetrahydrobiopterin (BH4), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) and others. In the presence of BH4 deficiency, this enzyme becomes uncoupled, and it is turned into a source of superoxide radicals instead of NO. Therefore, under these conditions which are present in patients with advanced atherosclerosis, eNOS in human vascular endothelium is largely a source of reactive oxygen species, inducing in this way atherogenesis. Therefore, the aim of future therapeutic strategies targeting atherosclerosis through regulation of eNOS physiology, should take into account that up-regulation of this enzyme in the vascular wall may not lead to a respective increase of NO bioavailability and improvement of vascular homeostasis, but it may actually induce intravascular oxidative stress, if intracellular bioavailability of eNOS co-factors is not simultaneously elevated. In conclusion, eNOS plays a critical role in the regulation of vascular homeostasis, and it is a therapeutic target against atherogenesis.
Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency
2011, Free Radical Biology and Medicine
Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase (eNOS), and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production (eNOS coupling). Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I (GTPCH). However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but whether DHFR is functionally important in maintaining eNOS coupling remains unclear. To investigate the mechanism by which DHFR might regulate eNOS coupling in vivo, we treated wild-type, BH4-deficient (hph-1), and GTPCH-overexpressing (GCH-Tg) mice with methotrexate (MTX), to inhibit BH4 recycling by DHFR. MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice. These effects were magnified in hph-1 but diminished in GCH-Tg mice. Attenuated eNOS activity was observed in MTX-treated hph-1 but not wild-type or GCH-Tg mouse lung, suggesting that inhibition of DHFR in BH4-deficient states leads to eNOS uncoupling. Taken together, these data reveal a key role for DHFR in regulating the BH4 vs BH2 ratio and eNOS coupling under conditions of low total biopterin availability in vivo.
GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression
2009, Journal of Biological Chemistry
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Altered GCH1 Expression in the hph-1 Mouse—Having established quantitative relationships between GCH1 expression, GTPCH, and BH4 in a reductionist cell culture system, we next investigated how primary alterations in GCH1 expression in vivo would alter GTPCH protein, enzymatic activity, and BH4 levels and the effect of these changes on GFRP levels. We determined these parameters in the hph-1 ENU mutant mouse model of reduced GCH1 expression, comparing matched Wt, heterozygote (+/-) and hph-1 (hph) littermates from heterozygote breeding pairs (13). Using real time RT-PCR, we observed a graded reduction in mouse GCH1 mRNA expression in livers of Wt, +/-, and hph mice, by 33% in +/- and by 83% in hph mice (Fig. 8a), confirming that the breeding of heterozygote pairs of hph-1 mice provides an in vivo model of a quantitative reduction in GCH1 expression, across an ∼10-fold range, on an otherwise identical genetic background.
GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r² = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r² = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

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¹: These authors contributed equally to this work.

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Brief CommunicationCongenic mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgements

Brain Res.

Biochem. Biophys. Res. Commun.

Mol. Genet. Metab.

Brain Dev.

Tetrahydrobiopterin biosynthesis, regeneration and functions

Biochem. J.

GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia

Eur. J. Pediatr.

Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries

Circulation

Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I over-expression

J. Clin. Invest.

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors

Proc. Natl. Acad. Sci. USA

Brief Communication
Congenic mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse